Kinase assays performed by Nicholas Ting, Dr. Susan Lees-. Miller and Dr. David Litchfield. ...... Reece and Platt, 1997; Drysdale et aL, 1998). 1.1.4 Activated ...
NOTE TO USERS
The original manuscript received by UMI contains pages with indistinct print. Pages were microfilmed as received. This reproduction is the best copy avaiiable
Composition, Regulation and Function of the Yeast ADA Complex
Ayman M. Saleh Department of Biochemistry r
/
Submitted in partial fulfillrnent of the requirement for the degree of Doctor of Philosophy
Facuity of Graduate Studies The University of Western Ontario London, Ontario July 1998
O
Ayman M. Saleh
1998
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ABSTRACT
The yeast ADAs are dual function regulators that stimulate or inhibit a set
of yeast transactivator proteins. Initial studies with three proteins, ADA2p, NGGl/ADA3p and GCNSp, demonstrated that they associate in vitro. I have analyzed the interaction between NGGl p and ADA2p from whole yeast extracts (MNE). NGGl p and ADA2p coimmunoprecipitate from WYE. In agreement with
their association in vivo, the stability of ADA2p and NGGl p depend on the presence of each other. In addition, a combination of ion-exchange and gelfiltration chromatography columns resolved four complexes containing ADA2p and NGG1p. Two of these had estimated sizes of -2 MDa; the other two were -900 and -200 kDa (ADA complexes). The basal factor TBP
coimmunoprecipitates with NGGl p, suggesting a role of the complexes in regulating TBP activity. We identified a new component of the ADA complexes, TRAl p, through the tandem mass spectrometry of proteins associated with NGGl p. TRAl p is a member of group of putative protein kinases, with a carboxyl-terminal region related to phosphatidylinositol 3-kinases. The interaction between TRAl p and ADA components was verified by the reciprocal coimmunoprecipitation of ADA2p and TRAl p from WYE and coelution of TRAl p with complexes that also containad SPT7p (ADAISPT complexes) on ion-exchange and DNA cellulose columns. I also provide evidence that the yeast hect-domain protein TOM1p regulates transcriptional activation through effects on the ADA proteins. Single and double nuIl mutations of tom7 and nggl result in a similar alteration of transcription from ADH2, HlS3 and GAL10 promoters, implying that TOM1 and
iii
NGGl act through the same pathway. In the absence of TOMlp, the normal
associations of the ADA proteins with SPT3p and TBP are reduced. The action of TOMl p is likely mediated through ubiquitination since mutation of Cys3235 to Ala, corresponding residues of which are required for thioester bond formation with ubiquitin in other hect-domain proteins, results in similar changes in transcription as the nuil mutation. A direct role for TOMl p in regulation of ADAassociated proteins was further supported by the finding that SPT7p was ubiquitinated in a TOMl p-dependent fashion and that TOMl p coimmunoprecipitated with the ADA proteins.
Keywords: Coactivators, Transcription, ADA complex, SPT, TBP, Ubiquitin ligase, TRAI. TOM1.
CO-AUTHORSHIP Chapter 2 of this thesis contains materials from a previously published manuscript CO-authoredby Ayman Saleh, Vema Lang, Robert Cook and Christopher J. Brandl. All the experiments presented in this chapter were performed by Ayman Saleh. Technical assistance and reagents for FPLC were provided by Vema Lang and Dr. Robert Cook. The results presented in chapter 4 have been accepted for publication in the Journal of Molecular Biology (in press; see appendix 3). This manuscript is CO-authoredby Ayman Saleh, Collart, M., Martens, J.A., Genereaux, J., Allard,
S., Cote', J. and Brandl, C.J. All the experimental work was performed by Ayman Saleh, with the exception of the measurements of histone acetyltransferase activity of the immunoprecipitated ADA complexes which was done by Dr. J. Cote and S. Allard. Dr. M. Collart perfomed the Western blot with the SPT3p antibody, and J. Martens provided the UBP3 construct. A version of Chapter 3 has been submitted recently for publication to the Journal of Biological Chemistiy. All the figures presented in this chapter were the result of work by Ayman Saleh. The tandem mass spectrometric analysis was carried out by Dr. D. Schieltz and Dr. J. Yates III, and the kinase assays by
Dr. D. Litchfield, Nicholas Ting and Dr. S. Lees-Miller. Original manuscripts, which appear in Chapters 2-4, were written by Ayman Saleh and Christopher Brandl. For copyright releases see appendices 2 and 3.
ACKNOWLEDGMENTS For many, many, hours of guidance and assistance, for a Stream of ideas that laid the foundation of this work and helped bring it ta completion, for continuous encouragement, especially in those moments when I needed it most, to Dr. Christopher J. Brandl, the advisor and above al1 the friend, I express my deepest gratitude. My CO-workersin the Brandl lab deserve much recognition for their helpful suggestions relating to this work. Thanks to Joe Martens and Julie Genereaux. I am indebted to my advisory cornmittee Dr. David Haniford and Dr. Eric Ball for their superb guidance and mentoring. Special thanks go to Dr. Peter Grant , Jacques Cote' and Dr. Jerry Workman for communicating unpublished results. Also I am grateful to Dr. George Chaconas, Dr. Eric Ball, Dr. Mark Watson, Dr. BR Lavoie and Dr. David Litchfield for their useful comments during the pwparation of manuscripts for publication. I am further humbled to the kindness of Dr. Jim Ingles, Dr. Michael Schultz, Dr. Morris Manolson, Dr. Fred Winston and Lisa Pacella for providing some plasmids, yeast strains and antibodies. I obtained assistance and advice with much of the experiments
presented in this thesis. Anne Brickendon prepared the Myc ascites used in these studies. Dr. Christopher Brandl was responsible for generating several constructs, including myc-NGG1 derivatives, Cys-Ala mutations of TOM7 and myc-TRA 1. Joe Martens provided the GAL4-UBP3 fusion molecule. Tandem
mass spectrometry analyses were expertly done by Dr. David Schieltz and Dr. John Yates III. Kinase assays performed by Nicholas Ting, Dr. Susan LeesMiller and Dr. David Litchfield. The contribution of Dr. Jacques Cote' to the histone acetyltransferase activity and Dr. Martine Collart with the SPT3p
antibody are also gratefully acknowledged. Vema Lang and Dr. Robert Cook graciously assisted in the early stages of the project by providing various FPLC chromatography columns. The work of this thesis would not be complete without the remarkable contribution of the above collaborators. Some acknowledgments are hard to put into words and certainly never adequate. To my wonderful wife, Neam, I express my deepest gratitude. Without your laughter, patience and understanding, I could not have completed this work. Finally, I want to acknowledge my parents, sisters and young brother for
love and support right from the start and for lightening my load in so many ways. ARhough thousand of miles are separating between us, distance wise, your names and love will always remain there in my heart. Thanks to my family in Canada; my sister Ibtisam, her husband Mefleh, Uncles Ali, Mohammed and Jehad, Aunt Hayat and grandfather Mefleh who helped me get settled into this city and made my stay here enjoyable. My friend Bassam Saddiqe, Ayman Thaher and Fadhel Al-thaher and his family also deserve much of appreciation. This thesis is dedicated to my mother and father whose hard work and love remain a continual inspiration.
TABLE OF CONTENTS Page
CERTIFICATE OF EXAMINATION ....................................................................... ii AûSTRACT ................................................................................................................ iii CO-AUTHORSHIP .................................................................................................... v ACKNOWLEDGMENTS ......................................................................................... vi
TABLE OF CONTENTS ..........................................................................................
...
WII
LIST OF TABLES ..................................................................................................... xii
...
LIST OF FIGURES ................................................................................................... xiii LIST OF APPENDICES ........................................................................................ xv
ABBREVlGlïONS .................................................................................................... xvi
CHAPTER 1 INTRODUCTION ............................................................................1 1.1 Transcription by RNA Polymerase II ......................................................... 2 1.1.1 Eukaryotic promoter elements ........................................................2 1.1.2 Components of the basal transcription machinery ..................... 3 1.1.3 DNA-specific transcriptional activators ......................................... 6 1.1.4 Activated transcription ...................................................................... 8 1.2 Coactivator Complexes .............................................................................10 1.2.1 TFllDcomplex ................................................................................. 10 1.2.2 Components of the Pol II holoenzyme complexes ...................... 12 1.2.3 SWVSNF complex ............................................................................ 15 1.2.4 SPT protein .................................................................................. 16 1.3 Components and Functions of the ADA Complexes ............................. 19 1.3.1 NGGl protein ..................................................................................... 19 1.3.2 Structure/function properties of NGGlp ........................................ 22 1.3.3 NGGl p foms a complex with ADA2p and GCN5p ..................... 23 1.3.4 ADA 1and ADA5 indicate a functional link between ADA and SPT genes ........................................................................ 25 viii
1.3.5 ADA proteins fonn multiple complexes in vivo ............................26 1.3.6 Role of the ADA complexes in contending with nucleosomal DNA ............................................................................ 27 1.3.7 Evidence that ADNSPT complexes regulate TBP function ...... 31 1.3.8 Regulation of the ADA complexes ................................................. 32 1.4 The Ubiquitin Conjugating System: Structure and Functions .............. 34 1.4.1 Components of the ubiqulin-conjugatingsystem .......................35 1.4.2 Proteasomes and ubiquitin proteases ........................................ 39 1.4.3 Functions of ubiquitination ............................................................. 40 1.5 Significance ................................................................................................... 42 1.6 References .................................................................................................... 46
CHAPTER 2 IDENTIFICATIONOF NATIVE COMPLEXES CONTAlNING THE YEAST COACTlVATOFVREPRESSOR PROTEINS NGG1 / ADA3 AND ADA2 .................................................................................. 67 2.1 Introduction ....................................................................................................67 2.2 Materials and Methods ................................................................................ 71 2.2.1 DNAconstructs .................................................................................. 71 2.2.2 Yeast strains, media and growth conditions ................................ 71 2.2.3 Nuclear extracts ................................................................................ 73 2.2.4 Preparation of whole Cell Extract ................................................... 73 2.2.5 Fractionation of NGG 1p and ADA2p ............................................. 74 2.2.6 lmmunoprecipitationof NGG 1p and ADA2p complexes ........... 75 2.2.7 Western blotting ................................................................................. 76 2.3 Results ............................................................................................................ 77 2.3.1 Coimmunoprecipitation of NGGl p and ADA2p ........................... 77 2.3.2 NGGl p and ADA2p cofractionate on ion-exchange and gel-filtration columns ...............................................................80 2.3.3 Stability of NGGl p and ADA2p is CO-dependent........................ 89 2.3.4 NGG1p 1s a nuclear Protein ............................................................ 89 2.3.5 Amino Acids 274-307 of NGGl p are required for interaction with TBP .......................................................................... 92 2.4 Discussion .....................................................................................................98 2.4.1 NGGl p and ADA2p Are associated in rnulticomponent complexes ..........................................................................................98
2.4.2 Arnino Acids 274-307 of NGG1p Are Required for
Interaction witt.i TBP ..........................................................................100 2.4.3 Coactivators in Activation and Repression ................................... 102
CHAPTER 3 TRAI :A YEAST PROTEIN RELATED TO THE CATALYTIC SUBUNIT OF HUMAN DNA-DEPENDENT PROTEIN KINASE IS A COMPONENT OF THE A D N S P T TRANSCRIPTIONAL REGULATORYCOMPLEXES ................................................................................. 108 3.1 Introduction .................................................................................................... 108 3.2 Matenals and Methods ................................................................................ 110 3.2.1 DNA conçtructs and yeast strains .................................................. 110 3.2.2 Preparation of whole cell extract .................................................... 110 111 3.2.3 Purification of HM-NGGl p .............................................................. 3.2.4 Protein identification ........................................................................ 111 3.2.5 Fractionation of TRAl p NGG1p. ADA2p and SPT7p ................ 112 3.2.6 Immunoprecipitation and immunoblotting .................................... 113 3.3 Resutts ........................................................................................................... 114 3.3.1 Association of T M 1p with NGGI p ............................................... 114 3.3.2 Coirnmunoprecipitation of TRAl p and ADA2p ............................ 115 3.3.3 TRAl p cofractionates with ADA and SPT proteins ..................... 118 3.3.4 Binding of TRAI p to DNA-cellulose requires ADA proteins ...... 121 3.4 Discussion ................................................................................................. 127 3.5 References ..................................................................................................... 131
.
.
CHAPTER 4 TOM1p A YEAST E3 UBlQUlTlN LIGASE WHlCH MEDIATES TRANSCRIPTIONAL REGULATION AND TARGETS THE ADNSAGA COACTIVATOR COMPLEXES ........................................................ 135 4.1 Introduction .................................................................................................. 135 4.2 Materials and Methods ............................................................................... 140 4.2.1 DNA constructs ............................................................................... 140 4.2.2 Yeast strains. media and growth conditions ............................... 140 4.2.4 B-galactosidase assays .................................................................. 143
4.2.5 Preparation of whole cell extract .................................................... 143 4.2.6 Analysis of histone acetyltransferase activrty ...............................143
4.2.7 lmmunoprecipitation of NGGlp and TOM1p ................................ 144 4.2.8 lmmunoprecipitation of SPT7p under denaturing conditions .... 146 4.2.9 Western blot analysis of proteins ................................................... 146 4.3 Resuks ............................................................................................................ 149 4.3.1 TOM1p has a role in transcriptional activation ............................ 149 4.3.2 An active site cysteine is required for TOMl p's function as a transcriptional regulator ................................................................ 152 4.3.3 The two-hybrid interaction of UBP3p and NGGl p requires TOM1 ................................................................................. 155 4.3.4 TOMl p is required for ubiquitination of a protein that irnmunoprecipitates with HA-NGGlp .......................................... 158 4.3.5 SPTip is ubiquitinated in a TOM1p-dependent fashion .......... 161 4.3.6 Coimmunoprecipitation of TOM1p with NGG1p .......................... 166 4.3.7 TOM1p is required for the association of the ADA components with SPT3p and TBP but not with SPT7p ............. 169 4.3.8 Histone acetyltransferase activity of the ADA complexes is unchanged in the absence of TOM1p .........................................172 4.4 Discussion ..................................................................................................... 178 4.4.1 TOMl p regulates the function of the ADA complexes through ubiquitination .................................................................... 178 4.4.2 Ubiquitinated component(s) of the ADA complexes ................... 179 4.4.3 What is the role of ubiquitination of ADA component(s) ............ 181 4.5 References ..................................................................................................... 184
CHAPTER 5 GENERAL DISCUSSION ............................................................... 191 5.1 ADA proteins fonn multiple complexes in vivo ........................................ 191 5.2 TRAl p is a component of the ADAfSPT complexes .............................. 195 5.3 ADA complexes interact with TBP ............................................................ 198 5.4 Amino acids 274-307 are critical for transcriptional function of the ADA complexes ..................................................................................... 199 5.5 Mechanisms of regulation by The ADA complexes ............................... 200 5.6 Post-translationalmodification of ADAEPT components ..................... 203 5.7 Conclusions ................................................................................................... 206 5.8 References ..................................................................................................... 207 APPENDICES ......................................................................................................... 215 CURRICULUM VlTA ............................................................................................. 218
LIST OF TABLES Page
Table 1.1 Table 2.1 Table 4.1 Table 4.2
Components of the ADA- and SPT-containing complexes ........... 20 Relevant genotypes of yeast strains used in this study ................. 72 Yeast strains used in this study ......................................................... 141 Transcription frorn ADH2 and GAL 110 promoters in wild type. tom 1. nggl. and tom 1 nggl yeast drains ...................... 1 5 4
LIST OF FIGURES Page Figure 1.1 Fornation of the transcriptional initiation cornplex on eukaryotic promoters ............................................................................. 4 Figure 2.1 Coimmunoprecipitation of NGG1p with ADA2p from yeast whole œll extract .................................................................................. 78 Figure 2.2 ADA2p and NGGlp coelute during chromatography on a FPLC Fast Q column .......................................................................... 81 Figure 2.3 ADA2p and NGGlp coelute on a Superose 6 gel-filtration column ............................................................................... 84 Figure 2.4 Fractionation of ADA complexes on Sephacryl S-500 HR ............ 87 Figure 2.5 Stability of ADA2p and NGGl p depends on the presence of each other ............................................................................................... 90 Figure 2.6 Detection of NGGlp in yeast nuclear extracts ................................. 93 Figure 2.7 Amino acids 274-307 of NGGl p are required for interaction with TûP .................................................................................................. 96 Figure 3.1 Coimrnunoprecipitation of TRAl p and ADA2p ................................ 116 Figure 3.2 NGGl p. ADA2p. SPT7p and TRAl p coelute from a FPLC Mono Q column ...................................................................................... 119 Figure 3.3 Cofractionation of TRAlp with the ADAISPT proteins on DNA-cellulose ....................................................................................... 122 Figure 3.4 Binding of TRAlp to DNA cellulose column requires ADA2 ......... 125 Figure 4.1 Transcription from ADH2, HlS3 and GAL 70 promoters in wild type (CY922). tom 1 (SY11) and ngg1 (JY335) yeast strains ........................................................................................... 150 Figure 4.2 The expression GAL4p is not affected by disruption of TOM1p .... 153 Figure 4.3 Transcription frorn the ADH2 and GAL IO promoters in the ............................................................. 156 preçence of TOM1pCys3235Ala Figure 4.4 Loss of interaction between NGGl p and UBP3p in the absence of TOM1p ............................................................................... 159 Figure 4.5 Ubiquitination of an ADA-associated protein requires TOM1p ..... 162 Figure 4.6 SPT7p is ubiquitinated in a TOM1p-dependent fashion ................ 164 Figure 4.7 Coimrnunoprecipitation of TOM1p with NGGl p from yeast m o l e cell extracts ..................................................................... 167 xiii
Figure 4.8 Coimmunoprecipitation of NGGl p with TOMl p frorn yeast M o l e cell extracts ......................................................................170 Figure 4.9 Association of the TBP class of SPT gene products in the presence and absence of TOM1p ............................. . ..................... 173 Figure 4.10 Histone acetyitransferase activity of ADA complexes in the presence and absence of TOM1p .................................................... 176
xiv
LIST OF APPENDICES
Page Appendix I NGG1p interacting proteins revealed by coimmunoprecipitation ................................................................... 215 Appendix II Copyright release from the Journal of Biological Chemistry ..... 216 Appendix III Copyright release from the Journal of Molecular Biology .......... 217
ABBREVIATIONS
aa Ab anti-HA BSA DTT EDTA EGTA 5-FOA FPLC HEPES
IgG IP kDa Ni 2 + -TA ~ NP-40 PAGE
PCR PMSF Pol II PVDF SDS TBS TCA Tris YPD
amino acids antibody anti-hemagglutinin antibody bovine serum albumin dithiothreitol ethylenediaminetetraacetic acid Ethylene bis-oxyethylennitnlotetraaceticacid 5-fluoroorotic acid fast performance liquid chromatography NQ-hydroxyethyl piperazine-Ni-2-ethane sulfonic acid immunoglobulin G immunoprecipitation kiloDalton b4i2+-nitriloaceticacid-agarose Nonidet P-40 poly-acrylamide gel electrophoresis polymerase chain-reaction phenylmethylsulphonyl fluoride RNA polymerase II polyvinylidine difluoride sodium dodecyl sulfate Tris-buffered saline Trichloroacetic acid 2-amino-2-hydroxymethyl-1,3-pro panedi 1% yeast extract, 2% peptone, 2% glucose
CHAPTER 1 INTRODUCTION
Transcriptional regulation is important in ail eukaryotic organisms for ceii growth, development, and response to environmental changes (reviewed in Conaway and Conaway, 1993). Saccharomyces cerevisiae has provided a powerful system for genetic and biochemical analysis of transcriptional regulation, and findings from the study of this system have proven broadly applicable to other organisms. Transcriptional regulation requires the concerted assembly of several multiprotein complexes on promoters. The central players in this process are gene specific activator proteins, components of the general transcription machinery, and auxiliary factors, termed coactivators or adaptors. that enable activator proteins to contend with chromatin ternplates andor interface effectively with various components of the general transcription apparatus. Interestingly, like al1 cornponents required for transcription, most coactivator proteins are highly conserved among eukaryotic species, suggesting that their regulatory functions are fundamentally important (reviewed in Kingston et a/., 1996; Orphanides et al., 1996). Biochemical approaches have proven effective in demonstrating the functions of coactivators, and the combination of biochemical, genetic, and molecular analyses has, in many cases, provided evidence for their fomation into huge protein assemblies. Protein-protein interactions play a key role in the fomation of the complexes as well as in their functions. The complex nature of these associations provide virtually unlimited possibilities for regulation and result in an elaborate mechanism for controlling gene expression. In several instances, coactivator complexes are involved in both positive and negative
regulatory responses in vivo. However, the precise mechanisms that signal either activation or repression are not clear, partly because of insuffkient information on the exact composition of any of the identified complexes. The ADA complex (elteration oeficiency in Activation) is one of the best charactenzed coactivator complexes, yet its mechanisms and even exact composition is still unclear. Components of the ADA complex are involved in both positive and negative regulatory responses in vivo. Recent reports indicate that the ADA complex regulates transcription by different mechanisms including chromatin remodeling and interactions with the basal machinery (reviewed in Guarente, 1995; Hampsey, 1997). The ADA cornplex, therefore, represents an interesting model for investigating the transcriptional roles of coactivator complexes and their relationships with other regulatory systems. Before presenting the results of this thesis on the composition, regulation and function of the yeast ADA complex, I will summarize the knowledge on coactivaton concentrating on the ADA complex. In addition, the ubiquitin conjugation pathways will be discussed, since results from this work indicate a
key role for ubiquitination in modulating the transcriptional activity of the ADA cornplex.
1.1 Transcription by RNA Polymerase II 1.1 .1
Eukaryotic promoter elements Most promoters of genes transcribed by eukaryotic RNA polymerase II
(Pol II) contain three essential DNA elements: core promoter sequences, initiator sequences, and upstream activating sequences (UAS, in yeast) or enhancers (in higher eukaryotes). Transcription initiation by Pol II is precisely regulated by factors that interact with these DNA targets and also with each
other (reviewed in Conaway and Conaway,l993 ). Core promoter sequences are necessary for the assembly of the transcriptional initiation complex and most are composed of a TATA element, consensus sequence of TATAAA (Davison et al., 1983; Guarente, 1988; Nakajirna et al., 1988). In yeast, TATA elements are located between 40 and 120 base pairs upstream the transcription start site (+1 in Fig. 1.1), whereas in higher eukaryotes they are typically located 25-30 base pairs upstream from the initiation site (reviewed in Struhl, 1989). Initiator sequences are rich in pyrimidine and considered as the primary deteminant of where transcription begins (Hahn et al., 1985; Nagawa and Fink, 1985; McNeil and Smith, 1986; reviewed in Struhl, 1989). Finally, UAS elements or enhancers, which can be found far from the transcription
initiation site in either direction or orientation, constitute the promoter targets for factors rnodulating transcription (Guarente and Hoar, 1984; Struhl, 1984). 1.1.2
Components of the basal transcription machinery Biochemical developrnents in yeast, Drosophila, and mammalian cells
have led to reconstitution of the basal transcription machinery in vitro with Pol II and so-cailed general or basal transcription factors, namely TFIID, TFIIA, TFIIB, TFIIE, TFIIF, and TFllH (reviewed in Ghysdael and Yaniv, 1991; Conaway and Conaway, 1993; Zawel and Reinberg; 1993). These components are highly conserved, and in some cases, functionally interchangeable among the above organisms. The model which ernerged from these studies (Fig. 1.1) indicated that the general transcription factors assemble on the core promoter before transcription begins. TFllD is the only one of these components capable of sequence-specific DNA binding. TFllD coiltains the TATA binding protein (TBP), and in the most general case the orderly process of transcription initiation starts with binding of this factor to the TATA element (Buratowski et al., 1989). TFllA is
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required to stabilize this binding (Ranish and HahnJ991). Thereafter, the TFIID-DNA complex directs recmitment of TFllB to the promoter. The resulting TFIIB-TFIID-DNA platforni is in tum recognized by a complex of Pol II and TFIIF. Mutations in TFllB alter Pol II start sites in yeast, as do mutations in the large subunit of Pol II, providing compelling evidence for its function as a precise spacerlbridge between TBP and Pol II that determines the transcription start sites (Li et al., 1994; Pinto et al., 1994). TFllF is also involved in the accurate
binding of Pol II to the promoter (Sopta et al., 1985). Finally, transcription initiation complex assembly is completed with recruitment of TF1lE and TF1IH. TF1lH contains endogenous helicase and protein-kinase activities which are required for promoter clearance (Drapkin and Reinberg, 1994) and elongation (Bentley, 1995). In the presence of nucleoside triphosphates, strand separation at the transcription start sites occurs to give an open complex, the C-terminal domain (CTD) of the large subunit of Pol II is phosphorylated, and Pol II initiates transcription and is released from the promoter. During elongation in vitro, TFllD can remain bound to the core promoter supporthg rapid re-initiation of transcription by Pol II and the other general factors (Fig. 1.1 ; Zawel and Reinberg, 1993). An abbreviated complex assembly mechanism has also been proposed, following recent discoveries of various Pol II holoenzymes (discussed below) containing many if not al1 of the general transcription factors (Koleske and Young, 1995). 1.1.3
DNA-specific transcriptional activators The general transcription factors mentioned above are essential for
transcription initiation, and are sufficient to allow a basal level of transcription from the core promoter. For regulated transcription, however, promoter-specific proteins are required which bind to upstream activating sequences. These
proteins are bipartite in structure and contain both a DNA-binding and an activation domain (reviewed in Ptashne, 1986, 1989). The activation domain is believed to modulate the activity of Pol II by direct or indirect interactions with cornponents of the transcriptional machinery (reviewed in Patikoglou and Burley, 1997). While perhaps an oversimplification, transcriptional activation domains have been divided into three major classes according to a predorninance of particular amino acid residues: acidic, proline-rich, or glutamine-rich (Mitchell and Tjian, 1989) . Of these classes, the acidic activation domains appear to be unique in that they can function in al1 eukaryotes tested from yeast to human (Attardi and Tjian, 1993). Three examples of transcriptional activators most central to this discussion are the yeast GAL4, GCN4 and ADRl proteins. GAL4p is required
for transcription of the galactose metabolizing (GAL) genes, a process that occurs in galactose medium (Laughon and Gesteland, 1984). GAL4p binds specifically to its upstream activating sequence to activate transcription of the adjacent GAL 1and GAL 10 genes. The N-teminal region of GAL4p contains both a Zn finger DNA-binding domain, and a dimerization domain (reviewed in Reece and Platt, 1997). In response to amino acid starvation, GCN4p binds to promoters and activates transcription of multiple genes encoding amino acid biosynthetic enzymes (reviewed in Hinnebusch, 1994). GCN4p binds to DNA as a homodimer, and its DNA binding and dimerization (leucine zipper region, bZIP) dornains are located at the extreme C-terminus of the protein (O'Shea et al., 1989; Ellenberger et al.. 1992). Finally, ADRl p is also a DNA-binding protein whose activity is required to enhance transcription of the alcohol dehydrogenase II encoding gene, ADH2 (Hartshorne et al., 1986; Yu et al., 1989). Recent reports on GAL4p, GCN4p and ADRl p indicate that their
activation domains are complex in nature, because of multiple clusters of hydrophilic and hydrophobic amino acids required for functions (Lohr, 1997; Reece and Platt, 1997; Drysdale et aL, 1998).
1.1.4
Activated transcription Transcriptional activation results from the ability of activator proteins to
stimulate the assembly or function of the transcription initiation complex. This stimulation is thought to depend on direct or indirect protein-protein interactions between transcriptional activators and components of the basal transcription machinery. Binding of TFllD to the core promoter appears to be rate limiting for initiation, and certain activators stimulate this step in initiation complex formation (Ingles et ab, 1991; Caron et al., 1993; Blair et aL, 1994; Metz et al., 1994). Several activators interact with TBP in vitro in a manner that depends on amino acids in the activation domain that are critical for transcriptional activation in vivo, suggesting that direct interactions between activators and TBP are
essential in recruiting TFllD to the core promoter (Ingles et al., 1991; Metz et al., 1994). Certain activation domains also bind TFllB in vitro in a sequence-specific manner (Baniahmad et al., 1993; Kim and Roeder, 1994) and recruitment of this factor to the initiation complex as well (Chiang et ab, 1996). Interestingly, many transcriptional activators contain multiple redundant activation domains which target several basal factors. The viral activator VP16 is one of the examples of this phenornenon, demonstrating many closely
spaced activation subdomains (Lai and Herr, 1997). VP16 has been reported to make specific protein-protein contacts with TBP (Ingles et al., 1991), TFllB (Roberts et al., 1993), and TFllH (Xiao et al., 1994). The contribution of each of these interactions to the full level of activation by VP16 has not been determined, but abolishing the contacts for in vitro interaction with TBP has little
effect on activation in vivo (Tansey and Herr, 1995). The yeast GCN4p has as many as seven clusters of amino acids that contribute to activation (Jackson et ab, 1996). GCN4p interacts with TFllD and many other factors that are bound to the large subunit of Pol II (Drysdale et al., 1998 ). Furthemore, GAL4p and ADRl p also have multiple activation sequences which form several contacts with the basal machinery (reviewed in Chang and Jaehning, 1997). Whether the observed multiple direct interactions between these activators and the general transcription factors are important to confer an efficient activation process (Chang and Jaehning, 1997), represent functional redundancy, or result frorn the evolution of these molecules from a common ancestor (Tansey and Herr, 1995) is a topic of much debate. Clearly, however, the interaction with
basal factors is not sufficient for activated transcription. Additional protein factors, as a group called coactivators, are required (reviewed in Guarente. 1995).
1.2
1.2.1
Coactivator Complexes
TFllD complex The identification of coactivators came from the observation that TBP
could replace TFllD for basal transcription in vitro, but did not facilitate activated transcription (Pugh and Tjian, 1990). Molecular analyses of human and Drosophila transcription factors have established that TFllD exists as a large multisubunit complex containing TBP and at least eight stably associated TAFs ( D P bssociated Eactors) of approximate sizes ranging from 30 to 250 kDa (reviewed in Goodrich and Tjian, 1994; Roeder, 1996). The yeast TFllD complex is not as tightly associated as in higher eukaryotes. Therefore, the yeast TBP was initially purified as a single 25 kDa protein corresponding to TBP (Buratowski et al., 1988). Yeast TAFs, however, have subsequently been identified by affinity isolation with TBP and shown to be essential for viability. (Reese et al., 1994; Poon et ab, 1995; Apone et al., 1996; Sauer and Tjian, 1997).
In Drosophila and hurnan, the TFllD complex has been reassembled from the purified components, and distinct protein-protein interactions have been identified between a variety of transcriptional activators and individual TAFs (reviewed in Burley and Roeder, 1996; Sauer and Tjian,1997). These include the human TAFl10 which interacts with certain glutamine-rich activators. such as SPI, and TAF40/60 which targets the acidic activator VP16 (reviewed in Chang and Jaehning, 1997; Sauer and Tjian, 1997). Drosophila TAF40 and TAF32 also physically associate with VP16 in vitro (Buriey and Roeder, 1996). A partially purified TAF complex from yeast was shown to be required for transcriptional activation of a GCN4p-regulated promoter in vitro
(Klebanow et al., 1996); however, recent studies indicate that yeast TAFs are not essential for transcriptional activation in vivo by GCN4p and by several other activator proteins (Moqtaderi et al., 1996; Walker et al., 1996). Although these findings raised a great deal of debate about the in vivo role of yeast TAFs,
R is clear that they are crucial for activated transcription of important subsets of genes (Apone et al., 1996; Sauer et aL, 1996). As more and more activators and repressors have been used in in vitro reconstituted transcription systems it has become apparent that there is not just
a single form of TFIID, but instead multiple foms with different functions. The first report of multiple foms of TFllD was from Timmers and Sharp (1991), who identified two large TBP-containing complexes with different protein components as well as transcriptional properties. Brou et al. (1993) and Jacq et
al. (1994) also identified multiple TFllD complexes capable of responding to different classes of activators. In particular, the latter investigators identified human TAF30 as a factor associated with only a subset of TFllD complexes but required for activation by estrogen receptors (Jacq et al., 1994). Subsequently, Mengus et al. (1995) found that human TAF18 is uniquely present in the subform that lacks TAF30. ln further studies it became evident that different foms of TFllD appear to share a core of TAFs, with coexisting subpopulations that contain or lack one or more additional TAF (reviewed in Tansey and Herr, 1997). Based on the observation that the human TAFSO-containing form of
TFllD is required for activation by the estrogen receptors, it is probable that these different foms interact with or respond to different subsets of transcriptional regulators. Different foms of TFllD also play a role in repression of transcription. Wade and Jaehning (1996) identified a subpopulation of yeast TFi ID required for repression from a LEU3 promoter. The repression-component form of TFllD
contained several of the core yeast TAFs but lacked TAFlSOp, encoded by the essential TSMl gene (Wade and Jaehning, 1996). Instead, this cornplex of TFllD was associated with the product of the essential MOTl gene. MOTl p has been shown to be a component of one f o m of the yeast TFllD complex (Poon et al., 1994) and it plays a critical role for expression of a subset of yeast genes (Collart, 1996; Madison and Winston, 1997). MOTl p may exert its repressive effect on transcription of same promoters via its ability to dissociate TBP from the TATA box in a ATP dependent process (Auble et al., 1994; Poon et al., 1994). Thus yeast TAF150p and MOTl p, like human TAF30 and TAFI 8, are associated with different subpopulations of TFIID, where they may direct the interactions of TBP with different promoters or regulatory factors.
1.2.2
Components of the Pol II holoenzyme complexes A second group of coactivators in yeast is the mediator complex found
associated with the C-terminal domain (CTD) of the largest subunit of Pol II (the holoenzyme complex; reviewed in Carlson, 1997). The holoenzyme complex is a large protein complex containing the catalytic core of Pol II itself, tightly
associated products of the SRB genes ( Suppressors of RNA polymerase B; Kim et al., 1994; Koleske and Young, 1994), the general transcription factor TFIIF, and a dissociable subcomplex of GALl 1p,
SUG 1p, RGRl p and SIN4p
(Kim et al., 1994; Koleske and Young, 1994 and 1995). The presence of some of the basal factors (TBP, TFllB, TFIIH) and components of the chromatin remodeling apparatus is still controversial (Koleske and Young, 1994; Liao et ai., 1995; Cairns et al., 1996). Two other components have recently been reported as members of this complex, ROX3p ( Song et al., 1996) and MEDGp (Lee et al., 1997).
The SR8 genes were initially identified by Young and colleagues from mutations that suppress the cold-sensitive growth of mutants bearing tnincations in the CTD of Pol II (Koleske and Young, 1994; 1995). Extracts from srb mutants show a reduced response to activators in vitro, suggesting a
coactivator function for the SRB gene products (Kim et ab, 1994). Consistent with their involvement in activated transcription, the SRBps can physically interact with the activation domain of VP16 (Hengartner et al., 1995). Several genetic studies have also demonstrated a repressive effect of some of the SRBps on transcription (reviewed in Carlson, 1997). Mutations in the genes encoding SRB8p to SRBl 1p appear to relieve negative regulation of diversely regulated genes (Kuchin et al., 1995; Wahi and Johnson, 1995). Kuchin and Carlson (1998) have recently indicated that SRBl Op and SR81 1p are essential for the transcriptional function of the global corepressor SSNG-TUPI . GALl1 p SIN4p, RGRlp and ROX3p have also been shown to play positive and negative roles in the transcription of many genes (Carlson 1997). RGRl p and SIN4p, for instance, were originally identified as repressors of glucose-regulated genes, and both proteins are required for maximal induction of these genes as well (Li et al., 1996; Song et al., 1996). These findings strongly argue that the
holoenzyme complex is involved in both transcriptional activation and repression. It is believed that the complex exerts its effect on transcription by mechanisms that either stimulate or inhibit the binding of Pol II to promoters (Carlson, 1997). Affinity purification of the CTD of Pol II and its associated proteins identified another f o m of the holoenzyme complex (Wade et al., 1996). In this case, the putified complex included some of those initially described components of the holoenzyme (TFIIB, TFW and GALl1p) but lacked the SRBps. In addition, proteins not found in the previously characterized
holoenzyme were identified, including PAFl pl CCR4p and CDC73p (Wade et al., 1996). Isolation of tagged foms of PAFl p and CDC73p confimed the existence of a distinct Pol II cornplex lacking the SRBps (Shi et al., 1997). The two f o n s of holoenzyme have been shown to CO-existin yeast cells (Shi et aL, 1997). PAFI, CCR4 and CDC73 are not essential for cell viability. However. mutations in these genes cause temperature sensitivity and alteration of transcription from a subset of yeast promoters (Shi et al., 1996; 1997). The two holoenzyrne complexes are portrayed as transcribing overlapping major and minor subsets of genes. This model is based on the fact that some of the SR& are essential (e.g. SRB4, SRB6, and SRB7; Thompson and Young, 1995) and affect transcription of most yeast genes, while PAFI, CCR4 and CDC73. in addition to the shared components GAL11, SIN4 and RGRI, are non essential and appear to affect only a subset of transcripts
(Carlson. 1997). The overlapping nature of the effects of the two complexes is based on the fact that expression of some genes is affected by mutations in either complex (Suzuki et al., 1988; Draper et al. 1994; Li et al., 1995; Zhu et al., 1995; Shi et al., 1997). In addition, many combinations of mutations in these factors are lethal (for example srb5/pafl and sr65/ccr4; Chang and Jaehning, 1997). Holoenzyme complexes and homologues of some of the SRBps have recentiy been identified in mammalian cells. In contrast to the cornpiexes identified in yeast, these mammalian complexes contain some or al1 of the general transcription factors and several known coactivators (Ossipow et al., 1995; Chao et aL, 1996; Maldonado et al., 1996). Without the addition of any exogenous basal factor , one of these complexes is capable of activatorresponsive transcription in vitro (Ossipow et al., 1995). Perhaps, as in the case of TFIID. the stability of the Pol Il-general transcription factors complexes in
yeast is less than that in mammalian cells. Substoichiometric amounts of TBP and TFllH have been reported in some preparations of the yeast holoenzyme in support of this idea (Thompson et al., 1993; Koleske and Young, 1994). In addition, mammalian Pol II holoenzyme complexes have been described containing DNA repair proteins (Maldonado et al., 1996).
1.2.3
SWVSNF complex A third group of coactivators are the genes encoding subunits of the
SWVSN F complex (SWltch/Sucrose Non-Eermenting), originally identified on the basis of their requirement for induction of genes involved with mating type switching and fermentation of sucrose (reviewed in Kruger et al., 1995). Biochemical studies of SWIfSNF proteins led to the purification of an approximate 2 MDa complex from yeast and mammalian cells (Peterson et al., 1994). As predicted genetically, the purified complex comprises an estimated 11 polypeptides, which include SWIl/ADR6p, SWWSNFZp, SW13pl SNF5p.
SNFGp, SNFl 1p, TFG3/ANCl p and SWP73p (reviewed in Kruger et al., 1995; Peterson, 1996; Pazin and Kadonaga, 1997a, 199713). Genetic evidence first suggested that SWVSNF proteins affect transcriptional activation by relieving the repressive effect of chromatin (reviewed in Winston and Carlson, 1992). Transcriptional defects of swü5nf mutants are suppressed by mutations in histone genes and genes encoding other chromatin related proteins (Kruger and Herskowitz, 1991). In addition, in s w i 2 h f 2 mutants, the chromatin structure at the SUC2 promoter is altered at sites near the TATA box (Carlson and Laurent, 1994). The relationship between the function of the SWVSNF complex and chrornatin structure was then established by biochemical experiments in which it was found that the purified complex possesses a DNA-stimulated ATPase activity that is able to destabilize histone-DNA interactions as well as to
enhance binding of GAL4p to its DNA binding site in reconstituted nucleosomes (Cote et ai., 1994). SWWSNF2p contains a conserved ATP-binding motif which appears to be an important component of the chromatin remodeling activity of the SWllSNF complex (Laurent et al., 1993). Mutagenesis of the conserved NTP-binding motif of SWIZ/SNF2p results in losç of the ATPase activity and facilitated binding of GAL4p to mononucleosomes (Cote et al., 1994). Other multisubunit coactivator complexes including the yeast RSC complex, Drosophila NURF, CHRAC, ACF and BRM complexes and mammalian BRGl complexes are placed in the same category as the SWIISNF complex because they each contain a homologous subunit to the SWWSNF2p (reviewed in Pazin and Kadonaga, 1997a, 1997b).
These complexes have a DNA-stimulated ATPase activity and are able to alter histone-DNA interactions in reconstituted nucleosomes. Although the exact nature of these structural changes is not yet clear, the available data on this group of related complexes suggest that they may function as ATP-driven motors which translocate along DNA to destabilize nucleosomes and then either modify or remove histones to allow transcription to occur (reviewed in Pazin and Kadonaga, 1997a, 1997b).
1.2.4
SPT proteins The SPT proteins, were identified as suppressors of Ty and 6
(retrotransposon elements) insertion mutations (reviewed in Winston and Carlson,1992). These Ty and 6 insertion mutations, generally in the promoter regions of genes, abolish or alter transcription of the adjacent gene, causing an auxotrophy (Winston et al., 1984, 1987; Fassler and Winston, 1988; Winston and Carlson, 1992). Mutations in SPTgenes alter transcription in vivo to restore transcription of the Ty disrupted genes. spt mutants also cause a variety of
additional transcriptional effects in vivo (reviewed in Winston and Carlson, 1992).
Genetic and molecular studies place SPTgenes into two classes. The first class, the histone class, includes SPT4, SPTS, SPT6, SPT1VHTA 1, and SPT12RITB1. SPTl 1, and SPTl2 encode histones H2A and H2B. respectively, (Clark-Adams et al., 1988; Winston and Carlson,1992). Mutations in SPT4,
SPT5 and SPT6 derepress transcription of a diverse spectrum of genes, indicating that their encoded proteins function as general transcriptional repressors (Clark-Adams and Winston, 1987; Malone et al., 1993). SPT4, SPT5 and SPT6 gene products are thought to control transcription by causing changes in chromatin structure (Winston and Carlson,I 992; Malone et al., 1993). Recent genetic and biochernical studies indicate that SPT4p, SPT5p and SPT6p are also involved in regulating transcriptional elongation by Pol II in yeast (Hartzog et al., 1998). The second class of SPTgenes, the TBP class, includes SPT3, SPT7, SPTB, SPT15, and SPT20 (see Table 1.1). SPT15 encodes TBP itself (Eisenrnann et al., 1989), while the other four gene products have been hypothesized to help TBP function at particular promoters (Eisenmann et al., 1992). This model is based on several lines of evidence. First, single or double nuIl mutations of these genes cause similar slow growth and transcription defects at particular promoters (Winston et al., 1984, 1987; Hirschhom and Winston, 1988; Eisenmann et al., 1989, 1994; Gansheroff et al., 1995; Roberts and Winston, 1996, 1997). Second, these phenotypes are similar to those caused by certain missense mutations in SPTlS/TBP (Eisenmann et al., 1989, 1992, 1994). Moreover, SPT3p was shown to associate directly with TBP (Eisenmann et ab, 1992). SPT3p also interacts genetically with MOT1p and TFIIA, two factors known to associate with TBP (Madison and Winston, 1997).
Finally, SPT3p has been recently indicated to be 26% identical to human TAF18 (Mengus et al.. 1995). Based upon these findings, it was suggested that the TBP class of SPT factors displays similar roles to those of TAF proteins in assisting andor stabilizing the binding of TBP to its DNA element (Eisenmann et al., 1992, 1994).
1.3
Components and Functions of the ADA Complexes
It is clear from the above examples that coactivators f o m multiple complexes with potential for different functions. The ability of different forrns of any of these assemblies to signal either activation or repression depends on the identity of their constluents. In addition, the finding that the transcriptional activity of a certain group of coactivator proteins requires their association together in a complex suggests that protein-protein interactions between members of the same complex play a crucial role in determining whether the complex will activate or repress transcription. The yeast ADA proteins form such dual function complexes and represent a valuable model for investigating the effect of protein-protein associations on the transcriptional activity of coactivator1corepressor complexes.
1.3.1
NGGl protein
The ADA factors (summarized in Table 1. l ) were identified genetically in
S. cerevisiae based on their requirement for the regulated activation and repression of a subset of yeast activator proteins (Berger et al., 1992; Georgakopoulos and Thireos, 1992; Brandl et al., 1993: Pina et al., 1993; Marcus et al., 1994 ). NGGl (Negative in Glucose and Galactose) was discovered in Our laboratory on the basis of its involvernent in repression of the GAL4p dependent promoter GAL 10 (Brandl et al., 1993).Expression of a
GAL IO-lad reporter is increased 300-fold in glucose media in a gai80 nggl background. Approximately 10 to 15-fold of this effect was attributable to nggl (Brandl et al., 1993). Based on several observations, we believe that GAL4p is a direct target for NGGl p action. The repressive effect of NGGl p is dependent on
Table 1.1 Components of the ADA and SPT-containing complexes
SGD ORF Name
Chromosome ORF Number Seciuence
Reference
ADAl
YPL254W
XVI
Horiuchi et al. ( 1997)
ADA2
YDR448W
IV
Berger et al. (1992)
ADA3/NGG 1
YDR176W
IV
Brand et al. (1993), Pina et al. (1993)
GCNS/ADA4
YGR252W
VII
ADAS/SPT20
YOL148C
xv
Georgakopoulos and Thireos ( 1992), Marcus et al. (1994) Marcus et al. (l996), Roberts and Winston ( 1996)
SPT3
YDR392W
IV
Winston et al. (1984)
SPT7
YBR081C
II
Winston et al. ( 1987)
SPT8
YLR055C
XII
Winston et ai. ( 1987)
Components of the ADAISPT complexes. The Saccharomyces Gemme Database (SGD) open reading frame name of the ADA and SPT genes, their chromosomal location (chromosome number), and the total number of amino acids encoded by these genes are indicated.
the presence of GAL4p but does not require the GAL4 promoter (Brandl et al., 1993). NGGl p thus does not appear to regulate transcription of GALA
Regulation by NGGl p is seen for promoters containing independent GAL4p binding sites (Brandl et al., 1993),thus excluding a role for other upstream regulatory sequences within GALIO. Consistent with its role in repression, NGGlp interacts with GAL4p in a two-hybrid analysis (Martens and Brandl; unpublished result). Finally, the synergistic effect of nggl with gai80 is in agreement with NGGl p regulating DNA binding or the activity of GAL4p. The negative effect of this molecule on gene expression was further substantiated with the finding that the transcriptional activation by PDRl p, an activator in the yeast pleiotropic drug resistance pathway (reviewed in Balzi ana Goffeau, 1995), is also enhanced in a mutant strain lacking NGGlp (Martens et al., 1996). The involvement of NGGl p in regulating PDRl p activity was supported by coimmunoprecipitation of both proteins frorn whole yeast extracts (Martens et
al., 1996). These results, taken together, indicate that
NGGl p acts more likely
as a general repressor as opposed to having a role specifically in glucose repression. In fact, NGGl p also inhibits transcriptional activation of GAL4p in galactose media (Brandl et al., 1993). In an independent selection for genes encoding putative transcriptional activator, Guarente and CO-workersidentified nggl/ada3 as a suppressor of the toxicity of overexpression of a GAL4-VP16p chimeric activator (Pina et al., 1993). Mutations within this gene are thought to alleviate the toxicity of VP16 by inhibiting its ability to sequester the general transcription factors away from productive transcriptional machineries (Berger et al., 1992; Pina et al., 1993). Subsequently, these investigators showed that NGGl p is required for maximal transcriptional activation by a group of activators which includes GAL4-VP16p and LexA-GCN4p (Pina et a/., 1993). These data, cornbined with the above
results from Our laboratory (Brandl et al., 1993), raised the interesting hypothesis that NGGl p is a dual function regulator having both positive and negative roles in transcription.
1.3.2
Structurelfunction properties of NGGl p. Additional evidence for the role of NGGl p in regulating transcription
comes from the structural and functional properties of this factor. Brandl et al. (1996) generated several deletion denvatives of NGGl p and analyzed their ability to repress the activity of GAL4p. One of the stable derivatives of NGGl p, which lacks a 33 amino acid region (residues 274 to 307),was found to be unable of repressing the activity of GAL4p. The role of sequences between amino acids 274 and 307 in repression was further substantiated by finding more specific mutations within this region that can either stimulate or inhibit the action of NGG1p on GAL4p activity (Brandl et al., 1996). Interestingly. the essential 33 amino acids region contains a Phe-rich segment with homology to a diverse group of proteins including the viral HIV-GAG and KEXl p in yeast (Brandl et al., 1993; Pina et al., 1993). A functional basis for this homology, however, remains to be clarified. Structural predictions suggest that this region may form an amphipathic cx-helix (Brandl et al., 1993; Pina et al., 1993).
The deletion studies of NGGlp are also consistent with a role of the Pherich region in transcription activation. The N-terminal 373 amino acids of NGGlp can activate transcription when fused to the DNA binding domain of GAL4p (Brandl et al.. 1996). This activation also requires the amino acids
between 274 and 307 (Brandl et al., 1996). Furthemore, the requirement of this region for both activation and repression by NGGl p is suggested by the fact that a reciprocal relationship exists between the ability of NGGl p mutants to activate transcription as GAL4p-NGGl p chimera and their ability to repress GAL4p
(Brandl et ab, 1996). Cumulatively, the transcriptional defects seen with the
nggl mutants provide additional evidence for the requirernent of NGGl p in transcriptional activation and repression, and demonstrate that. at least the protein segment between residues 274 and 307 is crucial for both functions.
1.3.3 NGGlp forms a complex with ADA2p and GCN5p Early genetic and in vitro biochemical evidence suggests that NGG1p comprises part of a complex containing two additional components, ADA2p and GCN5p (Berger et al., 1992; Pina et al., 1993; Marcus et al., 1994; Georgakopoulos et ai., 1995). ada2 was also identified by its ability to suppress the toxic effect of overexpression of the chimeric activator GAL4-VP16 (Berger et al., 1992). Strains with mutations in GCNS were initially isolated by their
ability to reduce transcriptional activation from GCN4p-responsive promoters (Georgakopoulos and Thireos, 1992). GCN5 was subsequently shown to be allelic with ADA4, another gene isolated as a suppresser in the toxicity screen (Berger et al., 1992; Marcus et al., 1994). ad&, nggl and gcn5 mutant strains al1 show similar slow growth phenotypes, temperature sensitivity, and reduced transcriptional activation by GAL4-VP16p and LexA-GCN4p, with double mutants having no more severe properties than the individual mutants ( Berger
et al., 1992; Brandl et al., 1993; Pina et al., 1993; Marcus et al., 1994; Georgakopoulos et al., 1995; Horiuchi et al., 1995). Transcriptional activation from an ADH2-driven promoter is also decreased in mutant strains lacking either of the three ADA proteins, suggesting that the activity of ADRI p also requires the ADA factors (Chiang et ai., 1996). ADMp, NGGl p and GCNSp interact with each other in al1 the pairwise combinations by two-hybrid analyses (Horiuchi et al., 1995; Candau et al., 1996). In addition, association of the Cterminal 250 amino acids of NGGlp with ADA2p was shown in vitro by far
Western blotting and immunoprecipitation (Hoduchi et al., 1995). Based upon the above evidence, Honuchi et al. (1995) have proposed that the ADA complex constitutes a physical link to allow productive interactions between the activation domains of certain activators and the basal transcription apparatus. This model is supported by the finding that ADA2p interacts directly with the activation domains of VP16 (Silveman et al., 1994; Barlev et al., 1995), GCN4p (Barlev et al., 1995), GAL4p (Melcher and Johnston, 1995), and ADRl p (Chiang et al., 1996). Whereas the downstream target for the complex is unknown, the
finding of Barlev et al. (1995) that ADA2p from crude yeast extracts is retained on a TBP affinity column suggests that the target may be TBP. It is important to emphasize that ADA2p is also required for the negative regulation of transcription. The finding that single and double disruptions of nggl and ada2 have similar effects on inhibition of GAL4p and PDRl p suggested that the same or related ADA complexes are involved in transcriptional activation and repression (Brandl et al., 1996, Martens et al., 1996). Interestingly, human genes (hADA2 and hGCNS), whose products are similar in sequence and function to the yeast ADA2p and GCNSp, have been identified (Candau et al., 1996). Human ADA2 and GCN5 proteins were shown to interact by a two-hybrid analysis and to complement transcriptional defects caused by nuIl mutations of the yeast counterparts (Candau et al., 1996). implicating the existence of both structural and functional conservation among the ADA complexes of different organisms. Additional support for a functional role for associations between ADA2p, NGGlp and GCN5p comes from dissecting the structure/function of ADA2p. An extensive deletion mutagenesis through ADA2p indicates that two regions are essential for the protein function in vivo (Candau et al., 1996; Candau and Berger, 1996). These regions are the amino terminus 166 arnino acids and the
central portion of the protein, the sequences between amino acids 200 and 300. Deletion of elher sequence results in phenotypes similar to those of the ada2 disruption strain. Strikingly, the same two regions are required for ADA2p to interact with GCNSp and NGGl p ni vitro (Candau and Berger, 1996). Amino acids 1-166 were sufficient for the interaction with purified recombinant GCN5p in coimmunoprecipitation and glutathione S-transferase-ADA2p-binding assays. The first 77 amino acids of this region contain a Cys-rich segment that is strongly conse~edbetween the human and yeast ADA2 proteins (Candau et al., 1996). The middle part of ADA2p (amino acids 200-300) is required for interaction with the in vitro translated C-terminal half of NGGl p (Candau and Berger, 1996). ADA2p is therefore important to bridge the association between
NGGl p and GCN5p. Cumulatively, the above genetic data, along with the correlation between regions of ADA2p required for the in vitro interaction with NGGl p and GCNBp, and regions required for function in vivo, strongly argue for the existence of a physiologically relevant complex containing the three proteins.
1.3.4 A DA 7 and ADA5 indicate a functional link between A DA and
S P f genes In addition to ADA2, NGGl and GCNS, the initial toxicity screen of GAL4VP16p identified two other ADA genes, ADA 1 and ARA5 (Berger et al., 1992).
ADA5 (Marcus et al., 1996) and ADA 1 (Horiuchi et al., 1997) have recently been characterized and shown to be similar to the other ADA proteins except that mutations result in some broader transcriptional defects. For exarnple, transcription from the ADHl promoter is not affected by mutations in ADA2, NGGI, or GCN5 but is reduced in adal and ada5 mutants. Interestingly, unlike ada2, nggl, and gcn5, both ada 1 and ada5 mutants display sirnilar phenotypes
to mutations in the TBP class of SPT genes which include inositol auxotrophy and suppression of 6 insertion into a specific region of the H E 4 promoter (Marcus et al., 1996; Roberts and Winston, 1996; Horiuchi et al.. 1997). Furthemore, ADA5 has been shown to be identical to SPT20 (Roberts and Winston, 1996), suggesting a link between ADA and the SPT genes. Despite these differences, ada5 nggl and ada 1 nggl double mutants do not dernonstrate any phenotypes more severe than that of ada5 and ada 1 single mutants. There is also biochemical evidence that ADAS/SPT20p and ADAl p interact with
NGGl p (Marcus et ai., 1996; Honuchi et al.,
1997), suggesting that
they too are part of the ADMNGGlIGCN5 complex. The genetic and biochemical similarities between ADA and SPT genes suggest that they may function in related pathways.
1.3.5
ADA proteins form multiple complexes in vivo Recent biochemical analyses of ADA2p and NGGl p in native yeast
extracts have demonstrated that these proteins are associated in four complexes, two with approximate sizes of 2 MDa, and others of 900 kDa and 200 kDa (Saleh et ab, 1997). The subsequent finding of Workman and
colleagues that a comparable 1.8 MDa ADA complex contains ADAZp, NGGl pl GCN5p, SPTJp, SPT7p, SPT8p and SPT20p/ADA5p1defines at least in part, the composition of one of these complexes, the SAGA-complex (SPT/ADA/GCNS histone Acetyltransferase; Grant et al., 1997). The associations between the ADA and SPT proteins in this complex confinn the previous genetic observations which suggested a link between the two regulatory groups (Marcus et al., 1996; Roberts and Winston, 1996). Interestingly, the capability of GCNSp within the ADA and SAGA complexes to
acetylate nucleosomal histones in vitro suggested that one role of these complexes is in modulating chromatin structure (Grant et al., 1997). In addition to the SAGA, current biochemical experiments indicate that ADA and SPT proteins are also associated in at least one more complex (Saleh and Brandl; Grant and Workman unpublished resuits). The absence of the SPT proteins in some of the other ADA-containing complexes, along with the overlapping but non identical phenotypes of the different nul1 alleles, suggests that the SPT and ADA proteins also act independently (Marcus et al., 1996; Roberts and Winston, 1996, 1997; Grant et al., 1997). It is important to emphasize that each of the identified ADA and ADNSPT complexes is chromatographically distinct from the SWVSNF and the Pol II holoenzyme assemblies (Grant et al., 1997; Horiuchi et al., 1997; Roberts and Winston, 1997, Saleh et al., 1997). Therefore, the high molecular-weight does not result from any association of ADA and SPT proteins with either of these multisubunit complexes characterized previously. Based upon these findings, one conclusion would be that the large complexes contain additional components. other than those described in Table 1.1. The exact relationship between the different f o n s of ADA and ADNSPT complexes will be clarified on the identification of the unknown subunits of each complex and the degree to which they are cornmon and unique. It is only through defining the components and determining their individual functions that it will be possible to determine how the cornpiexes are regulated and how they affect the transcriptional machinery.
1.3.6
Role of the ADA complexes in contending with nucleosomal
DNA The wrapping of the DNA duplex around an octamer of histones to form the nucleosome can seiectively occlude the binding of certain transcription
factors to their DNA recognition sites (Hager et al., 1993). In contrast to the repressive effects of chromatin, nucleosornes can also facilitate transcription by bnnging together distant DNA sequences, allowing protein-protein interactions between transcription factors (Thomas and Elgin, 1988; Wolffe, 1994). On the basis of the impact of chromatin structure on protein-DNA interactions. it is not surpnsing that chromatin modifying enzymes play a crucial role in transcriptional regulation. One group of these enzymes is the nuclear type-A histone acetyltransferases (HATs; reviewed in Stnihl. 1998) which have been shown to regulate chromatin structure and transcription. The core histones, particularly H3 and H4, can be acetylated at the eamino groups of conserved lysine residues in the N-teminal tails that extend outwardly from the globular core of the histone octamer (Loidl, 1994). It has been postulated that the charge neutralization that occurs upon acetylation of the lysine side chains may disrupt electrostatic interactions between the histone tail and the phosphodiester backbone of DNA. Thus, a functional consequence of acetylation can be increased access of transcription factors to their nucleosornal recognition sites (Lee et al., 1993; Loidl, 1994; Vettese-Dadey et al., 1996).
Recently, there has been considerable progress in the identification of enzymes that can catalyze the acetylation of histones (reviewed in Brownell and Allis, 1996; Wolffe and Pruss, 1996; Grunstein, 1997; Struhl, 1998). These studies revealed that the HATs include the transcriptional coactivators HAT1pl GCN5p, TAF250, p300/CBP, PlCAF and SRC-1. Biochemical analyses of the different factors have led to a model in which recniitment of HATs to genes is accomplished through protein-protein interactions with DNA-bound activator proteins (Ogiyzco et ai., 1996). This model also suggests that the HAT activity is
brought to promoters as part of a large rnulimeric protein complex (Grunstein, 1997).
In parallel with the identification of HAT enzymes, studies of histone deacetylase (HDAC) enzymes in human, yeast, and Drosuphila have also advanced significantly. Afinity chromatography with the ligand trapoxin, a high affinity, irreversible inhibitor, resulted in the purification and cloning of a human deacetylase (Tauton et al., 1996) composed of a catalytic subunit, HD-1pl renamed HDACl p ( Hassig et al., 1997), and a tightly associated WD repeat containing protein, RbAp48. The sequence of the HDAClp showed very strong sequence similarity to yeast RPDSp, previously identified genetically to be necessary for full repression and activation of a subset of genes (reviewed in Grunstein, 1997). There are five members of the RPD3 family in yeast, two of which (HDAI and RPD3) are components of the major histone deacetylase activities, which f ractionate as 350 kDa (HDA) and 600 kDa (HDB) complexes (Camen et al., 1996; Rundlett et al., 1996). The initial studies of Allis and colleagues dernonstrated that recombinant yeast GCNSp can acetylate lysine residues in the flexible N-terminal domains of histones H3 and H4 (Brownell et al., 1996; Kuo et al., 1996). However, recombinant GCNSp does not acetylate histones that are assembled into nucleosomes (Kuo et al., 1996), which led to the suggestion that acetylation of physiological substrate might require an intact ADA complex. Consistent with this view, Candau et al. (1997) have shown that the in vitro enzyme activity of GCNSp requires both the putative HAT catalytic domains (amino acids 95-253)
and the binding region of ADA2p (amino acids 253-350). Pollard and Peterson (1997) found that al1 the partialiy purified GCNSp-dependent HAT activities from
yeast extracts cofractionate with NGGl p in three distinct complexes. Moreover, ADA2p and NGGl p and ADAS/SPT20p are crucial for the in vitro HAT activity
and the integrity of SAGA complex (Grant et al., 1997) . Thus. other ADA components may also be required for proper functioning of GCNSp in vivo. These observations, taken together with the other results of direct interactions of ADA proteins with transcriptional activators (Marcus et al., 1994; Silverman and Guarente, 1994; Bariev et aL, 1995; Horiuchi et al., 1995; Melcher and Johnston, 1995; Chiang et al., 1996; Martens et al., 1996; Henriksson et al., 1997), indicate that proteinprotein contacts between transactivators and components of the ADA complexes may be important for targeting the HAT activity of GCN5p to specific prornoters. In addition to the catalytic HAT domains and ADA2p-binding region, most striking is the finding of a bromodomain sequence in GCNSp (residues of 350440; Tamkun et al., 1992), having exceedingly high conservation (80% identity)
between human and yeast, that is not necessary for the interaction of GCNSp with ADA2p or the in vitro HAT activity (Candau and Berger, 1996; Candau et al., 1996; 1997). However, mutant alleles of GCN5 lacking this region fail to complement gcn5 nuIl mutants in either growth or transcriptional reporter assays, implicating that the brornodomain is crucial for the full transcriptional function of GCN5p (Candau et al., 1996; 1997). Secondary structure prediction suggests that the bromodomain may f o m a surface for protein-protein contacts (Marcus et al., 1994). Indeed, Barlev et al. (1998) have demonstrated an interaction between the bromodomain of human GCN5 and the p70 subunit of Ku autoantigen. The Ku autoantigen p70lp80 heterodimer is the DNA binding component of the DNA-PK holoenzyme (Lees-Miller, 1996). This interaction may be regulatory, as DNA-Pi& was found to phosphorylate and inhibit the
HAT activity of human GCNSp in vitro (Barlev et al., 1998). Interestingly, the bromodornain is also present in many other transcriptional coactivators, including CBPlp300 (Eckner et al., 1994), human TAF250 (Ruppert et al., 1993).
Droçophila TAF230, yeast SPT7p (reviewed in Tamkun et al., 1W2),and
mernbers of the SWWSNF2p family (reviewed in Pazin and Kadonaga, 1997b). However, the role of this conserved motif in regulating transcriptional functions of the different protein remains to be identified. A critical question related to GCNSp is whether the histone acetylase
activity is relevant for the transcriptional effects in vivo. Two recent reports describe detailed mutational analyses of GCNS, leading to the conclusion that there is a strict correlation between the HAT activity in vitro and transcriptional function in vivo (Kuo et al., 1998; Wang et ai., 1998). In one of these studies (Wang et al., 1998), histone acetylation by the various GCNSp derivatives was perfomied in the context of the ADA and SAGA complexes on nucleosomal
substrates. In the other study, additional evidence for physiological relevance was obtained by analyzing directly the acetylation state of chromatin in yeast cells (Kuo et al., 1998). Overexpression of GCN5p leads to increased
acetylation of core histones. More interestingly, GCN5 increases histone acetylation at promoter regions in a manner that is correlated with GCN5dependent transcriptional activation and histone acetylase activity in vitro.
1.3.7
Evidence that ADA/SPT complexes regulate TBP function The size of the ADA complexes suggests that they contain other
attributes to their regulatory functions in addition to the HAT activity of GCNSp. The association between the ADA proteins and the TBP class of SPT proteins suggests that the large ADA complexes may regulate transcription by modulating TBP function. This view is supported by the finding that TBP from native yeast extracts interacts with components of the ADA complexes in coimmunoprecipitation (Saleh et al., 1997) and affinity chromatography experiments (Roberts and Winston, 1997). In addition, in vitro
coimrnunoprecipitation assays demonstrate an interaction between TBP and SPT3p (Eisenmann et al., 1992). The requirement for SPT8p in stabilizing the TBP-SPT3p interaction (Eisenmann et a/., 1994), along with the genetic link between SPT3p and MOT1p and TFllA (Madison and Winston, 1997), provides additional evidence for a functional connection between the SPT proteins and TBP. Based upon the above results, it is tempting to speculate that the ADNSPT complexes may control the binding of TBP to specific promoters andfor its interactions with other components of the basal machinery.
1.3.8 Regulation of the ADA complexes
Recent reports suggest that the transcriptional activity of the ADA complexes is regulated by post-translational modifications of their components. In chapter 4, we provide biochemical evidence for the role of a ubiquitin conjugating system in modulating the transcriptional activity of ADA complexes. This finding, dong with the results from Barlev et al. (1998) that the HAT activity of human GCNS may be regulated by the phosphorylation activity of D N A - P b , indicates that the transcriptional functions of the ADA complexes are targets for regulation by post-translational mechanisms. Interestingly, the Ku autoantigen is also a component of one of the human Pol II holoenzyme complexes (Maldonado et ai., 1996). The association of this DNA repair factor with the holoenzyme suggests that it may have a role in transcriptional regulation. This mode1 is supportad by the finding that the human
Ku homologues in yeast, HDFl p and HDFPp, are involved in both DNA repair and silencing at the HM loci and telomeres (Milne et ab, 1996; Tsukamoto et al.. 1997). In addition, the catalytic subunit of DNA-PK (DNA-Pt&) has been
reported to phosphorylate, in vitro, a long list of transcription factors including
The CTD of Pol II, TBP, TFllB and many transcriptional activators (e.g. HSPSO,
c-fos, c-jun, Oct1, 0ct.2, c-myc, p53, S P I , and steroid receptors) (reviewed in
Lees-Miller, 1996; Jin et al., 1997). However, a correlation between the activity
DNA-pKcsand transcriptional regulation remains to be demonstrated.
1.4
The Ubiquitin Conjugating System: Structure and Functions
The connection between the ADA complexes and ubiquitin conjugating systems was initially proposed in Our laboratory with the isolation of the ubiquitin protease enzyme UBPSp (Baker et al., 1992) as one of the components that interacted with the N-terminal 373 amino acids of NGGlp in a No-hybrid screen (Martens et al., 1996). The interaction between UBP3p and NGGI p suggested that UBP3p may be part of an ADA complex or that a component of an ADA complex, either NGGlp or a factor(s) associated wlh
NGGl p is ubiquitinated and recognized by UBP3p as a substrate. Consistent with this model, we demonstrate that the enzymatic activity of TOM1p, a ubiquitin ligase, is required to regulate transcriptional functions of the ADA complexes (see Chapter 4). Ubiquitin is a 76-amino acid protein that is covalently attached to a nurnber of cytoplasmic, nuclear, and integral membrane proteins (for reviews, see Hochstrasser, 1995; Wilkinson, 1995). Ubiquitination of proteins has been shown to play important roles in a number of cellular regulatory processes including transcription, DNA repair, induced mutagenesis, sporulation, cell cycle progression, stress resistance, peroxisome biogenesis, and programmed cell death (Hochstrasser, 1995). In particular instances, ubiquitination has been dernonstrated to be directly involved in the turnover of some proteins by the 26s proteasorne pathway (reviewed in Pickart, 1997). Several studies indicate that ubiquitination can also confer other fates on specific target proteins. Certain ribosomal proteins are synthesized as fusions with ubiquitin; the N-terminal (alinked) ubiquitin moiety functions as a chaperone in ribosome biogenesis (Finley and Chau, 1991). A significant fraction of histone H2A is mono-
ubiquitinated in higher eukaryotes (Busch and Goldknopf, 1981); although the exact function of this modification remains unclear, it is evidently not related to degradation. Ubiquitination of certain membrane proteins can be a signal for endocytosis (Hicke and Riezman, 1996), and in the case of the transcription factor NFKB,signal-induced attachment of muitiple ubiquitins is a signal for activation by limled processing, versus complete degradation, by the proteasorne (Palombella et al., 1994). These and other exarnples (reviewed in Pickart, 1997) indicate that ubiquitination is required for regulating certain functions of the substrate protein other than targeting its proteolysis.
1Al
Components of the ubiquitin-conjugating system
Ubiquitin Ubiquitin is expressed in al1 eukaryotic cells, where it is synthesized from multiple genes as an N-terminal fusion with other proteins, or as head to tail ubiquitin oligomers (Ozkaynak et ai., 1984; Lund et al., 1985). These are cleaved to mono-ubiquitin by a family of peptidases, one of which is the yeast UHlp (Miller et al., 1989). Individual ubiquitin molecules are linked to substrates through an isopeptide bond between the C-terminal glycine residue of ubiquitin and &-aminogroups of lysine residues on proteins. Branched chains of 20 or more protein-bound ubiquitins are not unusual and, because of the heterogeneity in size, poly-ubiquitinated species are typically visualized on SDS-PAGE as either a 'ladder" or a high molecular-weight "smear". It is generally believed that branched poly-ubiquitin chains are formed by the sequential addition of mono-ubiquitin to lysine 48 or 63 of substrate-bound ubiquitin.
Ubiquitination enzymes The ubiquitination of protein substrates, or the processive lengthening of substrate-bound poly-ubiquitin chahs, requires the concerted action of three classes of enzymes; the ubiquitin activating enzyme E l initially activates ubiquitin in an ATP-dependent reaction through the formation of a thiol ester bond between the C-terminus of ubiquitin and the thiol group of a specific cysteine residue of E l (reviewed in Pickart, 1997). Ubiqulin is then transferred to a specific cysteine residue on one of several ubiquitinconjugating enzymes (Ubcs or E2s). €2 enzymes in tum may transfer the ubiquitin either directly to a substrate or to the final class of enzymes known as ubiquitin protein ligases (or
€3~). The E3 enzymes catalyze the formation of an isopeptide bond between the C-terminus of ubiquitin and the €-amino group of lysine residues on a target protein (Scheffner et al., 1993, 1995; Hochstrasser, 1995). In order for this process to be efficient, it is likely that the E l , E2, and E3 enzymes involved form multiprotein complexes to allow rapid thiol ester transfer of ubiquitin molecules (Ciechanover,1994; Hochstrasser, 1996). While only one functional E l ubiquitin activating enzyme has been identified thus far (Finely and Chau, 1991). over 30 different E2 ubiquitinconjugating enzymes have been isolated frorn various organisms (reviewed by Jentsch, 1992). All E2s contain a conserved domain of approximately 14 kDa (130 amino acids) and an active site cysteine residue that is required for thiol ester formation with ubiquitin. E2 enzymes that consist almost exclusively of the conserved Ubc domain (class I E2s) are unable to transfer ubiquitin to protein substrates in vitro, suggesting that this class of E2s may require E3 ubiquitin ligases for substrate recognition. A second group of €2 enzymes (class II E2s) contain unique C-terminal extensions (e.g. CDC34p, RADGp, UBC6p) (Jentsch, 1992) that rnay contribute to substrate specificity (Jentsch, 1992). ln vitro studies
indicated that some members of this class of E2s are capable of ubiquitinating specific substrates in the absence of ubiquitin ligases (reviewed in Haas and Siepmann, 1997). The E3 ubiquitin protein ligaseç are thought to be the key enzymes that provide substrate specificity for the ubiquitin conjugation system. Although two E3 enzymes have been previously identified from rabbit reticulocytes ( E 3 a and
E3B) and one from yeast (UBRI p) (Hochstrasser, 1995), it was not until the cloning and characterization of E6AP that the structural and functional features of a new class of E3 enzymes was revealed (Reiss et al., 1989; Bartel et al., 1990; Reiss and Hershko, 1990; Jentsch, 1992). E6AP was initially identified as a 100 kDa cellular protein that in conjunction with the E6 oncoprotein of the human papillomavirus type 16 (HPV) constituted the E3 activity in the ubiquitination of p53 (Scheffner et al., 1990, 1995; Huibregtse et al., 1991, 1993). E6AP can also promote the ubiquitination of cellular proteins in the absence of E6, indicating that E6AP can function as an E3 independent of E6 (Scheffner et al., 1995). Sequence analysis of E6AP revealed a region of approximately 350 amino acids in the C- terminus that was highly conserved among a number of proteins from various organisms (Huibregtse et al., 1995). This region, temed the hect domain (Homologous to E6AP c-terminus), also contains a conserved cysteine residue that serves as the active site for thiol ester formation with ubiquitin (Huibregtse et al., 1995). In the case of the characterized hect domain proteins, mutation of the consewed cysteine residue abolishes their ability to accept ubiquitin, suggesting that these protein are functionally related to the €GAP ubiquitin ligase (Huibregtse et al., 1995). A total of six genes encoding hect domain proteins have been identified
in S. cerevisiae and several in D. melanogaster, C. elegans and mammals (Huibregtse et al., 1995). One of the yeast hect domain proteins is encoded by
the RSP5 gene (NPII; Hein et a/., 1995; MDPI; Zolladek et al., 1997). RSP5 was identified by the Winston laboratory as a suppressor of mutations in SPT3 (cited in Huibregtse et al., 1995) and has been found necessary for full transcriptional activation by human steroid receptors in yeast (Imhof and McDonell, 1996). The genetic link between RSPS and SPT3 suggests that ubiquitination rnay be important in regulating the function of SPT3p. This rnodel is further supported with the finding that RSPSp ubiquitinates the CTD of Pol II (Huibregtse et al., 1997). However, the functional role for this ubiquitination remains to be identified. Two of the other yeast hect protein encoding genes have recently been characterized; UFD4 is involved in the ubiquitin fusion degradation pathway (Johnson et al., 1995) while TOMl Qrigger Qfutosis) was identified as a temperature sensitive mutation that arrested the cell cycle at the G2/M transition (Utsugi et al., 1995). The mechanism by which TOMl p functions in cell cycle progression has not been reported. Possible roles of TOMl p in transcription have also been genetically suggested. A multicopy suppressor of the temperature sensitive phenotype of tom 1, STM1 (MPT4/G4p2) has been isolated (cited in Frantz and Gilbert, 1995; Utsugi et al., 1995). Interestingly, STMl p binds to nucleic acids in quadruplex f o n (Frantz and Gilbert, 1995).
STMl is also a multicopy suppressor of htrVmpt5 (Utsugi et al., 1995) and pop2 (cited in Frantz and Gibet?, 1995). POP2p regulates expression frorn the PGK and ADH2 promoters as part of a complex containing CCR4p (Sakai et al., 1992; Denis et al., 1994; Draper et al., 1995). The genetic ties between these
genes suggest that TOMlp rnay have a regulatory role in transcription. Despite these findings, the identity of TOMl p as a ubiquitin Iigase and its potential regulatory function have not been determined.
1.4.2
Proteasornes and ubiquitin proteases The most extensively characterized function for poly-ubiquitination is in
marking proteins for degradation in the 26s proteasome (reviewed in Pickart, 1997). The proteolytic core of the proteasorne is a 205 mulicatalytic particle, comprising 28 subunits in a barrel-shaped arrangement. The 26s structure is derived from the 20s particle by the addition of one or two multisubunit 19s structures. These 19 s structures are "cap-like" in appearance and constitute the regulatory components of the proteasorne. One subunit of the 19s cap is the binding site for poly-ubiquitin (Hochstrasser, 1996). Once proteins have been degraded to peptides in the 26s structure, a proteasome- associated ubiquitinspecific protease (UBP) cleaves poly-ubiquitin chains from residual proteasorne-bound peptides. Poly-ubiquitin chains are then released from the proteasorne and disassembled to mono-ubiquitin by members of a family of non-proteasomal UBPs. UBP3p is one of the yeast non-proteasomal ubiquitin-specific proteases (Baker et ab, 1992). UBP3p was shown to be capable of cleaving the ubiquitin moiety from different engineered substrates in vitro, suggesting that the protein may function as a ubiquitin protease in vivo (Baker et al., 1992). Recently, UBP3p has been reported to regulate silencing by interaction with SIR4p, which
is required for silencing of transcription at the silent mating type loci and at telomeres (Moazed and Johnson, 1996). This finding, along with the obsewed association between NGGlp and UBP3p in a two-hybrid study (Martens et al., 1996), strongly argues that UBP3p is involved in regulating the transcriptional functions of these cofactors. However, the key issue whether the deubiquitination activity of UBP3p is required for signaling such regulations has yet to be demonstrated.
1.4.3
Functions of ubiquitination As mentioned above, ubiquitin-dependent pathways have been shown to
play a crucial role in several cellular processes, among which are control of the cell cycle, DNA repair, cell differentiation and transcriptional regulation. (reviewed by Hochstrasser, 1995, 1996; Wilkinson, 1995; Weissman, 1997). During the cell cycle, cyclin 6 is targeted for ubiquitination and proteolysis to allow progression from metaphase to anaphase (Scherer et al., 1995). The components of this process are associated with the spindle itself and involve a large molecular-weight E3 ligase complex, called the anaphase promoting complex (APC) or cyclosome (reviewed in Pagano, 1997). Repair of UVdamaged DNA in yeast requires the ubiquitin-conjugating enzyme RADGp, which is targeted to single-stranded DNA by complex formation with the RAD18p (Bailly et al., 1994). The ubiquitin conjugating activity of RAD6p is also required for full silencing at the HM loci and at telomeres (Huang et ai., 1997). This result suggests that ubiquitination of certain silencing factors is essential for their function as transcriptional repressors. This mode1 is consistent with the evidence that the deletion of UBP3 can improve silencing in yeast (Moazed and Johnson, 1996), and that mutations in a putative deubiquitinating enzyme enhance position effect variegation in Drosophila (Henchoz et ai., 1996). However, in no case is the target transcriptional regulator for the action of these enzymes known. The possibility for multiple fates for ubiquitinated substrates raises the question of how might ubiquitination alter protein function without targeting to the proteasorne. The discovery of different types of poly-ubiquitin chains suggests one plausible mechanism in which the topology of the chain influences protein-protein interactions of the conjugated substrate (Pickart, 1997). It is known that targeting of proteins to the proteasorne complex is
achieved primarily through the interactions between one type of poly-ubiquitinbound chah and a specific cornponent in the 19s structure (vanNocker et al., 1996). Assigning the proteolytic signaling function to lysine 48-linked chain
provides documented benefits and probably allows mono-ubiquitinations to serve distinct interactions (Pickart, 1997). However, it remains uncertain whether additional diversity in protein-protein interactions is achieved through variations in chain topology or conformation (Pickart, 1997).
1.5
Significance
Coactivator proteins play crucial roles in regulating transcription of eukaryotic organisms. The complex mechanisms of their function become even more complicated with the finding that many coactivators affect transcription both positively and negatively. In many cases, related coactivators/corepressors have been shown to associate in large complexes, indicating that proteinprotein interactions between members of the same group appear to be the rule rather than the exception with this class of regulators. More complexity is also introduced with the finding that certain groups of coactivators do not only f o m one complex, but instead multiple assemblies with different composition and spectnim of transcripts whose expression they affect. The emergence of these facts raised several important questions. How does a coactivator complex regulate transcription? What are the roles of protein-protein contacts between components of a coactivator complex? Why do some groups f o m multiple assemblies? What are the mechanisms that control functions of coactivator molecules? Answers to these and other questions will surely yield important insights for the control of gene expression. The yeast ADA proteins provide an attractive mode1 for seeking answers to the above questions. Several components of this group of coactivators have been identified (see Table 1.1 ; reviewed in Guarente, 1995; Hampsey, 1997), three of which are well characterized (ADAZp, NGGlp and GCN5p). Roles of the characterized factors in both transcriptional activation and rapression have been established (Brandl et al., 1993; 1996; Guarente, 1995). A functional link with the basal machinery (TBP; Barlev et al., 1995; Saleh et al., 1997; Roberts and Winston, 1997) and chromatin structure (Brownell et al., 1996; Grant et al.,
1997) has been suggested. Target activators regulated by this group have been determined and provide probes for function (Berger et ab, 1992; Brandl et al., 1993; 1996; Pina et al., 1993; Marcus et al., 1994; Georgakopoulos et al., 1995;
Horiuchi et al., 1995; Chiag et al., 1996; Martens et al., 1996). The initial genetic and in vitro biochemical data provide a fertile ground for investigating the association between the ADA proteins in vivo. In addition, human homologues of yeast ADA2p and GCN5p have been identified, suggesting the existence of structural and functional conservation (Candau et al., 1996). Considering the above factors, the work in this thesis was initiated to analyze the ADA complex biochemically with special emphases on the in vivo associations between structural components. We reasoned that the identification of proteins that interact with an ADA complex in vivo would provide insight into the physiological roles of the complex and its relationship with other regulatory systems. To investigate the formation of ADA complex in vivo, we generated epitope-tagged versions of ADA2p and NGGl p to act as biochemical handles. In particular, Chapter 2 focuses on the identification of native complexes containing the yeast coactivator/repressor proteins NGGl p and ADA2p. 60th proteins are found to associate together in four complexes (ADA complexes), two with approximate sites of 2 MDa and single complexes of 900 kDa and 200 kDa. This work provided the first evidence for the existence of multiple ADA assemblies in vivo. In addition, we show that TBP coimmunoprecipitates with NGGl p from whole yeast extracts. The significance of the interaction with TBP is suggested by the fact that deletion of amino acids
274-307of NGGl p, which results in slow growth phenotype and loss in the repression of GAL4p seen in a nggl disruption (Brandl et al., 1996), also results in loss of the protein contact with TBP. In addition, the association of TBP with at least one of the huge complexes supports the previously suggested role for
ADA proteins in regulating the interactions between transcriptional activators and basal machinery (Horiuchi et ab, 1995).
A detailed understanding of precise functions and regulation of the ADA complexes requires the identification of component proteins. In Chapter 8,we document the gene product of TRA1, a previously uncharacterized open reading frame, as a component of two of the ADA complexes which also contain
SPT7p (ADA/SPT complexes). TRA 1 (YHR099W) is described as an essential gene in the Saccharomyces Genome Database (SGD) encoding a protein of 3744 amino acids with an estimated molecular mass of 433.2 kDa. TRAl p is a
mernber of a group of putative protein kinases, including the catalytic subunit of human DNA-PK holoenzyme (DNA-pKcs), which contain a C-terminal region related to phosphatidylinositol 3-kinases (P13K, reviewed in Zakian, 1995; LeesMiller, 1996). This finding parallels the recent report by Barlev et al. (1998) that Ku70 associates with human GCN5 protein. Finally, Chapter 4 provides evidence for regulation of coactivator functions by ubiquitination. Specifically, we show that the yeast hect-domain protein TOMl p regulates transcriptional activation through effects on the ADA proteins. Single and double nuIl mutations of tomllnggl result in similar defects in transcription from ADH2, HIS3 and GAL 10 promoters. The action of TOMl p is most likely mediated through ubiquitination since mutation of cysteine 3235 to alanine, conesponding residues of which are required for thiol ester bond formation with ubiquitin in other hect-domain proteins (Scheffner et al., 1995; Huibregtse et al., 1995), results in similar changes in transcription as the nuIl mutation. A direct role for TOMl p in regulation of ADA-associated proteins is further supported by the finding that SPT7p is ubiquitinated in a TOMl pdependent fashion and that TOMl p coimmunoprecipitates with the ADA proteins. In addition, a mechanistic role for ubiquitination by TOMl p is
suggested with the finding that in the absence of TOM1p the ability of the ADA proteins to interact with SPT3p and TBP is reduced. The later result confims that the interaction between TBP and components of ADAISPT complexes is an important facet in transcriptional functions and thus it is a target for regulation.
1.6
References
AlIison,L.A. and Ingles,C.J. (1989) Mutations in RNA polymerase II enhance or suppress mutations in GAL4. Proc. Natl. Acad. Sci. 86, 2794-2798. Apone,L.M., Virbasius,C.M., Reese,J.C., and Green,M.R. (1996) Yeast TAF(II)90 is required for ceIl-cycle progression through G2/M but not for general transcription activation. Genes Dev. 10, 2368-2380. Amason,T. and Ellison,M.J. (1994) Stress resistance inSaccharomyces cerevisiae is strongly correlated with assembly of a novel type of multiubiquitin chah. Mol. Cell. Biol. 14, 7876-7883. AttardiL. D. and Tjian, R. (1993) Drosophila tissue-specific transcription factor NTF-1 contains a novel isoleucine-rich activation motif. Genes Dev. 7, 13411353. Aub1e.D.T. and Hahn,S. (1993) An ATP-dependent inhibitor of TBP binding to DNA. Genes Dev. 7, 844-856. Auble,D.T., Hansen,K.E., Mueller,C.G., Lane,W.S., Thomer,J. and Hahn,S. (1994) Mot1, a global repressor of RNA polymerase II transcription, inhibits TBP binding to DNA by an ATP-dependent mechanism. Genes Dev. 8 1920-1934. Bailly,V., Lamb,J., Sung,P., Prakash,S. and Prakash,L. (1994) Specific cornplex formation between yeast RAD6 and RAD18 proteins: a potential mechanism for targeting RAD6 ubiquitin-conjugating activity to DNA damage sites. Genes Dev, 8, 811-820. Baker,R.T., Tobias,J.W. and Varshavsky,A. (1992) Ubiquitin-specific proteases of Saccharomyces cerevisiae. d Biol. Chem. 267, 23364-23375. Banerjee,A., Gregori, L., Xu,Y. and Chau,V. (1993) The bacterially expressed yeast CDC34 gene product can undergo autoubiquitination to form a multiubiquitin chain-linked protein. J. Biol. Chem. 268, 5668-75. Baniahmad,A., Hal, Reinberg,D., Tsai,S,. Tsai,M.J. and O'Malley,B.W. (1993) Interaction of human thyroid hormone receptor beta with transcription factor TFIlB may mediate target gene derepression and activation by thyroid hormone. Proc. Natl. Acad. Sci. U S A. 90, 8832-8836. Barbet, N.C., Schneider, U., Helliwell, SB.,Stansfield, 1. Tuite, M.F. and Hall, M.N. (1996) TOR controls translation initiation and early G1 progression in yeast. Mol. Biol. Cell. 7 , 25-42. Barlev,N.A., Candau,R., Wang,L., Darpino,P., Silverman,N. and Berger,S.L. (1995) Characterization of physical interactions of the putative transcriptional adaptor, ADA2, with acidic activation domains and TATA-binding protein. J.Biol. Chem. 270, 19337-19344.
Barlev,N.A., Poltoratsky,V., Owen-Hughes,T., Ying,C., Liu,L., Workman,J.L. and 6erger.S.L. (1998) Repression of GCNS histone acetyltransferase activity via bromodomain-mediated binding and phosphorylation by the KU-DNAdependent protein kinase complex. Mol Ce11 Biol. 18, 1349-1358. Bentley,D.L. (1995) Regulation of transcriptional elongation by RNA polymerase II. Curr. Opin. Genet. Dev. 5 210-216. Berger,S.L., PinalB., Silverman,N., Marcus,G.A., Agapite,J., Regier.J.L.. Triezenberg,S.J. and Guarente,L. (1992) Genetic isolation of ADA2 - a potential transcriptional adaptor required for function of certain acidic activation domains Ce11 70, 25 1-265. Bjorklund,S. and Kim,YJ. (1996) Mediator of transcriptional regulation. Trends Biochem. Sci. 21, 335-337. Blair,W.S., Bogerd,H.P., Madore,S.J. and Culler;,B.R. (1994) Mutational analysis of the transcription activation domain of RelA: identification of a highly synergistic minimal acidic activation module. Mol. Cell. Biol. 14, 722697234. Boulton, S.J. and Jackson, S.P. (1998) Cornponents of the Ku-dependent nonhomologous end-joining pathway are involved in telomeric length maintenance and telomeric silencing. EMBO J. 16, 1819-1828. Brandl,C.J., Furlanetto,A.M., Martens,J.A. and Hamilton,K.S. (1993) Characterization of NGGI, a novel yeast gene required for glucose repression of GAL4p-regulated transcription EMBO J. 12 , 5255-5265. Brandl,C.J., Martens,J.A., Margaliot,A., Stenning,D., Furlanetto A.M., Saleh,A., Harnilton,K.S. and Genereaux,J. (1996) Structure/functional properties of the yeast dual regulator protein NGG1 that are required for glucose repression. J. Biol. Chem. 270, 9298-9306. Brou,C., Chaudhary,S., Davidson,l., Lutz,Y., Wu,J., Egly,J.M., Tora,L. and Chambon,P. (1993) Distinct TFllD complexes mediate the effect of different transcriptional activators. EMBO J. 12, 489-499. Brown,E.J,, Albers,M.W., Shin,T.B,, IchikawaK, Keith,C.T., Lane,W.S. and Schreiber,S.L. (1994) A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature 369, 756-758. BrowneIl,J.E., Zhou,J., Ranalli,T., Kobayashi,R.. Edmondson,D.G., Roth,S.Y. and Allis,C.D. (1996) Tetrahymena histone acetyltransferase A: a homolog to yeast GcnSp linking histone acetylation to gene activation. Ce11 84, 843-851. Buratowski,S., Hahn,S., Sharp,P.A. and GuarentqL. (1988) Function of a yeast TATA element-binding protein in a mamrnalian transcription system. Nature 334, 37-42
Buratowski,S, Hahn,S., Guarente,L. and Sharp,P.A. (1989) Five intermediate complexes in transcription initiation by RNA polymerase II. Ce1156, 549-561. Burley,S.K. and Roeder,R.G. (1996) Biochemistry and structural biology of transcription factor IID (TFIID). Annu. Rev. Biochem. 65, 769-799.
-
Busch,H. and Goldknopf.l.1. (1981) Ubiquitin protein conjugates. Mol. Cell. Biochem. 40. 1 73-187. Caims,B.R., Levinson,R.S., Yamamot0,K.R. and Komberg,R.D. (1996) Essential role of Swp73p in the function of yeast SwVSnf complex. Genes Dev. 10, 2131 2144.
-
Candau,R. and Berger,S.L. (1996) Structural and functional analysis of yeast putative adaptors. Evidence for an adaptor cornplex in vivo. J.Biol.Chem. 271, 5237-5245. Candau,R., Moore,P.A., Wang, L., Barlev,N., Ying,C.Y., Rosen,C.A. and Berger,S.L. (1996) Identification of human proteins functionally conserved with the yeast putative adaptors ADA2 and GCNS. Mol. Cell. Biol. 16, 593-602. Candau,R., Zhou,J,-X., Allis,C.D. and Berger,S.L. (1997) Histone acetykransferase activity and interaction with ADA2 are critical for GCN5 function in vivo. EMBO J. 16, 555-565. Carlson,M. and Laurent,B.C. (1994) The SNF/SWI family of global transcriptional activators Curr. OOp.Cell Biol. 6, 396-402. Carlson, M. (1997) Genetics of transcriptional regulation in yeast: connections to the RNA polymerase II CTD. Annu. Rev. Cell. Dev. Biol. 13, 1-23. Carrnen,A.A., Rund1ett.S.E. and Grunstein,M. (1996) HDAl and HDA3 are components of a yeast histone deacetylase (HDA) complex. J. Biol. Chem. 271, 15837-15844. Caron.C., Rousset,R.. Beraud,C., Moncollin,V., Egly,J.M. and Jalinot,P. (1993) Functional and biochemical interaction of the HTLV-I Taxl transactivator with TBP. EMBO J. 12, 426904278. Chang,M. and JaehningJA. (1997) A multiplicity of mediators: alternative forms of transcription complexes communicate wit transcriptional regulators. Nucleic. Acids Res. 25,4861 -4865. Chao,D.M., Gadbois,E.L., Murray,P.J., Anderson,S.F., Sonu,M.S., Parvin,J.D. and Young,R.A. (1996) A marnmalian SRB protein associated with an RNA polymerase II holoenzyme. Nature 380, 82-85.
Chen,J.L., Attardi,L.D., Verrijzer,C.P., Yokomori,K. and Tjian,R. (1994) Assembly of recombinant TFllD reveals differential coactivator requirements for distinct transcriptional activators. Cell79, 93-105. Chen,W. and StnihllK. (1988) Saturation mutagenesis of a yeast his3 TATA elernent: genetic evidence for a specific TATA-binding protein. Proc. Natl. Acad. Sci. USA 85, 2691-2695. Chen,Z. and Pickart,C.M. (1990) A 25-kilodalton ubiquitin carrier protein (E2) catalyzes multiubiquitin chain synthesis via lysine-48 of ubiquitin. J. Biol. Chem. 265, 21835-21842. Chen,Z., Parent,L. and Maniatis,T. (1996) Site-specific phosphorylation of lkBa by a novel ubiquitination-dependent protein kinase activity. Ce// 84, 653-862. Chiang, Y.C., Komarnitsky, P., Chase, D.and Denis, C.L. (1996) ADRl activation domains contact the histone acetyltransferase GCN5 and the core transcriptional factor TFIIB. J. Biol. Chem.. 271, 32359-32365. Ciechanover,A. (1994) The ubiquitin-proteasorne proteolytic pathway. Ce1179, 13-21. Clark-Adams,C.D., Norris,D., Osley,M.A., Fassler,J.S. and Winst0n.F. (1988) Changes in histone gene dosage alter transcription in yeast. Genes Dev. 2, 150-159. Co1lartM.A. (1996) The NOT, SPT3, and MOT1 genes functionally interact to regulate transcription at core promoters. Mol. Cell. Biol. 16, 666806676. Conaway,R.C., Conaway,J.W. (1993) General initiation factors for RNA polymerase II. Annu. Rev. Biochem. 62. 161-190. Cote,J., Quinn,J., Workrnan,J.L. and Peterson,C.L. (1994) Stimulation of GAL4 derivative binding to nucleosomal DNA by the yeast SWVSNF complex. Science 265, 53-60. Denis,C.L., Draper,M.P., Liu,H.Y., Malvar,T., Vallari,R.C. and Cook,W.J. (1994) The yeast CCR4 protein is neither regulated by nor associated with the SPT6 and SPT10 proteins and forms a functionally distinct complex from that of the SNF/SWI transcription factors. Genetics 138, 1005-13. Draper,M.P., Salvadore,C. and Denis,C.L. (1995) Identification of a mouse protein whose homolog in Saccharomyces cerevisiae is a component of the CCR4 transcriptional regulatory cornplex. Mol. Cell. Bol. 15, 3487-95. Drapkin,R. and Reinberg,D. (1994) The multifunctional TFllH complex and transcriptional control. Trends Biochem. Sci. 19, 504-508.
Drysdale,C.M., Jackson,B.M., McVeigh,R., Klebanow,E.R., Bai,Y., Kokubo,T., Swanson,M., Nakatani,Y., Wei1,P.A. and Hinnebusch,A.G. (1998) The Gcn4p activation domain interacts specifically in vlro with RNA polyrnerase II holoenzyme TFIID, and the Adap-Gcn5p coactivator complex. Mol. Cell. Bol. 18, 1711-1724. Ellenberger,T.E., Brandl,C.J., Struhl,K. and Harrison,S.C. (1992) The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted alpha helices: crystal structure of the protein-DNA complex. CeIl 71, 1223-1237. Eisenmann,D.M., Dollard,C. and Winston, F. (1989) SPTIS, the gene encoding the yeast TATA binding factor TFllD is required for normal transcription in vivo. Ce11 58, 1183-1191. Eisenmann,D.M., Amt,K.M.. Ricupero,S.L., Rooney,J.W. and Winst0n.F. (1992) SPT3 interacts with TFllD to allow normal transcription in Saccharomyces cerevisiae. Genes Dev. 6,13 19- 1331. Eisenmann,D.M., Chapon,C., Roberts,S.M., Dollard,C. and Winston,F. (1994) The Saccharomyces cerevisiae SPT8 gene encodes a very acidic protein that is functionally related to SPT3 and TATA-binding protein. Genetics 137, 647657. Fassler,J.S. and Winston,F. (1988) Isolation and analysis of a novel class of suppressor of Ty insertion mutations in Saccharomyces cerevisiae. Genetics 118, 203-212. Feldmann,H., Driller,L., Meier,B., Mages,G., Kellenann,J. and Winnacker,E.L. (1996) HDF2, the second subunit of the Ku homologue from Saccharomyces cerevisiae. J. Biol. Chem. 271 , 27765027769. Finley,D., and Chau,V. (1991) Ubiquitination. Annu. Rev. CeIl Biol. 7 , 25-69 Frantz,J.D. and Gilbert,W. (1995) A yeast gene product, G4p2, with a specific affinity for quadruplex nucleic acids. J. Biol. Chem. 270, 9413-9419. Gansheroff,L.J., Dollard,C., Tan,P. and Winston,F. (1995) The Saccharomyces cerevisiae SPT7 gene encodes a very acidic protein important for transcription in vivo. Genetics 139, 523-536. Georgakopoulos,T. and Thireos,G. (1992) Two distinct yeast transcriptional activators require the function of the GCNS protein to promote normal levels of transcription EMBO J. 11, 4145-4152. Georgakopoulos,T., Gounalaki,N. and Thire0s.G. (1995) Genetic evidence for the interaction of the yeast transcriptional CO-activatorproteins GCN5 and ADA2 Mol. Gen. Genet. 246, 723-728.
Ghysdae1,J. and Yaniv,M. (1991) Nuclear oncogenes. Cun. Opin. Cell. Biol. 3 , 484-491. GiII,G., Sadowski, 1. and Ptashne,M. (1990) Mutations that increase the activity of a transcriptional activator in yeast and mammalian cells. Proc. Natl. Acad. Sci. USA 87, 2127-2131. Goodrich,J.A. and Tjian,R. (1994) TBP-TAF complexes: selectivity factors for eukaryotic transcription. Curr. Opin. Cell. Biol. 6 , 403-409. Gottlieb,T.M. and Jackson,S.P. (1993) The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen. Ce11 72, 131-142. Grant,P.A., Duggan,L., Cote,J., Roberts,S.M., Brownell,J.E., Candau,R., Ohba, R.. Owen-Hughes,T., Allis,C.D., Winston,F., Berger,S.L. and Workman,J .L. (1997) Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (SpVAda) complex. Genes Dev. 11, 1640-1650. Greenwell,P.W, K.ronmal,S.L., Porter,S.E., GassenhuberJ., Obermaier.B., Petes.T.D. (1995) TEL1, a gene involved in controlling telomere length in S. cerevisiae, is homologous to the human ataxia telangiectasia gene. Ce1182, 823-829. Grunstein M. (1997) Histone acetylation in chromatin structure and transcription. Nature 389, 349-352. Gu,J..Ren,K., Dubner,R. and ladarola,M.J. (1994) Cloning of a DNA binding protein that is a tyrosine kinase substrate and recognizes an upstream initiatorlike sequence in the promoter of the preprodynorphin gene. Mol. Brain Res. 24, 77-88. Gu,W., Roeder,R.G. (1997) Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell90, 595-606. Guarente,L. (1983) Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast.. Mefhods Enzymol. 101, 181-19 1. Guarente,L. and Hoar,E. (1984) Upstream activation sites of the CYCl gene of Saccharomyces cerevisiae are active when inverted but not when placed downstrearn of the ''TATA box". Proc. Natl. Acad. Sci. U S A. 81, 786007864. Guarente,L. (1988) UASs and enhancers: common mechanism of transcrÏptional activation in yeast and mammals. Ce1152, 303-305. Guarente,L. (1995) Transcriptional coactivators in yeast and beyond. Trends Biochem. Sci. 20, 5 17-21.
Haas,A.L., Reback,P.B. and Chau,V. (1991) Ubiquitin conjugation by the yeast RAD6 and CDC34 gene products. J. Biol. Chem. 266, 5 104-5 112. Haas.A.L. and Siepmann,T.J. (1997) Pathways of ubiquitin conjugation. FASEB J. 11, 1257-1268. Hager,G.L., Archer,T.K., Fragoso.G., Bresnick,E.H., Tsukagoshi,Y., John,S. and Smith,C.L. (1993) Influence of chrornatin structure on the binding of transcription factors to DNA. Cold Spring Harb. Symp Quant. Biol. 58, 63-71. Hahn,S., Hoar,E.T. and Guarente,L. (1985) Each of three "TATA elements" specifies a subset of the transcription initiation sites at the CYC-1 promoter of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U S A. 82, 8562-8566. Hahn,S., Buratowski,S., Sharp,P.A. and Guarente,L. (1989) Yeast TATAbinding protein TFllD binds to TATA elements with both consensus and nonconsensus DNA sequences. Proc. Natl. Acad. Sci. U S A. 86, 571 8-5722. Hampsey,M. (1997) A SAGA of histone acetylation and gene expression. Trends Genet. 13, 427-429. Hartley,K.O., Gell,D., Smith,G.C., Zhang,H., Divecha,N., Connelly,M.A., Admon,A., Lees-Miller,S..P., Anderson,C.W., and Jackson,S.P. (1995) DNAdependent protein kinase catalytic subunit: a relative of phosphatidylinositol 3kinase and the ataxia telangiectasia gene product. Ce11 82, 849-856. Hartshome,T.A., Blumberg,H. and Young,E.T. (1986) Sequence homology of the yeast regulatory protein ADRl with Xenopus transcription factor TF1IIA. Nature 320, 283-287. Hartzog,G.A., Wada,T., Handa,H. and Winston,F. (1998) Evidence that Spt4, SptS, and Spt6 control transcription elongation by RNA polymerase II in Saccharomyces cerevisiae. Genes Dev. 12, 357-369. Hassig,C.A., Tong,J.K., Fleischer,T.C., Owa,T., Grable,P.G., Ayer,D.E. and Schreiber,S.L. (1998) A role for histone deacetylase activity in HDAC1rnediated transcriptional repression. Proc. Natl. Acad. Sci. U S A. 95. 35193524. Hein,C., Springae1,J.-Y., Volland,C., Haguenaurer-Tsapis,R. and Andrel,B. (1995) NPII, an essential yeast gene involved in induced degradation of Gap1 and Fur4 permeases, encodes the RSPS ubiquitin-protein ligase. Mol. Microbiol. 18, 77-87.
Heitman,J., Mowa,N.R. and Hal1,M.N. (1991) Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science 253, 905-909.
Helliwell, SB.,Howald, I., Barbet, N. and Hall, M.N. (1998) TOR2 is part of two related signaling pathways coordinating cell growth in Saccharomyces cerevisiae. Genetics 1 48, 99-112. Henchoz,S., De Rubertis,F., Pauli,D. and Spierer,P. The dose of a putative ubiquitin-specific protease affects position-effect variegation in Drosophila melanogaster. Mol. Cell. Biol. 16, 5717-5725. Hengartner,C.J., Thompson,C.M., Zhang,J., Chao,D.M., Liao,S.M., Koleske,A. J., Okamura,S. and Young,R.A. (1995) Association of an activator with an RNA polymerase II holoenzyme. Genes Dev. 9, 897-910. Henry,N.L., Campbell,A.M., Feaver,W.J., Poon,D., Wei1,P.A. and Kornberg, RD. (1994) TFIIF-TAF-RNA polymerase II connection. Genes Dev. 8, 2868-2878. Hershko,A., and Ciechanover,A. (1992) The ubiquitin systern for protein degradation. Annu. Rev. Biochem. 61, 761-807. Hicke,L. and Riezman,H. (1996) Ubiquitination of a yeast plasma membrane receptor signals its ligand-stimulated endocytosis. Cell84, 277-287. Hinnebusch,A.G. (1994) Translational control of GCN4: an in vivo barorneter of initiation-factor activity. Trends Biochem. Sei. 1 9 , 409414. Hirschhom,J.N., Bortvin,A.L., Ricupero-Hovasse,S.L., Winston, F. (1995) A new class of histone H2A mutations in Saccharomyces cerevisiae causes specific transcriptional defects in vivo. Mol. Cell. Biol. 15, 1999-2009. Hochstrasser,M. (1995) Ubiquitin, proteosome, and the regulation of intracellular protein degradation. Curr. Opin. Ce11 Biol. 7, 215-213. Hochstrasser,M. (1996) Protein degradation or regulation: Ub the judge. Ce11 84, 813-815. Hope,l.A. and Struhl,K. (1986) Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of yeast Ce11 46, 885-894. Horiuchi,J., Silverman,N., Marcus,G.A. and Guarente,~.(1995) ADA3, a putative transcriptional adaptor, consists of two separable domains and interacts with ADA2 and GCNS in a trimeric cornplex. Mol.Cel1. Biol. 15, 1203-1209. Horiuchi,J., Silverman,N., Pina,B., Marcus,G.A. and Guarente,L. (1997) ADAI , a novel component of the ADAGCNB cornplex, has broader effects than GCNS, ADA2, or ADA3. Mol. Cell. Biol. 17 , 3220-3228. Huang,H., Kahana,A., Gottschling,D.E., Prakash.1. and Liebman,S.W. (1997) The ubiquitin-conjugating enzyme Rad6 (Ubc2) is required for silencing in Saccharomyces cerevisiae. Mol. Cell. Biol. 17 , 6693-6699.
Huang,Y., Baker,R., and Fischer-Vize,J.A. (1995) Control of ceIl fate by a deubiquitinating enzyme encoded by the fat facets gene. Science 270, 18281831. Huibregtse,J.M., Schefher,M., and Howley,P.M. (1991) A cellular protein mediates association of p53 with the E6 oncoprotein of human papillomavirus types 16 or 18. EMBO J. 13,29-4135. Huibregtse,J. M., Scheffner,M. and Howley,P.M. (1993) Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53. Mol. Cell. Biol. 13, 775-784. Huibregtse,J.M., Scheffner,M., Beaudenon,S. and How1ey.P.M. (1995) A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proc. Natl. Acad. Sei., USA, 92, 256302567. Irnhof,M.O. and M~Donne1l~D.P. (1996) Yeast RSPS and its human hornolog hRPFl potentiate hormone-dependent activation of transcription by human progesterone and glucocorticoid receptors. Mol. Cell. Bol. 16, 2594-2605. Imhof,A., Yang.X.J., 0gryzk0,V.V.~Nakatani,Y., Wo1ffe.A.P. and Ge,H. (1997) Acetylation of general transcription factors by histone acetyltransferases. Curr. Biol. 7, 689-692 Ingles,C.J., Shales,M., Cress,W.D., Triezenberg,S.J. and Greenblatt,J. (1991) Reduced binding of TFllD to transcriptionally compromiseci mutants of VP16. Nature 351, 588-590. Jackson,B.M., Drysdale,C.M., Natarajan,K. and Hinnebusch,A.G. (1996) Identification of seven hydrophobic clusters in GCN4 making redundant contributions to transcriptional activation. Mol. Cell. Biol. 16, 5557-5571. Jackson,S.P. (1996) The recognition of DNA damage. Curr. Opin. Genet. Dev. 6, 19-25. Jacq,X., Brou,C., Lutz,Y., Davidson,l., Chambon,P. and Tora,L. (1994) Human TAFIB O is present in a distinct TFllD complex and is required for transcriptional activation by the estrogen receptor. Ce1179, 107-117. Jentsch,S. (1992) The ubiquitinconjugation system. Annu. Rev. Genet. 26, 179-207. Jentsch,S. and Schlenker, S. (1995) Selective protein degradation: a joumey's end within the proteasome. Ce11 82, 881-884. Jin,S., Inoue,S. and Weaver,D. (1997) Functions of the DNA dependent protein kinase. Cancer Sem. 29, 221-261. Johnson,E.S., Ma,P.C.M., 0taJ.M. and Varshavsky,A. (1995) A proteolytic pathway that recognizes ubiquitin as a degradation signal. J. Biol. Chem. 270, 17442- 17456.
Kadosh,D. and Stnih1.K (1998) Histone deacetylase activity of Rpd3 is important for transcriptional repression in vivo. Genes Dev. 12, 797-805. Kato,R. and Ogawa,H. (1994)An essential gene, ESRI, is required for mitotic cell growth, DNA repair and meiotic recombination in Saccharomyces cerevisiae. Nucleic Acids Res. 22, 3104-3 112. Keith, C.T. and Schreiber,S.L. (1995) PIK-related kinases: DNA repair, recombination, and cell cycle checkpoints. Science 270, 50-51. Kim,T.K. and Roeder,R.G. (1994) Proline-rich activator CTFl targets the TF118 assembly step during transcriptional activation. Proc. Natl. Acad .Sci. U S A. 91, 4170-4174. Kim,Y .J., Bjorklund,S., Li,Y., Sayrt3,M.H. and Komberg,R.D. (1994) A multiprotein mediator of transcriptional activation and its interaction with the Cterminal repeat domain of RNA polymerase II. Ce1177, 599-608. Kingston,R.E., Bunker,C.A. and 1mbalzan0,A.N. (1996) Repression and activation by multiprotein complexes that alter chromatin structure. Genes Dev. IO, 905-920. Klebanow,E.R., Poon,D., Zhou,S. and Wei1,P.A. (1996) Isolation and characterization of TAF25, an essential yeast gene that encodes an RNA polymerase Il-specific TATA-binding protein-associated factor. J. Biol. Chem. 271, 13706-13715. Koleske,A.J. and Young,R.A. (1994) An RNA polymerase II holoenzyme responsive to activators. Nature 368, 466-469. Koleske,A.J. and Young,R.A. (1995) The RNA polymerase II holoenzyme and its implications for gene regulation. Trends Biochem. Sci. 20, 113-116. Kolodziej,P.A. and Young,R. (1989) RNA polymerase II subunit RPB3 is an essential component of the mRNA transcription apparatus. Mol. Cell. Bol. 9, 5387. Komitzer, D., Raboy, B., Kulka, R.G. and Fink G.R. (1994) Regulated degradation of the transcription factor Gcn4. EMBO J. 13, 6021-6030. Kruger,W. and Herskowitz,1. (1991) A negative regulator of HO transcription, SIN1 (SPT2), is a nonspecific DNA-binding protein related to HMGI. Mol. Cell. Biol. 11, 4135-4146. Kruger,W., Peterson,C,L., SiII,A., Cobum,C., Arents,G., Moudrianakis,E.N. and Herskowitz 1 (1995) Amino acid substitutions in the structured domains of histones H3 and H4 partially relieve the requirement of the yeast SWVSNF cornplex for transcription. Genes Dev. 9, 2770-2779.
Kuchin,S., Yeghiayan,P. and Carison,M. (1995) Cyclin-dependent protein kinase and cyclin homologs SSN3 and SSN8 contribute to transcriptional control in yeast. Proc. Natl. Acad. Sci. U S A. 92, 4006-4010. Kuchin,S. and Carlson,M. (1998) Functional relationships of Srbl O-Srbl 1 kinase, carboxy-terminal domain kinase CTDK-1, and transcriptional corepressor Ssn6-Tupl. Mol. Cell. Biol. 18, 1163-1171. Kunz,J., Henriquez,R., Schneider,U., Deuter-Reinhard,M., Mowa,N.R. and Hal1,M.N. (1993) Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression. Ce11 7, 585596. Kuo,M.H., Brownell,J., Sobel, R.E., Ranalli,T.A., Edmonson,D.G., Roth,S.Y. and Allis,C.D. (1996) Transcription-linked acetylation by GcnSp of histones H3 and H4 at specific lysines, Nature 383, 269-272. Kuo,M.H., Brownell,J.E., Sobel,R.E., Ranalli,T.A., Cook,R.G., Edmondson,D.G., Roth,S.Y. and Allis,C.D. (1996) Transcription-linked acetylation by GcnSp of histones H3 and H4 at specific lysines. Nature 383, 269-272.
Kuo, M. H., Zhou,J, Jambeck,P., Churchill,M. E. and Allis,C.D. (1998)Histone acetyltransferase activity of yeast Gcn5p is required for the activation of target genes in vivo. Genes Dev. 12, 627-639. Lai,J.S. and Herr,W. (1997) lnterdigitated residues within a small region of VP16 interact with Oct-1, HCF, and DNA. Mol. Cell. Biol. 17, 393703946, Laughon,A. Gesteland,R.F. (1984) Prirnary structure of the Saccharomyces cerevisiae GAL4 gene. Mol. Cell. Bol. 4, 260-267. Laurent,B.C., Treich,l. and Carlson, M. (1993) The yeast SNFUSW12 protein has DNA-stimulated ATPase activity required for transcriptional activation. Genes Dev. 7, 583-591. Lee,D.Y., Hayes,J.J, Pruss,D. and Wolffe,A.P. (1993) A positive role for histone acetylation in transcription factor access to nucleosomal DNA. Cell72, 73-84. Lee,Y.C., Min,S., Gim,B.S. and Kim,Y.J. (1997) A transcriptional mediator protein that is required for activation of many RNA polymerase II promoters and is conserved frorn yeast to humans. Mol. Cell. Biol. 17, 4622-4632. Lees-Miller,S.P., Chen,Y.R. and Anderson,C.W. (1990)Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen. Mol. Cell. Bol. 12, 6472-6481. Lees-Miller,SP. (1996) The DNA-dependent protein kinase, DNA-PK: 10 years and no ends in sight. Biochem Cell. Biol. 74, 503-512.
Li,Y., Flanagan,P.M., Tschochner,H. and Komberg,R.D. (1994) RNA polymerase II initiation factor interactions and transcription start site selection. Science 263, 805-807. Li, Y., Bjorklund, S., Jiang, Y.W., Kim, Y-J., Lane, W.S., Stillman, D. and Komberg, R.D. (1995) Yeast global transcriptional regulators Sin4 and Rgrl are components of mediator cornplex1RNA polymerase II holoenzyme. Proc. Natl. Acad. Sci. 92, 10864-10868. Liao,S.M., Zhang,J., Jeffery, D.A., Koleske,A.J., Thompson,C.M., Chao,D.M., Viljoen,M., vanVuuren,H.J. and Young,R.A. (1995) A kinase-cyclin pair in the RNA polymerase II holoenzyme. Nature 74, 193-196. Lohr,D. (1997)Nucleosome transactions on the promoters of the yeast GAL and P H 0 genes. J. Bol. Chem. 272, 26795-26798. Loidl,P. Histone acetylation: facts and questions. Chromosoma 103, 441-449. Lund,P.K., Moats-Staats,B.M., Simmons,J.G., Hoyt,E., D'Ercole,A.J., Martin,F., and Van Wyk, J.J. (1985) Nucleotide sequence analysis of a cDNA encoding human ubiquitin reveals that ubiquitin is synthesized as a precursor. J. Biol. Chem. 260, 7609-7613. Madison, J. M. and Winston, F. (1997)Evidence that Spt3 functionally interacts with Mot1, TFIIA, and TATA-binding protein to confer promoter-specific transcriptional control in Saccharomyces cerevisiae. Mol. Cell. Biol. 17, 287295. Mages,G.J., Fe1dmann.H.M. and Winnacker,E.L. (1996) Involvement of the Saccharomyces cerevisiae HDFl gene in DNA double-strand break repair and recombination. J. Biol. Chem. 271, 7910-7915. Maldonado,E., Shiekhattar,R., Sheldo, M., Cho,H., Drapkin,R., Rickert,P., Lees,E., Anderson,C.W., Linn,S. and Reinberg,.D. (1996) A human RNA polyrnerase II complex associated with SRB and DNA-repair proteins. Nature 381, 86-89. Malone,E.A., Fassler,J.S. and Winston,F. (1993) Molecular and genetic characterization of SPT4, a gene important for transcription initiation in Saccharomyces cerevisiae. Mol. Gen. Genet. 237, 449-459. Marcus,G.A., Silverman,N., Berger,S.L., Horiuchi,J. and Guarente,L. (1994) Functional similarity and physical association between GCN5 and ADA2: Putative transcriptional adaptors EMBO J. 13, 4807-4815. Marcus,G.A., Horiuchi,J., Silverman,N. and Guarente,L. (1996) ADAS/SPT20 links the ADA and SPTgenes, which are involved in yeast transcription. Mol. Cell. Bol. 16, 31 97-3205.
Martens,J.A. Genereaux,J., Sa1eh.A. and Brandl,C. J. (1996) Transcriptional activation by yeast PDRl p is inhibited by its association with NGG1p/ADA3p. J. Biol. Chem. 271, 15884-15890. McNei1,J.B. and Smith,M. (1986) Transcription initiation of the Saccharomyces cerevisiae iso-1-cytochrome c gene. Multiple, independent T-A-T-A sequences. J. Mol. Biol. 187, 363-378. Melcher, K. and Johnston, S.A. (1995) GAL4 interacts with TATA-binding protein and coactivators. Mol. Cell. Biol. 15, 2839-2848. Mengus,G., May,M., Jacq,X., Staub,A., Tora.L.. Chambon,P. and Davidson.l. (1995) Cloning and characterization of hTAF1118, hTAFII20 and hTAF1128: three subunits of the human transcription factor TFIID. EMBO J. 14, 1520-1531.
Metz,R., Bannister,A.J., Sutherland,J.A., Hagerneier,C., OtRourke,E.C., Cook.A., Bravo,R. and Kouzarides,T. (1994) c - fos-induced activation of a TATA-boxcontaining promoter involves direct contact with TATA-box-binding protein. Mol. Cell. Biol. 14, 6021-6029. Miller,H.I., Henzel,W.J., Ridgway,J.B.. Kuang.W.-J., Chisholm,V., and Liu,C.-C. (1989) Purification of a ubiquitin protein peptidase from yeast with efficient in vitro assays. Bio/Technology 7, 698-704. Milne,G.T., Jin,S., Shann0n.K.B. and Weaver,D.T. (1996) Mutations in two Ku homologs define a DNA end-joining repair pathway in Saccharomyces cerevisiae. Mol. Cell. Biol. 1 6 , 4 189-4 198. Mitchel1,P.J. and Tjian,R. (1989) Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science 245, 371-378. Mizzen,C.A., Yang,X.J., Kokubo,T., Brownell,J.E., Bannister,A.J., OwenHughes,T., Workman,J., Wang,L., Berger,S.L., Kouzarides,T., Nakatani.Y. and Allis,C.D. (1996) The TAF(11)250 subunit of TFllD has histone acetyltransferase activity. Cell87, 1261-1270. Moazed, D. and Johnson, D. (1996) A deubiquitinating enzyme interacts with SIR4 and regulates silencing in S. cerevisiae. Ce11 86, 667-677. Moqtaderi,Z., Bai,Y ., Poon,D., Wei1,P.A. and Struhl,K. (1996) TBP-associated factors are not generally required for transcriptional activation in yeast. Nature 383, 188-191. Morrow,D.M., Tagle,D.A.. Shiloh,Y., Collins,F.S. and Hieter,P. (1995) TEL 1, an S. cerevisiae homolog of the human gene mutated in ataxia telangiectasia, is functionally related to the yeast checkpoint gene MECI. Cell82, 831-840.
Nagawa,F. and Fink,G. R. (1985) The relationship behiveen the "TATA" sequence and transcription initiation sites at the HIS4 gene of Saccharomyces cerevisiae. Proc. Nat!. Acad. Sci. U SA. 82, 8557-8561. Nakajima,N., Horikoshi,M. and Roeder,R.G. (1988) Factors involved in specific transcription by mammalian RNA polymerase II: purification, genetic specificity, and TATA box-promoter interactions of TFIID. Mol. Cell. Biol. 8 , 4028-4040. Ogryzko,V.V., Schiltz,R.L., Russanova,V., Howard,B.H. and Nakatani,Y. (1996) The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell87, 953-959. OIShea,E.K., Rutkowski,R. and Kim,P.S. (1989) Evidence that the leucine zipper is a coiled coil. Science 243, 538-542. Ossipow,V., Tassan,J.P., Nigg,E.A. and Schibler,U. (1995) A mammalian RNA polymerase II holoenzyme containing al1 components required for promoterspecific transcription initiation. Ce1183, 137-146. Orphanides,G., Lagrange,T. and Reinberg,D. (1996) The general transcription factors of RNA polymerase II. Genes Dev. 10, 2657-2683. Ozkaynak, E., Finley, D., and Varshavsky, A. (1984) The yeast ubiquitin gene: head-to-tail repeats encoding a polyubiquitin precursor protein. Nature 31 2, 663-666. Pagano,M. (1997) Cell cycle regulation by the ubiquitin pathway. FASEB J. 11 1067-1075. Palombella,V.J., Rando,O.J., Go1dberg.A.L. and Maniatis,T. (1994) The ubiquitin-proteasome pathway is required for processing the NF-kappa B I precursor protein and the activation of NF-kappa B. Cell78, 773-785. Partel,B., Wunning, l., and Varshavsky,A. (1990) The recognition component of the N-end rule pathway. EMBO J. 9, 3179-3189. Patikoglou,G. and Burley,S.K. (1997) Eukaryotic transcription factor-DNA complexes. Annu. Rev. Biophys. Biomol. Struct. 26, 289-325. Pazin,M.J. and Kadonaga,J.T. (1997a) Nucleosome rnobility and the maintenance of nucleosome positioning. Science 276, 809-812. Pazin,M.J. and Kadonaga, J.T. (1997b) SW@/SNF2 and related proteins: ATPdriven motors that disrupt protein-DNA interactions? Cell 88, 737-740. Peterson,C.L., Dingwall,A. and Scott,M.P. (1994) Five SWVSNF gene products are components of a large multisubunit complex required for transcriptional enhancement. Proc. Natl. Acad. Sci. U S A. 91, 2905-2908.
Peterson,C.L. (1996) Multiple SWltches to tum on chromatin? Curr. Opin. Genet. Dev. 6, 171-175. Pickart,C.M. (1997) Targeting of substrates to the 26s proteasome. FASEB J. il,1055-1066. PinalB., Berger,S.L., Marcus,G.A., Silverman,N., Agapite,J. and Guarente,L. (1993) ADA3 a gene, identified by resistance to GAL4-VP16, with properties similar to and different from those of ADA2. Mol. Cell. Biol. 13, 5981-5989.
-
PintoJ., WU,W.H., Na,J.G. and Hampsey,M. (1994) Characterization of sua7 mutations defines a domain of TFllB involved in transcription start site selection in yeast. J. Biol. Chem. 269, 30569-30573. Pollard, K.J. and Peterson, C.L. (1997) Role for ADNGCNS products in antagonizing chromatin-mediated transcriptional repression. Mol. Cell. Biol.. 1 7 , 6212-6222. Poon, D., Campbell, A.M., Bjorklund, S., Kim, Y., Zhou, S., Komberg, R.D. and Weil, P.A. (1995) Identification and characterization of a TFIID-like multiprotein cornplex f rom Saccharomyces cerevisiae. Roc. Natl. Acad. Sci. USA. 92, 82248228. Ptashne,M. (1986) Gene regulation by proteins acting nearby and at a distance. Nature 322, 697-701. Ptashne,M. (1989) How gene activators work. Sei. Am. 260, 40-47. Ptashne,M. and Gann,A. (1997) Transcriptional activation by recruitment. Nature 386, 569-577. Pugh,B.F. and Tjian,R. (1990) Mechanism of transcriptional activation by Spl: evidence for coactivators. Ce11 61, 1187-1197. Ranish, J.A. and Hahn,S. (1991) The yeast general transcription factor TFllA is composed of two polypeptide subunits. J. Biol. Chem. 266, 19320-19327. Reece,R.J. and Platt,A. (1997) Signaling activation and repression of RNA polymerase II transcription in yeast. Bioessays 19, 1001-1010. Reese, J.C., Apone, L., Walker, S.S., Griffin, L.A. and Green, M.R (1994) Yeast TAF(ll)s in a multisubunit complex required for activated transcription Nature 371, 523-527, Reiss, Y., Heller, H., and Hershko, A. (1989) Binding sites of ubiquitin-protein ligase. Binding of ubiquitin-protein conjugates and of ubiquitin-carrier protein. J. Biol. Chem. 264, 10378-10383.
Reiss,Y., and Hershko,A. (1990) Affinity purification of ubiquitin-protein ligase on immobilized protein substrates. Evidence for the existence of separate NH2terminal binding sites on a single enzyme. J. Biol. Chem. 265, 368593690. Roberts,S.G., Ha,l., Maldonado,€., Reinberg,D. and Green,M.R. (1993) Interaction between an acidic activator and transcription factor TF116 is required for transcriptional activation. Nature 363, 741-744. Roberts,S.M. and Winston,F. (1996) SPT20/ADA5 encodes a novel protein functionally related to the TATA-binding protein and important for transcription in Saccharomyces cerevisiae. Mol. Cell. Biol. 16, 3206-3213. Roberts,S.M. and Winston,F. (1997) Essential functional interactions of SAGA, a Saccharomyces cerevisiae complex of Spt, Ada and Gcn5 proteins, with the Snf/Swi and SrUmediator complexes. Genetics 147, 451-465. Roeder, R.G. (1996) The role of general initiation factors in transcription by RNA polyrnerase II. Trends Biochem. Sci. 21, 327-335. Ruiz-Garcia, A.B., Sendra, R., Pamblanco, M. and Tordera, V. (1997) GCNSp is involved in the acetylation of histone H3 in nucleosomes. FEBS Le& 403, 186-190. Rundlett,S.E., Carmen,A.A., Kobayashi,R., Bavykin,S., Turner,B.M. and Grunstein.M.(1996) HDAI and RPD3 are mernbers of distinct yeast histone deacetylase complexes that regulate silencing and transcription. Proc. Natl. Acad. Sci. U S A. 93, 14503-14508. Ruppert,S., Wang,E.H. and Tjian,R. (1993) Cloning and expression of human TAF11250: a TBP-associated factor implicated in cell-cycle regulation. Nature 362, 175-179. Sakai,A., Chibazakura,T., Shiminzu,Y. and Hishinuma,F. (1992) Molecular analysis of POP2 gene, a gene required for glucose-derepression of gene expression in Saccharomyces cerevisiae. Nucleic Acids Res. 20, 622706233, Saleh,A., Lang,V., Cook,R. and Brandl,C. (1997) Identification of native complexes containing the yeast coactivator/repressor proteins NGG1/AD A3 and ADA2. J. Biol. Chem. 9, 5571-5578. Sanchez,Y ., Desany,B.A., Jones,W.J., Liu,Q., Wang,B. and Elledge,S.J. (1996) Regulation of RAD53 by the ATM-like kinases MEC1 and TEL1 in yeast cell cycle checkpoint pathways. Science 19, 271 357-360. Sauer,F., Hansen,S.K. and Tjian,R. (1995) Multiple TAFl ls directing synergistic activation of transcription. Science 270, 1783-1788.
Sauer,F. and Tjian,R. (1997) Mechanisms of transcriptional activation: differences and similarities between yeast, Drosophila, and man. Curr. Opin. Genet. Dev. 7, 176-181. Scheffner, M., Wemess, B. A., Huibregtse, J. M., and Howley,P.M. (1990) The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Celi 63, 1129-1 136. Scheffner,M., Huibregtse,J.M., Vierstra,R.D. and Howley,P.M. (1993) The HPV E6 and E6-AP complex functions as a ubiquitin protein ligase in the ubiquitination of p53. Ce1175, 495-505. Scheffner,M., Nuber,U. and Huibregtse,J.M. (1995) Protein ubiquitination involving an El-E2-E3 enzyme ubiquitin thioester cascade. Nature 373, 81-83. Scherer,D.C., Brockman,J.A., Chen,Z., Maniatis,T. and Ballard,D.W. (1995) Signal-induced degradation of I kappa B alpha requires site-specific ubiquitination. Proc. NatL Acad. Sci. U S A. 92, 11259-11263. Schultz,M.C., Choe,S.Y. andReeder,R.H. (1991) Specific initiation by RNA polymerase I in a whole-cell extract from yeast. Proc. Natl. Acad. Sci., USA, 88, 1004-1008. Seufert,W. and Jentsch,S. (1990) Ubiquitin-conjugating enzymes UBC4 and UBC5 mediate selective degradation of short-lived and abnonal proteins. EMBO J. 9, 543-550. Shi,X.. Finkelstein,A., Wolf,A.J., Wade,P.A., Burton,Z.F. and Jaehning, J.A. (1996) Paf1p, an RNA polymerase Il-associated factor in Saccharomyces cerevisiae, may have both positive and negative roles in transcription. Mol. Cell. Biol. 16, 669-676. Shi,X., Chang,M., Wolf,A.J., Chang,C.H., Frazer-Abel,A.A., Wade,P.A., Burton,Z.F. and Jaehning,J.A. (1997) Cdc73p and Paflp are found in a novel RNA polymerase Il-containing complex distinct from the Srbp-containing holoenzyme. Mol. Cell. Biol. 17, 1160-1169. Sidorova,J. M. and Breeden,L.L. (1997) Rad53-dependent phosphorylation of Swi6 and down-regulation of CLNl and CLNZ transcription occur in response to DNA damage in Saccharomyces cerevisiae. Genes Dev. 1 5 , 3032-3045. Siede,W., Allen,J.B., Elledge,S.J. and Friedberg,E.C. (1996) The Saccharomyces cerevisiae MEC1 gene, which encodes a homolog of the human ATM gene product, is required for G1 anest following radiation treatrnent. J. Bacteriol. 178, 5841-5843. Silveman,N., Agapite,J. and Guarente,L. (1994) Yeast ADA2 protein binds to the VP16 protein activation domain and activates transcription. Proc. Natl. Acad. Sci. , USA, 91, 11665-11668.
Song,W., Treich,l., Qian,N., Kuchin,S. and Carlson,M. (1996) SSN genes that affect transcriptional repression in Saccharomyces cerevisiae encode SIN4, ROX3, and SR8 proteins associated with RNA polymerase II. Mol. Cell. Biol. 16, 1 15-120. Sopta,M ., Carthew,R. W. G reenblatt,J (1985) Isolation of three proteins that bind to mammalian RNA polymerase II. J. Biol. Chem. 260. 10353-10360. Spence,J.. Sadis,S., Haas,A.L. and Finley,D. (1995) A ubiquitin mutant with specific defects in DNA repair and multiubiquitination. Mol. Cell. Biol. 15, 12651273. Struhl,K. (1984) Genetic properties and chromatin structure of the yeast gal regulatory element: an enhancer-like sequence. Proc. Nat/. Acad. Sei. U S A. 81, 7865-7869. StruhLK. (1985) Naturally occuring poly(dA-dT) sequences are upstream promoter elements for constitutive transcription in yeast Proc. Natl. Acad. Sci. 82, 8419-8423. Struhl,K. (1989) Molecular mechanisms of transcriptional regulation in yeast. Annu. Rev. Biochem. 58, 1O5 1-1077. Struh1.K. (1998) Histone acetylation and transcriptional regulatory mechanisms. Genes Dev. 12, 599-606. Suzuki,Y, Nogi,Y, Abe,A. and Fukasawa,T. (1988) GALI 1 protein, an auxiliary transcription activator for genes encoding galactose-metabolizing enzymes in Saccharomyces cerevisiae. M d CeIl. Biol. 8, 4991-4999. Tansey,W.P. and Herr,W. (1995) The ability to associate with activation domains in vitro is not required for the TATA box-binding protein to support activated transcription in vivo. Proc. Natl. Acad. Sci. U S A. 92, 10550-10554. Tansey,W.P. and Herr,W. (1997) TAFs: guilt by association? Ce11 88, 729-732. Taunton,J., Hassig,C.A. and Schreiber,S.L. (1996) A mammalian histone deacetylase related to the yeast transcriptional regulator Rpd3p. Science 272, 408-41 1. Taylor,S.S., Knighton,D.R., Zheng,J., TenEyck,L.F. and Sowadski,J.M. (1992) Structural framework for the protein kinase family. Annu. Rev. Cell. Biol. 8 , 429462. Thomas,G.H. and Elgin, S.C.(1988) ProteidDNA architecture of the DNase I hypersensitive region of the Drosophila hsp26 promoter. EMBO J. 7, 21912201.
Thompson,C.M., Koleske,A.J., Cha0,D.M. and Young,R.A. (1993) A multisubunit complex associated with the RNA polymerase II CTD and TATA-binding protein in yeast. Cd173, 1361-1375. Thompson,C.M. and Young,R.A. (1995) General requirement for RNA polymerase II holoenzymes in vivo. Proc. Natl. Acad. Sci. U S A., 92, 45874590. Timmers,H.T. and Sharp,P.A. (1991) The mammalian TFllD protein is present in two functionally distinct complexes. Genes Dev. 5, 1946-1956. Utsugi,T., Toh-e,A. and Kikuchi,Y. (1995) A high dose of the STMl gene supresses the temperature sensitivity of the tom 1 and htrl mutants in Saccharomyces cereviçiae . Biochim. Biophys. Acta 1263, 285-288. van Nocker,S. and Vierstra, R.D. (1991) Cloning and characterization of a 20kDa ubiquitin carrier protein from wheat that catalyzes multiubiquitin chain formation in vitro. Proc. Natl. Acad. Sci., USA, 88, lO29?-lO3Ol. van Nocker,S., Sadis,S., Rubin,D.M., Glickma,M., Fu,H., Coux,O., Wefesl, Finley,D. and Vierstra,R.D. (1996) The multiubiquitin-chain-bindingprotein Mcbl is a component of the 26s proteasome in Saccharomyces cerevisiae and plays a nonessential, substrate-specific role in protein turnover. Mol. Cell. Bol. 16, 6020-6028. Verrijzer,C.P. and Tjian,R. (1996) TAFs mediate transcriptional activation and promoter selectivity. Trends Biochem. Sci. 21, 338-342. Vettese-Dadey,M., Grant,P.A., Hebbes,T.R., Crane- Robinson,C., Allis,C.D. and Workman,J.L. (1996) Acetylation of histone H4 plays a primary role in enhancing transcription factor binding to nucleosomal DNA in vitro. EMBO J. 15, 2508-2518. Wade,P.A. and Jaehning,J.A. (1996) Transcriptional corepression in vitro: a Mot1p-associated f o m of TATA-binding protein is required for repression by Leu3p. Mol. Cell. Biol. 16, 164 1- 1648. Wade,P.A., Werel,W., Fentzke,R.C., Thompson,N.E., Leykam,J.F., Burgess,R.R., Jaehning,J.A. and Burton,Z.F. (1996) A novel collection of accessory factors associated with yeast RNA polymerase II. Protein Expr. Purit 8, 85-90. Wahi,M. and Johnson,A.D. (1995) Identification of genes required for alpha 2 repression in Saccharomyces cerevisiae. Genetics 140, 79-90. Walker,S.S., Reese,J.C., Apone,L.M. and Green,M.R. (1996) Transcription activation in cells lacking TAFIIS. Nature 1996, 383, 185-188.
Wang,L., Minen,C., Ying,C., Candau,R., Barlev, N., Brownell, J., Allis, C.D. and Berger, S.L.(1997) Histone acetyltransferase activity is conserved between yeast and human GCNS and is required for complementation of growai and transcriptional activation. Mol. Cell. Bol. 17, 519-527. Wang,L., Liu,L. and Berger,S.L. (1998) Critical residues for histone acetylation by Gcn5, functioning in Ada and SAGA complexes, are also required for transcriptional function in vivo. Genes Dev., 12, 640-653. Weinert,T.A., Kiser,G.L. and Hartwel1,L.H. (1994) Mitotic checkpoint genes in budding yeast and the dependence of mitosis on DNA replication and repair. Genes Dev. 8, 652-665. Weissman,A.M. (1997) Regulating protein degradation by ubiquitination. lmmunol Today. 18, 189-198. Wilkinson,K.D. (1995) Role of ubiquitination in proteolysis and cellular regulation. Annu. Rev. Nutr. 15, 161-189. Wilson,C. J., Chao,D.M., Imbalzano,A.N ., Schnitzler,G.R., Kingston,RE. and Young,R.A. (1996) RNA polymerase II holoenzyme contains SWIISNF regulators involved in chromatin remodelling. Ce11 84, 235-244. Winston,F., Durbin,K. J. and Fink,G.R. (1984) The SPT3 gene is required for normal transcription of Ty elements in S. cerevisiae. Cell39, 649-656. Winston, F., Dollard, C., Malone, E.A., Clare, J., Kapakos, J.G., Farabaugh, P. and Minehart, P.L. (1987) Three genes are required for trans-activation of Ty transcription in yeast. Genetics 115, 649-656. Winston, F. and Carlson,M. (1992) Yeast SNFISWI transcriptional activators and the SPTlSlN chromatin connection. Trends Genet. 8, 387-391. Wolffe,A.P. (1994) The role of transcription factors, chromatin structure and DNA replication in 5 S RNA gene regulation. J. Ce11 Sci. 107, 2055-2063. Wolffe,AP. and Pruss,D. (1996) Targeting chromatin disruption: Transcription regulators that acetylate histones. Ce1184, 817-819. Xiao,H., Pearson,A., Coulornbe,B., Truant,R., Zhang,S., Regier,J.L., Triezenberg,S.J., Reinberg,D., Flores,O. and Ingles,C.J. (1994) Binding of basal transcription factor TFllH to the acidic activation domains of VP16 and p53. Mol. Cell. Biol. 14, 7013-7024.
Yu,J., Donovie1,M.S. and Young,E.T. (1989) Adjacent upstream activation sequence elements synergistically regulate transcription of ADH2 in Saccharomyces cerevisiae. Mol. Cell. Biol. 9, 34-42.
Zawel,L. and Reinberg,D. (1993) Initiation of transcription by RNA polymerase II: a multi-step process. Prog. Nucleic. Acid. Res. Mol. Bol. 44, 67-108. Zawel,L., KumarK,P. and Reinberg,D. (1995) Recycling of the general transcription factors during RNA polymerase II transcription. Genes Dev. 9, 1479-1490. Zhu,Y.. Carroll,M., Papa,F.R., Hochstrasser,M., and DIAndrea,A.D. (1996) DUB1, a deubiquitinating enzyme with growth-suppressing activity. Proc. Natl. Acad. Sci. U.S. A. 93, 3275-3279. Zhu,Y., Peterson,C.L. and Christman,M.F. (1995) HPRl encodes a global poslive regulator of transcription in Saccharomyces cerevisiae. Mol. Cell. Biol. 15, 1698-1708. Zolladek,T., Tobiasz,A., Vaduva,G. Boguta,M., Martin,N.C. and Hopper,A.K. (1997) MDPI, A Saccharomyces cerevisiae gene involved in mitochondriaVcytoplasmic protein distribution is identical to the ubiquitin-protein ligase gene RSP5. Genetics 145, 595-603.
CHAPTER 2 IDENTIFICATION OF NATIVE COMPLEXES CONTAlNlNG THE YEAST COACTIVATOR/REPRESSOR PROTEINS NGG1 1 ADA3 AND ADA2'
2.1
Introduction
Activated transcription by RNA polymerase II requires the action of the basal transcriptional machinery, sequence-specific activator proteins and coactivator or mediator proteins (Zawel and Reinberg, 1995). Coactivators positively influence activator function either by providing a regulatory interface between the basal machinery and the activator protein or by enabling these components to contend with a DNA template in the f o m of chromatin. Coactivators have generally been found as components of regulatory complexes. The TBP (TATA-binding protein)-TBP associated factor (TAF) complex, the RNA polymerase II holoenzyme complex, and the SWVSNF complex represent principal examples (reviewed in Carlson and Laurent, 1994; Goodrich and Tjian, 1994; Koleske and Young, 1995). The mechanisms and component structure of these complexes may overlap as suggested by the finding of SWVSNF components (Treich et al., 1995) within the RNA polyrnerase II holoenzyme (Wilson et al., 1W6),the TAF complex and TFllF (Henry et al., 1994). l
A version of this chapter has been pubfished.
Ayman SaIeh, Vema Lang, Robert Cook and Christopher J. Brandl (1997) Identification of native complexes containing the yeast coactivator/repressor proteins NGGI/ADA3 and ADA2. J. Biol. Chem. 272, 5571-5578.
Probably through related mechanisms, coactivator complexes can also be involved in repression. We initially discovered NGGl based on its requirement for full inhibition of the GAL4 activator protein in glucose media (Brandl et al., 1993). NGGl p likely acts as a more general repressor because transcriptional activation by the carboxyl-terminal activation domain of PDR1p is enhanced in a nggl background (Martens et al., 1996). The Guarente laboratov independently isolated NGGUADA3 based on suppression of the toxicity of overexpression of VP16 in yeast (Pina et al., 1993). Mutations within four ADA genes, AûA2, NGGI/ADA3, GCN5/ADA4 and ADAS, suppress the toxicity of VP16 by inhibiting its ability to activate transcription (Berger et al., 1992; Marcus et al., 1994). These genes are also required for transactivation by GCN4p (Berger et al., 1992; Horiuchi et al., 1995); in fact, GCN5p had been
identified because it is required for maximal activation by GCN4p (Georgakopoulos and Thireos, 1992). Genetic and in vitro biochemical evidence suggests that the ADA proteins likely act in a complex which contains at least ADA2p, NGGlp, GCNSp and ADASp (Berger et al., 1992; Pina et al., 1993; Marcus et al., 1994; 1996; Horiuchi et al., 1995; Georgakopoulos et al., 1995; Candau et aL, 1996). Direct interaction, in vitro, has been observed
between ADA2p and both GCN5p and the carboxyl-terminal250 arnino acids of NGGl p (Horiuchi et al., 1995; Candau et al., 1996). Based on the finding that
single and double disruptions of nggl and ada2 have similar effects on inhibition of GAL4p and PDRl p, we suggested that the same or related ADA complexes are involved in transcriptional activation and repression (Brandl et al., 1996; Martens et ai., 1996). Other coactivator complexes also appear to be involved in the positive and negative regulation of transcription. The RNA polymerase II holoenzyme contains components, SIN4p, RGR l p and ROX3p,
which are required for transcriptional repression (Li et al., 1995; Song et al., 1996). The ADA proteins were predicted to interact with the basal transcriptional machinery and perhaps act as a regulatory bridge between activators and this machinery (Berger et al., 1992). An interaction between the ADA complex and the TATA binding protein (TBP) has been demonstrated by affinity chromatography (8arlev et ab, 1995). Recent genetic experiments showing that mutations in the gene encoding TBP, SPT15, lead to resistance to overexpression of GAL4p-VP16 (the ADA phenotype) also support a link between the ADA proteins and TBP (Marcus et al., 1996). However, the component(s) of the complex involved in the interaction with TBP is unknown. Components of the ADA complex have also been found to interact with activator proteins. PDRlp has been shown to interact with the amino-terminal 373 amino acids of NGGl p in a two-hybrid analysis and by coimmunoprecipitation (Martens et al., 1996). ADA2p in yeast extracts associates with the activation domains of VP16, GCN4p and GAL4p on affinity columns (Silverman et al., 1994; Bariev et al., 1995; Melcher and Johnston, 1995). The ability of recombinant ADA2p to interact with VP16 in vitro suggests that ADA2p may have a principal role in these interactions (Barlev et ab, 1995); disruption of ADA2 however did not totally abolish the two-hybrid interaction between NGGl p and PDRlp (Martens et al., 1996). To determine the functional associations of NGGl p and ADA2p that occur in vivo, we have begun a biochemical analysis of these proteins using epitope-tagged derivatives. We have found that virtually al1 of the cellular ADA2p is associated with NGGlp in multiple high rnolecular weight complexes. In addition, we have found that TBP coimrnunoprecipitates with
NGGl p. The
significance of the interaction with TBP is suggested by the fact that deletion of
amino acids 274 to 307 of NGGl p. which results in the slow growai phenotype
and loss in repression of GAL4p seen in a nggl disruption (Brandl et al., 1996), also results in loss of association with TB?,
2.2
2.2.1
Materials and Methods
DNA constructs To epitope-tag NGGlp, a Non restriction site was introduced into
YCplac33-NGGI directly upstream of the TAA stop codon (Brandl et al., 1993). An oligonucleotide encoding a triple hemagglutinin (HA) epitope ( kindly provided by M. Manolson) was cloned into this site. Y lplac211-HA-NGG 1 was constructed by Iigating a 2.7 kilobase pair Ssn-PsA fragment from this allele (including the NGGl p coding region), with an EcoM-Ssd fragment that contains 2.3 kilobase pairs of sequence upstream of NGGl into the EcoRl andPst1 sites
of YlplacZl 1 (Gietz and Sugino, 1988). Ylplac211-HA-ngglM74-307was constructed from Ylplac211-HA-NGGI by digestion of the intemal Bglll sites and religation. YCp88-HA-ADA2 was constructed using the identical strategy. The coding region was flanked by Hindlll and Ssll restriction sites and cloned into the equivalent sites of the URA3 centromeric plasmid YCp88 (Hope and Struhl 1986), to allow expression of tagged ADA2from a DEDl promoter.
2.2.2
Yeast strains, media and growth conditions Yeast strain CY756 (Brandl et al., 1993) is a derivative of KY320 (Chen
and Struhl, 1988) that contains a TRPl disruption of nggl (Table 2.1 ). Ylplac211-HA-NGG 1and Ylplac211-HA-ngglA 274-307 were digested with Kpnl and integrated into CY756 by selecting for ~ r a colonies + to generate SY6-2 and SY7-3, respectively. CY914 is isogenic to CY756 but contains a TnlO LUK disruption of the GALBO coding region (Brandl et ab, 1993). ~ r a colonies + were selected and verified for disruption by Southem blotting then selected for loss of URA3 on 5-fluoroorotic acid (Boeke et al., 1984). CY946 is a derivative of
Table 2.1 Relevant genotypes of yeast strains used in this study.
-
St rain
-
-
Nffil
A DA2
CY946
ngg 1 ::TRPl
ad&
CY947
myc- NGGI
ada2
CYl 077
myc-NGG1
ADA2
SY6-2
HA -NGGI
ADA2
SY7-3
~ - f w h 7 4 - 3 0 7
A DA2
KY320
Na1
ADA2
-
-
All strains are derivatives of KY320 (MA Ta ura3-52 ade2- 107 trp-A 1 lys29 801 his3-MOU leu2::PET56) (Chen and Struhl, 1988).
CY914 that contains a TnlO LUK disruption of the ADA2 coding region (Pina et
al., 1993; kindly provided by J. Horiuchi and L. Guarente). CY947 and CY1077 are derivatives of CY946 and CY914, respectively, that contain a myc-tagged
NGGl expressed from the DEDl promoter integrated at his3 (Brandl et a/., 1996).
Yeast strains were grown at 30°C in YPD broth (1% yeast extract, 2% peptone, 2% glucose) or in a minimal medium (0.67% yeast nitrogen base without arnino acids, 2% glucose and supplemented with amino acids as required).
2.2.3
Nuclear extracts Nuclei were prepared as described by Ponticelli and Struhl (Ponticelli
and Struhl 1990) and then disnipted for SDS-PAGE by the addition of SDS to a concentration of 2% and incubation at 70'~ for 10 minutes. Insoluble material was removed by centrifugation at 35,000 x g for 10 minutes.
2.2.4
Preparation of whole cell extract. Yeast whole cell extract was prepared as described by Schultz et al.
(1991) with minor modifications. Cells were grown in YPD broth to A600,,
=1.5-
2.0 and pelleted, and the wet weight was determined. Cells were washed
successively in water (4 voVg), extraction buffer (2 vollg) and extraction buffer containing protease inhibitors (1.2 vollg; 0.2 rnM phenylmethylsulfonyl fluoride; 0.1 mM benzamidine hydrochloride; 2 pg/ml pepstatin A; 2pg/ml leupeptin;
3pg/ml aprotinin; 0.1m g h l trypsin inhibitor). The paste from the pelleted ceils was extruded from a syringe into liquid nitrogen. Frozen cells were broken, under liquid nitrogen, with a ceramic rnortar and pestle. 0.6 volumes of extraction buffer was added to the powder of broken cells. The suspension was
cleared by centrifugation at 40,000 x g for 1 hour. Protamine sulphate was added to a final concentration of 0.2% to the supernatant (Badaracco et al., 1983). The extract was cleared by centrifugation at 27,000 x g for 15 min and the protein concentration of the supernatant was standardized to 40 mgfml (Bradford, 1976).
2.2.5
Fractionation of NGGlp and ADA2p. Ion-exchange chromatography: 200 mg of a mixture of whole cell extract
from yeast strains SY6-2, and CY947 containing YCp88-HA-ADA2, prepared in 40 mM Tris-HCI (pH7.9), 20 mM NaCI, 0.1% Nonidet P-40, and 10% glycerol, was applied to a Q Sepharose Fast Flow (Fast Q) HR10/10 column (Pharmacia Biotech Inc.) at a flow rate of 1.5 mllmin. After washing the column with 10 ml of buffer, protein was eluted with a 65 ml gradient of buffer containing 20 mM to 500 mM NaCI. Protein from 200 pl aliquots of 1.5 ml fractions was precipitated with trichloroacetic acid and separated by SDS-PAGE. Gel-filtration chromatography: 16 mg of a mixture of whole cell extract from yeast strains SY6-2, and CY947 containing YCp88-HA-ADA2, prepared in 40 mM Tris-HCI (pH7.7), 300 mM NaCI, 0.1% Nonidet P-40, and 10% glycerol, was filtered through a 0.22 ym membrane and applied at a flow rate of 0.2 mVmin to a Superose 6 HR10/30 column (FPLC, Pharmacia Biotech Inc.). Protein from 200 pl aliquots of 600 pl fractions was precipitated with 10% trichloroacetic acid, solubilized in SDS sample buffer and separated on a 7.5% SDS-PAGE. In a similar preparation, 120 mg of whole cell extract from strains expressing HA-NGGlp and HA-ADA2p was prepared in 40 mM HEPES (pH 7.4), 150 mM NaCI, 0.1 % Tween 20, 10% glycerol, 1 mM EDTA and 1 mM
dithiothreitol in the presence of the protease inhibitors and fractionated on a
100 ml open column of Sephacryl S-5OOHR (Pharmacia Biotech Inc.) at a flow rate of 0.2 mllmin. The protein in 200 pl aliquots of altemate 1.6 ml fractions were precipitated in the presence of 10% trichloroacetic acid, separated by SDS-PAGE and assayed for the presence of HA-ADA2p and HA-NGG1p by immunoblotting with anti-HA antibody. To chromatograph NGGl p and ADA2p-containing Fast Q peak fractions on Superose 6, fractions 2 to 5 (50 mM NaCl eluting peak) and fractions 9 to 12 (100 mM NaCl eluting peak) were concentrated to 200 pl using a Centricon 30 concentrator and then diluted to 500 pl with 40 mM Tris-HCI (pH7.7), 300 mM NaCI, 0.1 % Nonidet P-40, and 10% glycerol. Fractions 20 to 24 (250 mM NaCl eluting peak) were concentrated to 500 pl. Fractions 35 to 39 (450 mM NaCl eluting peak) were concentrated to 200 pl and then diluted with 40 mM Tris-HCI (pH7.7), 100 mM NaCI, 0.1% Nonidet P-40 and 10% glycerol. Individually, the concentrated samples were fractionated on Superose 6 column, as described above. 600 pl aliquots of altemate fractions were precipitated by 10% Trichloroacetic acid, separated by SDS-PAGE and assayed for the presence of HA-NGG1p by immunoblotting with anti-HA antibody.
2.2.6
lmmunoprecipitstion of NGGl p and ADAZp complexes For immunoprecipitation of myc-NGGlp with HA-ADAZp, whole cell
extracts were prepared from yeast strain CY947 containing YCp88-HA-ADA2, CY946 containing YCp88-HA-ADA2, and CYlOï7 in 50 mM HEPES (pH7.4), 150 mM NaCI, 16 mM magnesium acetate, 1 mM EGTA, 0.1 % Nonidet P-40, 0.5
mM dithiothreitol, 10% glycerol, and protease inhibitors (immunoprecipitation
buffer). 50 mg of extract was rotated for 1 hour at 4OC with 0.5 ml of Sepharose CL-4B (Pharmacia Biotech Inc.). Unbound protein was incubated with 4 pg of monoclonal antibody 12CA5 (Boehringer Mannheim) directed against the
hemagglutinin (HA) epitope and rotated at 4OC for 1 hour. The mixtures were added to 50 pl of Protein A Sepharose beads (Pharmacia Biotech Inc.), equilibrated in immunoprecipitation buffer and rotated for 4 hours at 4OC. The beads were washed five times with 1.5 ml of immunoprecipitation buffer, and the bound protein was eluted by incubation at 65OC for five minutes in SDS gel loading buffer. A similar procedure was used for the immunoprecipitation of -NGGlp from these extracts with the exception that the equivalent of 50 pl of ascites fluid (12 mg/ml) derived from the Mycl-SEI O cell line (Evan et al., 1985) was coupled to cyanogen bromide activated Sepharose (Pharmacia Biotech Inc.). For analysis of TBP in the complexes, whole cell extracts were prepared frorn SY6-2, SY7-3 and KY320. lmmunoprecipitations were performed with 200 mg of extract using monoclonal anti-HA antibody covalently bound to Nhydroxysuccinimide-activated Sepharose (Berkeley Antibody). Protein was
eluted from the beads by incubation at 40°C in 4.0 M urea.
2.2.7
Western blotting.
Western blotting with a primary antibody from ascites fluid derived from the Mycl-SEI0 cell line using polyvinylidene difluoride membrane and the Supersignal cherniluminescence kit (Pierce) has been described (Brandl et al., 1996). Probing with monoclonal anti-HA antibody, TBP polyclonal antibody (Upstate Biotechnology Inc.) and a monoclonal antibody to the carboxylterminal domain of RNA polymerase II (Allison and Ingles, 1989; kindly supplied by J. Ingles) was perforrned with dilutions of primary antibody of 1:4000, 1:2000, and 1:3000, respectively.
2.3
2.3.1
Results
Coimmunoprecipitation of NGGl p and ADAZp When expressed in vitro, NGGlp interacts with ADA2p (Horiuchi et al.,
1995). Genetic experiments including the similar slow growth phenotype and relief of inhibition on GAL4p shown by single and double mutants also suggest that NGGl p and ADA2p act in a cornplex (Horiuchi et al., 1995; Brandl et al., 1996). To address whether these proteins associate in vivo, we determined whether they can be coimmunoprecipitated from a yeast whole cell extract. Epitope-tagged derivatives were constructed to immunoprecipitate and identify NGGl p and ADA2p. NGGl p tagged with a Myc epitope at its amino terminus is functional (Brandl et al., 1996). ADA2p was tagged at its carboxyl terminus with a HA epitope (Field et al., 1988; Kolodzieg and Young, 1989). This derivative was also functional as determined by its ability to complement ada2 disruptions in restoring wild-type growth rates and repression of GAL4p
(data not shown). HA-ADA2, expressed from the DEDl promoter, was introduced into yeast strains CY947 (myc-NGG1 adaZ) and CY946 (ngg1::TRPl ad@ on a URA3 centromeric plasmid (YCp88-HA-ADAZ). Whole cell extracts
were prepared from these strains and from CYI O 7 7 (myc-NGG1ADAZ). The extracts containing HA-ADA2p were treated with anti-HA antibody, separated by SDS-PAGE, and Western-blotted with anti-Myc antibody to detect Myc-NGGlp
(Fig. 2.1 A). The presence of Myc-NGGlp in immunoprecipitates with HAADA2p (lane 1) shows that NGGl p and ADA2p expressed in vivo are associated. The specificity of the association was shown by the absence of a band of -1 16 kDa that reacts with anti-Myc antibody in immunoprecipitates from strains lacking HA-ADA2p (lane 3) or Myc-NGGl p (lane 2).
Fig. 2.1 Coimmunoprecipitation of NGGlp with ADA2p from yeast whole cell extract. A. 50 mg of protein in whole cell extract prepared from yeast strain CY947 containing YCp88- HA-ADA2 (myc-NGG 1 HA-ADA2; lane l), CY946 containing YCp88-HA-ADA2 (nggl HA-ADAZ lane 2),and CYl On (myc-NGGI AD= lane 3 were incubated with anti-HA antibody and Protein A Sepharose. After washing, protein was eluted by heating at 95OC in SDS loading buffer, separated by SDS-PAGE, and analyzed by Western blotting with anti-myc antibody as a probe for myc-NGGl p. The migration of molecular weight protein standards (kDa) is shown on the right. The band at approximately 67 kDa is the heavy chain of the myc-antibody which serves as a loading control. B. 600 pg of whole cell extract from yeast strain CY947 containing YCp88-HAADA2 (myc-NGG 1 HA4DA2) was incubated with myc-antibody coupled to Sepharose 48 (lanes 1 and 2) or to Sepharose 48 alone (lanes 3 and 4). The supernatant (Sup, lanes 2 and 3) was removed and after washing, protein was eluted from the beads by boiling in SDS-loading buffer (Bound, lanes 1 and 4). Protein in each of the fractions was separated by SOS-PAGE on an 8% gel and HA-ADA2p detected by Western blotting with anti-HA antibody. Lanes 5 and 6 contain 600 pg of whole cell extract from CY947 containing YCp88-HA-ADA2 (positive control) and KY320 (negative control), respectively. WT, wild type
NGGl
myc
-
*YC
To detemine the relative amount of ADA2p associated with NGGl p, we perfomed an immunodepletion experiment. 600 yg of whole cell extract containing Myc-NGGI p and HA-ADA2p (CY947 containing YCp88-HA-ADAZ) was incubated with anti-Myc antibody coupled to cyanogen bromide-activated Sepharose 48 or to Sepharose 48 alone. The amount of HA-ADA2p bound to the beads and thus associated with Myc-NGGlp and that found independent of Myc-NGGl p were detenined by Western blotting with anti-HA antibody (Fig. 2.1
B). As in the reciprocal experiment shown in figure 2.1 A, HA-ADA2p
coirnmunoprecipitated with Myc-NGGlp (lane 1). In fact, c2% of HA-ADA2p was found in the immune supernatant, not complexed with Myc-NGGl p (compare
lanes 1and 2). The specificity of the coimmunoprecipitation of HA-ADA2p was confimed by the absence of HA-ADA2p bound to the control Sepharose 4B beads (compare lanes 3 and 4).
2.3.2
NGGlp and ADAZp cofractionate on ion-exchange and gel-
filtration columns To further examine the interaction between NGGl p and ADA2p, we detemined their elution profiles after fractionation of whole cell extracts by ionexchange chromatography (Fig. 2.2). To enable detection of both proteins on the sarne Westem blot, we constnicted a derivative of NGGl p with a carboxylterminal HA tag. HA-NGGlp was functional as determined by its ability to cornplement ngg1::TRPI (data not shown). 200 mg of protein in a whole cell extract was applied to a FPLC Fast Q column in buffer containing 20 mM NaCl and eluted in a linear gradient of 20-500 mM NaCI. Equal volumes of altemate fractions were examined by SDS-PAGE, and the presence of ADA2p and NGGl p was detected by Westem blotting with anti-HA antibody (Fig. 2.2 A). Densitornetric scanning revealed that NGGl p eluted in four peaks centered at
Fig. 2.2 ADA2p and NGG1p coelute during chromatography on a FPLC Fast Q column. A. 200 mg of whole cell extract from strains expressing HA-NGGl p and HAADA2p was fractionated on a FPLC Fast Q column. After washing the column with extraction buffer, protein was eluted with a continuous gradient of 20-500 mM NaCI. Equal volumes of altemate fractions were separated by SDS-PAGE and assayed for HA-ADA2p and HA-NGGl p by immunoblotting with anti-HA antibody. The first two lanes contain 150 pg of whole cell extract from strains lacking or containing HA-ADA2p and HA-NGGl p and serve as negative and positive controls, respectively. These are followed by the column fractions numbered as they were eluted. The positions of HA-ADA2p and HA-NGGlp are labeled. Arrowheads indicate the peak fractions for elution of ADA2p and NGGl p. B. shown are the densitometric scanning profiles of ADA2p (solid line) and NGGlp (dashed line) obtained from the immunoblot shown in A. The relative amounts of ADA2p and NGGlp represent the intensity of each band expressed in densitometric units. W, wild type.
Fraction
fractions 4 (-50 mM NaCI), 10 (-100 rnM NaCI), 22 (-250 mM NaCI), and 36 (-450 mM NaCI) (Fig. 2.2 B). ADA2p cofractionated with NGGl p, with the
exception of the 450 mM NaCl eluting fraction. Individually, the 50, 100, and 250 mM NaCl complexes were reapplied to the Fast Q column to ensure that they were chromatographing true. Each NGGl ptontaining complex eluted in a single peak at the same position as it had first chrornatographed (data not shown). The occurrence of three separable fractions containing both NGGl p and ADA2p suggests that they are associated and found within multiple complexes. To provide additional support for the association of ADA2p and NGGl p in multiple complexes and to obtain approximate sizes of these complexes, the elution profiles of the tagged derivatives were examined after separation of whole cell extract on gel-filtration columns. Extract containing HA-ADA2p and HA-NGGlp was fractionated on a Superose 6 column in 300 mM NaCl (Fig. 2.3 A). Equal volumes of altemate fractions were exarnined by SDS-PAGE, and the
presence of ADA2p and NGGl p was detected by Westem blotting with anti-HA antibody. NGGl p eluted in three peaks centered at fractions 14, 34, and 40. By cornparison with protein standards, these fractions correspond to apparent molecular sizes of -1.5 MDa (this value was obtained by linear extrapolation from the calibration proteins and is an underestimate of its size; see below), 200 kDa, and 90 kDa, respectively. Approximately 5% of NGGl p fractionated at a
size indicative of a monomer (90-kDa peak); the amount of this f o n varied from
2.0 Mda.
Fig. 2.4 Fractionation of ADA complexes on Sephacryl S-500 HR. Shown are densitometrk scanning profiles of immunoblots of ADA2p and NGGl p obtained after fractionation of whole cell extract on a 100-ml open Sephacryl S-500 HR column. 120 mg of whole ce11 extract containing HANGGlp and HA-ADA2p was fractionated at a flow rate of 0.2 mumin. The protein in 200 pl of 1.6-ml altemate fractions was precipitated. separated by SDSPAGE, and analyzed for the presence of HA-ADA2p and HA-NGGlp by immunoblotting with anti-HA antibody. The dashed line represents HAADA2p; the solid line represents HA-NGGlp. The void volume was detemined wiai high molecular mass DNA and occurs at fraction 18. Arrowheads depict the peak fractions containing the calibration markers as indicated.
Fraction
2.3.3
Stability of NGGlp and ADA2p is CO-dependent The integrity of a protein complex is often critical for the stability of its
component members. To determine if the stability of ADA2p was dependent on NGGlp, we expressed HA-ADAZ from the constitutive DEDI promoter (Struhl, 1985) in yeast strains containing (CY947) or not containing (CY946) NGGI. The
DEDl promoter is not influenced by NGG1, allowing an analysis of protein stability (Brand1 et al., 1996). The presence of HA-ADA2p was detected by Western blotting with anti-HA antibody (Fig. 2.5 A). Alrnost no full-length ADA2p was found in CY946. To further support that NGGlp regulates the stability of ADA2p rather than its expression, we have observed a band of -10 kDa
reacting with anti-HA antibody in the CY946 extract, indicative of a proteolytic fragment of HA-ADA2p (data not shown). We conclude that the stability of ADA2p is highly dependent on NGGl p.
The reciprocal experirnent was perfoned in which Myc-NGGlp was expressed in strains containing or not containing ADA2p. Myc-NGGl p was detected By Westem blotting with anti-Myc antibody. As shown in figure 2.5 8, little or no NGG1p was detectable in the strain lacking ADA2p, even when 300 pg of total protein was analyzed. The dependence of the stability of ADA2p and
NGGI p on the presence of each other again supports their association in vivo.
2.3.4
NGGlp is a nuclear protein The action of NGGIp as a transcriptional coactivator/repressor predicts
that it should be found in the nucleus. ADA2p has been found in nuclear extracts (Bariev et al.. 1995); however, cytoplasmic extracts were not examined. Furtherrnore, ADA2p-independent
NGGI p might potentially be involved in a
cytoplasmic process. To detemine the cellular localization of NGGI p, nuclear and cytoplasmic fractions were prepared from a yeast strain expressing HA-
Fig. 2.5 Stability of ADA2p and NGGlp depends on the presence of each other. A. Whole cell extract from yeast strains CY946 containing YCp88-HA-ADA2 (ngg1 HA-ADA2; lanes 7 and 2),CY947 containing YCp88-HA-ADA2 ( m y e NGG7 HA-ADA2; lanes 3 and 4). and C Y l O T l (myc-NGG1 ADA2; lane 5) was separated by SDS-PAGE and analyzed by Western blotting with monoclonal anti-HA antibody to detect HA-ADA2p.
B. Whole cell extract from yeast strains CY 1On (myc-NGG1 AD= lane l), KY320 (NGG l AD= lane 2),CY947 containing YCp88-HA-ADA2 (myc-NGGI HA-ADA2; lanes 3 and 4). and CY947 (myc-NGGI ada2; lanes 5 and 6) was separated by SDS-PAGE and analyzed by Western blotting with anti-Myc antibody to detect myc-NGGlp. The NGGl and ADA2 alleles found in each strain and amount of protein analyzed are indicated at the top of each lane. WT, wild type.
rnyc
LE/^ myc rnyc myc myc
+ + + + - -
NGGl p and analyzed by Western blotting with anti-HA antibody (Fig. 2.6 A).
The integrity of the fractions was examined by reprobing the fiiter with antibody to the carboxyl-terminal domain of the large subunit of RNA polymerase II (Fig. 2.6 B). Like RNA polymerase II, HA-NGGl p was almost exclusively found in the
nuclear fraction. On extensive overloading, trace amounts of HA-NGGl p could be detected in the cytoplasmic fraction.
2.3.5
Amino Acids 274-307 of NGGlp are required for interaction
with TBP Barlev et al. (1995) have found that ADA2p contained iii nuclear extracts will associate with TBP. Their inability to find a direct interaction for recombinant proteins suggests that the interaction between TBP and ADA2p is indirect. A possible role for NGGl p in the interaction is supported by the finding that amino acids 1-308 of NGGl p, when fused to a GAL4p DNA-binding domain, will activate transcription independently from ADA2p (Brandl et a/., 1996). This activation requires amino acids 274-307 of
NGGl p. To test for the involvement
of NGGlp in the association of an ADA complex with TBP, we immunoprecipitated HA-NGG1p and HA-NGGlp~74-307from whole cell extracts with anti-HA antibody and probed for TBP by Westem blotting (Fig. 2.7 A). TBP coimmunoprecipitated with HA-NGGl p (compare lanes 1and 3). TBP was not found in the imrnunoprecipitate from the HA-NGGlp~74-307extract (lane 2); therefore, amino acids 274-307 of NGGl p are essential for interaction of an ADA complex with TBP. Although the expression of NGGlp~74-307is comparable to the fulllength protein (Brandl et al., 1996), the inability of TBP to coimmunoprecipitate 07 be indirect, resulting from the lack of association with HA-NGGI~ ~ 7 4 - 3 could
of NGG1~ ~ 7 4 - 3 0with 7 an ADA complex. Therefore, we analyzed for complex
Fig. 2.6 Detection of NGGlp in yeast nuclear extracts. A. Whole cell extract (lane l ) , cytosolic extract (lanes 3 and 4). and nuclear extract (lanes 5 and 6)from yeast strain SY6-2 (HA-NGG1) were separated by SDS-PAGE and analyzed by Western blotting with anti-HA antibody. Lane 2 contains a whole cell extract from yeast strain KY320 (NGGI), which serves as a negative controi for anti-HA antibody. Cytosolic extract is the supernatant obtained after pelleting the intact nudei from disrupted spheroplasts. The NGGl allele and the amount of protein loaded are shown at the top of each lane.
B. The same membrane was reprobed with antibody specific for the carboxylterminal domain of the largest subunit of RNA polymerase II (POL II). WT, wild type*
1
2
3 4 5 6 HA ANTIBODY
POL II ANTIBODY
formation by HA-NGGlpu74307 by detemining its elution profile from a Superose 6 colurnn. HA-NGG1pa74-307 was found in complexes of -900 and 200 kDa (data not shown). The largest cornplex containing the deletion
derivative eluted approximately four fractions later than that for the wild-type protein, suggesting that amino acids 274-307 are required for protein-protein contacts necessary in the formation of the largest ADA complexes.
We compared the elution profile of TBP with NGGl p from the Fast Q ionexchange column as a first step in detemining which of the NGGl p- and ADA2p-containing complexes associate with TBP (Fig. 2.7 6). TBP was found in two peaks centered at fractions 4 and 22. The profile of TBP from the Fast Q column paralleled the elution of NGGlp found within one of the two 2 MDa complexes and that contained within the 900/200-kDa peak. Since TBP immunoprecipitates with NGGl p (Fig. 2.7 A), this result indicates that TBP is associated with at least one of these complexes. Furthemiore, the absence of
TBP in the NGGl p peaks at fractions 12 and 36 indicates that TBP is not associated with one of the two largest ADA complexes or with free NGGl p.
Fig. 2.7 Amino acids 274-307 of NGGlp are required for interaction with TB?. A. 50 mg of whole cell extract prepared from strains SY6-2 (HA-NGG1; lane 1). SY7-3 ( H A + ~ g g llane ~ ~2). ~ and ~ ; KY320 (NGGI; lane 3 was incubated with anti-HA antibody covalently bound to N-hydroxysuccinimide-activated Sepharose. After washing, protein was eluted from the beads by incubation at 40 OC in 4 M urea, separated on a 12% SDS-polyacrylamide gel, and analyzed by Western blotüng using anti-TBP antibody. Lane 4 contains 150 pg of whole cell extract (WCE) frorn yeast strain SY7-3 (HA-nggld274-307). The NGG1 and ADA2 alleles found in each strain are shown at top of each lane. Relevant molecular mass protein standards (kDa) are indicated.
B. 200 pl of the protein fractions from the FPLC Fast Q column shown in Fig. 2.2 B was separated on a 12% SDS-polyacrylarnide gel and Western-blotted with anti-TBP antibody. The denslometric scanning profiles of this irnmunoblot have been superimposed on those of HA-NGGlp, with TB? represented by the dashed line and HA-NGGlp by the solid line. Shown in the inset is the elution profile of total protein from this column.
HA-NGGlp immunoprecipitates
O
10
20
Fraction
30
40
50
2.4
2.4.1
Discussion
NGGlp and ADA2p are associated in multicomponent
complexes Previous reports on components of the ADA cornplex demonstrated that ADA2p, NGGlp, GCNSp, and ADA5p associate in vitro and by two-hybrid analysis (Marcus et al., 1994; 1996; Horiuchi et a/., 1995; Candau et al., 1996).
The similar effects of single and double disruptions of nggl, ada2, and gcn5 on transcriptional activation and repression have been used to support the functional association of the proteins in vivo (Pina et al., 1993; Marcus et al., 1994; Horiuchi et al., 1995; Brandl et al., 1996). The finding of Candau et al. (1996) that the stability of ADA2p is dependent on GCNSp and Our finding that
the stability of ADA2p and NGGl p depends on the presence of each other provide additional evidence for their association in vivo. However, the instability of the proteins does Iimit the interpretation of previous gene disruption experiments. In this report, we provide biochemical evidence for the functional association of ADA2p and NGGl p. The association of NGGl p and ADA2p was revealed by their coimmunoprecipitation from yeast whole cell extracts. This
was supported by the coelution of the proteins in multiple peaks after fractionation on both gel-filtration and ion-exchange columns. It should also be noted that Candau and Berger (1996) have shown that the stability of LexAADA2p is dependent on the presence of NGGlp and that NGGl p and GCN5p coimmunoprecipitate with epitope-tagged ADA2p when the three proteins are overexpressed in vivo. The combination of gel-filtration and ion-exchange chromatography resolved four complexes containing both ADA2p and NGG1p (ADA complexes). Two of these had estimated sizes of -2 MDa; the other two were -200 and 900
km. The simple& explanation for the POO-kDa complex is that it represents a minimum complex containing ADA2p, NGGl p, and perhaps GCNSp. The presence of GCNSp would be consistent with the demonstrated interaction with ADA2p in vitro (Horiuchi et aL, 1995) and its stabilization of LexA-ADA2p (Candau and Berger, 1996).
The 2-MDa complexes and the 900-kDa ADA complex from the gelfiltration chromatography almost certainly contain additional NGGl p- and ADA2p-associated proteins. We do not believe that the high molecular mass ADA complexes anse as the resuk of the interaction of the 200-kDa complex with DNA. This possibility was suggested by the finding that the SWI/SNF
complex interacts with DNA (Quinn et al., 1996). First, the yeast whole cell extract used for the chromatography experiments was treated with protamine sulfate to remove DNA. Second, ADA complexes fractionated on the Fast Q column were subsequently chromatographed on Superose 6 and shown to have approximate sizes of >1.5 MDa, 900 kDa, and 200 kDa. These complexes are almost certainly free of DNA since any residual DNA not removed by protamine sulfate treatrnent would be retained at low sait concentrations on the cation-exchange column. Third, we have found the same elution profile on Superose 6 when the extract is chromatographed in the presence of ethidium bromide (data not shown). There are several potential reasons for the presence of four biochemically resolvable complexes containing ADA2p and NGGl p. Activation and repression may require complexes with different components. Stable subcomplexes (for example, the 200 kDa complex) may represent a core that is an intermediate in the formation of these forms rather than having an independent functional role. NGGl p and ADA2p may also function as components of non-ADA complexes. The finding of TBP in irnmunoprecipitates
with NGGl p might suggest that one of the complexes is a TBP-associated factor complex (TAF complex); however, the sizes of proteins that coimrnunoprecipitate with
NGGl p do not resemble the sizes of the yeast TB?-
associated factors (Reese et al., 1994; Poon et al., 1995; also see appendix 1)
The finding that ADA5pISPT20p may associate with NGGl p/ADA3p (Marcus et al., 1996) suggests that some of these complexes may contain the genetically related proteins SPT3, SPT7, and SPT8 (Marcus et ai., 1996; Roberts and Winston, 1996). While we cannot exclude the possibility that the smaller complexes dissociate from the larger complexes during the isolation procedure, independently, the Workman laboratory has found similar chrornatographically distinct, high molecular mass complexes containing GCN5p and ADA2p (J. Cote, P. Grant and J. Workman; personal communication). In addition, the high molecular mass complexes did not dissociate after a second chromatography step on either Superose 6 or Fast Q columns.
2.4.2
Amino acids 274-307 of NGGlp are required for interaction
with TBP As coactivators, the ADA proteins were predicted to provide a regulatory link between the basal transcriptional machinery and activator proteins. Interactions between activators including GCN4p, VP16, GAL4p, and PDRl p with components of the ADA complexes have been observed by affinity chromatography, coimmunoprecipitation, and two-hybrid analysis (Silverman et al., 1994; Barlev et al., 1995; Melcher and Johnston, 1995; Martens et al., 1996).
A link between the ADA complexes and the basal machinery was established with the finding that ADA2p in nuclear extracts associated with a GST-TBP fusion protein on an affinity column (Barlev et al., 1995) and is supported by the genetic findings that ADAS/SPTZU encodes a protein functionally related to TBP
(Marcus et al., 1996; Roberts and Winston, 1996). The inability of Barlev et al. (1995) to detect an interaction between TBP and recombinant ADA2p suggested that the association might be mediated by another component of the ADA complexes. Our finding of TBP in immunoprecipitates with NGGlp
demonstrates in vivo the interaction between the ADA complexes and TBP that was found by affinity chromatography. Furthemore, we have shown that the association with TBP requires amino acids 274-307 of NGG1p. The role of NGGl p in the interaction with TBP is indirect since TBP was not found associated with monomeric NGGl p. This result, along with the finding that recombinant ADA2p does not interact with TBP (Barlev et al., 1995) and the identification of GCNSp as a histone acetyltransferase (Brownell et al., 1996), suggests that none of these three ADA components interacts directly with TBP.
The requirement for amino acids 274-307 of NGGl p for association with TBP agrees with the region being crucial for function of the molecule. Deletion of this region results in a loss of repression of GAL4p and in the slow growth phenotype typical of disruption of ngg 1 (Brandl et ai., 1996). Amino acids 1-308 of NGGl p activate transcription as a GAL4p fusion independent of ADA2p (Brandl et al., 1996). This activation depends on amino acids 274-307 (Brandl et al., 1996). Mutations can be isolated in this region that either stimulate or
inhibit the action of NGGlp in repression of GAL4p and the activity of GAL4pNGGl p fusions in transcriptional activation (Brand1 et ai., 1996). Interestingly,
this region contains a Phe-rich segment with homology to a group of proteins including human immunodeficiency virus group-specific antigen protein that strongly predicts to be an amphipathic a-helix (Pina et al., 1993; Brandl et al., 1996). An involvement of amino acids 274-307 in protein-protein interactions is
also suggested by the finding that this region is required for a two-hybrid interaction with PDRl p (Martens et al., 1996).
2.4.3
Coactivators in activation and repression NGGl p, ADA2p, and likely other components of the ADA complexes fit
into a class of regulators that are able to stimulate or repress activator function. The link between activation and repression for NGGl p is particularly strong since a region essential for repression (amino acids 274-307) is also required for transcriptional activation as a GAL4p-NGGl p fusion (Brandl et al., 1996) and for association with TBP. Mechanisms for activation and repression may be closely related. An activator protein may signal an ADA complex to either stimulate or repress the activities of TBP. Differences leading to activation or repression could anse because specific activators elicit different conformational changes in the complex, perhaps by targeting different components or possibly by acting on different ADA complexes. The finding &y Brownell et al. (1996) that GCN5p is a histone acetyltransferase also suggests that some of the activities of
the cornplex rnay be rnediated by chromatin modification, with others being dependent on interaction with the basal transcriptional machinery. Other dual function regulators probably function to stimulate or inhibit the basal transcriptional machinery. PAF1p was isolated as a RNA polymerase IIassociated protein that differentially activates or represses transcription (Shi et al., 1996). Recently, SIN4p, ROX3p, and RGRl p, which had previously been
characterized with roles in activation and repression, were found as components of the RNA polymerase II holoenzyme (Li et ai., 1996; Song et al., 1996).
2.5
References
Allison,L.A. and Ingles,C.J. (1989) Mutations in RNA polymerase II enhance or suppress mutations in GAL4. Proc. Natl. Acad. Sci. 86, 279402798. Badaracco,G., Plevani,P., Ruyechan,W.T. and Chang,L.M.S. (1983) Purification and characterization of yeast topoisomerase 1. J. Biol. Chem. 258, 2022-2026. Bariev,N.A.. Candau,R., Wang,L., Darpino,P., Silvenan,N. and Berger,S.L. (1995) Characterization of physical interactions of the putative transcriptional adaptor, ADA2, with acidic activation domains and TATA-binding protein. J.Bio1. Chem. 270, 19337-19344. Berger,S.L., Pina,B., Silverman,N., Marcus,G.A., Agapite, J., Regier,J.L., Triezenberg,S.J. and Guarente,L. (1992) Genetic isolation of ADA2 - a potential transcriptional adaptor required for function of certain acidic activation domains Ce11 70, 25 1-265. Bjorklund,S. and Kim,Y-J . (1996) Mediator of transcriptional regdation. Trends Biochem. Sci. 21, 335-337. Boeke,J.D., Lacr0ute.F. and Fink,G.R. (1984) A positive selection for mutants lacking orotidine-S'-phosphate decarboxylase activity in yeast: 5-f luoro-orotic acid resistance. Mol. Gen. Genet. 197, 345-346. Bradford,M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of dye binding. Anal. Biochem. 72, 248-254. Brand1,C.J.. Fur1anett0,A.M.~Martens,J.A. and Hami1ton.K.S. (1993) Characterization of NGG1, a novel yeast gene required for glucose repression of GAL4p-regulated transcription EMBO J. 12, 5255-5265. Brandl,C.J., Martens,J.A., Margaliot,A., Stenning,D., Furlanetto,A. M., Saleh,A., Hamilton,K.S. and Genereaux,J. (1996) Structure/functional properties of the yeast dual regulator protein NGGl that are required for glucose repression. J. Bid. Chem. 270, 9298-9306. Brownell,J.E., Zhou,J., Ranalli,T., Kobayashi,R., Edmondson,D.G., Roth,S.Y. and Allis,C.D. (1996) Tetrahymena histone acetyltransferase A: a homolog to yeast Gcn5p linking histone acetylation to gene activation. CeIl 84, 843-851. Candau,R. and Berger,S.L. (1996) Structural and functional analysis of yeast putative adaptors. Evidence for an adaptor complex in vivo. J.BioLChem. 271, 523705245.
Candau.R., Moore,P.A., Wang,L-, Barlev,N., Ying,C.Y., Rosen,C.A. and Berger,S.L. (1996) Identification of human proteins functionally conserved with the yeast putative adaptors ADA2 and GCNS. Mol. Cell. Biol. 16, 593-602. Carlson,M. and Laurent,B.C. (1994) The SNF/SWI family of global transcriptional activators Curr. OOp. Cell Biol. 6 , 396-402. ChenW. and Struhl,K. (1988) Saturation mutagenesis of a yeast his3 TATA element: genetic evidence for a specific TATA-binding protein. Proc. Natl. Acad. Sci. USA 85, 2691-2695. Evan,G.I., Lewis,G.K., Ramsay,G. and Bishop,J.M. (1985) Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol. Cell. 8iol. 5, 3610-3616. Field,J., Nikawa,J., Broek,D., MacDonald,B., Rodgers,L., Wilson,A., Lemer,R., Wigler, M. (1988) Purification of a RAS-responsive adenylyl cyclase cornplex from Saccharomyces cerevisiae by use of an epitope addition method. Mol. Cell. Biol. 8, 21 59. Georgakopoulos,T. and Thireos,G. (1992) Two distinct yeast transcriptional activators require the function of the GCN5 protein to promote normal levels of transcription EMBO J. 11, 4145-4152. Georgakopoulos,T., Gounalaki, N. and Thireos,G. (1995) Genetic evidence for the interaction of the yeast transcriptional CO-activatorproteins GCNS and ADA2 Mol. Gen. Genet. 246, 723-728. Gietz,R.D. and Sugino,A. (1988) New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74, 527-534. GiII,G., SadowskiJ. and Ptashne,M. (1990) Mutations that increase the activity of a transcriptional activator in yeast and mammalian cells. Proc. Natl. Acad. Sci. USA 87, 2127-2131. Goodrich,J.A. and Tjian,R. (1994) TBP-TAF complexes: Selectivity factors for eukaryotic transcription Curr. 0pin.Cell Bol. 6 , 403-409. Henry,N.L., Campbell,A.M., Feaver,W.J., Poon,D., Wei1,P.A. and Komberg,R.D. (1994) TFIIF-TAF-RNA polymerase II connection. Genes Dev. 8, 2868-2878. Hope, I.A. and Struhl, K. (1986) Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of yeast Cell 46, 885-894. Horiuchi, J., Silveman, N., Marcus, G.A. and Guarente, L. (1995) ADA3, a putative transcriptional adaptor, consists of two separable domains and interacts with ADA2 and GCNS in a trimeric complex. Mol.Cel1. Biol. 15, 12031209.
Koleske, A.J. and Young, R.A. (1995) The RNA polymerase II holoenzyrne and its implications for gene regulation. Trends Biochem. Sci. 20, 113-116. Kolodziej, P.A. and Young, R. (1989) RNA polymerase II subunit RPB3 is an essential component of the mRNA transcription apparatus. Mol. Cell. Biol. 9 , 5387. Kuo,M.H., Brownell,J.E., Sobel,R.E., Ranalli,T.A., Cook,R.G., Edmondson,D.G., Roth,S.Y., Allis,C.D. (1996) Transcription-linked acetylation by Gcn5p of histones H3 and H4 at specific lysines. Nature 383, 269-72. Li,Y ., Bjorklund,S., Jiang,Y .W.,Kim,Y-J., Lane,W.S., Stillman,D. and Komberg,R. D. (1995) Yeast global transcriptional regulaton Sin4 and Rgrl are components of mediator complex/RNA polymerase II holoenzyme. Proc. Natl. Acad. Sci. 92, 10864-10868. Marcus,G.A., Silverman,N., Berger,S.L., HoriuchiJ. and Guarente,L. (1994) Functional similarity and physical association between GCN5 and ADA2: Putative transcriptional adaptors E M 8 0 J. 13, 4807-4815. Marcus,G.A., Horiuchi,J., Silverman,N. and Guarente,L. (1996) ADA5/SPT20 links the ADA and SPT genes, which are involved in yeast transcription. Mol. Cell. Biol. 16, 31 97-3205. Martens,J.A, Genereaux,J., Saleh,A. and Brand1,C.J. (1996) Transcriptional activation by yeast PDR1p is inhibited by its association with NGG1 pIADA3p.J. Biol. Chem. 271, 15884-15890. Melcher,K. and Johnston,S.A. (1995) GAL4 interacts with TATA-binding protein and coactivators. Mol. Cell. 8/01. 15, 283902848. Orphanides,G., Lagrange,T. and Reinberg,D. (1996) The general transcription factors of RNA polymerase II. Genes Dev. 10, 2657.2683. Peterson,C.L. (1996) Multiple SWltches to tum onchromatin? Curr Opin Genet Dev . 6 : 171-5 Pina,B., Berger,S.L., Marcus,G.A., Silverman,N., Agapite, . and Guarente,L. ADA3 a gene, identified by resistance to GAL4-VP16, with properties similar to and different from those of ADA2. (1993) Mol. Cell. Biol. 13, 5981-5989.
-
Ponticelli,A.S. and Struhl,K. (1990) Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription. Mol. Cell. Biol. 10, 2832-2839.
Poon,D., Campbell,A.M., Bjorklund,S., Kim,Y., Zhou,S.. Komberg,R.D. and Wei1,P.A. (1995) ldentlication and characterization of a TF1ID-like multiprotein complex from Saccharumyces cerevisiae. Proc. Natl. Acad. Sci. USA. 92, 8224-
8228. Reese,J.C., Apone,L., Wa1ker.S.S.. Griffin,L.A. and Green,M.R (1994) Yeast TAF(II)s in a multisubunit complex required for activated transcription Nature 371, 523-527. Roberts,S.M. and Winston,F. (1996) SPT2O/ADA5 encodes a novel protein functionally related to the TATA-binding protein and important for transcription in Saccharomyces cerevisiae. Mol. Cell. Biol. 16, 3206-3213. R0eder.R. (1996) The role of general initiation factors in transcription by RNA polymerase I1. Trends Biuchem. Sci. 21, 327-335. Schultz,M.C., Choe,S.Y. andReeder,R.H. (1991) Specific initiation by RNA polymerase I in a whole-cell extract from yeast. Proc. Natl. Acad. Sci. USA 88. 1004-1008. Shi,X., Finkelstein,A., Wolf,A.J., Wade,P.A., Burton,Z.F. and Jaehning,J.A. (1996) Paf1p, an RNA polymerase Il-associated factor in Saccharomyces cerevisiae, may have both positive and negative roles in transcription. Mol. Cell. Biol. 16, 669-676. Silveman,N., Agapite,J. and Guarente.L. (1994) Yeast ADA2 protein binds to the VP16 protein activation domain and activates transcription. Proc. Natl. Acad. Sci. USA. 91, 11665-1 1668. Song,W., TreichJ., Qian,N., Kuchin,S. and Carlson,M. (1996) SSN genes that affect transcriptional repression in Saccharomyces cerevisiae encode SIN4, ROX3, and SRB proteins associated with RNA polymerase II. Mol. Cell. Biol. 16, 1 15-1 20. Struhl,K. (1985) Naturally occuring poly(dA-dT) sequences are upstream promoter elements for constitutive transcription in yeast Proc. Natl. Acad. Sci. 82, 8419-8423. Treich, l., Caims.6. R., De Los Santos,T., Brewster,E. and Carlson,M.(1995) SNFI 1, a new component of the yeast SNF-SWI complex that interacts with a conserved region of SNF2 Mol. Cell. Biol. 15, 4240-4248. Verrijzer,C.P. and Tjian,R. (1996) TAFs mediate transcriptional activation and promoter selectivity. Trends Biochem. Sci. 21, 338-342. Wilson,C.J., Cha0,D.M.. Imbalzano,A.N., Schnitzler,G.R., Kingston,R.E. and Young,R.A. (1996) RNA polymerase II holoenzyme contains SWVSNF regulators involved in chromatin remodelling. Ce11 84, 235-244.
Zawel,L. and Reinberg,D. (1995) Common themes in assembly and function of eukaryotic transcription complexes Annu. Rev. Biochem. 64, 533-561.
CHAPTER
3
TRAI: A YEAST PROTEIN RELATED TO THE CATALYTIC SUBUNIT OF HUMAN DNA-DEPENDENT PROTEIN KINASE IS A
COMPONENT OF THE ADNSPT TRANSCRIPTIONAL REGULATORY COMPLEXES2
3.1
Introduction
The ADA genes were identified in S. cerevisiae based on their requirement for the regulated activation and repression of transcription (Berger et al., 1992; Georgakopoulos and Thireos, 1992; Brandl et ab, 1993). In initial
studies, ADAZp, NGGl p/ADA3p and GCN5p/ADA4p were shown to function in a complex (Horiuchi et al., 1995; Brandl et al., 1996; Candau and Berger, 1996). The identity of GCN5p and its human homolog hGCN5 as histone acetyltransferases suggested that one role of the complex was to modulate nucleosome structure (reviewed in Hampsey, 1997). Biochemical analyses of the ADA proteins demonstrated that they are found in at hast four high molecular weight complexes, two with sizes of more than 2 MDa, and others of 900 kDa and 200 kDa (Saleh et al., 1997; Grant et al., 1997; Pollard and Peterson, 1997). Other proteins identified within these complexes include the product of ADA 1 (Berger et al., 1992; Horiuchi et al., 1997), and the products of A version of this chapter has been submitted for publication to the Journal of Biological Chemistry.
Ayman Saleh, David Schieltz, Nicholas Ting, Steven B. McMahon, David W. Litchfield, John R. Yates III,Susan P. Lees-Miller, Michael D. Cole and Christopher J. Brandl(1998) TRAl p is a component of the yeast ADAEPT transcriptional regulatory complexes. J. Biol. Chem. submitted
the TBP-class of SPT genes, SPT3, SPT7, SPT8 and SPTZO/ADAS (Marcus et al., 1996; Roberts and Winston;1996; Grant et al., 1997). The latter group of proteins are found within at least two of the ADA-containing complexes, one of which being the SAGA-complex (Saleh and Brandl, unpublished results; Grant
and Workman, personal communication). The association between SPTs and ADAs agrees with the functional link between the ADA proteins and TBP which was suggested by immunoprecipitation and affinity chromatography (Barlev et al., 1995; Saleh et al., 1997; Roberts et al., 1997). A detailed understanding of the mechanisms and regulation of the ADA and SPT protein-containing complexes (ADAISPT complexes for simplicity) requires the identification of component proteins. We now identify TRAlp as a component of the ADAISPT complexes. TRAl p is a member of a group of putative protein kinases, including the catalytic subunit of human DNAdependent protein kinase (DNA-Pb), which contain a carboxyl-terminal region related to phosphatidylinositol 3-kinases (P13K; reviewed in Hunter, 1995; Keith and Schreiber, 1995; Zakian, 1995; Lees-Miller, 1996). This report parallels the recent finding by Barlev et al. (1998) that Ku70, which as a heterodimer with Ku80 regulates the DNA binding of DNA-PbS (Lees-Miller et al., 1990; Gottlieb and Jackson, 1993), associates with hGCN5.
3.2
3.2.1
Materials and Methods
DNA constructs and yeast strains Epitope-tagging of TRA7: A Bslll071-Bg/il fragment of YCp88-NGG 1
(Brandl et al., 1993), containing the DED1 promoter, myc tag and Nofi restriction site, was cloned into Hindll-BamH1 sites of YCplacl 11. A 11832 base pair Fspl fragment from ATCC cosmid 70897 was cloned into the Smal site of this construct placingTRA 1 downstrearn of the promoter. The 5' segment of TRA1 was synthesized by PCR, with a Nofi site at the position of the translational start. Alleles expressing myc-NGG 1 and HA-ADA2 have been described (Brandl et al., 1996; Saleh et al., 1997, see Chapter 2). Six histidine tagged myc-NGG1 (HM-NGGI) was constructed by inserting a Non fragment expressing 6-his into pOMYC-NGG1 (Brandl et al., 1996).
All strains are derivatives of KY320 (MA Ta ura3-52 ade2- 101 trp-d 1 lys2-801 hi's A 200 leuZ::PET56).
CY927 and CY947 contain Tn 1O LUK
disruptions of the ADA2 coding region (Brandl et al., 1996). CY947 and CY1077 contain myc-tagged N G G l integrated at his3 (Brandl et ab, 1996; see Chapter 2). CY979 contains disruptions of nggl and gai80 and has HM-NGGI inserted
at his3. Yeast were grown at 30°C in YPD broth (1% yeast extract, 2% peptone, 2% glucose) or in minimal media (0.67% yeast nitrogen base without amino
acids, 2% glucose).
3.2.2
Preparation of whole cell extract Yeast whole cell extract was prepared in liquid nitrogen as described by
Schultz et al. (1991) with minor modifications (Saleh et al., 1997; see Chapter
2).
3.2.3
Purification of HM-NGGl p Whole cell extract was prepared from 8 Iiter cultures of strains expressing
HM-NGGl p (CY979) or myc-NGGlp (CYlO77) in Buffer E (50 mM HEPES pH 7.6, 50 mM NaCI, 20 mM imidazole, 10% glycerol and 0.1% Nonidet P-40;
Saleh et al., 1997). Protease inhibitors (Saleh et al., 1997; see Chapter 2) and 1.O mM D l T were included in al1 solutions. 150 mg of protein (-4 ml) was rotated with 1.5 ml of Sepharose CL-4B for 30 min at 4OC. Unbound protein was rotated with 2.5 ml of ~i'+-nitriloaceticacid-agarose (N~~'-NTA;Qiagen) for one hr. The mix was applied to a column, washed with 50 ml of Buffer E then consecutively with 5 ml of Buffer E containing 50 mM and 350 mM imidazole. Fractions were concentrated (Centricon-30: Amicon) and separated on a 6% SDS-gel. From the Coomassie stained gel of the 350 mM imidazole fraction, gel-slices corresponding to a -400 kDa protein from the HM-NGGI strain and its parallel position from the myc-NGGI control were excised, and washed with water for 20 min.
3.2.4
Protein identification Protein was eluted and in-gel trypsin digested by the method of
Shevchenko et al. (1996). Protein was identified by micro-column high performance liquid chromatography (HPLC) coupled to electrospray ionization tandem mass spectrometry and database searching. A 100 pm by 200 pm fused silica capillary (Kennedy and Jorgenson, 1989; Polymetrics, Inc.) was packed to a length of -1 5 cm with 10 mm POROS 10 R2 reverse phase material (Perseptives Biosystems). The fritted end of the column was inserted into the needle of the electrospray ion source and sample loaded by helium pressurization in a stainless steel bomb (Yates III et al., 1994). Chromatography was performed with a dual syringe pump (Applied Biosystems). The mobile
phase consisted of 0.5% acetic acid (solvent A) and 80:20acetonitrilefwater containing 0.5% acetic acid (solvent B). A 100:l precolumn split was used to deliver a flowrate of 1 to 1.5 ml per minute. The HPLC pump was programmed to ramp solvent B from 0% to 60% in 30 min. Electrospray ionization was camed out at voltage of 4.6 kV. Tandem mass spectra were acquired automatically during the entire gradient run (Link et al.. 1997). Tandem mass spectra were searched against a S. cerevisiae protein database (Saccharomyces Genome Database) using the SEQUEST program (Eng et al., 1994). Parameters for the SEQUEST program were set to locate potential sites of phosphorylation at serine, threonine and tyrosine residues (Yates III et a/., 1995). Every sequence with high scores that matched a tandem mass spectrum was manually verified.
3.2.5
Fractionation of TRAlp, NGGlp, ADA2p and SPT7p Ion-exchange chromatography: 40 mg (1.O ml) of cell extract, prepared
in 40 mM Tris-HCI (pH 7.7), 20 mM NaCI, 0.08% Nonidet P-40 and 10% glycerol, from KY320 expressing myc-TRA 1 and HA-ADA2, and CY947 expressing HA-ADAZ, was applied to a FPLC Mono Q column ( 1 .O ml; Pharmacia Biotech Inc.) at a flow rate of 0.1 mumin. Afier washing with 2 ml of buffer, protein was eluted with a 15 ml linear gradient of buffer containing 20 mM to 1 .O M NaCI. 25 pl aliquots of 0.5 ml fractions were separated by SDS-
PAGE before irnmunoblotting. DNA-cellulose chromatography: 25 mg of extract from strains CY 1077, KY320 expressing mye-TRA 1, CY927 expressing myc-TRA 1 and KY320 was fractionated on Mono Q. Protein from Mono Q fraction 12 for ADA2 strains (-350 mM NaCI) or fraction 8 for CY927 (-250 mM NaCI) was dialyzed against DB-buffer (5 mM Tris-HCI [pH 7.41, 50 mM NaCI, 5 mM MgCl*, 0.025% Nonidet
P-40, 5% glycerol) then 100 pg of protein applied to a 0.5 cm diameter column
containing 0.4 ml of DNA-cellulose (Pharmacia Biotech Inc.) at a flow rate of 1.5 mühour. After washing with 10 ml of DB-buffer, protein was eluted with 1.O ml of buffer containing 100, 300, and 500 mM NaCI.
3.2.6
lmmunoprecipitation and immunoblotting lmmunoprecipitation with anti-HA and anti-Myc antibodies from whole
cell extracts: lmmunoprecipitation of HA-ADA2p and myc-TRAlp from whole cell extracts was perfomed as described in Saleh et al. (1997; see Chapter 2). lmmunoprecipitation from ion-exchange column fractions: lmmunoprecipitations from Mono Q fractions were done after adjusting the buffer to 25 mM Tris-HCI (pH 7 3 , 100 rnM NaCI, 0.1% Nonidet P-40, and 5% glycerol (IP buffer) in a volume of 1.5 ml. 150 pg of protein was rotated for 20 min with 50 pl of protein A-Sepharose (Pharmacia Biotech Inc.). Unbound protein was rotated with 7 pl (12 mg/ml) of ascites fluid derived from the Mycl 9E10 cell line (Saleh et al.. 1997, Chapter 2),or with 8 pl (7 mg/ml) of monoclonal anti-HA antibody of the 12CA5 ascites fluid, and 50 pl of protein ASepharose beads (Pharmacia Biotech Inc.) for 3 hr. Beads were washed four times with 1.5 ml of IP buffer, and twice with 1.5 ml buffer containing 25 mM
HEPES (pH 7.5). 50 mM KCI and 5% glycerol then suspended in 20 pl of buffer and assayed for kinase activity, or eluted in SDS loading buffer at 60 OC for 5 min and separated by SDS-PAGE. Western blotting with polyclonal anti-SPl7p antibody and monoclonal anti-myc and HA antibodies has been described (Brandl et ab, 1996; Saleh et al., 1997, see Chapter 2 & 4).
3.3.1
Association of TRAlp with NGGlp To search for components of the ADA complexes, we analyzed proteins
associated with NGGl p after affinity purification of NGGl p from whole cell extracts. A derivative of NGGl was constructed that codes for a protein with both six histidine and myc-epitope tags (HM-NGGlp). Extracts from a strain containing HM-NGG1 and as a control from a strain containing myc-NGGI were loaded ont0 parallel N~"-NTA columns. HM-NGGlp eluted from the fVi2'NTA column with buffer containing 350 mM imidazole as compared to mycNGGlp which eluted in 50 mM imidazole. 350 mM imidazole eluants from both strains were compared by SDS-PAGE, revealing potential components of the ADA complex (not shown). One of these proteins had an apparent molecular mass of -400 kDa and thus did not match previously described ADA components. With the purification of the ADA components obtained by chromatography on N~~'-NTAand the resolution afforded by its migration on
SDS-PAGE, the -400 kDa protein was isolated and its identity determined by micro-column HPLC coupled to tandem mass spectrometry and database searching.
The gene product TRAlp (YHR099W) was identified from 36 unique peptides which correspond to 15.2% of the protein's mass. In the Saccharomyces Gemme Database, TRAI is descnbed as an essential gene encoding a protein of 3744 amino acids (433 kDa) with homology to TR-AP, a human protein which associates with c-myc. The absence of TRAl p in the parallel gel slice from the control strain, suggested that its association with HMNGGl p was specific. In addition, mass spectrometry revealed that the peptide
AEQGDLDSPKEPQ-ADELLDEFSK between residues of 165 and 187 of TRAl p contained one phosphoserine.
3.3.2
Coimmunoprecipitation of TRAl p and ADA2p To verify the interaction of TRAl p wlh the ADA components, we
determined if TRAl p coimmunoprecipitates with ADA2p. TRA 1 was rnyctagged at its amino-terminus and inserted into a centromeric plasmid. Whole cell extracts were prepared from strains expressing myc-TRA 1 and HA-ADA2
independently, and from a strain expressing both mye-TRA 1 and HA-ADM. HA-ADA2p and associated proteins were imrnunoprecipitated with anti-HA antibody, separated by SDS-PAGE, and Western blotted with anti-myc antibody (Fig. 3.1 A). The presence of rnyc-TRAlp in imrnunoprecipitates with HA-ADA2p (lane 3),but not in immunoprecipitates lacking either HA-ADA2p (lane 2) or myc-TRAlp (lane 1) indicated that TRAl p associates with ADA2p. Reprobing the blot with anti-HA antibody indicated that equivalent amounts of HA-ADA2p were immunoprecipitated from the HA-ADA2 containing strains (not shown). The reciprocal experiment was performed in which myc-TRAl p was immunoprecipitated and the presence of HA-ADA2p assayed by Western blotting. As a positive control an extract containing rnyc-NGGl p and HA-ADA2p was inciuded. As shown in figure 3.1 B, HA-ADA2p was found in rnyc immunoprecipitates from strains expressing myc-TRA 1 (lane 7) or myc-NGG1 (lane 4) in combination with HA-AD&
but not from the strain lacking rnyc-
TRAl (lane 3). No band was detected in the strain lacking HA-tagged ADA2p
(lane 2). To determine if other components of the SAGA compiex associate with myc-TRAlp, the blot was reprobed with anti-SPT7p antibody. As with HA-
Fig. 3.1 Coirnmunoprecipitation of TRAl p and ADA2p. A. Coimmunoprecipitation of TRAl p with ADA2p. 25 mg of cell extract from yeast strain KY320 containing HA-ADA2 (lane l), myc-TRA 1(lane 2),or mycTRA1 plus HA4DA2 (lane 3 were incubated with anti-HA antibody. Precipitated protein was Western blotted with anti-myc antibody. HC indicates the antibody heavy chain.
B. Coimmunoprecipitation of ADA2p with TRAl p. 25 mg of cell extract from KY320 containing myc-TRA 1 and HA-AûA.2(lane l), myc-TM1 (lane 2),HAADA2 (lane 3,or CY947 containing HA-AûA2 ( T R I 1 myc-NGG 1 H A - A N ; lane 4) were incubated with anti-myc antibody. lrnmunoprecipitated protein was Western blotted with anti-HA and SPT7p antibodies. The positions of HAADA2p (top panel; lanes 1 & 4) and SPl7p (bottom panel; lanes 1,2 & 4) are labeled. Migration of molecular mass standards (kDa) are indicated on the right. LC indicates the anti-myc antibody light c h a h
TRAI
(
Mvc
Myc
WT
WT -
A
Myc
ADA2
I
ADA2p, SPT7p specifically coimrnunoprecipitated with myc-TRAl p and rnycNGGI p. In certain whole cell extracts SPT7p appeared as a doublet (Fig. 3.1 B, lanes 1 and 4). The doublet may anse as a result of the modification of SPT7p by ubiquitination (Saleh et al., 1998, see Chapter 4). The reason for variability in the appearance of the doublet is unclear.
3.3.3 T R A i p cofractionates with ADA and SPT proteins To examine if TRAl p interacts with one or more of the ADA complexes, we compared the elution of myc-NGGl p, HA-ADA2p, SPT7p, and myc-TRAlp after fractionation of a whole ceIl extract on a FPLC Mono Q column. Whole cell extract was eluted from a Mono Q column in a linear gradient of 20 mM-1.O M NaCl and equal volumes of each fraction were examined by Western blotting
(Fig. 3.2 A). Three ADA-containing complexes were distinguished, with rnycNGGlp and HA-ADA2p cofractionating in peaks centered at fraction 12 (-350 mM NaCI), 14 (-450 mM NaCI) and 16 (-550 mM NaCI). SPT7p and rnycTRAl p cofractionate with myc-NGGl p and HA-ADA2p, in two of the peak fractions, 12 and 16. myc-TRAlp was absent from fraction 14 and SPT7p was very much reduced, suggesting that the ADA components also exist independently (Grant et al.. 1997). The elevated level of SPT7p in fraction 13 may also indicate the existence of ADA-independent SPT7p. To exclude the possibility that the coelution of the ADAISPT molecules
was coincidental, HA-ADA2p was immunoprecipitated from the -350 mM NaCl Mono Q fraction and analyzed by immunoblotting for myc-TRAl p and SPT7p (Fig. 3.2 B). Both of these proteins specifically coimmunoprecipitated with HAADA2p from the -350 mM NaCl fraction (lane 2) as they did from a whole cell extract (lane 3).
Fig. 3.2 NGGlp, ADAZp, SPl7p and TRAI p coeiute from a FPLC Mono Q column. A. 40 mg of cell extract containing myc-TRAlp, myc-NGGlp, HA-ADA2p and SPT7p was fractionated on a Mono Q column. Protein was eluted with a gradient of 20 mM-1.O M NaCi. Equa! volumes of each fraction were separated on a 7.5% SDS-gel to blot for myc-NGGl p and HA-ADMp, and 5.5% SDS-gel to blot for SPf7p and myc-TM1p. Arrows indicate the peak fractions for elution of myc-NGGlp, HA-AD@, SPT7p and myc-TRAlp.
B. Coimmunoprecipitation of myc-TRAlp and SPT7p wRh ADA2p from the -350 mM NaCl fraction. 150 pg of protein from Mono Q fraction 12 containing myc-TRAl p and HA-ADA2p (lane 2),25 mg whole cell extract containing HAADA2p (lane I ) , HA-ADA2p and myc-TRAlp (lane 3), or myc-TRAl p (lane4) were incubated with anti-HA antibody. Protein was separated on a 5.5% SDSgel and Western blotted with anti-myc and SPT7p antibodies.
C. Densitometric scanning profiles of myc-TRAlp irnmunoblots after fractionation of cell extracts from wild-type (myc-TRA1 ADA2;WT. solid line) and ada2 deletion strains (myc-TRA 1 ad= dashed line) on a Mono Q column. The elution profile of total protein from the column is shown in the inset.
l7MI ADA2
UT HA
Mvc
H A -
Mvc
,UVC
Fraction
To address whether coelution of T M 7 p with the ADNSPT components in the -550 mM NaCl fraction 16 was indicative of their association, we analyzed whether removal of ADA2p. altered the elution profile of myc-TRAl p from the Mono Q column. As shown in figure 3.2 C, in the absence of ADA2p, myc-TRAl p eluted in a single peak centered at fraction 8 (-250 mM NaCI) and was absent from fractions in which it was found when prepared from the wildtype ADAO strain. This altered elution profile for myc-TRAl p from the ada2
strain supports the idea that it is a component of two ADAISPT complexes.
3.3.4
Binding of TRAlp to DNA-cellulose requires ADA proteins To determine if TRAlp could be further purified with components of the
ADNSPT complexes, we chromatographed the -350 mM NaCl fraction from Mono Q on a DNA-cellulose column. After dialysis, the -350 mM NaCl fraction 12 containing myc-TRAlp, myc-NGGl p and HA-ADA2p was loaded ont0 DNA-
cellulose, and bound protein eluted with buffer containing 50 mM (FT), 100
mM, 300 mM and 500 mM NaCl (Fig. 3.3). Both myc-TRAl p and myc-NGGl p coeluted in buffer containing 300 mM NaCI, as did HA-ADA2p and SPT7p. When the peak fraction containing myc-TRAl p from an ada strain (Mono
Q column fraction 8) was loaded onto DNA-cellulose, myc-TRAl p eluted in the fiow-through fraction (Fig. 3.4; compare top two panels). This suggests that the binding of TRAl p to DNA-cellulose is mediated by component(s) of the
ADNSPT complexes. To test this possibility, we addressed whether factors in Mono Q fraction 12 frorn a wild-type strain could restore DNA binding of mycTRAl p obtained from an ada strain. Mono Q fraction 8 containing myc-TRAl p from the ada2 deIetion strain was rnixed with Mono Q fraction 12 frorn a wild
type strain containing myc-NGGlp. As shown in the second to last panel,
Fig. 3.3 Cofractionation of TRAlp with the ADA/SPT proteins on DNA-cellulose. 100 pg of protein from the Mono Q column eluting fraction 12 containing mycTRAl p, rnyc-NGGlp, SPT7p and HA-ADA2p was chromatographed on DNAcellulose. After washing with 50 mM NaCl buffer, bound protein was eluted with a stepgradient of NaCi (100, 300 and 500 mM). The elution of myc-TRAl p, myc-NGGlp, SPT7p and HA-ADA2p was followed by Western blotting.
binding of approximately 25% of the myc-TRAl p to DNA-cellulose was restored upon mixing with the wild-type ADNSPT-containing fraction. The reconstitution of binding was due to ADABPT proteins because mixing of fraction 8 with fraction 12 obtained from the ad& strain did not restore binding of TRAl p to DNA-cellulose (lower panel). These results demonstrate that the binding of myc-TRAl p to DNA-cellulose requires ADAEPT cornponents.
Fig. 3.4 Bindlng of TRAlp to DNA cellulose column requires ADAZ. 100 pg of protein obtained from Mono Q fraction 12 of a wild-type extract containing myc-TRAlp was applied and eluted from a DNA cellulose column as descnbed in Fig. 3.3 (Fr. 12 mye-TRA 1; top panel). Similady, 150 pg of the Mono Q column fraction 8 containing myc-TRAlp frorn a ada2 strain was chrornatographed, individually (Fr.8 ada2; second panel), and after mixing with 150 pg of the Mono Q fraction 12 from an ADA2 strain (Fr.8 ada2 + Fr. 12 WT; third panel), or after mixing with 150 pg of Mono Q fraction 12 from the same ada2 strain (Fr. 8 + 12 ada2; bottom panel). The elution of myc-TM1p was assayed by Western blotting. The first lane (load) is 25 pg of the protein applied to the column, followed by the fiow-through (FT; 50 mM NaCI), 100 mM, 300 mM and 500 mM NaCl fractions.
fNaCfl mM Load
FT
100
300
C
Fr. 12 mye TRAI
Fr. 8 +12 ada2
I
w
500
3.4
Discussion
Our identification of TRAl p within the ADAISPT complexes was based on
its association with ADA cornponents after affinity purification on N~~+-NTA. Several lines of evidence confinn that TRAlp associates with the ADNSPT proteins. First, the association of TRAl p and ADAISPT components was shown
by their reciprocal coimmunoprecipitation from whole ceIl extracts. Chromatography on a FPLC Mono Q coturnn revealed al1 of the TRAlp coeluted with NGGlp, ADA2p and SPl7p in two distinct peaks. The validity of this cofractionation as a measure of association was verified by the finding that TRAlp coimmunoprecipitated with ADA2p in the -350 rnM NaCl Mono Q fraction and the demonstration that the slution profile of TRAlp was altered when perfoned with an extract from a strain lacking ADA2p. The latter suggests that TRAlp is found in two ADASPT complexes. Third. as well as their copurification on a Ni2+-NTAcolurnn, TRAl p, NGGl p and SPT7p remain associated through an approximately 300-fold purification over consecutive chrornatography on Mono Q and DNA-cellulose columns. Fourth. association of
TRAlp is supported by the ability of partially pun'fied ADAISPT proteins to reconstitute binding of TRAl p to DNA-cellulose.
TRAl p is a member of a group of molecules with carboxyl-terminal sequences similar to P13Ks (reviewed in Kapeller and Cantley, 1994). While related to the P13Ks these molecules can be distinguished from the P13Ks by having a common region directly at their carboxyl-terminus, their large size and in some cases an ability to phosphorylate proteins (Hartley et al., 1995; Keith and Schreiber, 1995). lncluded in this group are the mammalian proteins DNA-
P
, ATM. and FRAP; the Drosophila protein MEI-41; the S. cerevisiae
proteins TOR1p, TOR2p, MEC1p, TEL1p; from S. pombe, RAD3p (reviewed in
Hunter, 1995; Keith and Schreiber, 1995; Zakian, 1995; Lees-Miller, 1996) and the product of ORF C l F5.11C; the products of the C. elegans gene C47D12.1; and from Arabidopsis the product of 19K4.210. Of this group. C1F5.11 C is most closely related having 34% identity and 54% similam throughout its length. As
a group these molecules are involved in many key cellular processes including
DNA repair, meiotic recombination, V(D)J recornbination, cell-cycle regulation. DNA-damage recognition, and transcription (Hunter, 1995; Zakian, 1995; LeesMiller, 1996). In its carboxyl-terminal P13K region, TRAl p shares approximately
25% sequence similarity to ail members of the group. Short stretches of similarity between TRAl p and rnembers of the group, as identified through a
BLAST search, are also found throughout the molecule. TRAlp lacks the DFG sequence found in many other protein kinases (Taylor et ab, 1992); it does contain the DXXXXN kinase motif but in a flanking sequence context different from other family rnembers. BLAST and FASTA searches identify other sequence relationships that could relate to function. The P13K region shows similarity to the transcriptional repressor RGMl p (27% identity over a 136 arnino acid overlap; Estruch, 1991), and SIR4p (24% identity over a 244 amino acid overlap; Tsukamoto et al., 1997; Boulton and Jackson, 1998). Central regions of the protein are also similar to the transcriptional regulator TECl p (Laloux et al., 1990) and to MTRlp which is involved in nuclear protein import (Rosenblum et
al., 1997). The essential nature of TRAl has not allowed direct gene disniption experiments to determine its role in the ADAfSPT complexes. Many models reflect the possibility, based on sequence similarities, that TRAl p is a protein kinase and in turn, the identity of substrates. In the case of DNA-PkS, hGCN5 is a target in vitro and phosphorylation correlates with decreased histone
acetyltransferase activity (Barlev e l al., 1998). hGCN5 can thus be added to the
list of substrates for DNA-PK that are involved in transcription including activator proteins, components of the basal machinery, as well as HMG1 and HMGP (reviewed in Lees-Miller, 1996, see also Watanabe et al., 1994; Giffin et al., 1996; Chibazakura et al., 1997).
To test if TRAl p is a protein kinase, we have examined kinase activity in immunoprecipitates from partially purified ADAlSPT complexes. Under separate conditions in which DNA-Pbs, casein kinase II, or the cyclin dependent kinase p34Cdc2were active, we were unable to detect phosphorylation of pcasein, RPA, histone H l , myelin basic protein or the amino-terminus of p53 at a level above that found in control imrnunoprecipitates (not shown). Assays were also performed with and without DNA as well as withlwithout Ku with no significant activity detected. In addition, under these same conditions. no specific phosphorylation of endogenous components was noted. A general loss in the integrity of the irnmunoprecipitated complexes was unlikely because they retained histone acetyltransferaçe activity (not shown, see Chapter 4). Furthemore, it is unlikely that TRAl p is inhibited by components of the ADA complex because immunoprecipitates of TRAl p isolated from the ada2 deletion strain also lacked kinase activity. Cleariy, we can not at present exclude the possibility that TRAl p has unique substrates or assay conditions; however, the lack of kinase activity is consistent with the absence of the nonally conseived DFG sequence in the kinase motif. The sequence similarity of TRAl p to several key cellular regulators, its appearance in at least two of the ADAISPT complexes and even its large size suggests that TRAlp plays a key role in the structure, function or regulation of the ADNSPT complexes. Since al1 the detectable TRAl p cofractionated with myc-NGGl p, it appears to function principally through its association with the ADNSPT proteins. The fact that unlike other ADNSPT complex proteins, TRAl p
is essential, does predict that it has a broader range of function(s) than the other components.
3.5
References
Bariev,N.A., Candau,R., Wang,L., Darpino,P., Silverman,N. and Berger,S.L. (1995) Characterization of physical interactions of the putative transcriptional adaptor, ADA2, with acidic activation domains and TATA-binding protein. J.Bio1. Chem. 270, 1 9337-19344. Barlev,N.A., Poltoratsky,V., Owen-Hughes,T., Ying,C., Liu,L., Workman,J.L. and Berger,S.L. (1998) Repression of GCNS histone acetyltransferase activity via bromodomain-mediated binding and phosphorylation by the KU-DNAdependent protein kinase cornplex. Mol Cell Biol. 18, 1349-1358. J.Biol. Chem. 270, 19337-19344. Berger,S.L., Pina,B., Silverman,N., Marcus,G.A., Agapite,J., Regier,J.L., Triezenberg,S.J. and Guarente,L. (1992) Genetic isolation of ADA2 - a potential transcriptional adaptor required for function of certain acidic activation domains Cell70, 251-265. Bou1ton.S.J. and Jackson,S.P. (1998) Components of the Ku-dependent nonhomologous end-joining pathway are involved in telomeric length maintenance and telomeric silencing. EMBO J. 16, 1819-1828. Brandl,C.J., Furlanetto,A.M., Martens,J.A. and Hamilton,K.S. (1993) Characterization of NGG1, a novel yeast gene required for glucose repression of GAL4p-regulated transcription EMBO J. 12. 5255-5265. Brandl,C.J., Martens,J.A., Margaliot,A.. Stenning,D., Furlanetto A.M., Saleh,A., Hamilton,K.S. and Genereaux,J. (1996) Structurelfunctional properties of the yeast dual regulator protein NGGl that are required for glucose repression. J. Biol. Chem. 270, 9298-9306. Candau,R. and Berger,S.L. (1996) Structural and functional analysis of yeast putative adaptors. Evidence for an adaptor complex in vivo. J.8iol.Chem. 271, 5237-5245. Chibazakura,T., Watanabe,F., Kitajima,S., Tsukada,K., Yasukochi,Y. and Teraoka, H. (1997) Phosphorylation of human general transcription factors TATA-binding protein and transcription factor llB by DNA-dependent protein kinase--synergistic stimulation of RNA polymerase II basal transcription in vitro. Eur. J. Biochem. 247, 11 66-1173. Eng,J.K., McCorrnack, A.L. and Yates II1,J.R. (1994) Am. Soc. Mass Spectrom. 5 , 976-989.
Estnich,F. (1991) The yeast putative transcriptional repressor RGMl is a proline-rich zinc finger protein. Nucleic Acids Res. 1 9 , 487394877.
Georgakopoulos,T. and Thireos,G. (1992) Two distinct yeast transcriptional activators require the function of the GCNS protein to promote normal levels of transcription EMBO J. 11, 4145-4152. Giffin,W., Torrance, H, Rodda,D.J., Pref0ntaine.G .G ., Pope,L. and Hache, R.J. (1996) Sequence-specific DNA binding by Ku autoantigen and its effects on transcription. Nature 380. 265-268. Gottlieb,T.M. and Jackson,S.P. (1993) The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen. Cell72, 131-142. Grant,P.A., Duggan,L., Cote,J., Roberts,S.M., Brownell,J.E., Candau,R., Ohba,R., Owen-Hughes,T., Allis,C.D., Winston,F., Berger,S.L. and Workman,J.L. (1997) Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (SptlAda) complex. Genes Dev. 11, 1640-1650. Hampsey,M. (1997) A SAGA of histone acetylation and gene expression. Trends Genet. 13, 427429. Hartley,K.O., Gell,D., Smith,G.C., Zhang,H., Divecha,N., Connelly,M.A., Admon,A., Lees-Miller,S..P., Anderson,C.W.. and Jackson.S.P. (1995) DNAdependent protein kinase catalytic subunit: a relative of phosphatidylinositol 3kinase and the ataxia telangiectasia gene product. Ce1182. 849-856. Horiuchi,J., Silverman,N., Marcus,G.A. and Guarente,L. (1995) ADA3, a putative transcriptional adaptor, consists of two separable domains and interacts with ADA2 and GCNS in a trimenc compiex. Mol. Cell. Biol. 15, 1203-1209. Horiuchi,J., Silverman,N., Pina,B., Marcus,G.A. and Guarente,L. (1997) ADAI, a novel component of the ADNGCNB complex, has broader effects than GCN5, ADA2, or ADA3. Mol. Cell. Biol. 17, 3220-3228. Hunter, T. (1995) When is a lipid kinase not a lipid kinase? When it is a protein kinase. Cell83,l-4. Kapeller,R. and Cantley,L.C. (1994) Phosphatidylinositol 3-kinase. Bioessays 16, 565-576. Keith,C.T. and Schreiber,S.L. (1995) PIK-related kinases: DNA repair, recombination, and cell cycle checkpoints. Science 270, 50-5 1. Kennedy, R.T. and Jorgenson,J.W. (1989) Quantitative analysis of individual neurons by open tubular liquid chromatography with voltammetric detection. Anal. Chem. 61, 1128-1135. Laloux,l., Dubois,E., Dewerchin,M. and Jacobs,E. (1990) TECI, a gene involved in the activation of T y l and T y l -mediated gene expression in Saccharomyces cerevisiae: cloning and molecular analysis. Mol. Ceil. Bhl. 10, 3541-3550.
Lees-Miller,S.P., Chen,Y.R. and Anderson,C.W. (1990) Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen. Mol. Cell. Biol. 12, 647206481. Lees-Miller,SP. (1996) The DNA-dependent protein kinase, DNA-PK: 10 years and no ends in sight. Biochem Cell. Biol. 74, 503-512. Link, A. J., Hays, L.G., Carmack, E.B. and Yates III, J.R. (1997) ldentifying the major proteorne components of Haemophilus influenzae type-strain NCTC 8 143. Electrophoresis 18, 1314- 1334. Marcus,G.A., Horiuchi,J., Silverman,N. and (3uarente.L. (1996) ADASSPT20 links the ADA and SPTgenes, which are involved in yeast transcription. Mol. Cell. Biol. 16, 3197-3205. Pollard, K.J. and Peterson, C.L.(1997) Role for ADNGCN5 products in antagonizing chromatin-mediated transcriptional repression. Mol. Cell. Biol.. 17, 6212-6222. Roberts,S.M. and Winston,F. (1996) SPT20/ADA5 encodes a novel protein functionally related to the TATA-binding protein and important for transcription in Saccharomyces cerevisiae. Mol. Cell. Biol. 16, 3206-3213. Roberts,S.M. and Winst0n.F. (1997) Essential functional interactions of SAGA. a Saccharomyces cerevisiae complex of Spt, Ada and Gcn5 proteins, with the SnWSwi and SrVrnediator complexes. Genetics 147, 451-465. Rosenblurn,J.S., Pembert0qL.F. and Blobel,G. (1997) A nuclear irnport pathway for a protein involved in tRNA maturation. J. Cell Biol. 139, 1645-1653. Saleh,A., Lang,V., Cook,R. and Brandl,C. (1997) Identification of native complexes containing the yeast coactivator/repressor proteins NGG1/ADA3 and ADA2. J. Biol. Chem. 9 , 5571-5578. Saleh, A., Collait, M., Martens, J.A., Genereaux, J., Allard, S., Cote', J. and Brandl, C.J. (1998) TOMlp, a potential yeast E3 ubiquitin ligase which mediates transcriptional regulation and targets the ADA/SAGA coactivator complexes.J. Mol. Bol. in press Shevchenko,A., Wilm M., Vorm, 0. and Mann,M. (1996) Mass spectrornetric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850-858. Taylor,S.S., Knighton,D.R., Zheng,J., TenEyck,L.F. and Sowadski,J.M. (1992) Structural framework for the protein kinase family. Annu. Rev. Cell. Biol. 8, 429-462.
Tsukamoto,Y., Kato,J. and Ikeda,H. (1997) Silencing factors participate in DNA repair and recombination in Saccharomyces cerevisiae. Nature 388, 900-903. Watanabe, F., Shirakawa, H., Yoshida, M., Tsukada, K.and Teraoka, H. (1994) Stimulation of DNA-dependent protein kinase activity by high mobility group proteins 1 and 2. Biochem. Biophys. Res. Commun. 202, 736-742. Yates III, J.R., McCormack, A.L., Hayden, J.B. and Davey, M.P. (1994) in Ce11 Biology: A laboratory Handbook, Vol. 3, ed. Celis, JE. (Academic Press, San Diego) pp. 380-388. Yates III,J.R., Eng, J.K., McComack, A.L. and Schieltz, D. (1995) Method to correlate tandem mass spectra of rnodified peptides to amino acid sequences in the protein database. Anal. Chem. 67, 1426-1436. Zakian,V.A. (1995) ATM-related genes: what do they tell us about functions of the human gene? Ce1182, 685-687.
CHAPTER 4
TOMlp, A YEAST E3 UBlQUlTlN LIGASE WHlCH MEDIATES TRANSCRIPTIONAL REGULATION AND TARGETS THE ADAISAGA COACTIVATOR COMPLEXES3
4.1
Introduction
Coactivators enhance the function of activator proteins either by providing a regulatory interface with the basal machinery andor facilitating the activity of the transcription machinery on its chromatin template (Orphanides et
al., 1996). As these are two key steps in transcriptional initiation, it is not surpnsing that as well as having a positive effect on gene induction, coactivators can also play significant roles in repression (for examples see Song et al., 1996; Li et al.. 1996). The ADA genes were identified in Saccharomyces cerevisiae based on their requirement for the regulated activation and repression of transcription (Guarente, 1995). Initial studies identified three genes ADA2, NGGUADA3 (referred to as NGGl for simplicity) and GCN5 which as single or double disruptions resulted in both decreased transcriptional activation by GCN4p and GAL4p-VP16 fusions (Berger et al., 1992; Pina et ab, 1993; Marcus et ai., 1994; Georgakopoulos and Thireos, 1992) and increased transcriptional activation by GAL4p and PDRl p (Brandl et al., 1993; 1996; Martens et al.. 1996). The - --
.
--
A version of this chapter has been accepted for publication.
Ayman Saleh, Collart, M., Martens J., Genereaux, J, Allard, S., Cote, J. and Brandl, C.J. (1 998) TOM1p. a yeast hect domain protein which mediates tanscriptional regulation and targets the ADNSAGA coactivator complexes. J. Mol. Biol. (in press).
phenotype of strains containing double disniptions as well as affinity chromatography and coimrnunoprecipation suggested that these proteins act in a complex (Pina et al., 1993; Marcus et al., 1994; Georgakopoulos et al., 1995; Horiuchi et al., 1995; Brandl et al., 1996; Candau et al., 1996; Saleh et al., 1997). Of this group of proteins, GCN5p is a histone acetyltransferase, implying that at least one role of the ADA proteins is in regulating nucleosome function (Brownell et al., 1996; Kuo et al., 1996; Wang et al., 1997; Candau et aL, 1997; Grant et al., 1997; Pollard and Peterson, 1997). The regulatory functions of the ADA proteins anse as the result of their presence in multiple high molecular weight complexes containing additional components. Analysis of ADA2p and NGGlp in native yeast extracts revealed that these proteins are associated in four complexes, two with approximate sizes of greater than 2 MDa, and others of 900 kDa and 200 kDa (Saleh et al., 1997). The finding that a comparable 1.8 MDa ADA complex contains SPT3p, SPT7p, and SPT20p/ADA5p defines at least in part, the composition of one of these complexes, the SAGA-complex (Grant et al., 1997). The absence of the
SPT proteins in some of the other ADA containing complexes along with the overlapping but nonidentical phenotypes caused by the different nuIl alleles suggests that the SPT and ADA proteins also act independently (Marcus et al., 1996; Roberts and Winston, 1996, 1997; Grant et al., 1997). The same is likely true for ADAI p which associates with ADA2p, GCN5p and NGGl p but whose disniption results in broader effects than those seen for the other ADA genes (Horiuchi et al., 1997). The connection between the ADA and SPT genes is particularly intriguing because the class of SPTgenes including SPT3, SP77, SPT8 and
SPTZU have been linked to function of the TATA-binding protein (TBP; SPT15p) Winston et al., 1984; Eisenmann et al., 1989, 1994; Gansheroff et al., 1995). In
fact, SPT3p interacts directly with TBP (Eisenmann et al., 1989, 1992). The Iink between the TBP class of SPT proteins and ADA gene products suggests that the ADA proteins likely affect the basal transcriptional machinery. A role in modulating the activity of TBP had also been predicted based on the ability of the ADA proteins ta interact with TBP (Barlev et al., 1995; Saleh et al.. 1997; Roberts and Winston, 1997). To identify factors that associate with or perhaps regulate the ADA proteins, we previously performed a yeast two-hybrid analysis using the aminoterminal 373 amino acids of NGGl p (Martens et al., 1996). This screen identified four genes, including the ubiquitin protease UBP3p which functions to remove ubiquitin moieties from substrate proteins (Baker et al., 1992). The interaction between UBP3p and NGGIp suggested that UBP3p may be part of an ADA complex or that a ubiquitinated component of an ADA complex, either
NGGl p or a factor(s) associated with NGGl p is ubiquitinated and recognized by UBP3p as a substrate.
Ubiquitination is a key elernent in the regulation of transcription as it is for other cellular processes (reviewed by Jentsch, 1992; Hochstrasser, 1995, 1996; Wilkinson, 1995). In some instances this regulation is through the direct signalling for rapid degradation by the 26s proteasorne of a modified activator protein (eg GCN4p; Komitzer et al., 1994; see reviews by Ciechanover, 1994; Jentsch and Schlenker, 1995). In other cases ubiquitination may signal events other than direct protein degradation (Hochstrasser, 1996). Ubiquitin is joined to lysine residues of acceptor proteins via a thioester cascade catalyzed by three classes of enzymes:
E 1 ubiquitin-activating
enzymes, E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases (Chen and Pickart, 1990; Haas et al., 1991; van Nocker and Vierstra, 1991; Banerjee
et al., 1993; Scheffner et al. , 1995). The €3 proteins play a major role in
detenining the substrate specificity of the system and are involved in the final transfer of ubiquitin to the target protein. Characterization of the mechanism by which E6 proteins of papilloma viruses promote degradation of p53 led to the identification of the E3 ubiquitin ligase, E6-AP (Scheffner et al., 1993). There are several eukaryotic proteins, including five yeast open reading frames, that show sirnilarity to E6-AP over its carboxyl-terminal 350 amino acids (Huibregtse et al., 1995). One of the yeast hect-domain (homologous to E6-AP ~arboxyl-terminus)proteins, RSP5p
(NP11p; Hein et al., 1995; MDPl p; Zolladek et al., 1997), was identified by the Winston laboratory as a suppressor of mutations in SPT3 (cited in Huibregtse et al., 1995) and has been found necessary for full transcriptional activation by human steroid receptors in yeast (Imhof and McDonell, 1996). Two of the other yeast hect-domain proteins have been characterized. UFD4p is involved in the ubiquitin fusion degradation pathway (Johnson et al., 1995) while TOM1 p (Irigger pf Mitosis) was identified based on its involvement in the G2/M transition (Utsugi et al., 1995). Interestingly, UreB1, a rat hect-domain protein of 310 amino acids, may have a role in transcriptional regulation through direct
DNA binding (Gu et al., 1994). This report demonstrates that the hect-domain protein TOMl p has a role in transcriptional regulation. The effects of TOMl p are likely mediated through the ADA complexes since single and double mutations in tom1 and nggl result in similar changes in transcription from ADH2, HlS3, and GAL 10 promoters. Ubiquitination appears essential for the regulation by TOMl p as a cysteine residue conserved amongst hect-domain proteins and found necessary for thioester bond formation with ubiquitin (Scheffner et al., 1995; Huibregtse et al., 1995), is required for transcriptional regulation by TOMl p. Furthemore, TOM1p associates with the ADA proteins and is required for the ubiquitination of a 210
kDa protein, likely SPT7p, that coimrnunoprecipitates w l h the ADA components. A mechanistic role for ubiquitination by TOM1p is suggested by the finding that in the absence of TOM1p the ability of the ADA proteins to interact with SPT3p and TBP is reduced.
4.2
4.2.1
Materials and Methods
DNA constructs Molecules were constnicted using standard cloning techniques and
verified by restriction and sequence analysis. l a d reporter constnicts were cloned as his3-lacZfusions in the LEU2 centrorneric plasrnid YCp87 (Brandl et al., 1993) or transferred as BamHI to San fragments into the ADE2 centromeric vector pASZ11 ( S t o b and Linder, 1990). The HlS3 promoter was from his3189 (Hill et ai.,1986). The GAL 1O promoter region contains GAL 1- 10 sequences from 299-649 (Johnston and Davis, 1984) fused to a TATA element sequence of TATAAA at -25 relative to the his3 transcriptional start site (his3G25, Brandl et al., 1993). Similarly, the ADH2 prornoter contains sequences from 759 to 1052 as defined by Yu et al. (1989), including the regions required for ADRI p activation, fused at -25 of his3.
4.2.2
Yeast strains, media and growth conditions Yeast strain CY922 (relevant genotype, MAT a ade2- 101 ga14-A ga180-
A his3-MO0 leu2-3,112 trp 1-A901ura3-52 L YS2::GAL 1-HlS3; see Table 4.1 ), is
a derivative of Y190 (Durfee et al., 1993). JY335 is isogenic to CY922 but contains nggl- 1 (Brandl et al., 1993; Martens et al., 1996). A Tnl OLUK disruption of the coding region of TOM1 from amino acids 244 to 2952 was introduced in CY922 and JY335 by double gene replacement method to generate SYl O and CY 1004, respectively. ~ r a colonies + were selected and verified for disruption by ?CR and Western blotting. SY11 was generated from SY 10 by selecting for loss of URA3 on 5-fluoroorotic acid (Boeke et ai., 1984). Ylplac211-HA-NGGI (Saleh et al., 1997) was digested with Kpnl and integrated into SY 11 to generate SY12. Similarly, Ylplac211-HA-NGG1 and
Table 4.1 Y e a s t strains used i n this study.
e
CY922
FY 1093 N335
CY1004 CY 1005 SYH sY12
SY13 SY14
644Ta a&?-101 go[#-d gal8GA his3-d7ûU leul-3,2 ~p l-A9O/ura3-52L IS2::GU 1-HISj A44 Ta sp17MO?:tELr2 his9178js2-173R2 ieuhAl ura3-52 npl A63 ode8 Isogenic io CY922 uccept nggl-1 Isogenic to JY3 3 5 excepi tom 1:: L'Rd 3 Isogenic 10 CY922 cscept tom& p 5 3 - . 4 b Isogenic to CY922 excepi t 0 r n / ~ 4 4 - 2 9 5 2 Isogenic io SY 1 1 e , ~ c q HcZ-XGGI t Isogenic to CY922 except f i 4 4%Gl Isogenic to CY922 esœpt It-i-nggfa73-307
Brandl el al.,19% Gansheroff el al., 1995
Martens et al., 1996
ïhk study This midy This study This study This study This study
Ylplac211-HA-nggld 274-307 were integrated into yeast strain CY922 to generate SY13 and SY 14. To constnict the Cys-Ala mutation at amino acid 3235 of TOMl p, a 1.1 kilobase pair HinB II fragment containing URA3 was cloned in the terminal Kpnl fragment of TOM1, downstream of the TOMl p coding region. The mutation was introduced by site directed mutagenesis as described by Kunkel (1985) using the oligonucleotide 5'-cagttgattgaaaGCggtatgtgatgat-3'. This allele was introduced into yeast strain CY922 by double strand gene replacement to generate CY1005. URA+ colonies were verified for introduction of the mutation by PCR analysis and the incorporation of a novel Acrl restriction site. In general, yeast strains were grown at 30'~ in YPD broth (1% yeast extract, 2% peptone, 2% glucose) or in minimal media (0.67% yeast nitrogen base without amino acids, 2% glucose and supplemented with amino acids as requirad).
4.2.3
Two-hybrid analysis GAL4oso-NGG1i-373 (DBD; QNA mnding Domain of GAL4p) contains a
Ndel-Nsd (blunt) fragment of NGG1, encoding the initiator ATG to codon 373
into the Ndel-Smal site of pASl (Durfee et ai., 1993; Martens et al., 1996). Yeast strains CY922, SY 11 and CY 1005 containing the plasmid pAS1-GAL~DBDNGG 11-373and a GAL IO-lac2 reporter on an ADE2containing centromeric
plasmid (Martens et al., 1996) were transformed with a cDNA fragment of UBP3 encoding amino acids 733-912 (Baker et al., 1992) fused to the GAL4p activation domain in pACT (Durfee et ai., 1993).
4.2.4 j3-galactosidase assays
Yeast strains were grown in liquid culture in minimal media to an ODsoo of 1.O to 1.5. For the induction of ADRl p, starter cultures grown in glucose were pelleted, washed twice with water and diluted 10-fold into media containing 3% ethanol and 0.05% glucose. Cells were pelleted, washed and concentrated in lacZ buffer (Guarente, 1983). fbgalactosidase activity was determined after disruption of the cells with glass beads (Himmelfarb et al., 1990). Activity was standardized to protein concentration. For yeast strains expressing potent activator proteins, fl-galactosidase activity was assayed according to Ausubel et
al. (1990) and standardized to cell density. To ensure the presence of GCN4p for the analysis of the HIS3 promoter, GCN4 was expressed from the DEDl promoter on the centromeric plasmid YCp88 (Hope and Struhl, 1986). GAL4 expressed from its native promoter was introduced into yeast strains on the TRP 1 centromeric plasmid YCplac22 (YCPG4trp, kindly provided by Ivan Sadowski).
4.2.5
Preparation of whole cell extract Yeast whole cell extract was prepared in liquid nitrogen as described by
Schultz et al. (1991) with minor modifications (Saleh et al., 1997; see Chapter 2)
4.2.6
Analysis of histone acetyltransferase activity Isolation of ADA complexes: Whole cell extracts were prepared from
cells harvested from one liter cultures of CY922, SY 12, and SY 13 grown to an optical density of 1.6 to 1.8 at 600 nm by grinding the cells in liquid nitrogen.
The grinding buffer containing 25 mM Tris-HCI (pH 7 3 ,1.5 mM EDTA, 10% glycerol, 140 mM NaCI, 1.O mM dithiothreitol, 0.1 % Nonidet P-40, and protease
inhibitors (as described in Chapter 2). After adjusting the pH of the solution to 7.5 and removing cellular debris by centrifugation at 40,000 g, 25 mg of protein ~ 30 was incubated with 200 pl of Sepharose CL-4B and rotated at 4 ' for
minutes. Unbound protein was measured as was the HA-NGGl p in each extract by analyzing serial dilutions on a Western blot. 10 pl of anti-HA antibody (12CA5, 12 mg/ml) was incubated for two hours w l h two and five mg of protein in a total volume of 1.O ml. Immune complexes were isolated by a second two hour incubation with 50 pl of protein A Sepharose that had been incubated with 20 pg of rabbit anti mouse IgG and igM (1.O mg/ml). The beads were washed
four tirnes with 1.5 ml of extraction buffer followed by two washes with buffer containing 40 mM Tris-HCI (pH7.5). 40 mM NaCl, 10% glycerol, 0.1 mM EDTA, 0.08% Nonidet P-40 and protease inhibitors.
Histone acetyltransferase activity: Liquid histone acetyltransferase assays were performed as described by Grant et al. (1997). Briefly 1.O pg of HeLa Hl-depleted oligonucleosornes (Cote et ai., 1995) was incubated with
each immunoprecipitated fraction (6 pl of beads) and [ 3 ~ ] a c e t y l - (0.25 ~ o ~ pCi) in 30 pl of HAT buffer (50 mM Tris-HCI, pH 8.0, 50 mM KCI, 50h glycerol. 0.1 mM EDTA, 1.O mM DTT, 1.O mM phenylmethylsulphonyI fluoride [PMSF], 10 mM sodium butyrate) at 3 0 ' ~for 60 minutes with frequent mixing. One-half of the reaction (15 pl) was separated by electrophoresis on a 18% SDSpolyacrylamide gel. The gels were stained to ensure loading of equivalent amounts of histone in each lane, destained and fluorographed with Enhance (Dupont NEN).
4.2.7
lmmunoprecipitation of NGGlp and TOM1p lmrnunoprecipitation with anti-HA antibody: For immunoprecipitation of
HA-NGGl p, whole cell extracts were prepared from yeast strain SY12, SY13
and CY922 in a buffer containing 50 mM HEPES (pH 7.4), 100 mM NaCI, 16 rnM magnesium acetate, 1 mM EGTA, 0.1% Nonidet P-40, 0.5 rnM dithiothrelol, 10% glycerol, 2.0 rnM PMSF, 0.1 mM benzamadine hydrochloride, 2 pg/ml
pepstatin A, 2 yg/ml leupeptin, 3 pg/ml aprotinin, and 0.1 mg/ml trypsin inhibitor (IP buffer). 75 mg of extract was rotated for 1 hour at 4 ' with ~ 0.5 ml of
Sepharose CL-46 (Pharmacia Biotech Inc.). Unbound protein was incubated with 10 pg of monoclonal antibody 12CA5 (Wilson et al., 1984) directed against the hemagglutinin (HA) epitope and rotated at 4 ' ~ for 1 hour. The mixtures were added to 75 pl of Protein A Sepharose beads (Pharmacia Biotech Inc.) equilibrated in IP buffer, and rotated for 3 hours at 4 ' ~ . The beads were washed five tirnes with 1.5 ml of IP buffer and bound protein was eluted by incubation at 6 5 ' ~for five minutes in SDS gel loading buffer. For irnrnunoprecipitates that were to be probed with anti-TBP or anti-SPMp antibodies, the HA-antibody was covalently coupled to cyanogen bromide activated Sepharose 4 6 (Parmacia Biotech Inc.) at a concentration of 8.0 rngfml, with a volume of 40 pl used per immunoprecipitation. lrnmunoprecipitation with anti-TOM1p antibody: A similar procedure using Protein A Sepharose was used for the immunoprecipitation of TOM1p frorn SY12, SY13, SY14 and GY922 extracts, substituting an equivalent of 30 pg of affinity purified polyclonal antibody raised against the peptide sequence
CKFSIHRDFGSSERL, located from amino acids 3215 to 3229 at the carboxylterminus of TOM1p. This antibody was purchased from the Western ImmunoTechnology Setvice (WITS). The extracts were incubated with antibody for 2 hours at 4 ' ~followed by rotating the mixture with Protein A Sepharose at the same temperature for 3 hr.
4.2.8 Immunoprecipitation of S P n p under denaturing conditions
The denaturing immunoprecipitation for SPT7p was performed by a procedure adapted from Ausubel et al. (1990). Whole cell extracts were prepared from yeast strains CY922, SY 11 and FY1093 (kindly provided by Fred Winston) in a buffer containing 50 mM Tris-CI (pH 7.5),50 mM NaCl and protease inhibitors (2.0 mM PMSF, 2 pg/ml pepstatin, 2 pg leupeptin and 0.1 mM benzamadine hydrochloride). Sodium dodecyl sulfate (final concentration 1.5%) was added to an aliquot from each extract that contained 10 mg of protein
and the samples placed in a boiling water bath for 10 minutes. The extracts were transferred to ice for 20 minutes then debns pelleted by centifugation at 20,000 g for 15 minutes. The supematant was diluted 1bfold in RIPA buffer (50
mM Tris-CI, pH 7.5, 140 mM NaCI, 1% Na-deoxycholate, 1mM dithiothreitol, 1% Nonidet P-40, 1% bovine serum alburnin [BSA] and protease inhibitors) and centrifuged at 12,000 g for 20 minutes. 50 pg of polyclonal antibody to SPT7p was added to the supematant, which was then rotated for 3 hours at 4OC. This
was followed by the addition of 75 pl of Protein A Sepharose and rotation for 2 hours at 4 ' ~ . The beads were washed five times with 2 ml of RIPA buffer. then twice with RlPA buffer without dithiothreitol, Na-deoxycholate and bovine serurn albumin. Bound protein was eluted by incubation at 6 5 ' ~for five minutes in
SDS gel loading buffer and Westem blotted as descdbed below using antiubiquitin antibody (Research Diagnostics, Inc.)
4.2.9
Western blot analysis of proteins
Westem blotting with a prirnary antibody from Ascites fluid derived frorn the 12CA5 cell line using PVDF membrane and the Supersignal cherniluminescence kit (Pierce) has been described (Brandl et al.. 1996).
lmrnunoprecipitations processed for Westem blots with anti-TOM1p antibody were separated on 5% SDS gels and transferred to PVDF membrane by semi-
dry blotting at 2.0 mA/cm2 for 2.5 hr. After blotting, membranes were incubated in Tris-buffered saline (TBS: 20 mM Tris, pH 7.5; 0.3 M NaCI) ovemight at OC, and then blocked in a TBS containing 5% casein for 2 hr at room temperature. Membranes were incubated with anti-TOM1p polyclonal antibody diluted 1:200 in 0.5 X TBS containing 0.5% Tween 20 for 2 hr at room temperature followed by six 20 min. washes in 20 mM TrisHCl (pH 7.5), 75 mM NaCI, 0.5% Tween 20 and 0.04% sodium dodecyl sulphate).
The Westem blot with the anti-ubiquitin antibody was carried out as described by Avantaggiati et al. (1996) with minor modifications. After electroblotting, membranes were incubated in 6 M guanidine-HCI; 20 mM TrisHCI, pH 7.5; 5 mM J3-mercaptoethanol; 1 mM PMSF for 30 min at 4 ' ~ .then washed extensively in phosphate buffered saline (PBS). Anti-ubiquitin antibody
(Sigma) was diluted to a concentration of 5 rng/ml in PBS and further diluted 1:100 in blocking solution.
Probing with rabbit polyclonal antibodies to the GAL4p DNA binding dornain (kindly provided by Ivan Sadowski), the amino-terminal 198 amino acids of SPT3p, SPl7p (kindly provided by Lisa Pacella and Fred Winston) and to TBP (Upstate Biotechnology, Inc.) were perfotmed at dilutions of the primary antibody of 1 :1000, 1:3000, 1: 500. and 1: 2000, respectively. With the exception of the SPT3p blots the secondary antibody was goat anti-rabbit IgG (Promega) conjugated to home radish peroxidase and used at a dilution of 1:4000. For the SPT3p blots, detection was through the use of secondary goat anti-rabbit antibodies conjugated to alkaline phoshatase (BioRad) used at a dilution of 1:3000. When the same membrane was probed sequentially with different antibodies, the membrane was stripped according to the
manufacturer's instructions and reprobed with secondary antibody to venfy that the first antibody was removed.
4.3
4.3.1
Results
TOMlp has a role in transcriptional activation The interaction of UBP3p with NGGl p in a two-hybrid analysis (Martens
et ab, 1996) suggested that
NGGl p itself, or a protein directly or indirectly
associated with NGGl pl as part of an ADA complex, may be ubiquitinated. In tum, this predicted that an E3 ubiquitin Iigase may be involved in regulation of the ADA complexes. Wlh the availability of the yeast genome sequence, we could analyze identified yeast E3 ubiquitin ligases for a possible role in regulation of the ADA proteins. TOMl p (SachDB ORF: YDR45NV) was a strong candidate since it was suggested to be involved in nuclear processes, contains a potential nuclear localization signal from amino acids 199-210 (Dingwell and
Laskey, 1991), and like the ADA proteins, is not essential for viability (Utsugi et al., 1995). TOMl encodes a 3268 amino acid protein with an estimated molecular mass of 374 kDa and has a high degree of sequence sirnilarity to the hect-domain E3 ubiquitin ligases (Huibregtse et al., 1995).
To determine if TOMlp has a role in transcriptional activation related to the ADA proteins, we compared transcription from promoters activated by
ADRl p (ADHZ), GCN4p (HIS3) and GAL4p (GAL 10) in the wild-type strain CY922, an isogenic tom1 disruption strain, S Y l 1, and in an isogenic strain
JY335 which carries a nonfunctional alleie of nggl (see Table 4.1 for yeast strains). Plasmids containing IacZfusion genes were introduced into these strains. The strains were also transformed with plasmids expressing the appropriate activator protein or grown under relevant inducing conditions to achieve expression of the activator (see materials and methods). Disruption of nggl resulted in a decrease in induction of the ADH2 promoter (Fig. 4.1 A) and
HIS3 promoter (Fig. 4.1 6) of approximately 12-fold and 2-fold, respectively.
Fig. 4.1 Transcription from ADH2, HIS3, and GAL IO promoters in wild type (CY922), tom1 (SYI 1) and nggl (JY335) yeast strains. A. Transcription from the ADH2 prornoter. The indicated strains containing the ADH2 promoter as a lacZfusion were grown in 3% ethanol 0.05% glucose. P-
galactosidase activity was assayed according to Ausubel et al. (1990) and standardized to cell density.
B. Transcription from the HIS3 promoter. B-galactosidase activity for strains containing a HIS3-lacZfusion and GCN4p expressed from the DEDl promoter were detemined after disniption of cells with glass beads and standardized to total protein. C. Transcription from the GALlO promoter. Strains were transfomed with a GAL 10-lacZ reporter plasmid and a plasmid expressing GAL4p. Cells were grown in 2% glucose and p-galactosidase activity detemined as for the ADH2 promoter. Measurements are the averages of eight trials for the above three promoters.
St rain TOM1 NGGI
St rain TOM1
NGGl
Strain TOM1 NGGt
CY922 JY335
+ +
+
O
SV11
.. +
Similarly, disruption of tom l in SY 1 1 (NGG7 tom 1) resulted in a decrease in transcription of ADH2 and HlS3 of 13-fold and 2-fold. As shown previously transcription from the GALIO promoter in glucose media is enhanced 3-fold in a
nggl background (Brandl et al., 1993; Fig. 4.1 C). Again, disruption of tom 1 resulted in a comparable induction of the GALIO promoter. As shown in figure 4.2, increased expression from the GALlO promoter was not due to an increase
of GAL4p in SY 11 as compared to the wild-type strain CY922. These experiments show that disruptions of tom7 and nggl have similar effects on transcriptional activation. This suggests that TOMlp plays a role in transcriptional activation and that there is a link between TOMlp and the ADA proteins. Further parallels between TOM1 and NGG7 were suggested by the similar slow growth phenotype and temperature sensitivity of strains with disruptions of tom 1and nggl (not shown). To analyze whether NGGl p and TOMl p are acting through the same pathway, transcription from the ADH2 and GAL 10 promoters was determined in CY 1004, a strain carrying a double disruption of tom 1 and ngg 1 (Table 4.2). Transcription from the ADH2 and GAL I O promoters in the double disruption background was virtually identical to that in each single disruption background. This result suggests that TOMlp is regulating transcriptional activation through the same pathway as the ADA proteins.
4.3.2
An active site cysteine is required for TOMlp's function as a
transcriptional regulator
TOMl p contains a hect-domain at its carboxyl-terminus related in sequence to those found in E6-AP and RSP5p. The hect-domain is characterized by having a conserved Cys residue that is essential for the formation of a thioester linkage with ubiquitin (Scheffner et ai., 1995;Huibregtse
FIG. 4.2 The expression GAL4p is not affected by disruption of TOM1p. 100 or 200 pg (as indicated) of whole cell extract from yeast strains CY922 (lanes 2 and 4) and SY11 (lanes 3 and 5) containing GAL4 on a centromeric plasmid were separated by SDS-PAGE and analyzed by Western blotting with antibody to the DNA binding domain of GAL4p. Lane 1 is an extract prepared frorn CY922 without the GAL4 expression plasmid. The migration of molecular mass protein standards (kDa) is shown on right.
Table 4.2 Transcription from ADH2 and GALlO promoters in Wild type,
toml, nggl, and toml nggl yeast strains
B-Gatactosidase Units
PROMOTI STRAIN: TOM1 Na37
CY922
JY335
SYI I
+ +
+
-
-
+
CY1004
-
The indicated strains containing the ADH2 or GAL 10 promoters as lac-Z fusions were assayed for activation by their appropriate transactivator protein (ADRl p and GAL4p, respectively). &galactosidase activity was measured according to Ausubel et al. (1990) and standardized to cell density. Measurernents are the average of eight trials for each promoter.
et al., 1995). To determine if the role of TOMl p in transcription is dependent on the consenred active site cysteine residue, we constructed yeast strain CY 1005, carrying a gene encoding a derivative of TOMl p with a Cys to Ala substitution at was present at a level equivalent to that of amino acid 3235. TOM1Pcys3235~fa the wild-type protein as determined by Western blotting (not shown). Transcription from the ADH2 prornoter was detemined in CY1005 and similar to that in the tom1 deletion strain SY11, was 9-fold less than in the wild-type strain CY922 (Fig. 4.3 A). Transcription from the GAL10 promoter was also compared in CY1005, SY11 and CY922 (Fig. 4.3 8). In this trial, deletion of
tom 7 resulted in a 5-fold increase in expression of GAL IO as compared to the wild-type. The presence of TOM1pcys3235-~la resulted in slightly less of an increase, approximately 3.5-fold. These results suggest that the effect of TOMl p on transcription is principally mediated through its ubiquitination function.
The two-hybrid interaction of UBP3p and NGGlp requires
4.3.3
TOM1
In a two-hybrid analysis, the carboxyl-terminal 180 amino acids of the yeast ubiquitinspecific protease, UBP3p, associates with amino acids 1-373 of NGGl p (Martens et al., 1996). We believe that this interaction is not directly 1pl-373 and UBPJp, but rather mediated by additional between GAL4DBD-NGG
factors that assemble with GAL4DBD-NGG1 pl-373 because the endogenous copy of ADA2 is required to observe the interaction (not shown). One model would then suggest that UBP3p may recognize a ubiquitinated protein in an ADA complex as a substrate. If this component is ubiquitinated in a TOMl pdependent manner, then the UBP3p-NGGlp two-hybrid interaction may be absent in a tom1 deletion strain.
Fig. 4.3 Transcription from the ADH2 and GALlO promoters in the presence of TOM1P ~ ~ ~ 3 2 3 5 - ~ 1 ~ .
A. Transcriptional activation by ADRl p of a ADH2-lac2 fusion promoter in wild type (CY922), tom 1 (SY11) and tom IQsCysAIa (CY1005) strains. f3-galactosidase activity was measured as in Figure 4.1. Measurements are the averages of assays perfomied on five individual transfomants. Standard errors were less than 20% of the means. B. Transcriptional activation by GAL4p of a GAL 10-lac.fusion in CY922, SY11, and CY1005. Measurements are the averages of assays perfomed on five individual transformants. Standard enors were less than 20% of the means.
Activation by ADRlp
Activation by GAL4p
In the wild-type background (CY922) the two-hybrid interaction between is observed as a 7-fold increase in expression of NGG1p l-373 and UBP3p733-912 the GAL10-lacZ reporter when GAL4Deo-NGG1pl-373 is CO-expressedwith GAL4A~-UBP3p733.912 as compared to coexpression with GAL4nD-SNF4p (Fig. 4.4). This two-hybnd interaction was absent in the tom1 disruption strain (SY11) and in the strain containing TOM1Pcy&235-Ala(SY1005). Absence of the interaction did not result from reduced expression of GAL4DeD-NGG1pl-373 in the toml strains, as demonstrated by Westem blotting (not shown). As a control for potential nonspecific effects of the tom1 disniption, increased expression of GAL 10-lac2 resulting from the interaction between G A L ~ D ~ ~ - p S and NF~ GAL~AD-SNF was ~ ~found , to be similar in wild-type and toml strains. These results demonstrate that the two-hybrid association of UBP3p with NGGl p requires TOMl p and in tum suggests that there may be a substrate for TOMl p in one or more of the ADA complexes.
4.3.4
TOMlp is requiied for ubiquitination of a protein that
immunoprecipitates with HA-NGGl p To determine if a component of an ADA complex is ubiquitinated in a
TOM1p-dependent manner, ubiquitination of ADA components was examined in wild type and toml backgrounds by Westem blotting with anti-ubiquitin antibody. Whole cell extracts were prepared from yeast strains CY922(NGG1
TOMI), SY12 (HA-NGG1 toml), and SY13 (HA-NGGI TOMI). HA-NGGlp and the associated ADA components were immunoprecipitated with anti-HA antibody, separated by SDS-PAGE, and Western-blotted with anti-ubiquitin antibody (Fig. 4.5 A). A major band at approximately 210 kDa was detected by the anti-ubiquitin antibody in the SY13 immunoprecipitate (lane 3). This band
Fig. 4.4 Loss of interaction between NGGlp and UBP3p in the absence of TOMlp. Plasmids encoding the indicated pairs of GAL4p DNA binding domain (GAL4,,,; black box) and GAL4p activation domain (GAL4AD;striped box)
fusion proteins were introduced into CY922 (wild type), SY11 (toml) and CY1005 ( t 0 m l c ~ 3 Cells ~ ~ ~were ~ ~grown ~ ) .in minimal media containing 2% glucose and assayed for expression of a GALIO-lac2 reporter introduced on a ADE2 centromeric plasmid. &galactosidase assays were perfomed after glass bead disruption of cells and standardized to protein concentration. Each value represents the average from five independent transfomants.
-
G A L ~ A DFusion Prot eins
GAL4 Fusion Prot eins
NCGl
SNFl SNFl
1 1 1
SNF4
1
P-galact osidase Act ivit y (Ulmg total protein)
0.7 70.2 0.4 f0.1 14 I2.5
0.4 k0.1 0.3 10.1 16 f2.2
0.5 10.2 0.2 IO.05 2 2 k4.5
was specifically immunoprecipitated with the ADA proteins as it was absent from CY922 which lacks HA-NGGlp (lane 1). Furthemore the band was absent from the tom1 deletion strain SY12 (lane 2), thus indicating that its ubiquitination is dependent on TOMl p. To establish that the absence of the ubiquitinated proteins from SY12 was not due to variations in the amount of immunoprecipitated HA-NGGlp. the same filter was reprobed with anti-HA antibody (Fig. 4.5 B). The intensity of the 116 kDa band, corresponding to HANGGl p, in immunoprecipitates from SY 13 and SY12 was almost identical. Thus an ADA-associated protein is ubiquitinated in a TOMl p dependent fashion. This agrees with the prediction based on sequence cornparisons that TOMlp is an E3 ubiquitin ligase. In addition, this result suggests that the transcriptional
effects of TOMlp, which closely parallel those of the ADA proteins, could be mediated through its ubiquitination of a component(s) of the ADA complexes.
4.3.5
SPT7p is ubiquitinated in a TOMl p-dependent fashion
SPT7p which migrates with an estimated mass of approximately 205 kDa on SDS-PAGE (Gansheroff et a/., 1995) and associates with at least one form of ADA complex (SAGAtomplex; Grant et al., 1997; see Chapter 3), was an obvious candidate for the ubiquitinated protein. To determine if SPT7p is ubiquitinated in a TOMl p dependent fashion, SPTip was isolated by immunoprecipitation after whole ceil extracts from wild-type and tom 1 deletion strains were boiled in 1.5% SDS to dissociate protein complexes. The immunoprecipitates were fractionated by SDS-PAGE and Western blotted with anti-ubiquitin and anti-SPT7p antibody. As shown in figure 4.6, SPl7p was immunoprecipitated frorn both TOMl (CY922) and tom 1 (SY 11) strains. The identity of this band as SPT7p was verified by its absence in the immunoprecipitate from the spt7A402::LEUZ strain, FY1093 (generously
Fig. 4.5 Ubiquitination of an ADA-associated protein requires TOM1 p. A. 70 mg of whole cell extract prepared from yeast strains CY922 (TOM1 NGGI; Iane l ) , SY12 (tom1 HA-NGGI; lane 2),and SY13 (TOM1 HA-NGG 1; lane 3 were incubated with anti-HA antibody and Protein A Sepharose. After washing, the irnmunoprecipitatedADA cornplex was eluted at 6 5 ' ~in SDS loading buffer, separated on 5.5% SDS-polyacrylamide gel, and analyzed by Western blotting with anti-ubiquitin antibody. The principal ubiquitinated band within the ADA complex migrates with an approximate mass of 210 kDa (lane 3) in cornparison to protein standards shown on the right. B. The same membrane was reprobed with anti-HA antibody to ensure that equivalent amounts of HA-NGGI p were immunoprecipitated from SY 12 and SY 13 extracts (lanes;2 and 3, respectively).
UBtQUITIN ANTIBODY
HA-NGGI p 1
2
3
HA ANTIBODY
Fig. 4.6 S P f i p is ubiquitinated in a TOMlp-dependent fashion. 10 mg of whole cell extract from yeast strains GY922 (wild-type; Iane l ) , SY11 (toml;Iane 2) and ml093 (spiz lane 3) was boiied in extraction buffer containing 1.5% SDS and diluted 15-fold in RIPA buffer minus SDS. The samples were irnmunoprecipitated with anti-SPT7p antibody, separated by SDS-PAGE in a 5.5% gel and Western blotted wiai anti-SPT7p and antiubiquitin antibody.
1
2
3
SPT7p ANTIBODY
1
2
3
UBIQUITIN ANTIBODY
provided by Fred Winston; lane 3). SPT7p was ubiquitinated in the wild-type
TOMl background but not in the tom1 deletion background. Reprobing of the blot with a polyclonal antibody to NGGl p revealed that no NGGl p was present in the immunoprecipitates, thus helping exclude the possibility that other proteins may associate with SPT7p even under the denaturing conditions (not shown).
4.3.6
Coimmunoprecipitation of TOMl p with NGGl p. If the action of TOMl p is direct, that is an ADA-associated protein is a
substrate for TOMl p, it might be possible to detect TOM1p in irnmunoprecipitates of the ADA complexes. ADA associated proteins were immunoprecititated with anti-HA antibody from 75 mg of whole cell extracts prepared from SY 13 (HA-NGG1 TOMI),CY922 (NGG1 TOMl),and SY 12 (HA-
NGG 1 tom 1). lmmunoprecipitates were probed by Western blotting with antibody directed against a 15 amino acid peptide found within TOM 1p. As shown in figure 4.7 A, TOMl p specifically immunoprecipitated with HA-NGG1p (compare lanes 1 and 2). The identity of the high molecular weight protein as
TOMl p was confirmed by the absence of this band in the immunoprecipitate from SY12 (HA-NGG1 toml; lane 3). To ensure that equal amounts of HA-
NGGl p were immunoprecipitated from extracts of the different strains, the filter was reprobed with anti-HA antibody to detect HA-NGGl p (Fig. 4.7 6).There was very little variation in the amount of HA-NGGl p in these extracts (compare lanes 1 and 3). These results support the idea that TOMl p acts directly to
ubiquitinate an ADA-associated protein. The amount of extract required to visualize the interaction between TOMl p and HA-NGG1p was substantially more than that required to see a core ADA component such as ADA2p. This suggests that TOMl p is not likely an integral component of the ADA complexes
Fig. 4.7 Coimmunoprecipitation of TOMl p with NGGl p from yeast whole cell extracts. A. 75 mg of whole cell extract prepared from yeast strain SY13 (TOM1 HANGG 1; lane l ) , CY922 (TOM1 NGG1; lane 2)and SY 12 (tom1 HA-NGGI; lane 3) were incubated with anti-HA antibody and Protein A Sepharose. Protein was eluted ai 6 5 ' ~ in SDS loading buffer, separated by SDS-PAGE, and analyzed by Western blotting with affinity purified polyclonal antibody specific for a 15 arnino acids peptide within the carboxyl-terminus of TOMl p. The position of TOM1p (lane 1) is labeled. Relevant molecular mass protein standards (kDa) are indicated on the right.
B. The same membrane was reprobed with anti-HA antibody to detect irnmunoprecipitated HA-NGG1p.
TOM1 -1 TOMlp
-
but rather is involved in transient associations with the ADA proteins or is restricted to a subset of the complexes. To confirm the interaction between TOMlp and NGGl p, the reciprocal experiment was perfonned in which TOMl p was immunoprecipitated from whole cell extracts and the presence of HA-NGGl p assayed by Western blotting with anti-HA antibody (Fig. 4.8 A). A deletion derivative of NGGl p lacking amino acids 274-308 (HA-NGGIP
~
~ was ~also-examined ~ ~ for~
)
interaction with TOM1p to determine if this essential region of NGG1p (Brandl et al., 1996; Saleh et al., 1997) was required for interaction with TOMl p. HAreactive bands of 116 kDa and 110 kDa, corresponding to HA-NGGl p and HANGGlpa74-308, were found in immunoprecipitates from the wild type TOM7 strains SY 13 and SY 14, respectively, but not in immunoprecipitates from the
tom 1 deletion strain, SY 12 (compare lanes 1 and 4 with lane 2). The identity of HA-NGGl p and HA-NGGlp~74-308,were confirmed by their absence in the immunoprecipitate from CY922 (NGGI TOMI; lane 3). As shown in figure 4.8 B, approximately equal amounts of TOMl p were immunoprecipitated from the TOM1 containing strains. As well as confining the interaction of TOMl p and
NGGl p, this experiment indicates that the central region of NGGl p from amino acids 274-308 is not required for interaction with TOMl p.
4.3.7
TOMlp is required for the association of the ADA
components with SPT3p and TBP but not with SPT7p. One mechanism by which TOMlp could influence the activity of the ADA proteins would be to alter the protein-protein associations within the complexes. This could occur by targeting components of the complex for degradation or by signaling conformational changes. As the ADA proteins associate with the TBP
class of SPT proteins and SPWp is ubiquitinated in a TOMl p-dependent
Fig. 4.8 Coimmunoprecipitation of NGGlp with TOMlp from yeast whole cell extracts. A. 75 mg of whole cell extract prepared from yeast strains SY13 (TOM1 HANGGI;lane l ) , SY 12 (tom1 HA-NGG1; lane 2),GY922 (TOM1 NGG 7; lane 3) and SY 14 (TOM1 HA-NGGlGT4-rn7; lane 4) were incubated with polyclonal antibody directed to aie carboxyl-terminus of TOM1p followed by incubation with Protein A Sepharose. Bound protein was eluted at 6 5 ' ~in SDS loading buffer, separated on a 5.5% SDS-polyacrylamide gel, and analyzed by Western blotting with anti-HA antibody as a probe for HA-tagged derivatives of NGGl p. The indicated bands at approximately 116 and 110 kDa represent HA-NGGl p (lane 1) and HA-NGG 1pu74307 (lane 4), respectively. The migration of molecular mass protein standards are indicated on the nght. B. The same membrane was reprobed with the anti-TOMlp antibody.
fashion, we chose to analyze if TOMl p alters the protein-protein interactions between the ADA proteins and SPT7p, SPT3p or SPTI 5p (TBP). We immunoprecipitated HA-NGG1p from cell extracts of SY13 (HA-NGG1 TOM),
SY12 (HA-NGG1 toml) and as a negative control GY922 (NGGI TOMI). As shown in figure 4.9, the immunoprecipitation of HA-NGGlp was equally efficient from SY12 and SY13. Likewise, the amount of SPT7p associated with HA-
NGGI p was virtually identical in both strains. This suggests that ubiquitination of SPT7p is not required for its association with the ADA components. In contrast, the amount of both SPT3p and TBP associated with HA-NGGlp was reduced to undetectable levels in the toml deletion background. This was not due to a general reduction of SPT3p and TBP in SY12 as determined by Western blotting of the crude extracts (not shown). These results suggest that
TOMl p may exert its effects on transcription by altering the associations of the ADNSAGA complexes with TBP.
4.3.8
Histone acetyltransferase activity of the ADA complexes is
unchanged in the absence of TOMlp. The ADA complexes have histone acetyltransferase activity mediated through ADA4p/GCNSp. To detemine whether TOMl p may additionally affect transcription by altering the histone acetyltransferase activity of the ADA complexes, we immunoprecipitated HA-NGGlp and associated proteins from the yeast strains CY922 ( M G 1 TOMl), SY12 (HA-NGG1 tom 1), SY 13 (HA-
NGG1 TOM1) and SY14 (HA-ngglA274-307 TOM) with anti-HA antibody. These complexes were assayed for histone acetyltransferase activity using oligonucleosomes as substrates (Fig. 4.10). ADA-associated histone acetyltransferase activity can be identified by its preference for histone H3 and to a lesser extent histone H2B (Wang et al., 1997; Grant et ai., 1997;
Fig. 4.9 Association of the TBP class of SPT gene products in the presence and absence of TOMlp. 15 mg of whole cell extract prepared from yeast strains CY922 (TOM1 NGG1; lane l), SY13 (TOM1 HA-NGG1; lane 2) and SY12 (tom1 HA-NGGI; lane 3 were incubated with anti-HA antibody and Protein A Sepharose. After washing, protein was eluted by heating at 6 5 ' ~in SDS loading buffer, separated in a 6% gel and analyzed by Western blotting with anti-HA antibody to detect for HANGGl p. The same filter was reprobed with anti-SPnp antibody. The indicated bands at approximately 116 and 210 kDa represent HA-NGGl p and SPT7p, respectively. Similady, 30 mg of whole cell extract was incubated w l h anti-HA antibody covalently coupled to Sepharose 48. Bound protein was separated in a 12.5% gel and analyzed by Westem blotting with a polyclonal antibody to TBP. The same filter was probed with a polyclonal antibody to SPT3p. The immunoreactive bands at approximately 35 and 27 kDa represent SPT3p and TBP, respectively.
Ruiz-Garcia et ai., 1997). No ADA-associated histone acetyltransferase activity was apparent in the control immunoprecipitates from CY922 extracts (lanes 1 and 2). The amount of ADA-associated histone H3 acetyltransferase activity was similar in irnmunoprecipitates from the tom 1 deletion strain SY 12 and the wild-type TOMl strain SY1 3 (compare lane 3 with lane 5 and lane 4 with lane 6). Histone H3 acetylation was calculated for tom1 and TOM1
immunoprecipitates by densitometric scanning of five independent experiments performed with 2 and 5 mg of protein. When standardized to the concentration of HA-NGGl p in the extracts, the H3 acetyltransferase activity in immunoprecipitates from the tom1 strain was found to be 90% of that in the wildtype TOMl strain. By cornparison, the histone acetyltransferase activity in
immunoprecipitates from SY14 (HA-nggI* 274-307) was 10% that found in the wild-type (compare lanes 7 and 8 with lanes 5 and 6 , respectively). This experiment indicates that disruption of tom 7 has little influence on the histone acetyltransferase activity of the ADA complexes, and contrasts with a dramatic loss in acetyltransferase activity seen in strains containing the nggld274-307
allele.
Fig. 4.10 Histone acetyltransferase activity of ADA complexes in the presence and absence of TOM1p. HA-NGG1p-containing complexes were immunoprecipitated f rom 2 mg (lanes 1, 3. 5 and 7) or 5 mg (lanes 2,4, 6, 8) of whole cell extract prepared from yeast strains CY922 (TOM1 NGG 1; lanes 1 and 2), SY 12 (tom 7 HA-NGG 1; lanes 3 and 4), SY13 (TOM1 HA-NGGI; lanes 5 and 6) and SY14 (TOM1 HANGGlM7e30e; lanes 7 and 8). Liquid histone acetyltransferase assays were performed using HeLa Hl -depleted oligonucleosomes (Cote et al., 1995) and P~lacetyl-COA. Products of the reaction were separated by electrophoresis on a 18% SDS-polyacrylamide gel. The gels were stained with Coomassie brilliant blue to ensure loading of equivalent amounts of histone in each lane, destained and subjected to fluorography. The positions to which histones migrate are indicated.
4.4
4.4.1
Discussion
TOMlp regulates the function of the ADA complexes through
ubiquitination Post-translational modification of proteins by ubiquitin has been
implicated in many cellular processes among which are signal transduction, cell cycle progression, DNA repair, receptor mediated endocytosis, cell
differentiation and regulation of transcription (Hochstrasser, 1995; Wilkinson, 1995). This document shows that the hect-domain containing protein TOMlp is
required for the function of the ADA regulatory proteins. A tom1 deletion strain has the same slow-growth phenotype, temperature sensitivity, and defects in transcription as a nggl deletion strain. Enhanced transcription of promoters activated by ADRl p (ADHZ) and GCN4p (HlS3). both of which require ADA proteins for full activity (Georgakopoulos and Thireos, 1992; Pina et al., 1993; Marcus et ab, 1994; Chiang et al., 1996), also require TOMl p. A tom 1 deletion displays a similar relief of transcriptional repression on the GAL 10 promoter as
a nggl deletion. In addition, the slow growth and altered transcriptional phenotypes are no more severe in nggl tom1 double mutants than in either single mutant. Regulation of the ADA complexes by TOMlp is likely direct and dependent upon the predicted E3 ubiquitin ligase function of this protein. First. TOM1p can be irnmunoprecipitated with HA-NGGl p. Second, TOMl p is
required for the ubiquitination of a protein that associates with the ADA proteins, SPT7p. Third, mutation of a cysteine residue within the hect-domain of TOMl p, corresponding to a residue essential for ubiquitination by E6-AP and RSPSp (Scheffner et al., 1995; Huibregtse et al., 1995), results in a strain with changes in transcription from ADH2 and GALIO promoters which are qualitatively similar,
although slightly less in magnitude, as strains with the toml nuIl mutation. This suggests that the role of TOMl p in transcription is principally mediated through its ubiquitination function and contrasts with the observation from RSP5p in which the active site cysteine was not required to potentiate transcriptional activation by human steroid receptors in yeast (Imhof and McDonell, 1996). tom 1was previously identified as a gene containing temperature sensitive mutation that arrested the cell cycle at the G2/M transition (Utsugi et al., 1995). The mechanisrn by which TOMl p functions in cell cycle progression
has not been reported. A multicopy suppressor of the temperature sensitive phenotype of toml, STMl (MPT4/G4p2)has been isolated (cited in Frantz and Gilbert, 1995; Utsugi et al., 1995). Interestingly, STMl p binds to nucleic acids in quadruplex form (Frantz and Gilbert, 1995). STMl is also a multicopy suppressor of htrVmpt5 (Utsugi et al., 1995) and pop2 (cited in Frantz and Gibert, 1995). POP2p regulates expression from the PGK and ADH2 promoters as part of a cornplex containing CCR4p (Sakai et al., 1992; Denis et al., 1994; Draper et al., 1995). This suggests additional ties between transcriptional regulation and TOM1p.
4.4.2
Ubiquitinated component(s) of the ADA complexes The finding that the two-hybrid interaction between the carboxyl-terminus
of the ubiquitin protease UBP3p and NGGl p requires TOMl p suggested that TOMl p is required to ubiquitinate a component(s) of the ADA complexes. We found a protein of 210 kDa that immunoprecipitates with HA-NGGl p, to be ubiquitinated specifically in the presence of TOMl p. Proteins of the TBP-class of SPTs, including SPT7p, SPT3p and ADASpEPT20 have been found to associate with the ADA proteins in at least one high molecular mass complex called the SAGAcomplex (Grant et al., 1997; Roberts and Winston, 1997).
Based on its estimated size on SDS-PAGE of 205 kDa (Gansheroff et a/., 1995; Roberts and Winston, 1997), SPl7p was an obvious candidate to be the protein ubiquitinated in the TOMlp-dependent process. As an initial step to determine if SPT7p is ubiquitinated, we have found that SPT7p immunoprecipitates with HA-NGGlp and that S P n p migrates at a position identical to the ubiquitinated protein (not shown). Furthemiore, we found that SPT7p immunoprecipitated from a denatured yeast extract is ubiquitinated in a TOMl p-dependent fashion. At this time our data strongly support the view that S P l 7 p is the 210 kDa ubiquitinated protein that immunoprecipitates with the ADA proteins. We do note the possibility, that the ubiquitinated SPT7p is not associated with the complex but rather a second ADA-associated protein of 210 kDa is ubiquitinated in a TOM 1p-dependent fashion. The possibility of additional ubiqutinated proteins within the ADA complexes exists. We analyzed immunoprecipitated complex on high percentage polyacrylamide gels to visualize lower molecular mass proteins. Unfortunately, numerous background bands that reacted with anti-ubiquitin antibody were found for proteins below approxirnately 80 kDa, complicating the interpretation of these gels. It is also possible that TOMl p has substrates relative to its role in transcriptional regulation that are not directly part of the ADA complexes. The coimmunoprecipitation of TOM 1p and NGGl p suggests that TOMl p is most likely involved directly in ubiquitination of the 210 kDa protein that associates with the ADA proteins. The amount of input protein required to visualize the association of HA-NGGlp and TOMl p was more consistent with a transient association of the proteins or association in a small subset of complexes rather than with TOMlp being an integral member of one or more of the principal ADA complexes. This agrees with our finding that the fractionation
profile on both Mono Q and Superose 6, for ADA complexes prepared from
toml deletion and wild-type strains are virtually identical (not shown). 4.4.3
What is the role of ubiquitination of ADA component(s)? Many examples for the regulatory role of protein ubiquitination involve
targetting for degradation by the 26s proteasorne (Ceicheanover, 1994). Several lines of evidence suggest that the regulation of the ADABAGA complexes by TOM1p may not involve protein degradation. The amount of SPT7p associated with the ADA proteins was not increased in the absence of TOM1p as would be expected if ubiquitination of SPT7p was a signal for its degradation. In addition, neither the levels of NGGI p, ADAZp, nor the overall levels of the ADA complexes as judged after their fractionation on gel-filtration and ion-exchange columns, were affected by the absence of TOM1p (not shown). To further support that TOM1p is not involved in general protein turnover, we find that the growth of toml strains is not affected by the presence of 1.5 pg/ml of the amino acid analog canavanine (Seufert and Jentsch, 1990). While these observations do not eliminate the possibility that a minor population or a component of one of the ADA complexes is degraded as the result of ubiquitination, they are consistent with the idea that ubiquitination of an ADA component may lead to alteration in function in a way similar to those often described for other protein modifications such as phosphorylation. Similar roles for ubiquitination not directly involving protein turnover have been proposed (Amason and Ellison, 1994; Spence et ai., 1995; Chen et ai., 1996; Hicke and Riezman, 1996). The nonlinear branching of ubiquitin chains as seen in the yeast stress response (Arnason and Ellison, 1994) and DNA repair (Spence et al., 1995), may signal this type of regulatory event.
The above model would suggest that ubiquitination of an ADA complex component alters one of the activities of the complex. The ADA proteins have been found to associate with both the basal transcription factor TBP and activator proteins (Silverman et al., 1994; Barlev et al., 1995; Martens et al., 1996; Melcher and Johnston, 1995; Saleh et al., 1997; Chiang et al., 1997). The
role of the ADA proteins in regulating transcription positively and negatively rnay, at least in part, anse through these protein-protein interactions. Our finding that TOM1p is required for the association of the ADA components with SPT3p and TBP suggests that ubiquitination of one of the ADNSAGA components may regulate the interaction with the basal transcriptional machinery. Indeed. the loss of the association of TBP with the complex in the absence of TOM1p is consistent with the TOM1p-dependent ubiquitination of one of the TBP-class of
SPT proteins. In light of the genetic evidence linking the TBP class of SPTs (Winston et al., 1987; Eisenmann et al., 1994; Gansheroff et al., 1995; Marcus et al., 1996; Roberts et al., 1996, 1997; Grant et al., 1997) and the previously described physical association of SPT3p and TBP (Eisenmann et al.. 1992),it is attractive to predict that ubiquitination of SPT7p may result in a loss of interaction with SPT3p. In tum this would result in the loss of interaction of the
ADA components with TBP. A second principal functioti ascribed to the ADA proteins is based on the identification of GCN5p/ADA4p as a histone acetyltransferase (Brownell et al., 1996). The ADAISAGA complexes have the capability of acetylating
nucleosomal histone H3 (Grant et al.. 1997; Ruiz-Garcia et ab, 1997) and recently, SIN 1p has also been shown to be a substrate for acetylation (Pollard and Peterson, 1997). Although we can not exclude that ubiquitination by TOM1p may alter targeting of the acetyltransferase or its substrate specificity,
the activity of the acetyltransferase as demonstrated by its acetylation of histone
H3 was virtually unchanged in a toml deletion background. This contrasted with the effects of NGGlp~743ogin which acetylation of histone H3 was reduced 10-fold. This result is particulariy noteworthy since the NGG lM74-308 allele results in similar transcriptional defects as disruptions of toml yet gives rise to an ADA complex which is defective in both histone acetylation and interaction with TBP (Brandl et al., 1996; Saleh et al., 1997). The finding that an E3 ubiquitin ligase regulates the activity of the complex probably through ubiquitination of a component protein supports a possible functional significance for our original observation that UBP3p associates with NGGl p (Martens et al., 1996). UBP3p could play an obvious role with TOMlp in cycling one or more of the ADA complexes from an active ubiquitinated f o m to an inactive deubiquinated forrn. Experiments are currently underway to test this model. It is interesting to note that a role for UBP3p in regulating silencing of the yeast mating type loci has recently been described (Moazed and Johnston, 1996).
4.5
References
Amason, T. and Ellison,M. J. (1994) Stress resistance inSaccharomyces cerevisiae is strongly correlated with assembly of a novel type of multiubiquitin chain. Moi. CeII. Biol. 14, 7876-7883. Ausubel,F.A., Brent,R., Kingston,R.E., Moore,D.D., Seidrnan,J.G., Smith,J.A. and Stnihl,K. (1990) Protocols in Molecular Biology, John Wiley & Sons, New York, pp. 13.62-13.64. Avantaggiati, ML., Carbone,M., Graessmann,A., Nakatani,Y. Howard,B. and Levine,A. (1996) The SV40 large T antigen and adinovirus E l a oncoproteins interact with distinct isofoms of the transcriptional CO-activator,p300. EMBO J. 15, 223602248. Baker,R.T., Tobias,J.W. and Varshavsky,A. (1992) Ubiquitin-specific proteases of Saccharomyces cerevisiae. J. Biol. Chem. 267, 23364-23375. Banejee,A., Gregori, L., Xu,Y. and Chau,V. (1993) The bacterially expressed yeast CDC34 gene product can undergo autoubiquitination to f o n a multiubiquitin chain-linked protein. J. Biol. Chem. 268, 5668-75. Bariev,N.A., Candau,R., Wang,L., Darpino,P.. Silverman,N. and Berger,S.L. (1995) Characterization of physical interactions of the putative transcriptional adaptor, ADA2, with acidic activation domains and TATA-binding protein. J.Biol. Chem. 270, 19337-19344. Berger,S.L., Pina,B., Silveman,N., Marcus,G.A., Agapite,J., Regier,J.L., Triezenberg,S.J. and Guarente,L. (1992) Genetic isolation of ADA2 - a potential transcriptional adaptor required for function of certain acidic activation domains Ce1170, 251-265. Boeke,J.D., LaCroute, F. and Fink, G.R. (1984) A positive selection for mutants lacking orotidine+phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol. Gen. Genet. 197: 345-346. Brandl,C.J., Furlanetto,A.M., MartensJA. and Hamilton,K.S. (1993) Characterization of NGG 1, a novel yeast gene required for glucose repression of GAL4p-regulated transcription EMBO J. 12, 5255-5265. Brandl,C.J., Martens,J.A., Margaliot,A., Stenning,D., Furlanetto A.M., Saleh,A., Hamilton,K.S. and Genereaux,J. (1996) Stnicture/functional properties of the yeast dual regulator protein NGGl that are required for glucose repression. J. Biol. Chem. 270, 9298-9306. Brownell,J.E., Zhou,J., Ranalli,T., Kobayashi,R., Edmondson,D.G., Roth,S.Y. and Allis, C.D. (1996) Tetrahymena histone acetyltransferase A: a homolog to yeast GcnSp linking histone acetylation to gene activation. Ce11 84, 843-851.
Candau,R. and Berger,S.L. (1996) Structural and functional analysis of yeast putative adaptors. Evidence for an adaptor complex in vivo. J.BioLChem. 271, 5237-5245. Candau,R., Zhou,J,-X., Allis,C.D. and Berger,S.L. (1997) Histone acetyltransferase activity and interaction with ADA2 are critical for GCN5 function in vivo. EMBO J. 16, 555-565. Chen,Z. and Pickart,C.M. (1990) A 25-kilodalton ubiquitin carrier protein (E2) catalyzes rnultiubiquitin chain synthesis via lysine-48 of ubiquitin. J. Biol. Chem. 265, 21835-21842. Chen,Z., Parent.1. and Maniatis T. (1996) Site-specific phosphorylation of lkBa by a novel ubiquitination-dependent protein kinase activity. Ce1184, 653-862. Chiang, Y.C., Komamitsky, P., Chase. D.and Denis, C.L. (1996) ADRl activation domains contact the histone acetyltransferase GCNS and the core transcriptional factor TFIIB. J. Biol. Chem.. 271, 32359032365. Ciechanover,A. (1994) The ubiquitin-proteasome proteolytic pathway. Ce1179, 13-21. Denis,C.L., Draper.M.P., Liu,H.Y., Malvar,T., ValIari,R.C. and Cook,W.J. (1994) The yeast CCR4 protein is neither regulated by nor associated with the SPT6 and SPT10 proteins and f o n s a functionally distinct complex from that of the SNFISWI transcription factors. Genetics 138, 1005-13. Dingwall, C. and Laskey, R A . (1991)Nuclear targeting sequences-a consensus? Trends Biochem. Sci. 16, 478-48 1
.
Draper,M.P., SaIvadore,C. and Denis,C.L. (1995) Identification of a mouse protein whose homolog in Saccharomyces cerevisiae is a component of the CCR4 transcriptional regulatory complex. Mol. Cell. Biol. 15, 3487-95. Durfee,T., Becherer,K., Chen,P.L., Yeh,S.H. Yang,Y., Kilbum,A.E. Lee,W.H. and Elledge,S.J. (1993) The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev. 7, 555-569. Eisenmann,D.M., Dollard,C. and Winst0n.F. (1989) SPTl5, the gene encoding the yeast TATA binding factor TFllD is required for normal transcription in vivo. CeIl 58, 1183-1191. Eisenmann,D.M., Amt,K.M., Ricupero,S.L., Rooney,J.W. and Winst0n.F. (1992) SPT3 interacts with TFllD to allow normal transcription in Saccharomyces cerevisiae. Genes Dev. 6,1319-1331.
Eisenrnann,D.M., Chapon,C., Roberts,S.M., Dollard,C. and Winston,F. (1994) The Saccharomyces cerevisiae SPTB gene encodes a veiy acidic protein that is functionally related to SPT3 and TATA-binding protein. Genetics 137, 647657. Frant2,J.D. and Gilbert,W. (1995) A yeast gene product, G4p2, with a specific affinity for quadruplex nucleic acids. J. Biol. Chem. 270, 9413-9419. Gansheroff,L. J., Dollard,C., Tan,P. and Winston,F. (1995) The Saccharomyces cerevisiae SP77 gene encodes a very acidic protein important for transcription in vivo. Genetics 139, 523-536. Georgakopoulos,T. and Thireos,G. (1992) Two distinct yeast transcriptional activators require the function of the GCN5 protein to promote normal levels of transcription EMBO J. 11, 4145-4152. Georgakopoulos,T., Gounalaki,N. and Thireos,& (1995) Genetic evidence for the interaction of the yeast transcriptional CO-activatorproteins GCN5 and ADA2 Mol. Gen. Genet 246, 723-728. Grant,P.A., Duggan,L., Cote,J., Roberts,S.M., Brownell,J.E., Candau,R., Ohba,R., Owen-Hughes,T., Allis,C.D., Winston,F., Berger,S.L. and Workman,J.L. (1997) Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosornal histones: characterization of an Ada complex and the SAGA (SpüAda) complex. Genes Dev. 11, 1640-1650. Gu,J.,Ren,K., Dubner,R. and ladarola,M.J. (1994) Cloning of a DNA binding protein that is a tyrosine kinase substrate and recognizes an upstream initiatorlike sequence in the promoter of the preprodynorphin gene. Mol. Brain Res. 24, 77-88. Guarente,L. (1983)Yeast promoters and lac2 fusions designed to study expression of cloned genes in yeast.. Methods Enzymol. 101, 181-191. Guarente,L. (1995) Transcriptional coactivators in yeast and beyond. Trends Biochem. Sci. 20, 517-21. Haas,A.L., Reback,P.B. and Chau,V. (1991) Ubiquitin conjugation by the yeast RAD6 and CDC34 gene products. J. Biol. Chem. 266, 5104-5112. Hein,C., Springael,J.-Y., Volland,C., Haguenaurer-Tsapis,R. and Andrel,B. (1995) NPII, an essential yeast gene involved in induced degradation of Gap1 and Fur4 permeases, encodes the RSP5 ubiqulin-protein ligase. Mol. Microbiol. 18, 77-87. Hicke,L. and Riezman,H. (1996) Ubiquitination of a yeast plasma membrane receptor signals its ligand-stimulated endocytosis. Ce11 84, 277-287.
HiII,D.E., Hope,l.A., Macke,J.P. and Struh1,K. (1986). Saturation mutagenesis of the yeast HIS3 regulatory site: requirements for transcriptional induction and for binding by GCN4 activator protein. Science 234, 45 1-457. Himmelfarb,H.J., Pearlberg,J., Last,D.H. and Ptashne,M. (1990) GALl1P: a yeast mutation that potentiates the effect of weak GAL4-derived activators. Cell 63, 1299-1309. Hochstrasser,M. (1995) Ubiquitin, proteosorne, and the regulation of intracellular protein degradation. Curr. Opin. Ce11 Biol. 7, 215-213. Hochstrasser,M. (1996) Protein degradation or regulation: Ub the judge. Cell 84, 813-815. Hop8,l.A. and Struhl,K. (1986) Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of yeast. Ce11 46, 885-894. Horiuchi,J., Silvenan,N., Marcus,G.A. and Guarente,L. (1995) ADA3, a putative transcriptional adaptor, consists of two separable domains and interacts with ADA2 and GCN5 in a trimeric complex. MoLCell. Biol. 15, 1203-1209. Horiuchi,J., Silveman,N., PinalB., Marcus.G.A. and Guarente,L. (1997) ADAI, a novel component of the ADNGCNS complex, has broader effects than GCN5, ADA2, or ADA3. Mol. Cell. Biol. 17, 3220-3228. Huibregtse,J.M., Scheffner,M., 8eaudenon.S. and Howley,P.M. (1995) A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proc. Natl. Acad. Sci., USA, 92, 256302567, Imhof,M.O. and McDonnel1,D.P. (1996) Yeast RSPS and its human homolog hRPFl potentiate hormone-dependent activation of transcription by human progesterone and glucocorticoid receptors. Mol. Cell. Biol. 16, 2594-2605. Jentsch,S. (1992) The ubiquitin-conjugation system. Annu. Rev. Genet. 26, 179-207. Jentsch,S. and Schlenker, S. (1995) Selective protein degradation: a joumey's end within the proteasorne. Cell 82, 881-884. Johnson,E.S., Ma,P.C.M., Ota,l.M. and Varshavsky,A. (1995) A proteolytic pathway that recognizes ubiquitin as a degradation signal. J. Biol. Chem. 270, 17442-17456. Johnson,M. and Davis,R.W. (1984) Sequences that regulate the divergent GAL 1-GAL 10 promoter in Saccharomyces cerevisiae. Mol. Cell. Biol. 4, 14401448. Komitzer, D., Raboy, B., Kulka, R.G. and Fink G.R. (1994) Regulated degradation of the transcription factor Gcn4. EMBO J. 13, 6021-6030.
Kunke1,T.A. (1985) Prenatal diagnosis and carrier detection of Duchenne muscular dystrophy with closely linked RFLPs. Proc. Natl. Acad. Sci., USA, 82. 488-492. Kuo,M.H., Brownell,J., Sobel, R.E., Ranalli,T.A., Edmonson,D.G., Roth,S.Y. and Allis,C.D. (1996) Transcription-linked acetylation by GcnSp of histones H3 and H4 at specific lysines, Nature 383, 269-272. Li, Y., Bjorklund, S., Jiang, Y.W., Kim, Y.J., Lane, W.S., Stillman, D.J. and Komberg, R D . (1996) Yeast global transcriptional regulators Sin4 and Rgrl are components of mediator complexlRNA polymerase II holoenzyme. Proc. Natl. Acad. Sci., USA, 21, 10864-10868. Marcus,G.A., Silverrnan,N., Berger,S.L., Horiuchi,J. and Guarente,L. (1994) Functional similarity and physical association between GCNS and ADA2: Putative transcriptional adaptors EMBO J. 13, 4807-48 15. Marcus,G.A., Horiuchi,J., Silveman,N. and Guarente,L. (1996) ADA5lSPT20 links the ADA and SPT genes, which are involved in yeast transcription. Moi. Cell. Biol. 16, 3197-3205. Martens,J.A, Genereaux,J., Saleh,A. and Brandl,C. J. (1996) Transcriptional activation by yeast PDRl p is inhibited by its association with NGGl piADA3p. J. Biol. Chem. 271 , 15884-15890. Melcher, K. and Johnston, S.A. (1995) GA14 interacts with TATA-binding protein and coactivators. Mol. Cell. Biol. 15 , 2839-2848. Moazed, D. and Johnson, D. (1996) A deubiquitinating enzyme interacts with SIR4 and regulates silencing in S. cerevisiae. Ce11 86, 667-677. Orphanides,G., Lagrange.T. and Reinberg,D. (1996) The general transcription factors of RNA polymerase II. Genes Dev. 10, 2657-2683. Pina,B., Berger,S.L., Marcus,G.A., Silverman,N., Agapite,J. and Guarente,L. (1993) ADA3 - a gene, identified by resistance to GAL4-VP16, with properties similar to and different from those of ADA2. Mol. Cell. Biol. 13, 5981-5989. Pollard, K.J. and Peterson, C.L. (1997) Role for ADNGCN5 products in antagonizing chromatin-mediated transcriptional repression. Mol. Cell. Biol.. 17, 6212-6222. Roberts,S.M. and Winston,F. (1996) SPT20/ADA5 encodes a novel protein functionally related to the TATA-binding protein and important for transcription in Saccharomyces cerevisiae. Mol. Cell. Bol. 16, 3206-3213. Roberts,S.M. and Winston,F. (1997) Essential functional interactions of SAGA, a Saccharomyces cerevisiae complex of Spt, Ada and Gcn5 proteins, with the Snf/Swi and Srtfmediator complexes. Genetics 147, 451 -465.
Ruiz-Garcia, A.B., Sendra, R., Pamblanco, M. and Tordera, V. (1997) GCNSp is involved in the acetylation of histone H3 in nucleosomes. FEBS Lett. 403, 186-190. Sakai,A., ChibazakuraJ., Shiminzu,Y. and Hishinuma,F. (1992) Molecular analysis of POP2 gene, a gene required for glucose-derepression of gene expression in Saccharomyces cerevisiae. Nucleic Acids Res. 20, 6227-6233. Saleh,A., Lang,V., C0ok.R. and Brandl,C. (1997) Identification of native complexes containing the yeast coactivator/repressor proteins NGG1/ADA3 and ADA2. J. Biol. Chem. 9 , 5571-5578. Scheffner,M., Huibregtse,J.M., Vierstra, R.D. and Howley,P.M. (1993) The HPV E6 and €6-AP complex functions as a ubiquitin protein ligase in the ubiquitination of p53. Ce1175, 495-505. Scheffner,M., Nuber,U. and Huibregtse,J.M. (1995) Protein ubiquitination involving an E1-E2-€3 enzyme ubiquitin thioester cascade. Nature 373, 81-83. Schultz,M.C., Choe,S.Y. andReeder,R.H. (1991) Specific initiation by RNA polymerase I in a whole-cell extract from yeast. Proc. Natl. Acad. Sci.. USA, 88. 1004-1008. Seufert,W. and Jentsch,S. (1990) Ubiqulin-conjugating enzymes UBC4 and UBC5 mediate selective degradation of short-lived and abnormal proteins. EMBO J. 9, 543-550. Silverman, N., Agapite, J. and Guarente, 1. (1994) Yeast ADAZ protein binds to the VP16 protein activation domain and activates transcription. Proc. Natl. Acad. Sci. , USA, 91, 11665-11668. Song, W., Treich, I., Qian, N., Kuchin, S. and Carlson. M. (1996) SSN genes that affect transcriptional repression in Saccharomyces cerevisiae encode SIN4, ROX3, and SRB proteins associated with RNA polymerase II. Mol. Cell. Biol. 16, 115-120. Spence,J., Sadis,S., Haas,A.L. and Fin1ey.D. (1995) A ubiquitin mutant with specific defects in DNA repair and multiubiquitination. Mol. Cell. Biol. 15, 12651273. Sto1tz.A. and Linder,P. (1990) The ADE2 gene from S. cerevisiae: sequence and new vectors. Gene 95, 91-98. Utsugi,T., Toh-e,A. and Kikuchi,Y. (1995) A high dose of the STMl gene supresses the temperature sensitivity of the tom 1and htrl mutants in Saccharomyces cerevisiae . Biochim. Biophys. Acta 1263, 285-288.
van Nocker,S. and Vierstra,R.D. (1991) Cloning and characterization of a 20kDa ubiquitin carrier prutein from wheat that catalyzes multiubiquitin chain formation in vitro. Proc. Natl. Acad. Sci., USA, 88, 10297-10301. Wang, L., Minen,C., Ying,C., Candau,R., Barlev, N., Brownell, J., Allis, C.D. and Berger, S.L. (1997) Histone acetyltransferase activity is conserved between yeast and human GCN5 and is required for complementation of growth and transcriptional activation. Mol. Cell. Biol. 17, 5 19-527. Wilkinson,K.D. (1995) Role of ubiquitination in proteolysis and cellular regdation. Annu. Rev. Nutr. 15, 161-189. Wilson,l., Niman,H., Houghten,R., Cherenson,A., Connolly,M. and Lemer,R. (1984) The structure of an antigenic determinant in a protein. Ce1137, 767- 7ï8. Winston,F., Durbin,K.J. and Fink,G.R. (1984) The SPT3 gene is required for normal transcription of Ty elements in S. cerevisiae. Ce11 39, 649-656. Winston, F., Dollard, C., Malone, E.A., Clare, J., Kapakos, J.G., Farabaugh, P. and Minehart, P.L. (1987) Three genes are required for trans-activation of Ty transcription in yeast. Genetics 115, 649-656. Yu, J., Donovie1,M.S. and Young,E.T. (1989) Adjacent upstream activation sequence elernents synergistically regulate transcription of ADH2 in Saccharomyces cerevisiae. Mol. Cell. Bol. 9, 34-42.
Zolladek,T., Tobiasz,A., Vaduva,G. Boguta,M., Martin,N.C. and Hopper,A.K. (1997) MDPI, A Saccharomyces cerevisiae gene involved in mitochondrial/cytoplasmic protein distribution is identical to the ubiquitin-protein ligase gene RSPS. Genetics 145, 595-603.
CHAPTER 5 GENERAL DISCUSSION
5.1
ADA proteins form multiple complexes in Vivo Initial reports on components of the yeast ADA complex demonstrated
that ADAZp, NGGl p and GCN5p associate in vitro and in vivo in a yeast twohybrid analysis (Marcus et al., 1994, 1996; Horiuchi et al., 1995; Candau et al., 1996). The observations that single and double disniptions of ada2, nggl and
gcn5 have similar effects on transcriptional activation and repression support the functional association of the three proteins in vivo (Berger et ab, 1992; Pina et al., 1993 Marcus et al., 1994, 1996; Horiuchi et a/., 1995; Brandl et al., 1996). The finding of Candau et al. (1996) that the stability of ADA2p is dependent on the expression of GCN5 and Our finding that the stability of ADA2p and NGGl p depend on the presence of each other (Saleh et aL, 1997, Chapter 2) provide additional evidence for their association in vivo. The association of NGGl p and ADA2p was revealed more directly by their coirnmunoprecipitation from whole cell extracts. This was further supported by the coelution of the proteins in multiple peaks after fractionation on both gel-filtration and ion-exchange chromatography columns (Saleh et a/., 1997, Chapter 2). It should also be noted that Candau and Berger (1996) have shown that NGGl p and GCN5p coimmunoprecipitate with epitope-tagged ADA2p when the three proteins are overexpressed in v h . Taken together. these results indicate that ADA2p, NGGlp and GCN5p associate together in vivo. The combination of gel-filtration and ion-exchange chromatography experiments resolved four native complexes containing both ADA2p and
NGG1p (ADA complexes) (Saleh et a/., 1997, Chapter 2). Two of the complexes had estimated sizes of -2 MDa; the other two were 200 kDa and 900 kDa. The
simplest explanation for the 200 kDa complex that it represents a minimum complex containing ADA2p, NGGlp and probably GCN5p. The presence of GCN5p in this complex would be consistent with the results from the Peterson laboratory (Pollard and Peterson, 1997) which demonstrated the in vivo existence of a comparable -150 kDa ADA complex containing NGGlp and GCN5p. It is also important to emphasize that most likely there is only one copy of NGGl p in each of the ADA complexes. This is suggested by the observation that myc-NGGl p and HA-NGGlp did not associate with each other by immunoprecipitation assays when both proteins were CO-expressedin yeast (Saleh and Brandl, unpublished results). Similady, it is likely that ADA2p does not exist as a multimer. When two forms of the ADA2p were CO-expressed,they did not interact with each other by immunoprecipitation and yeast two-hybrid experiments (Martens and Brandl, unpublished results). Thus, the high molecular size of the ADA complexes results from the association of ADA2p and NGGl p with other components rather than their presence as multimers within the complexes. Our biochemical analyses of the association of NGGl p and ADA2p provide the first evidence for the existence of at least four chromatographically distinct ADA complexes. The subsequent reports from other laboratories also support this finding. Grant et al. (1997) have partially purified two of the ADA complexes containing ADA2p, NGGl p and GCN5p; one of which, is the SAGA complex, has a molecular size of -1.8 MDa while the other is -800 kDa. The SAGA complex also contains the gene products of the TBP class of SPTgenes; SPT3p, SPT7p, SPT8p and SPT20/ADA5p. These complexes are likely to correspond to two of the large complexes identified in Our laboratory (-2 MDa and -900 kDa). The association of the products of SPTgenes with ADA proteins in the SAGA cornplex confims the previously proposed functional link
between the two groups of coactivators with the finding that SPT20 is identical to ADA5 (Marcus et al., 1996; Roberts and Winston, 1996). However. the absence of the SPT proteins from the 800 kDa complex (Grant et al., 1997), along with the overlapping but non identical phenotypes caused by different nuIl alleles, suggests that the SPT and ADA proteins also act independently (Marcus et al., 1996; Roberts and Winston, 1996, 1997). Another report on the formation of multiple complexes by ADA components came from Pollard and Peterson (1997), who identified three complexes containing NGGl p and GCN5p. The sizes of these complexes, -2 MDa, -700 kDa and -1 50 kDa, are similar to those of three complexes that I have described. These authon also suggested the existence of additional ADA complexes, since the first step of their purification strategy relies on the fortuitous binding of ADA proteins on Ni2+-NTAagarose. Indeed, current results from the Workman laboratory suggest the in vivo presence of at least one more huge ADA complex that does not bind th8 N~~+-NTA resin nonspecifically as
found with the SAGA (Grant and Peterson, personal communication). Interestingly, the preliminary data on this uncharacterized complex suggest that it contains both ADA and SPT proteins. This finding is also in parallel with our results which demonstrated the cofractionation of SPT7p with NGGl p and ADA2p in two of the ADA complexes using a Mono Q column (ADNSPT complexes, Chapter 3). Furthemore, Honuchi et al. (1997) have demonstrated
that the five ADA proteins (ADAI p to ADA5p) associate together in one of the -2 MDa complexes. Taken together, the above results strongly support Our
initial finding which indicates that ADA proteins form multiple complexes in vivo (Saleh et al., 1997). The relationship between the ADA complexes is not yet clear. Most likely, the iargest N o complexes contain al1 the five ADA proteins as well as SPT
proteins (Grant et aL, 1997; Hofluchi et al., 1997; Roberts and Winston, 1997).
One possibility is that the -900 kDa complex also contains al1 the ADA proteins but lacks the SPT gene products and is a subassembly of the -2 MDa complexes. In this case, the larger complexes would then likely posses additional attributes to the function, conferred by the SPT proteins or other unidentified factors. An alternative possibility is that, although these complexes contain ADA proteins, each form might represent a distinct complex with unique transcriptional activities (i.e. activation and repression may require complexes with different components). Therefore, the additional components would dictate the transcriptional mechanisms of the ADA complexes and how their activities are regulated. The exact relationship between the different ADA complexes will be clarified on the identification of al1 subunits of each complex and the degree to which they are common and unique. Many other transcription factors have been shown to associate in large-molecular-weight complexes, such as the TBP-TAFs complexes (reviewed in Tansey and Herr, 1997), SWVSNF complex (reviewed in Peterson, 1996; Pazin and Kadonaga, 1997a, 1997b), and the Pol II holoenzyme complexes (reviewed in Carlson, 1997). It is unlikely that the ADA proteins could be part of any of these complexes. The size of proteins that coimmunoprecipitate with NGGlp (see appendix 1) do not resemble the sizes of the yeast TAFs (Poon et al., 1994; Reese et al., 1994). Probing the partially purified ADA fraction from the various chromatography columns with an antibody to the large subunit of Pol II demonstrated that the protein neither cofractionates nor immunoprecipitates with ADA proteins (not shown). Other reports have also demonstrated that the ADA complexes are chromatographically distinct from the yeast SWVSNF and the Pol II holoenzyme complexes (Grant et a/., 1997; Pollard and Peterson; 1997). Therefore, the ADA
complexes are novel and distinct from these multisubunit transcription regulatory complexes charactenzed previously.
5.2
TRAlp is a component of the ADAKPT complexes In line with the identification of additional components of the ADA
complexes, we provide evidence that the gene product of TRA 1is a member of two of the ADNSPT complexes (Chapter 3). First, the identification of TRAl p within the ADAISPT complexes was based on 1s association with HM-NGGl p (6 x His-Myc-NGGlp) after affinity purification on a ~i*+-NTA column. Second, the association of TRAl p and ADNSPT components was demonstrated by their reciprocal coimmunoprecipitation from native yeast extracts. Third, ionexchange chromatography demonstrated that al1 of the TRAl p coeluted with ADAZp, NGGl p and SPT7p in two distinct peaks. The validity of this coelution as an indication of interaction was verified by coimmunoprecipitation of TRAl p with ADA2p from one of the peak fractions. Fourth, the elution profile of TRAl p was altered when the same ion-exchange chromatography experiment was performed with native yeast extracts lacking a ADA2p. Fifth, TRAl pl NGGl p and SPT7p remain associated together throughout an approximately 1200-fold purification over three successive chromatography columns of N~~+-NTA, Macro prep 50 Q and DNA cellulose (not shown). Finally, the requirernents of partially purified ADNSPT components to reconstitute binding of TRAl p to DNA cellulose provide additional support for the association. These lines of evidence, taken together, indicate that TRAl p is a member of two of the
ADAISPT complexes. The molecular site of TRAlp (433 kDa) suggests that the protein may
serve as a scaffold for the formation of ADAISPT complexes. Such a mode1 predicts TRAl p to form multiple protein-protein interactions with components of
the ADNSPT complexes. Examples from other coactivator complexes are consistent with this model. The largest TAF in Drosophila and human, TAF250, is critical in the assembly of TFllD and is an important anchor for TBP in the TFllD complex (Chen et al., 1994: see review by Tansey and Herr 1997). Interestingly, unlike the previously characterized components of
ADAEPT complexes, TRAl p is essential, suggesting that it is crucial for transcription of more or different genes than the other components. Several other coactivator proteins are essential for yeast viability. Three of the SRB proteins in the Pol II holoenzyme complex are essential; SRB4p, SRB6p and SRB7p (Thompson and Young, 1995). These SR8 proteins are required to regulate transcription of a broad range of yeast genes (reviewed in Carlson, 1997). In addition, six components of the yeast TBP-TAF complex are critical for
viability (Moqtaderi et al., 1996). Surprisingly, studies of these TAFs containing conditional mutation indicate that they are not required for transcriptional activation by GCN4p and many other activators, implicating that they probably have other regulatory functions (Apone et al., 1996; Moqtaderi et al., 1996; Walker et al., 1996). The essential nature of TRA1 also predicts that the protein may be involved in other regulatory processes in addition to transcription. The sequence similarities between TRAl p and members of a related group of proteins in the P13K family support this possibility (reviewed in Jin et al., 1997). This group includes the S. cerevisiae proteins DRR1TTORlp (Heitman et al., 1991; Cafferkey et ab, 1993), TOR2p (Kunz et al., 1993; Brown et al., 1994),
ESRlIMECl p (Weinsett et al., 1994; Kato and Ogawa 1994) and TEL1p
(Greenwell et al., 1995; Morrow et al., 1995). Each of these proteins, including TRAl p. has a single kinase homology domain of approximately 25% to 40%
identity with other members of the P13K family (reviewed in Jin, 1997). The
kinase homology domain is located at the C-terminal end of the protein in every case. While related to the P13Ks. members of this group of proteins are distinguished from the true lipid kinases by having a common motif in their Cterminus regions (Hartley et al., 1995; Keith and Schreiber, 1995), their large size (>200 kDa) and in some cases their capability to phosphorylate proteins. As a group these molecules are involved in many key cellular processes, such as DNA recombination (reviewed in Jackson, 1996; Sidorova et al., 1997) cell cycle regulation (Helliwell et al., 1998), maintenance of telorneres length (Sanchez, et al., 1996), protein translation (Barbet e l al., 1996). and transcription (Boulton et al., 1998). The sequence similarity between TRAl p and DNA-PK, in the P13K kinase domain raises the possibility that TRAlp is a protein kinase whose activity may be required to regulate the transcriptional function of ADNSPT complexes. The recent finding of Barlev et al. (1998) that D N A - P L phosphorylates hGCN5 in vitro and thereby inhibiting its HAT activity provides additional support for the possibility that TRAl p may function as a protein kinase within the ADA complexes. However, Our results suggest that TRAl p does not have a protein kinase activity ( see Chapter 3). Our inability to detect a kinase activity by T M 1p is consistent with the absence of the conserved DFG sequence in the putative kinase motif (Taylor et al., 1992) and the fact that DXXXXN motif is found in a different flanking sequence context than the other P13K proteins. lt is also important to emphasize that we could not detect phosphorylated components of the immunoprecipitated ADA/SPT complexes, suggesting that the structure of the complexes does not include protein kinases (Chapter 3). Analogies with DNA-PK, lead to the question of whether Ku-like molecules are present in the ADA cornplex. The Ku heterodimer is required for
DNA targeting of DNA-PK, (Lees-Miller et al., 1990; Gottlieb et al., 1993) while
components of the ADNSPT complexes enable the binding of TRAl p to DNA cellulose (Chapter 3). Obvious mediators of this process are the yeast proteins HDFlp and HDF2p which are related in sequence to the p70 and p80 subunits of Ku and likewise bind DNA (Feldmann et al., 1996; Mages et al., 1996; Milne et al., 1996). The pattern of proteins that coimmunoprecipitate (see appendix 1)
and copurify (Grant et al., 1997) with the ADAISPT complexes does not rule out the possibility that HDFlp and HDF2p may be present; however, disruption of either of these genes has not been reported to result in an ada -like phenotype. At the present, we do not know the function(s) of TRAl p. The sequence similarity of TRAl p to several key cellular regulators, its coelution with ADA/SPT complexes and even its large size suggest that TRAlp plays a key role in the structure, function, and regulation of the ADAISPT complexes. The fact that
TRAl is essential has not allowed for direct gene disruption experiments to determine these roles. Further genetic and biochemical studies of TRAl p are required to investigate its functions.
5.3
ADA complexes interact with TBP Early studies on the ADA gene products led to the hypothesis that these
proteins functioned as transcriptional regulators that might bridge activation domains to components of the general transcription machinery (Berger et al., 1992; Horiuchi et al., 1995). This model was initially supported by genetic studies in which the ADA encoding genes were isolated on the basis of their positive and negative effects on the activity of a subset of transcriptional activators (Berger et al., 1992; Georgakopoulos and Thireos, 1992; Brandl et al., 1993; Pina et al., 1993; Marcus et al., 1994), and biochemical studies in which ADA2p, NGGl p andor GCN5p were shown to interact with the activation
domains of VP16, ADRl pl GAL4p, GCN4p and PDRl p (Silveman et al., 1994; Bariev et al., 1995; Melcher and Johnston, 1995; Chiang et a/., 1996; Martens et a/., 1996). Subsequently, the link between the ADA complexes and the basal
machinery was demonstrated with Our observation that TBP associated with
NGGl p by immunoprecipitation from whole cell extracts (Saleh et al.. 1997. Chapter 2). The later finding of Roberts and Winston (1997) that TBP from whole yeast extract was retained into a GST-SPT20/ADA5p affinity column confimis the in vivo interaction between TBP and ADA complexes. The association between ADA and SPT proteins in at least two of the complexes, combined with the finding that SPT3p interacts directly with TBP (Eisenmann et a/., 1992), provides additional evidence for a functional link between the ADA proteins and TBP. SPT3p also interacts genetically with MOT1p and TFIIA, two factors known to associate with TBP (Madison and Winston, 1997). Taken together, both types of interactions with TBP and activator proteins, suggest that the ADA complexes serve as an intemediary between transcriptional activators and TBP, possibly providing a means of recruiting TBP to specific promoters independently of the yeast TAF complex.
5.4
Amino acids 274-307 are critical for transcriptional function of
the ADA complexes Our finding of the interaction between NGGl p and TBP by immunoprecipitation requires amino acids 274-307 agrees with the region being essential for the function of NGGl p. Deletion of this region results in loss of repression of GAL4p and in the slow grovvth phenotype typical of disruption of
nggl (Brandl et al., 1996). Amino acids 1-308of NGGl p activate transcription as a GAL4p fusion (Brandl et a!., 1996). This activation depends on amino acids 274-307. Mutations can be isolated in this region that either stimulate or inhibit
the action of NGGlp on repression of GAL4p and the activity of GAL4p-NGGl p fusions in transcriptional activation (Brandl et al., 1996). Interestingly, deletion of this region of NGGl p results in alteration in the size of 2 MDa complexes (Chapter 2; Saleh et al., 1997), suggesting that arnino acids 274-307 are required for protein-protein contacts with other components of the large ADNSPT complexes. Indeed, this segment of NGGlp is important for the association of SPT3p with the complexes, since ADA immunoprecipitates from
a mutant extract of NGGlp~74-307lacked SPT3p (not shown). This finding provides compelling evidence for the involvement of residues 274-307 of NGGl p in protein-protein interactions with TBP and other components of the ADAfSPT complexes, and explains functional defects seen in the ngg1s7e307 strain. However, the key issue of whether the residues 274-307 of NGGl p interact directly with TBP, or indirectly through its association with SPT3p and other components of the complexes has yet to be resolved.
5.5
Mechanisrns of regulation by the ADA complexes One possible mechanism, based on the interaction with TBP, is that the
ADA complexes are required to regulate the activity of TBP at specific promoters. Since binding of TBP to the TATA element is believed to be one of the rate-limiting steps in the transcription process (reviewed in Conaway and Conaway, 1993), then modulating the formation of a stable TBP-DNA complex by ADA proteins will either promote or repress transcription from specific promoters. The function of certain other cofactors is consistent with this model. For instance, several TAFs in the human TFllD complex are recognized as important regulatory components in the TBP recruitment process. TAFI 8, TAFI50 and TAF250 recognize certain initiator elements in the core promoter
and stabilize binding of TB? to DNA (reviewed in Goodrich and Tjian, 1994;
Roeder, 1996). On the other hand, the mammalian repressors DR1 and NC2 destabilize TBP-TATA box interaction by their direct binding to TBP and excluding the transcription factor TFllA (Auble and Hahn, 1993). The yeast MOTl p binds and removes TBP from promoters in an ATP-dependent process (Auble et al., 1994; Poon et al., 1994). The genetic interactions between SPT3,
MOTl and TFllA (Madison and Winston, 1997) provide compelling evidence for the role of ADNSPT complexes in regulating the TBP-DNA binding processes. Based on this information, it is tempting to speculate that the ADA complexes regulate transcription positively and negatively by controlling the recruitrnent of TBP to specific promoters or regulating its interactions with other basal factors. The identity of yeast GCN5p and its hurnan homologue as histone acetyltransferases implies that transcriptional regulation by the ADA complexes also involves a chromatin remodeling mechanism (reviewed in Hampsey, 1997). Consistent with this rnodel, Grant et al. (1997) and Pollard and Peterson (1997) have shown that al1 their partially purified ADA complexes are capable of acetylating nucleosomal histones in vitro. The finding that acetylation of nucleosomal histones by GCN5p depends on its presence within an intact ADA complex suggests that ADA components are required to regulate this activity in
vivo (Kuo et ab, 1998; Wang et al., 1998). One possibility is that the ADA components are important for targeting the HAT activity of GCN5p to specific promoters through their interactions with transcriptional activators. Interestingly, the enzymatic activity of GCNSp, within the context of ADA complexes, has been shown to acetylate the yeast negative regulator SIN1p in vitro (Pollard and Peterson, 1997). This suggests that the acetylase activity of
GCN5p may target non-histone proteins. We have preliminary evidence which indicates the endogenous acetylase activity of ADA complexes targets the acetylation of a -100 kDa component of the complexes (Saleh, Brandl and
Cote; unpublished result), although the identity of this protein remains to be uncovered. Several recent reports have shown that core histone are not the only substrates for the acetylase activity of coactivators (reviewed in Struhl. 1998). For instance, acetylation of HMG proteins, p53 (Gu and Roeder. 1997) and the basal transcription factors TFIlE and TFllF by the human and Drosophila TFllD complexes have been demonstrated (Imhof et ab, 1997). These findings indicate that the physiological function of histone acetyltransferases probably extend beyond the acetylation of core histones. It is important to note that the transcriptional function of ADA complexes involves both the interaction with the basal factor TBP and modulation of nucleosomal structure. Our results in chapter 4 are consistent with the ADA complexes functioning by these two mechanisms. A mutant strain lacking TOM1p displays similar transcriptional defects as disruption of ada2 and nggl. The transcriptional changes correlate with the absence of TBP and SPT3p from the immunoprecipitated ADA complexes. The same immunoprecipitates, however, showed only a marginal decrease when they were tested for their ability to acetylate nucleosomal histones. In contrast, immunoprecipitated ADA complexes from mutant extracts of NGGl p lacking the amino acids 274-307 demonstrated loss of the association with TBP and SPT3p and considerable reduction of the HAT activity ( 4 0% of that from a wild type NGG1p immunoprecipitates). Thus, the amino acid sequences between 273-308are required for both functions while TOMlp is involved in regulation of the interaction between the ADA complexes and TBP. The correlation between the alterd transcription in tom1 deletion strains and the loss of TBP interaction, without affecting the HAT activity, supports functioning of the ADA complexes by a TBP-dependent mechanism in addition to acetylation of core histones.
5.6
Post-translational modification of ADNSPT components Ubiquitination is a key element in the regulation of transcription as it is for
other cellular processes (reviewed by Jentsch, 1992; Hochstrasser, 1995, 1996; Wilkinson, 1995). We have shown that the hect domain protein TOM1p is a potential ubiquitin ligase whose activity is required for the transcriptional function of ADA complexes. A toml deletion strain has the same slow-growth, temperature sensitivity, and defects in transcription from the GAL IO, ADH2 and
HlS3 promoters as a nggl deletion strain. In addition, the slow growth and altered transcriptional phenotypes are no more severe in nggl toml double mutants than in either single mutant. The identity of TOMlp as an E3 ubiquitin ligase is suggested with the finding that mutation of the cysteine residue, corresponding to a residue essential for ubiquitination by €6-AP and RSPSp (Huibregtse et al., 1995), results in a strain with similar transcriptional properties as toml nuIl mutants. This view was further supported with the demonstrated requirement of TOMl p to ubiquitinate a 210 kDa component (SPT7p) of the ADA complexes. These results, taken along with the observation that TOMlp can be irnmunoprecipitated with NGGl p, imply that the role of TOMl p in transcription is principally mediated through its ubiquitination function and contrasts with the obsenration from RSP5p in which the active site cysteine was not required tu potentiate transcriptional activation by human steroid receptors in yeast (Imhof and McDonell, 1996). It is important to emphasize that ubiquitination of SPT7p by TOM1p is unlikely to be involved in the protein degradation. The amount of SPT7p associated with the ADA proteins was not increased in the absence of TOMl p as would be expected if ubiquitination of SPT7p was a signal for its degradation. While this observation does not eliminate the possibility that a
minor population of SPTïp is degraded as the result of ubiquitination, the finding that the growth of tom7 strains are not affected by the presence of the amino acid analog canavanine (Seufert and Jentsch, 1990) supports that TOMl p is not involved in general protein tumover (not shown). Similar roles for ubiquitination not directly involving protein tumover have been proposed (Amason and Ellison, 1994; Spence et al., 1995; Chen et al., 1996; Hicke and Riezman, 1996). The nonlinear branching of ubiquitin chains as seen in the yeast stress response (Amason and Ellison, 1994) and DNA repair (Spence et al., 1995), may signal this type of regulatory event.
The finding that TOM1p is required for the association of TBP and SPT3p
with the ADA complexes ied us to propose that the transcriptional function of the complexes is regulated by ubiquitination events. This model indicates that ubiquitination of SPT7p, and probably other components, by TOMl p is a signal for the complexes to interact with TBP andor SPT3p, thereby stimulating the transcnptional activity of ADAISPT complexes. Contrasting this positive effect, deubiqutination of component(s) of the ADAISPT complexes should result in destabilization of these interactions. Our original observation that UBP3p associates with NGGl p (Martens et al., 1996) suggests that UBP3p could play an obvious role with TOMl p in cycling one or more of the ADA complexes from an active ubiquitinated f o m to an inactive deubiquinated form. Experiments are currently underway to test this mode1 in Our laboratory. lnterestingly UBP3p has been recently shown to associate with SIR4p and regulate silencing at the HM silent mating type loci and telomeres (Moazed and Johnson, 1996). This result, along with the finding that NGGlp interacted with UBP3p in a two-hybrid study (Martens et ai., 1996), suggests a possible link between the ADA and SIR complexes. Indeed, SIR4p associates with
affinity purified ADA complexes, suggesting a regulatory role of the ADA complexes in silencing (Saleh and Brandl, unpublished results).
5.7
Conclusions Biochemical approaches have and will continue to facilitate studies into
many aspects of the ADA complexes. Identification of al1 the components of each complex will permit detailed investigations into stmcture/function relationships and the exact mechanisms of activation and repression by these regulatory complexes. We have also begun analyses of regulation of the ADA complexes by ubiquitination. Further studies will determine the factors involved
in this post-translational process and in tum their role in controlling functions of the ADA complexes. Furthemore, isolation of conditional mutants of TRAl p and addressing their effect on transcription will allow the detemination of the physiological roles for the association of this molecule with ADAISPT complexes.
5.8
References
Apone,L.M., Virbasius,C.M., Reese,J.C., and Green,M.R. (1996) Yeast TAF(II)SO is required for cell-cycle progression through G21M but not for general transcription activation. Genes Dev. 10, 236802380. Amason, T. and Ellison,M.J. (1994) Stress resistance inSaccharomyces cerevisiae is strongly correlated with assembly of a novel type of multiubiquitin chain. Mol. Cell. Biol. 14, 787607883. Auble,D.T. and Hahn,S. (1993) An ATP-dependent inhibitor of TBP binding to DNA. Genes Dev. 7, 844-856. Auble,D.T., Hansen,K.E., Mueller,C.G., Lane,W.S., Thomer,J. and Hahn,S. (1994) Mot1, a global repressor of RNA polymerase II transcription, inhibits TBP binding to DNA by an ATP-dependent mechanism. Genes Dev. 8 1920-1934. Baker,R.T., Tobias,J.W. and Varshavsky,A. (1992) Ubiquitin-specific proteases of Saccharomyces cerevisiae. J. Biol. Chem. 267, 23364-23375. Barbet, N.C., Schneider, W., Helliwell, S.B., Stansfield, 1. Tuite, M.F. and Hall, M.N. (1996) TOR controls translation initiation and early G1 progression in yeast. Mol. Biol. Cell. 7, 25-42. Barlev,N.A., Candau.R., Wang.1.. Darpino,P., Silverman,N. and Berger,S.L. (1995) Charactenzation of physical interactions of the putative transcriptional adaptor, ADA2, with acidic activation domains and TATA-binding protein. J.Biol. Chem. 270, 19337-19344. Barlev,N.A., PoRoratsky,V., Owen-Hughes,T., Ying,C., Liu,L., Workrnan,J.L. and Berger,S.L. (1998) Repression of GCN5 histone acetyltransferase activity via bromodomain-mediated binding and phosphorylation by the KU-DNAdependent protein kinase complex. Mol Ce11 Biol. 18, 1349-1358. Berger,S.L., Pina,B.. Silverman,N., Marcus,G.A., Agapite,J., Regier,J.L.. Triezenberg,S.J. and Guarente,L. (1992) Genetic isolation of ADA2 - a potential transcriptional adaptor required for function of certain acidic activation domains Ce1170, 251-265. Boulton, S.J. and Jackson, S.P. (1998) Components of the Ku-dependent nonhomologous end-joining pathway are involved in telomeric length maintenance and telomeric silencing. EMBO J. 16, 1819-1828. Brandl,C. J., Furlanetto,A.M., Martens,J.A. and Hamilton,K.S. (1993) Characterization of NGG1, a novel yeast gene required for glucose repression of GAL4p-regulated transcription EMBO J. 12, 5255-5265. Brandl,C.J., Martens,J.A., Margaliot,A., Stenning,D., Furlanetto A.M., Saleh,A., Hamilton,K.S. and Genereaux,J. (1996) Structurelfunctional properties of the
yeast dual regulator protein NGGl that are required for glucose repression. J. Biol. Chem. 270, 929809306. Brown,E.J., Albers,M.W., Shin,T.B., IchikawaK, Keith,C.T., Lane,W.S. and Schreiber,S.L. (1994) A marnrnalian protein targeted by G1-arresting rapamycin-receptor complex. Nature 369, 756-758. Brownell,J.E., Zhou,J., Ranalli.T., Kobayashi, R., Edmondson,D.G., Roth,S.Y. and Allis.C. D. (1996) Tetrahymena histone acetyltransferase A: a homolog to yeast Gcn5p linking histone acetylation to gene activation. Cell84, 843-851. Candau, R. and Berger. S.L. (1996) Structural and functional analysis of yeast putative adaptors. Evidence for an adaptor complex in vivo. JBioLChem. 271, 523795245. Candau,R., Moore,P.A., Wang,L., Barlev,N.,Ying,C.Y., Rosen,C.A. and Berger,S.L. (1996) Identification of human proteins functionally conserved with the yeast putative adaptors ADA2 and GCNS. Mol. Cell. Biol. 16, 593-602. Candau,R., Zhou,J,-X., Allis,C.D. and Berger,S.L. (1997) Histone acetyltransferase activity and interaction with ADA2 are critical for GCNS function in vivo. EMBO J. 16, 555-565. Carison,M. (1997) Genetics of transcriptional regulation in yeast: connections to the RNA polymerase II CTD. Annu. Rev. Cell. Dev. Biol. 13, 1-23. Chen,J.L., Attardi,L.D., Verrijzer,C.P., Yokomori,K. and Tjian,R. (1994) Assembly of recombinant TFllD reveals differential coactivator requirements for distinct transcriptional activators. Ce11 79, 93-105. Chen,Z. and Pickart,C.M. (1990) A 25-kilodalton ubiquitin carrier protein (E2) catalyzes multiubiquitin chain synthesis via lysine-48 of ubiquitin. J. Biol. Chem. 265, 21835-21842. Chiang, Y.C., Komamitsky, P., Chase, D.and Denis, C.L. (1996) ADRl activation domains contact the histone acetyltransferase GCNS and the core transcriptional factor TF1IB. J. Biol. Chem.. 271, 32359-32365. Ciechanover,A. (1994) The ubiquitin-proteasorne proteolytic pathway. Ce11 79, 13-21. Conaway. R.C., Conaway.J.W. (1993) General initiation factors for RNA polymerase I1. Annu. Rev. Biochem. 62, 1 61-190. Eisenmann,D.M., Dollard,C. and Winston,F. (1989) SPT15, the gene encoding the yeast TATA binding factor TFllD is required for normal transcription in vivo. CeIi58, 1183-1191.
Eisenmann,D.M., Amt,K.M., Ricupero,S.L., Rooney,J.W. and Winst0n.F. (1992) SPT3 interacts with TFIlD to allow normal transcription in Saccharomyces cerevisiae. Genes Dev. 6,131 9- 1331. Eisenmann,D.M., Chapon,C., Roberts,S.M., Dollard,C. and Winston,F. (1994) The Saccharomyces cerevisiae SPTB gene encodes a very acidic protein that is functionally related to SPT3 and TATA-binding protein. Genetics 137, 647657. Feldmann,H., Driller,L., Meier,B., Mages,G., Kellerrnann,J. and Winnacker,E.L. (1996) HDF2, the second subunit of the Ku homologue from Saccharomyces cerevisiae. J. Biol. Chem. 271, 27765-27769. Georgakopoulos,T. and Thireos,G. (1992) Two distinct yeast transcriptional activators require the function of the GCN5 protein to promote normal levels of transcription EMBO J. 11, 4145-4152. Georgakopoulos,T., Gouna1aki.N. and Thire0s.G. (1995) Genetic evidence for the interaction of the yeast transcriptional CO-activatorproteins GCNS and ADA2 Mol. Gen. Genet. 246, 723-728. Goodrich,J.A. and Tjian, R. (1994) TBP-TAF complexes: selectivity factors for eukaryotic transcription. Curr. Opin. Cell. Biol. 6 , 403-409. Gottlieb,T.M. and Jackson,S.P. (1993) The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen. Cell72, 131-142. Grant,P.A., Duggan,L., Cote,J., Roberts,S.M., Brownell,J.E., Candau,R., Ohba,R., Owen-Hughes,T., Allis,C.D., Winston,F., Berger,S.L. and Workman,J.L. (1997) Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (SpVAda) complex. Genes Dev. 11 , 1640-1650. Greenwell,P.W, K.ronmal,S.L., Porter,S.E., Gassenhuber,J., Obermaier,B., Petes,T.D. (1995) TELI, a gene involved in controlling telomere length in S. cerevisiae, is homologous to the human ataxia telangiectasia gene. Ce11 82, 823-829. Gu,J.,Ren,K., Dubner,R. and ladarola,M.J. (1994) Cloning of a DNA binding protein that is a tyrosine kinase substrate and recognizes an upstrearn initiatorlike sequence in the promoter of the preprodynorphin gene. Mol. Brain Fies. 24, 77-88. GU,W., Roeder,R.G. (1997) Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal dornain. Ce1190, 595-606. Guarente,L. (1995) Transcriptional coactivators in yeast and beyond. Trends Biochem. Sci. 20, 5 17-21.
Hampsey,M. (1997) A SAGA of histone acetylation and gene expression. Trends Genet. 13, 427-429. HartIey,K.O., Gell,D., Srnith,G.C., Zhang,H., Divecha,N., Connelly,M.A., Admon,A., Lees-Miller,S..P., Anderson,C.W., and Jackson,S.P. (1995) DNAdependent protein kinase catalytic subunit: a relative of phosphatidylinositol 3kinase and the ataxia telangiectasia gene product. Cell82, 849-856. Heitman,J., Mowa,N .R. and Hal1,M.N. (1991) Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science 253, 905-909. Helliwell, S.B., Howald, I., Barbet, N. and Hall, M.N. (1998) TOR2 is part of two related signaling pathways coordinating cell growth in Saccharomyces cerevisiae. Genetics 148, 99-1 12. Hicke,L. and Riezman,H. (1996) Ubiquitination of a yeast plasma membrane receptor signals its ligand-stimulated endocytosis. Ce1184, 277-287. Hochstrasser,M. (1995) Ubiquitin, proteosorne, and the regulation of intracellular protein degradation. Curr. Opin. Ce11 Biol. 7, 215-213. Hochstrasser,M. (1996) Protein degradation or regulation: Ub the judge. Ce11 84, 813-815. Horiuchi,J., Silvemian,N., Marcus,G.A. and Guarente,L. (1995) ADA3, a putative transcriptional adaptor, consists of two separable domains and interacts with ADA2 and GCN5 in a trimeric complex. MoLCell. Biol. 15, 1203-1209. Horiuchi,J., Silveman,N., Pina,& Marcus,G.A. and Guarente,L. (1997) ADAI , a novel component of the ADNGCN5 complex, has broader effects than GCNS, ADA2, or ADA3. Mol. Cell. Bol. 17, 3220-3228. Huibregtse,J.M., Scheffner,M., Beaudenon,S. and Howley,P.M. (1995) A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proc. Natl. Acad. Sci, USA, 92, 2563-2567. Imhof,M.O. and McDonnel1,D.P. (1996) Yeast RSP5 and its human homolog hRPFl potentiate hormone-dependent activation of transcription by human progesterone and glucocorticoid receptors. Mol. Cell. Biol. 16, 2594-2605. Imhof,A., Yang,X.J., 0gryzk0,V.V.~Nakatani,Y., Wolffe,A.P. and Ge,H. (1997) Acetylation of general transcription factors by histone acetyltransferases. Curr. Bid. 7, 689-692 Jackson,S.P. (1996) The recognition of DNA damage. Curr. Opin. Genet. Dev. 6, 19-25. Jentsch,S. (1992) The ubiquitin-conjugation system. Annu. Rev. Genet. 26, 179-207.
Jentsch,S. and Schlenker, S. (1995) Selective protein degradation: a joumey's end within the proteasorne. CeIl 82, 881-884. Jin,S., Inoue,S. and Weaver,D. (1997) Functions of the DNA dependent protein kinase. Cancer Sem. 29, 221-261. Kadosh,D. and Struhl,K (1998) Histone deacetylase activity of Rpd3 is important for transcriptional repression in vivo. Genes Dev. 12 , 797-805. Kato,R. and Ogawa,H. (1994)An essential gene, ESRI, is required for mitotic cell growth, DNA repair and meiotic recombination in Saccharomyces cerevisiae. Nucleic Acids Res. 22, 3 104-3 112. Keit, C.T. and Schreiber,S.L. (1995) PIK-related kinases: DNA repair, recombination, and cell cycle checkpoints. Science 270, 50-51 . Kunz,J., Henriquez,R., Schneider,U., Deuter-Reinhard,M., Mowa,N.R. and Hal1,M.N. (1993) Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression. Ce11 7,585596. Kuo,M.H., Brownell,J., Sobel, R.E., Ranalli,T.A., Edmonson,D.G., Roth,S.Y. and Allis,C.D. (1996) Transcription-linked acetylation by Gcn5p of histones H3 and H4 at specific lysines, Nature 383, 269-272. Kuo,M.H., Zhou,J, Jambeck,P., Churchill,M.E. and Allis,C.D. (1998) Histone acetyltransferase activity of yeast GcnSp is required for the activation of target genes in vivo. Genes Dev. 12, 627-639. Lees-Miller,S.P., Chen,Y.R. and Anderson,C.W. (1990) Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen. Mol. Cell. Biol. 12, 6472-6481. Lees-Miller,SP. (1996) The DNA-dependent protein kinase, DNA-PK: 10 years and no ends in sight. Biochem Cell. Bol. 74, 503-512. Madison, J.M. and Winston, F. (1997)Evidence that Spt3 functionally interacts with Mot1, TFIIA, and TATA-binding protein to confer promoter-specific transcriptional control in Saccharomyces cerevisiae. Mol. Cell. Bol. 17, 287295. Mages,G.J., Feldmann,H.M. and Winnacker,E.L. (1996) Involvernent of the Saccharomyces cerevisiae HDFl gene in DNA double-strand break repair and recombination. J. Biol. Chem. 271, 7910-7915. Marcus,G.A., Silvemian,N., Berger,S.L., Horiuchi,J. and Guarente,L. (1994) Functional similarity and physical association between GCN5 and ADA2: Putative transcriptional adaptors EMBO J. 13, 4807-4815.
Marcus,G.A., Horiuchi,J., Silveman,N. and Guarente,L. (1996) ADAS/SPT20 links the ADA and SPT genes, which are involved in yeast transcription. Mol. Cell. Biol. 16, 3197-3205. Martens,J.A, Genereaux,J., Saleh,A. and Brandl,C. J. (1996) Transcriptional activation by yeast PDRIp is inhibited by its association with NGG1p/ADA3p. J. Bioi. Chem. 271 , 15884-15890. Melcher, K. and Johnston, S.A. (1995) GAL4 interacts with TATA-binding protein and coactivators. Moi. Cell. Biol. 15, 2839-2848. Milne,G.T., Jin,S., Shannon-,K.B. and Weaver,D.T. (1996) Mutations in two Ku homologs define a DNA end-joining repair pathway in Saccharomyces cerevisiae. Mol. Cell. Biol. 16, 4 189-4 198. Moazed, D. and Johnson, D. (1996) A deubiquitinating enzyme interacts with SIR4 and regulates silencing in S. cerevisiae. Cell 86, 667-677. Moqtaden,Z., Bai,Y., Poon,D., Wei1,P.A. and Struhl,K. (1996) TBP-associated factors are not generally required for transcriptional activation in yeast. Nature 383, 188-191. Morrow,D.M., Tagle,D.A., Shiloh,Y., Collins,F.S. and Hieter,P. (1995) TEL 1, an S. cerevisiae homolog of the human gene mutated in ataxia telangiectasia, is functionally related to the yeast checkpoint gene MECI. Cell82, 831-840. OrphanideqG., Lagrange,T. and Reinberg,D. (1996) The general transcription factors of RNA polymerase II. Genes Dev. 10, 2657-2683. Pazin,M.J. and Kadonaga,J.T. (1997a) Nucleosome mobility and the maintenance of nucleosome positioning. Science 276. 809-812. Pazin,M.J. and Kadonaga, J.T. (1997b) SWWSNF2 and related proteins: ATPdriven motors that disrupt protein-DNA interactions? Cell 88, 737-740. Peterson,C.L. (1996) Multiple SWltches to tum on chromatin? Curr. Opin. Genet. Dev. 6, 171-175. Pina,B., Berger,S.L., Marcus,G.A., Silverman,N., Agapite,J. and Guarente,L. (1993) ADA3 - a gene, identified by resistance to GAL4-VP16, with properties similar to and different from those of ADA2. Mol. Cell. Biol. 13, 5981-5989. Pollard, K.J. and Peterson, C.L. (1997) Role for A DNGCNS products in antagonizing chromatin-mediated transcriptional repression. Mol. Cell. Biol.. 17, 621206222. Poon, D., Campbell, A.M., Bjorklund, S., Kim, Y., Zhou, S., Komberg, R.D. and Weil, P.A. (1995) Identification and characterization of a TFIID-like multiprotein
complex from Saccharomyces cerevisiae. Froc. Natl. Acad. Sci. USA. 92, 82248228. Reese, J.C., Apone, L., Walker, S.S., Griffin, L A . and Green, M.R (1994) Yeast TAF(1l)s in a multisubunit complex required for activated transcription Nature 371, 523-527. Roberts,S.M. and Winston,F. (1996) SPT20/ADA5 encodes a novel protein functionally related to the TATA-binding protein and important for transcription in Saccharomyces cerevisiae. Mol. Cell. Biol. 16 , 3206-3213. Roberts,S.M. and Winston, F. (1997) Essential functional interactions of SAGA, a Saccharomyces cerevisiae complex of Spt, Ada and Gcn5 proteins, with the Snf/Swi and Srtlmediator complexes. Genetics 147, 451-465. Roeder, R.G. (1996) The role of general initiation factors in transcription by RNA polymerase I1. Trends Biochem. Sci. 21 , 327-335. Ruiz-Garcia, A.B., Sendra, R., Pamblanco, M. and Tordera, V. (1997) GCN5p is involved in the acetylation of histone H3 in nucleosomes. FEBS Lett. 403, 186-190. Saleh,A., Lang,V., Cook,R. and Brand1,C. (1997) Identification of native complexes containing the yeast coactivator/repressor proteins NGGllADA3 and ADA2. J. Biol. Chem. 9, 5571-5578. Sanchez, Y., Desany, B.A., Jones, W.J., Liu, Q., Wang, B. and Elledge, S.J. (1996) Regulation of RAD53 by the ATM-like kinases MEC1 and TEL1 in yeast cell cycle checkpoint pathways. Science 19, 271 357-360. Scheffner,M., Huibregtse,J.M., Vierstra,R.D. and Howley,P.M. (1993) The HPV E6 and E6-AP cornplex functions as a ubiquitin protein ligase in the ubiquitination of p53. Ce1175, 495-505. Scheffner,M., Nuber,U. and Huibregtse,J.M. (1995) Protein ubiquitination involving an E l -E2-E3 enzyme ubiquitin thioester cascade. Nature 373, 81-83. Seufert,W. and Jentsch,S. (1990) Ubiquitin-conjugating enzymes UBC4 and UBC5 mediate selective degradation of short-lived and abnormal proteins. EMBO J. 9, 543-550. Sidorova, J.M. and Breeden, L.L. (1997) Rad53-dependent phosphorylation of Swi6 and down-regulation of CLNl and CLNP transcription occur in response to DNA damage in Saccharomyces cerevisiae. Genes Dev. 15, 3032-3045.
Siede,W., Allen, J.B., Elledge,S. J. and Friedberg,E.C. (1996) The Saccharomyces cerevisiae MEC1 gene, which encodes a homolog of the human ATM gene product, is required for G1 arrest following radiation treatment. J. Bacterid. 178, 5841 -5843. Silverrnan, N., Agapite, J. and Guarente, L. (1994) Yeast ADA2 protein binds to the VP16 protein activation domain and activates transcription. Proc. Natl. Acad. Sci. , USA, 91, 11665-11668. Spence,J., Sadis,S., Haas,A.L. and Finley,D. (1995) A ubiquitin mutant with specific defects in DNA repair and multiubiquitination. Mol. Cell. Biol. 1 5 , 1 2651273. Struhl,K. (1998) Histone acetylation and transcriptional regulatory mechanisms. Genes Dev. 12, 599-606. Tansey,W.P. and Heu, W. (1997) TAFs: guilt by association? Ce11 88, 729-732. Tay10r~S.S.. Knighton,D.R., Zheng,J., TenEyck,L.F. and Sowadski,J.M. (1992) Structural framework for the protein kinase family. Annu. Rev. Cell. Biol. 8, 429-462. Thompson,C.M. and Young,R.A. (1995) General requirement for RNA polymerase II holoenzymes in vivo. Proc. Natl. Acad. Sci. U S A., 92, 45874590. Walker,S.S., Reese,J.C., Apone,L.M. and Green,M.R. (1996) Transcription activation in cells lacking TAFIIS. Nature 1996, 383, 185-188. Wang, L., Miuen,C., Ying,C., Candau,R., Barlev, N., Brownell, J., Allis, C.D. and Berger, S.L. (1997) Histone acetyltransferase activity is conserved between yeast and human GCN5 and is required for complementation of growth and transcriptional activation. Mol. Cell. Biol. 1 7 , 5 19-527. Wang,L., Liu,L. and Berger,S.L. (1998) Critical residues for histone acetylation by Gcn5, functioning in Ada and SAGA complexes, are also required for transcriptional function in vivo. Genes Dev., 12, 640-653. Weinert,T.A., Kiser,G.L. and Hartwell,L.H. (1994) Mitotic checkpoint genes in budding yeast and the dependence of mitosis on DNA replication and repair. Genes Dev. 8, 652-665. Wilkinson,K.D. (1995) Role of ubiquitination in proteclysis and cellular regulation. Annu. Rev. Nutr. 15, 161-189. Winston,F., Durbin,K.J. and Fink,G.R. (1984) The SPT3 gene is required for normal transcription of Ty elements in S. cerevisiae. Ce1139, 649-656.
Winston, F., Dollard, C., Malone, E.A., Clare, J., Kapakos, J.G., Farabaugh, P. and Minehart, P.L. (1987) Three genes are required for trans-activation of Ty transcription in yeast. Genetics 115, 649-656.
NGG1p associated proteins revealed by immunoprecipitation
NGGl p interacting proteins revealed by coimmunoprecipitation. A whole cell extract from cells grown in 35Smethionine and
3%
-cysteine was
prepared by glass bead lysis from yeast strains expressing myc-NGGl p (lane 1) or NGGl p (negative control, lane 2). Approximately 8 mg of protein (108 trichloroacetic acid countshin) from each extract was incubated with anti-myc antibody and 25 ml of Protein A Sepharose. After washing in extraction buffer containing 450 mM NaCI, protein was eluted from the beads by heating at 95OC in SDS loading buffer, separated on 6.5% SDS-PAGE and detected by fluorography. Bands corresponding to
the mobilities of ADA2p and NGGl p are indicated as such. Proteins interacting with the cornplex are labelled with their approximate molecular masses in kDa.
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