without a requirement for galactose or GAL4, and an up- stream activation sequence (UAS)-less GAL] or CYCl pro- moter is constitutively expressed (5).
Vol. 13, No. 2
MOLECULAR AND CELLULAR BIOLOGY, Feb. 1993, p. 831-840 0270-7306/93/020831-10$02.00/0 Copyright © 1993, American Society for Microbiology
TSF3, a Global Regulatory Protein That Silences Transcription of Yeast GAL Genes, Also Mediates Repression by CL2 Repressor and Is Identical to SIN4 SHIMING CHEN,1 ROBERT W. WEST, JR.,'* STEPHEN L. JOHNSON 2t HAYLEY GANS,1 BRIAN KRUGER,1 AND JUNLI MA1 Department of Biochemistry and Molecular Biology, State University of New York Health Science Center, Syracuse, New York 13210,1 and Department of Genetics, University of Washington,
Seattle, Washington 981952 Received 10 September 1992/Returned for modification 6 October 1992/Accepted 30 October 1992
TSF3 encodes one of six (TSF1 to TSF6) recently identified global negative regulators of transcription in Saccharomyces cerevisiae. Mutant tsaf strains exhibit defects in transcriptional silencing of the GAL) promoter, allow expression from upstream activation sequence-less promoters, and exhibit pleiotropic defects in cell growth and development. Here we show that TSF3 is involved in transcriptional silencing mediated by the at2 repressor and demonstrate that specific systems of transcriptional silencing may depend on the more global role of TSF3. Cloning and sequencing of TSF3 allowed us to predict a 974-amino-acid gene product identical to SIN4, a negative regulator of transcription of the HO (homothallism) mating type switching endonuclease. TSF3 disruptions are not lethal but result in phenotypes similar to those of the originally isolated alleles. Our results, together with those of Y. W. Jiang and D. J. Stillman (Mol. Cell. Biol. 12:4503-4514, 1992), suggest that TSF3 (SIN4) affects the function of the basal transcription apparatus, and this effect in turn alters the manner in which the latter responds to upstream regulatory proteins.
Transcription of the Saccharomyces cerevisiae GALI and GAL10 genes is controlled by positive and negative (silencing) regulatory factors (5, 13-15, 33, 37, 40, 57). We recently obtained over 30 recessive mutations that alleviate silencing of the GAL1 and GAL1O promoters by GAL operator sequences (5). These mutations were subsequently identified as alleles of six different regulatory genes, designated TSFI to TSF6. Further analyses indicated that all six regulatory genes have pleiotropic effects on yeast gene expression and suggested that they encode general, rather than gene-specific, transcriptional regulatory factors. One of the six genes, TSF3, appears to play a major role in controlling the expression of transcriptionally silenced genes. In tsf3 strains, the wild-type GAL] promoter is transcribed at a detectable level without a requirement for galactose or GAL4, and an upstream activation sequence (UAS)-less GAL] or CYCl promoter is constitutively expressed (5). Moreover, tsf3 strains
MATERIALS AND METHODS Strains and plasmids. The S. cerevisiae strains used in this study are listed in Table 1. Eschenchia coli DH5a was used for routine cloning work. E. coli DIH101 (HB101 F+ Kan'; 29; a gift from David Mitchell) was used as the host strain to isolate single-stranded DNA for sequencing. A LEU2+disrupted merl strain used for genetic mapping of tsf3 (JE102-13D; Table 1) was provided by JoAnne Engebrecht and Shirleen Roeder. JF395 (galll [sptl3]) and the isogenic wild-type strain JF15 (GALI] [SPT13]) were provided by Jan Fassler. The yeast genomic DNA library in YEp24 (3) was provided by Janet Schultz and Marian Carlson. pRS316 (51; a gift from Robert Sikorski and Phil Hieter) and YEp352 (20; a gift from Mary Crivellone) were used to subclone parts of TSF3. pGEM4 containing the yeast actin gene was used as a control for mRNA levels in Northern (RNA) analysis and was a gift from Mary Crivellone. pCMSRB1-9 harboring the KEX2 gene (34) was a gift from Chris Martin and Rick Young. pBS+ and pBS- sequencing vectors were from Stratagene Inc. To determine the complementation of tsf3 by TSF3+ (on a URA3 vector), we constructed a HIS3 reporter plasmid, pSC-HllOA, from CYCI-UASG-110A (5, 14, 57) by inserting a 1.8-kb BamHI fragment containing the wild-type HIS3 gene into the StuI site of URA3. CYC1-oa2-1 and CYCl-ot2-4 were constructed by inserting one and four copies, respectively, of a 38-bp oligonucleotide harboring the MATTa2 operator (see below) between UASC and the TATA box of the CYCJ-lacZ fusion in plasmid pLGA-312 (5). Plasmids M467 harboring ALT6c2 (a gift from David Stillman) and KS-MCM1-NdeI harboring MCMI (a gift from Chantal Christ and Bik Tye) were used to generate labeled probes for Northern blot analysis of MAT4Ta2 and MCM1 expression in TSF3+ versus tsf3-10 strains. Media and chemicals. Media and chemicals were the same as those described by Finley et al. (13). Yeast transformations and fl-galactosidase assays. Yeast
are severely growth defective, showing temperature-sensi-
tive lethality, flocculence, and abnormal cell morphology, and exhibit defects in mating and in sporulation. Previously, analysis of TSF3-mediated transcriptional silencing was limited to systems in which few specific repressor molecules have been demonstrated (5). We examine here the role of TSF3 in a2-mediated repression of transcription. Cloning and sequencing of the TSF3 gene (this report) demonstrate it to be identical to SIN4, a gene identified from its role in the negative regulation of transcription of the HO mating type switching endonuclease (24). Together, these results indicate that the action of a wide variety of transcriptional repressors may depend on the activity of TSF3 (SIN4). (TSF3 was formerly designated GAL22 [4a].)
*
Corresponding author.
t Present address: Institute of Neuroscience, University of Ore-
gon, Eugene, OR 97403.
831
832
CHEN ET AL.
MOL. CELL. BIOL.
TABLE 1. S. cerevisiae strains useda Strain
Haploid YM335 YM256 BWY3 BWY54
BWY55 BWY102 BWY104
Genotype
..M....VATa ura3-52 lys2-801 his3-200 ade2-101 met 4al4g536 ..M....MATa ura3-52 lys2-801 his3-200 ade2-101, 101 met GAL4 ..M....MATa ura3-52 lys2-801 his3-200 ade2-101 met Agal4-536 leu2::HIS3+ ..M....AL4Ta Atsf3-2::LEU2' ura3-52 lys2-801 his3-200 ade2-101 met GAL4+ leu2::HIS3+ .. MATot .. Atsf3-2::LEU2' ura3-52 lys2-801 his3-200 ade2-101 met GAL4+leu2::HIS3+ ..M VATa tsf4.1 ura3-52 iys2-801 his3-200 ade2-101 Agal4-536 Met' ..MTa tsf2-5 ura3-52 iys2-801 his3-200 ade2-101 Agal4-536 Met' ..M....MATa tsf3-7 ura3-52 iys2-801 his3-200 ade2-101 met Agal4-536 ..MA X4Ta tsf3-10 ura3-52 lys2-801 his3-200 ade2-101 met Agal4-536 ..M....A4Ta tsf3-12 ura3-52 lys2-801 his3-200 ade2-101 met Agal4-536 ..M.... JATa tsf3-13 ura3-52 lys2-801 his3-200 ade2-101 met Agal4-536 ...... MATa tsf3-14 ura3-52 lys2-801 his3-200 ade2-101 met Agal4-536 ..M....MATa tsfl-1 ura3-52 iys2-801 his3-200 ade2-101 met Agal4-536 ..M.... JATa tsfS-1 ura3-52 Iys2-801 his3-200 ade2-101 Agal4-536 MET ..M....MATa tsf6-1 ura3-52 lys2-801 his3-200 ade2-101 met Agal4-536 .. MATa tsf3-27 ura3-52 iys2-801 his3-200 ade2-101 met Agal4-536 ..M..4ToTa tsf3-10 ura3-52 iys2-801 his3-200 ade2-101 met GAL4' .. MATa tsf3-lj- ade2 ade6 leu2-3,112 ura3-52 gall trpl his7
BWY113 BWY115 BWY117 BWY118 BWY119 BWY124 BWY132 BWY133 BWY135 BWY136 SJ1031-7 ..MAMTa merl::LEU2 met2 ura3 his4 leu2 JE102-13D JF15 ..M JTa lys2-128d ura3-52 ieu2 his4-917 JF395 ....... MA Ta sptl3-1 ura3-52 leu2 his4-917
Diploid BWY5002 ....... BWY5 x BWY6 BWY7003 ........ SJ5899-8a x SJ5899-18d SJ1300 ....... V4Ta/AL4 Ta tsf3-lj-ITSF3 met4/MET4 SJ1301 ..A 4Ta/MA4Ta tsf3-1-ITSF3 lys9/LYS9 SJ1303 ..AMA Ta/MA4Ta tsf3-lj-/TSF3 pha2/PHA2 SJ1304 ..A 4Ta/A4Ta tsf3-ljf/TSF3 met2/MET2 leu2-3,1121leu2-3,112 merl::LEU2/MER1 a YM and BWY strains are isogenic, except for BWY7003. All strains, except for YM, JF, and JE strains (see Materials and Methods), originated in this study. tsf3-lj- was isolated in an independent screening (5).
transformations and 3-galactosidase assays were performed as described by Finley et al. (13). For ,B-galactosidase assays, multiple identical constructs were tested simultaneously, individual samples were analyzed in duplicate in each experiment, and the results of at least four independent determinations performed on different days were averaged. The standard error was less than 15%. Values lower than 0.02 unit were not determined with precision. Oligonucleotides. A 34-bp synthetic oligonucleotide harboring a single copy of the wild-type AL4Ta2 operator sequence (25) was obtained from Genetic Designs, and the DNA was sequenced to confirm its fidelity (see below). The sequence contains Sall ends for cloning purposes as well as an asymmetrically located NlaIII restriction site for determining orientation and copy number. The oligonucleotide was inserted into the unique XhoI site of pLGA-312 (between UASC and the TATA box of the CYCl promoter; see Results). NlaIII tcgaCATGTAATTACCTAATAGGGAAATTTACACGCTG Sal SaiI GTACATTAATGGATTATCCCTTTAAATGTGOCGACagct Cloning of TSF3. Strain BWY118 (tsf3-13; 5) was transformed with YEp24 library DNA (3) to an efficiency of about 1,000 transformants per mg. Approximately 10,000 transformants were obtained and subsequently screened for a nonclumpy phenotype by the method of Schultz and Carlson (46), with slight modifications. In brief, transformants were recovered from regeneration agar by homogenization in a blender (48) and resuspended in liquid YEP-D medium. Cells were allowed to settle at room temperature for 30 to 60 min. Thereafter, cells were taken from the top of the culture and
passed into fresh YEP-D (48) medium. After settling-passaging was repeated several times, the enriched culture of nonclumpy cells was plated on synthetic defined (SD) medium lacking uracil, and then individual Ura+ colonies were tested for the presence or absence of the mutant phenotype (temperature sensitivity, flocculence, and derepression of the UASc-GAL hybrid promoter in pSC-HllOA, etc.). Transformants showing the wild-type phenotype were further analyzed by segregation analysis and by recovery of the YEp24 yeast library plasmid DNA in E. coli DH5a (by a modification of the yeast miniprep method of Sherman et al. [48]; provided by Keith Bostian) and transformation into various tsf3 strains (5) to test for complementation of the tsf3 phenotype. Subcloning of portions of TSF3. To delimit the location of the TSF3 coding sequence, we subcloned portions of the original clone in pSC-TSF3-1 (see Fig. 3) by using the single-copy vectors YCp5O and pRS316 and the multicopy vectors YEp352 and YEp24. pSC-6S contains a 6-kb Sall fragment of TSF3 in YEp24. pSC-2B contains a 2-kb BamHI fragment of the 3' portion of TSF3 in YEp24. pSC-5B contains a 5-kb BamHI fragment of TSF3 in YCp5O (pSC5B-S) or YEp24 (pSC-5B-M). pSC-Ap5B is identical to pSC-5B-S, except that it harbors a deletion of both the 0.4and the 0.7-kb PvuII fragments in the middle of TSF3. pSC-4XS contains a 4-kb XbaI-SalI fragment of TSF3 in YEp352. pSC-2BC harbors a 2.5-kb BamHI-ClaI fragment of TSF3 in pRS316. pSC-Atsf3-1 is a pBR327 derivative harboring a 5-kb BamHI fragment of TSF3 and in which both the 0.4- and the 0.7-kb PvuII fragments in the middle of the open reading frame (ORF) are deleted and replaced with a 2.2-kb
YEAST TRANSCRIPTIONAL SILENCING GENE TSF3 (SIN4)
VOL. 13, 1993
XhoI-SalI fragment of the LEU2 gene. pSC-Atsf3-2 is similar to pSC-Atsf3-1, except that the LEU2 gene replaces a 1,989-bp NheI-PvuII fragment encompassing the 5' portion of TSF3. DNA sequence analysis. For
pBS+-5B was obtained by
sequence analysis, inserting a 5-kb BamHI
subclone
tsf
subjected to tetrad analysis to recover haploid segregants harboring a tsf3 disruption. For TSF3 linker insertion mutagenesis, plasmid pSC-4XS (see Fig. 3) was cut with BclI or ClaI, and the sticky ends were filled in with the Klenow fragment and deoxynucleotide triphosphates. An 8-bp XhoI linker (New England Biolabs) was ligated into the filled-in BclI site to generate plasmid pSC-4XS-BX8. A 10-bp BglII linker was ligated into the filled-in ClaI site to generate plasmid pSC-4XS-CB10. That the TSF3 reading frame was restored in both cases was verified by DNA sequencing of both constructs and by transforming strain BWY54 (Atsf3-2) with pSC-4XS-BX8 or pSC-4XS-CB10 and observing the complementation of tsf3 defects (see Fig. 3). Poly(A)+ RNA isolation and Northern blot analysis. Total cellular RNA was prepared by the method of Sherman et al. (48). Poly(A)+ RNA was selected on an oligo(dT) column (Boehringer Mannheim Co.) prepared and used in accordance with the manufacturer's instructions. Total RNA or poly(A)+ RNA was loaded onto a 1% formaldehyde-agarose gel and, following electrophoresis, transferred to a GeneScreen Plus nylon membrane (New England Nuclear Co.). Radioactive probes were prepared by the method of random primer labeling (Boehringer). A 600-bp EcoRI-HindIII fragment control probe containing the yeast actin gene was sequence re-
ported here has been entered into the GenBank and EMBL data bases, under accession number X64516. RESULTS TSF3 is required for ci2 repressor function. Previous results suggested that TSF3 encodes a pleiotropic negative
partially suppresses ARTO2 operator activity
P-Galactosidase activity (U) produced in Glu Plasmida
fragment
from the original plasmid containing TSF3 (pSC-TSF3-1) into sequencing vector pBS+. Subclone pBS--6S was constructed by inserting a 6-kb Sall fragment from pSC-TSF3-1 into sequencing vector pBS-. Exonuclease III deletion derivatives from pBS+-5B and pBS--6S were obtained by use of the Erase-a-base protocol of Promega Corp. DNA sequencing was performed by the dideoxy chain termination method with a Sequenase kit (U.S. Biochemical Corp.). Computer analysis. Nucleic acid and protein sequences were analyzed by use of University of Wisconsin Genetics Computer Group software version 7.0 (8) run on a VAX 6310 minicomputer at the State University of New York Health Science Center, Syracuse, computer center (default settings were used for the analysis). TSF3 gene disruption and linker insertion mutagenesis. For TSF3 gene disruption, plasmids pSC-Atsf3-1 and pSCAtsf3-2 (described above; see Fig. 3) were digested with BamHI to release a 5.7-kb fragment of TSF3 harboring a LEU2 disruption. Purified fragment DNA was used to transform diploid strains BWY5002 (pSC-Atsf3-l and pSCAtsf3-2) and BWY7003 (pSC-Atsf3-2) to Leu+ prototrophy. Transformants in which the TSF3 gene on one of the two homologs was disrupted were identified by Southern blot analysis with the 2.2-kb 32P-radiolabeled XhoI-SalI fragment of LEU2 and the 0.7-kb 32P-radiolabeled BamHI-XbaI fragment of TSF3 as probes. Leu+ diploids were sporulated and
prepared from plasmid pGEM4. Nucleotide sequence accession number. The
TABLE 2.
833
YM335 (M Ta
medium-grown strain: BWY3 BWY115 (MA Ta (MA Ta
BWY136
TSF3+)
TSF3+)
tsf3-10)
tsf3-10)
130 254 380
178 16 2
CYCI (wild type) CYCl-a2-1
255
0.4
CYCl-at2-4
300
50 >50 >50
SJ1304
tsf3-merl::LEU2
B
No. of the following tetrad typea: PD NPD TT
SJ1303
PD, parental ditype; NPD, nonparental ditype; TI, tetratype. Map distances were calculated by the formula [50(T + 6NPD)/[total tetrads (PD + NPD + T)] (41). tsf3 was scored on the basis of temperaturesensitive growth and rough colony morphology, defects that cosegregate with relaxed repression of the GAL1O promoter (data not shown). a
b
FIG. 1. tsf3 does not alter AMTa2 orMCMI mRNA levels. RNA prepared for Northern blot analysis from yeast strains BWY3 (AMTot TSF3) and BWY136 (MATa tsf3-10) grown in Glu medium. (A) Approximately 15 p,g of poly(A)+ RNA from each strain was loaded onto a 1% denaturing agarose gel, transferred to a nylon membrane following electrophoresis, and probed with a 32P-radiolabeled 1.1-kb EagI-NdeI fragment of MA4Ta2. A 32P-radiolabeled 0.6-kb EcoRI-HindIII fragment of the ACTI gene was used as an internal standard for the RNA concentration (40-fold-lower specific activity of the control probe than of the MATa2 probe was used). (B) Fifty micrograms of total RNA from each strain was blotted and probed with a 32P-radiolabeled 0.6-kb EcoRI-NdeI fragment of MCM1. Several distinct MCMI transcripts were detectable (40a). was
map units from met2 (Fig. 2 and Table 3). The gene order, . telomere-met2-tsf3-merl-centromere, is suggested by the finding that the met2-merl recombination distance in this cross (28 map units) is longer than either of the former distances. This conclusion is supported by strand analysis of the four merl-tsf3 recombinant tetrads, since the proposed order requires only one four-strand double crossover, whereas two four-strand double crossovers would need to be postulated if the gene order were telomere-met2-merl-tsf3centromere (data not shown). Cloning of TSF3. ts3 mutations cause a variety of growth defects, such as clumpiness, temperature-sensitive lethality, and abnormal cell morphology (5). We exploited the clumpy phenotype of strain BWY118 (tsf3-13; 5) to clone the wildtype TSF3 gene by using YEp24 yeast gene library plasmid DNA (a gift from Marian Carlson). Three nonclumpy strains were isolated from 10,000 original colonies recovered upon transformation of BWY118 spheroplasts with YEp24 library plasmid DNA. Segregation analysis subsequently showed that each of the three strains relied on the presence of the YEp24 library plasmid DNA to maintain a wild-type (nonclumpy) phenotype. Plasmid DNA isolated from these three strains complemented the mutant phenotype of seven originally isolated tsf3 strains (BWY113, BWY115, BWY117, BWY118, BWY119, BWY135, and SJ1031-7b) and failed to complement the mutant phenotype of tsfl, tsf2, tsf4, tsf5, and tsf6 strains (BWY124, BWY104, BWY102, BWY132, and BWY133, respectively). Plasmids recovered from two of these strains, pSC-TSF3-1 and pSC-TSF3-3, were shown by restriction mapping studies to contain an identical 8-kb DNA
pha2
merl
met2
met4
Iys9
I
(
I kex2
FIG. 2.
tsf
I
)
tsf3
maps
distal to merl
on
chromosome 14.
insert, while a plasmid recovered from the third strain, pSC-TSF3-2, contained a 6.5-kb overlapping DNA fragment (Fig. 3). A 5.0-kb BamHI fragment common to the three yeast inserts was sufficient to complement all of the phenotypic defects of tsf3 strains. Complementation was not due to multicopy suppression, since the 5.0-kb fragment complemented tsJ3 defects when present on either single-copy centromeric vector YCpS0 (pSC-5B-S) or multicopy vector YEp24 (pSC-5B-M) (Fig. 3; see Materials and Methods). The map position for tsf3, 6 centimorgans distal to merl, suggested that the TSF3 gene should be near the KEX2 gene, which maps to the region. Restriction map analysis of TSF3 plasmids pSC-TSF3-2 and pCMSRB1-9 (KEX2; 34) suggested an extensive overlap between these plasmids. Subsequent sequencing of the insert in pSC-TSF3-2 confirmed this overlap and indicated that the TSF3 coding region lies 2 kb 3' of the KEX2 coding region (Fig. 3). The physical proximity of the cloned gene to the genetically mapped position confirms that the clone corresponds to the tsf3 mutations. Nucleotide sequence analysis and identification of the TSF3 coding region. The DNA sequence of most of the 5.0-kb BamHI fragment containing TSF3 was determined by use of the sequencing strategy shown in Fig. 3. A long ORF designated ORF1 encompasses 2,922 bp of this sequence, starting with an ATG codon at nucleotide +1 and terminating with a TAG codon at nucleotide +2,923 (Fig. 4). A shorter ORF, ORF2 (Fig. 3), overlaps ORF1 on the opposite strand at its carboxy terminus, between nucleotides +3050 (ATG) and +2625 (TAA) (Fig. 3). To determine which of these two ORFs represented TSF3, we subcloned portions of the 5.0-kb BamHI fragment into multicopy plasmid YEp352 or centromeric plasmid YCp5O (or pRS316; see Materials and Methods) and transformed various tsf3 strains with the respective plasmid. A 4.0-kb XbaI-SalI fragment containing ORF1 in plasmid pSC-4XS (Fig. 3) complemented the derepressed phenotype, clumpiness, temperature sensitivity, and abnormal cell morphology of tsf3 strains, suggesting that ORF1 corresponds to TSF3. Consistent with this result, the removal of a 1.1-kb PvuII fragment from the middle of ORF1 (plasmid pSC-Ap5B; Fig. 3) precluded the ability to complement the tsf defects, and a 2.5-kb BamHI-ClaI fragment containing ORF2 (plasmid pSC-2BC; Fig. 3) failed to complement the tsfl defects. TSF3 is identical to SIN4. The predicted translation product for TSF3 (Fig. 3 and 4) is 974 amino acids long, a size that would correspond to a protein of 111,316 daltons. The deduced TSF3 protein is moderately charged, comprises 13.1% basic and 9.75% acidic amino acids, and has a
YEAST TRANSCRIPTIONAL SILENCING GENE TSF3
VOL. 13, 1993
0
1
2
3
1
1
1
I
Sal YEp24
I
I
4
Bam Bcl Xba Pvu
5
6
7
l
I
I
1I
I
I
nd rYEp24
__ '
::-::*-
835
8
Cia Bcl Pvu/ Pvu Nhe Eco Sal Bam
>1
(SIN4)
pSC -TSF3 -1, -3
*-'..
YEp24 I
.YEp24 pSC
i
-TSF3 - 2 TSF3
Subclone pSC-6S pSC-2B
4--
(KEX2)
pCMSRB1 -9 pSC-5B
+
pSC-4XS
+
pSC-2 BC pSC-Ap5 B
,2!
LEU2
pSC-Atsf3
LEU2
pSC-Atsf3- 2
BgI-1 0
pSC-4XS-CB1 0
7
Xho-8
Sequenced
--lo.z-*T-00.
ORFI
OR
2922bp (974aa)
+
pSC-4XS-BX8
7
-.A
- 1
.
1377
ORF3
-5
portion
Predicted ORFs
bp (459 aa)
429bp (143aa) FIG. 3. Cloning and mutagenesis of TSF3. pSC-TSF3-1, -2, and -3, derived from the YEp24 library, are drawn approximately to scale. Relevant restriction sites are noted; unique sites are in boldface type. The scale at the top is in kilobase pairs. Subcloned TSF3 sequences are shown below; bars designate the presence of the corresponding DNA sequence. In pCMSRB1-9, the position of the KEX2 carboxy terminus is shown. In pSC-Ap5B, parentheses denote the portion of TSF3 deleted. In pSC-Atsf3-1 and -2, the positions of insertion of LEU2+ are indicated, and in pSC-4XS-CB10 and pSC-4XS-BX8, the positions of linker insertions are denoted (see the text). Results of complementation for the respective TSF3 subclones are shown at right: +, complementation of tsf3 strains; -, no complementation. The sequencing strategy and predicted ORFs (ORF1, ORF2 [(TSF3)], and ORF3) are shown at the bottom. The single long ORF of 1,377 bp, designated ORF3, that resides between TSF3 and KEX2 predicts a deduced protein of 438 amino acids (aa) that harbors little sequence similarity to other proteins in the SwissProt data base.
predicted isoelectric point of 8.9. The deduced TSF3 protein contains overall 16.2% Ser and Thr and concomitantly harbors a number of consensus sites for possible recognition by several different protein kinases (28), including six consensus sites for cyclic AMP-dependent protein kinase [(RI K)2X(S/T)] and 14 each for protein kinase C [(S/T)X(R/K)] and casein kinase II [(S/T)XX(D/E)]. Additionally, between positions 63 and 81 is a stretch of 19 amino acids, of which 14 are Ser or Thr, that may play a role in transcriptional repression (6). The sequence NX(S/T), which is a potential
site for N-glycosylation (32), appears 13 times. The signifiof any of these sites remains to be determined. The TSF3 gene shows little codon use bias, a result suggesting that the product is not abundant, consistent with its predicted role as a regulatory molecule. No striking homology was found between the deduced TSF3 protein and proteins in the SwissProt data base (June 1991 release; FASTA program). Sequence analysis with the personal data base of Mark Goebl (1Sa), however, revealed that TSF3 is identical to SIN4, a negative regulator of the cance
836
CHEN ET AL.
MOL. CELL. BIOL.
EcoRI -512 -456 45I 6 -336
_ Nh.l
-216 ATAAAT
-96 91Tu
ATCcTrAGrcicCrTGTA lICT
_
_
_
PAGAAAAAAAA
lllGMTG
_
M M L G E H L M 25
8
C TGAT AM S W S K T G I I A Y S D S Q S S N A N I C L T F L E S I N G I N W R F H T P Q K
48
Y V L H P a L H E V Q Y Q E S SAS T L S T H S T T T S V N G S T T A G V G S T P
88
145 265
N F G G N S N K S P P Q F F Y N
S S
H W N N W F S L P G D M L A V C D E L G
128
385 N M T M L I T G CQ R P D R A T T Y E K L T M V F Q D N V Y K I Y N H V M P L K P
168
V D K L K P M N I E R K 0 T R K E Y N T S 625
A F D S S S N T Y R S R A 00
L E F R W L T S S K S V I V S
O F C
208
TA V P P Y G V Y H P P F I K Y A C L A I R K N G Q I 248
745
kTrACA= 0 L L D T S N Q R F K D L 0 W L E F A R I T P
288
865 AnAA M N D D Q C M L I T T Y S K L S K N I S F Y K L H V N W N L N A T K P N V L N D
328
D F W Y a F S N SK D H K K I T L BcIl
985 1105
P S L K I a F I L S T T L D P T D D E G H V L K L E N L H V V S K S S I E K D P
368
A CTW TA _ _ _ T S P E I L V L Y N V C D T S K S L V K R Y R L A P T Q L S A E Y L V I L K P D L
408
1 225 MTAT Iz iTCTC1GATGTCi ;lrTafACATATCTCGTL~IU,~WI~X N I D R N N S T N Q I F Q S R R Y N L R R H S D I V L D K K V T L T S E M F D 1 345
C lii IC 13 3P aM=ACAATATTATr A F V S F Y F E D G T I E S Y N Q N D W K L E T E R L I S Q S a L G K F K N I I
448 488
1465
A S P L S A G F N Y G K L P L P P S V E W M K V S P S M C G V
V K0
Y N K K W
528
1585 P 0 F Y A A V Q K N Y A D P E K D S I N A T A L A F G Y V K S L H K Q I S A E D
L T I A A K T P I L R I S F L D R K R A K E F 1825
568
IcGT
1705
T T L L K S L Y S F F N I S P D
608
IGCMAAAC"7TAY;YMX3GcXW
A P K E I M D K
T S R P L 0 K I M L L 0 L E L G S C F S 0 E N I E E M A R V
648
0 0 0 N P K L F Q T
688
P V A K W F V K F I T Y L T 0 E I L I L I N D P T N K E
728
1 945
I L Y L K N V L F A F N G V A R N F H F A I E
0 IS N N S N
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I F S K 0 D L I H S L 2185
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V A K F P E T S Y P
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2305 2425
L N E I K K V T 0
I L N E S S T F L K L V L S E S P V D F E K F E T F L V D V N N K F I A L C E Q
808
CCA 0 P S Q E R E F S L L V K A E
Y A N N A V I S H A N
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S N S E F F N P E I F H L L Q P L E E G L I I D T D
888
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2545
A A A V Y F A D T S G L K 2665
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928
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K L P I K N R T S K S F S K L L Y D D V T C D K L S V S E 2785
G C G S V T R A G N
I SS D K T
AM 2905 UG l AAC 3 IWlCGGM TTTCTT L F E M D G974
Xbal I 3025 lCl 3146 3265 G 3385_
aaIIlc
VOL. 13, 1993
YEAST TRANSCRIPTIONAL SILENCING GENE TSF3 (SIN4)
yeast HO gene (24; see Discussion). Motifs present in other known general transcriptional regulatory proteins, such as tracts of acidic amino acids (43, 54, 55), polyglutamine or poly(glutamine-alanine) (46, 53), and tetratricopeptide (TPR) repeats (16, 47, 50), etc., were not detected in the deduced TSF3 protein. TSF3 is not regulated by carbon catabolite repression. Northern blot analysis was performed on poly(A)+ RNA isolated from wild-type and Atsf3 strains grown under catabolite-repressing and non-catabolite-repressing conditions. Figure 5 shows that the TSF3 probe taken from an internal fragment of the coding region hybridized with a 3-kb band in lanes of RNA from wild-type cells but not mutant cells. The observed band at 3 kb is consistent with that expected for an ORF of 2,922 bp. We observed no differences in the expression of TSF3 transcripts under catabolite-repressing or noncatabolite-repressing conditions, a result suggesting that there is no role for control of the expression of TSF3 itself through the catabolite repression pathway in transcriptional silencing at the GALl-GAL10 locus. TSF3 gene disruption shows that TSF3 is not essential. We disrupted the chromosomal copy by gene transplacement (45) with the LEU2+ gene as a selectable marker. Two different null mutations were constructed by use of plasmids pSC-Atsf3-1 and pSC-Atsf3-2 (Fig. 3). In plasmid pSCAtsf3-1, the LEU2 gene replaced a 1.1-kb PvuII fragment in the middle of TSF3, while in plasmid pSC-Atsf3-2, the LEU2 gene replaced a 1,989-bp NheI-PvuII fragment encompassing the 5' portion of TSF3. Both disrupted genes were transplaced into the genomes of two diploid strains, BVVY5002 and BWY7003, which have different genetic backgrounds. Southern analysis confirmed that only one of the two homologs was disrupted in each of the four resulting diploid strains (data not shown). When the respective diploids were sporulated and 6 to 10 tetrads were dissected for each sporulated strain, phenotypes similar to those of tsf3 point mutants (temperature-sensitive lethality, flocculence, abnormal cell morphology, and mating and sporulation defects; Table 4; see also below) cosegregated 2:2 with the LEU2+ phenotype. Southern analysis confirmed that two LEU2+ spores from each tetrad contained a tsf3::LEU2 disruption (data not shown). Northern analysis confirmed that TSF3-specific poly(A)+ RNA was missing from Atsf3-2 (Fig. 5) and AtsJ3-1 (data not shown) null mutant strains. Table 4 shows that in addition to causing defective growth, the Atsf3-1 mutation, like the original tsf3 point mutations (Table 4) (5), caused elevated expression from the wild-type GAL] promoter under noninducing conditions (glycerol- and lactate-containing medium), from a CYCl-GAL hybrid promoter (CYCl-UASG-110A) containing operator sequences (GALO3 and GALO5; 5), and from UAS-less GALl and CYC1 promoters. Altogether, these results suggest that TSF3 is important, but not essential, for growth and development. tsJ3 and gall) mutants share pleiotropic transcriptional defects. We found that galll (sptl3), like tsf3, causes UASless promoters to be constitutively expressed and derepresses the uninduced level of expression of the wild-type
837
A tsf3 G R G R ..- 5
TSF3 t
TS F3
'All. -i "W, * ;W,
-
-z. .4.
_~~5.3 2.8
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FIG. 5. Identification of a TSF3-specific poly(A)+ RNA. Poly(A)+ RNA was prepared from YM256 (wild type; TSF3) and BWY55 (/v tsf3-2) grown in glucose (G)- or raffinose (R)-containing medium. Approximately 10 Fg of poly(A)+ RNA from each source was loaded onto a 1% denaturing agarose gel, transferred to a nylon membrane following electrophoresis, and probed with a 32P-radiolabeled 0.7-kb PvuII fragment of TSF3 (Fig. 3). A 32P-radiolabeled 0.6-kb EcoRI-HindIII fragment of the actin gene was used as an internal standard for the RNA concentration. Tenfold less control probe (< 1 ng/ml) than TSF3 probe was used. The sizes (in kilobases) of RNA molecular weight markers (Boehringer) are indicated at right.
GALl promoter. Table 5 shows that expression in a galll strain (JF395) in Glu medium of the UAS-less GALl promoter (fused to lacZ) occurred at a level at least 160-fold higher than that in the isogenic GAUl + strain (JF15). This amount of derepression was proportional to that caused by tsf3 (Table 5). A similar result was observed with a UAS-less CYCl promoter (Table 5). galll further caused the wild-type GAL] promoter to be expressed at a level at least 10-fold higher than normal in the absence of galactose (Table 5) (5). Combined with the facts that GALl] and TSF3 have similar pleiotropic transcriptional regulatory effects (5, 11, 12, 39) and that mutations in these genes cause similar growth defects (5, 11, 12, 39), these results raise the possibility that the respective proteins have common regulatory functions (see below). DISCUSSION
Transcription of GAL] and GALJO when galactose is absent, or when glucose is present in addition to galactose, is significantly impaired by negative control elements in UASG (13-15, 33, 37, 57). tsf3 mutations were obtained (5) and the TSF3 gene was cloned and characterized (this report) as part of our effort to determine the molecular basis of this negative regulation. Flick and Johnston (15) have similarly characterized mutations in three URR genes that override the repression by GAL negative control elements of a UASLEU2-HIS3 promoter fusion. Unlike tsf3 mutants, however, none of the urr mutants exhibits a temperature-sensitive growth phenotype, suggesting that the URR genes are unique from TSF3.
FIG. 4. Nucleotide sequence of TSF3 and its deduced amino acid sequence. Nucleotides are numbered at left, with +1 designated as the first base of the initiation codon. Amino acids are numbered at right. TATA boxes, putative initiation (TAAG; ATG; 2, 4, 9) and termination and/or polyadenylation (22, 23, 42, 58) signals, restriction sites, a Ser- and Thr-rich region between amino acids 63 and 81, a sequence between amino acids 889 and 893 that is homologous to the nuclear localization signal of the a2 repressor, and a potential zinc finger domain between amino acids 926 and 965 are underlined (see the text).
838
CHEN ET AL.
MOL. CELL. BIOL.
TABLE 4. Phenotypes of tsf3 null versus ts# point mutants 1-Galactosidase activityb Strain
YM256 BWY54 BWY113 BWY115 BWY118 a b
Temperature sensitivity
Allele
TSF3+ tsf3-7
+ +
tsf3-10
+c
tsf3-13
+
Atsf3-2
Flocculencea
++ ++ ++ ++
Mating efficiency
MATa
MATot
1.00 0.09 0.02 0.02 ND
1.00 0.06 0.01 0.01 ND
Wild type
GALl .0.1 80 150 35 32
(4,400) (3,560) (ND) (3,700) (3,500)
Hybrid
(CYC1-
CYCI
UASG-11OA)
165 158 150 178 193
5 40 35 44 50
UAS-less
GALI 0.2 38 ND 11 26
CYCI 0.3 52 ND 23 32
+vr 1_ -, nonclumpy (wild type); + +, very clumpy.
1-Galactosidase activities (U) are from isogenic strains having the indicated TSF3+ (YM256) or tf3 allele (Table 1) and harboring a multicopy plasmid that
contains (fused to lacZ) the wild-type GALl promoter (pRY131), the wild-type CYCI promoter (pLGA-312), a CYC-GAL hybrid promoter (CYCl-UASG-11OA), the UAS-less GALl promoter (plasmid 121-632), or the UAS-less CYCI promoter (pLG67OZ). Values shown are from cells grown in Glu medium, except that those for the wild-type GALI promoter are from cells grown in SD medium containing glycerol and lactate. Values in parentheses are from cells grown in SD medium containing galactose plus glycerol and lactate and are shown for comparison. ND, not determined. c Leaky temperature-sensitive (36'C) phenotype.
Our previous data suggesting that TSF3 encodes a global negative transcriptional regulatory protein (5) are consistent with our present finding that TSF3 is identical to SIN4, a negative transcriptional regulator of the HO mating type switching endonuclease (24). Other S. cerevisiae transcriptional control proteins having pleiotropic negative effects on transcription have been characterized and include SIN3 (SDI1, UME4, or RPD1; 36, 52, 56) and SSN20 (SPT6 or CRE2; 7, 38, 54). Global regulatory proteins that play positive as well as negative regulatory roles in transcription also have been characterized and include SIN1 (SPT2; 31, 44), which encodes an HMG-like chromatin protein, and GAL11 (SPT13; 11, 12, 53), which is postulated to encode a transcriptional potentiator protein (21). The functional role of TSF3 (SIN4) may be more like that of the latter category of proteins. For example, Jiang and Stillman (24) found that a SIN4-LEXA fusion protein moderately activates transcription from test promoters containing LEXA binding sites, suggesting that TSF3 (SIN4) can contribute to promoter activation as well as repression. Moreover, tsf3 (sin4) strains demonstrate constitutive expression of UAS-less promoters as well as elevated expression of the wild-type GALl promoter (5), and we demonstrate here that strains harboring a mutated version of the putative transcriptional potentiator protein GAL11 cause similar transcriptional defects (Table 5). These results, taken together, favor a model in which TSF3 (SIN4) and perhaps GAL11, potentiates transcripTABLE 5. galll (sptl3) activates constitutive expression of UAS-less promoters and the wild-type GALl promoter ,-Galactosidase activity in strains harboring the
following promoter': Wild type UAS-less
Strain
GALl
JF15 (GAL11+) JF395 (galll) YM335 (TSF3+) BWY118 (tsf3-13)
CYC1
GALl
2.0 (1)
