Functional interaction of general transcription ... - Wiley Online Library

Functional interaction of general transcription initiation factor TFIIE with general chromatin factor SPT16/CDC68 Seung-Woo Kang1,a, Takashi Kuzuhara1 and Masami Horikoshi1,2,* 1

Laboratory of Developmental Biology, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan 2 Horikoshi Gene Selector Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Corporation ( JST), 5-6-9 Tokodai, Tsukuba, Ibaraki 300-2635, Japan

Abstract Background: Transcriptional initiation of class II genes is one of the major targets for the regulation of gene expression and is carried out by RNA polymerase II and many auxiliary factors, which include general transcription initiation factors (GTFs). TFIIE, one of the GTFs, functions at the later stage of transcription initiation. As recent studies indicated the possibility that TFIIE may have a role in chromatin transcriptional regulation, we isolated TFIIE-interacting factors which have chromatin-related functions. Results: Using the yeast two-hybrid screening system, we isolated the C-terminal part of the human homologue of Saccharomyces cerevisiae (y) Spt16p/ Cdc68p, a general chromatin factor. The C-terminal part of human SPT16/CDC68 directly interacts with TFIIE, and ySpt16p/Cdc68p also interacts with yTFIIE (Tfa1p/Tfa2p), thus indicating the

Introduction Accurate transcription initiation of class II genes in vitro requires a set of components: RNA polymerase II and several auxiliary factors, including general transcription initiation factors (GTFs) (Matsui et al. 1980). Functional roles of each GTF, including TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH in transcription initiation Communicated by: Akira Ishihama * Correspondence: E-mail: [email protected] a Present address: Microbial Chemistry, Center for Basic Research, The Kitasato Institute, 5-9-1 Shirokane, Minatoku, Tokyo 108-8642, Japan. q Blackwell Science Limited

existence of an evolutionarily conserved interaction between TFIIE and SPT16/CDC68. Functional interaction of yTFIIE and ySpt16p/Cdc68p was examined using a conditional yTFIIE-a mutant strain. Over-expression of ySpt16p/Cdc68p suppressed the phenotype of cold sensitivity of the yTFIIE-a-cs mutant strain, and in vitro binding assays revealed that yTFIIE-a-cs mutant protein showed diminished binding af®nity to ySpt16p/ Cdc68p. Conclusions: These observations indicate that general transcription initiation factor TFIIE functionally interacts with general chromatin factor SPT16/ CDC68, a ®nding which provides new insight into the involvement of TFIIE in chromatin transcription. This may well lead to a breakthrough in relationships between the transcription initiation process and structural changes in chromatin.

have been relatively well-characterized by in vitro interactions and transcription assays using a reconstituted system and naked DNA as a template (for reviews, see Zawel & Reinberg 1995; Roeder 1996). In eukaryotes, chromatin structure has an inhibitory effect on many processes through preventing factors from gaining access to DNA (for review, see Kornberg & Lorch 1999b). Therefore, initiation of eukaryotic transcription is likely to be regulated at the level of chromatin (for review, see Struhl 1999). Chromatin remodeling is considered to be a prerequisite event in most reactions occurring on the DNA template (for review, see Kornberg & Lorch 1999b). Several chromatin remodeling factors were found to be Genes to Cells (2000) 5, 251±263

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required for transcription (for review, see Workman & Kingston 1998), hence GTFs may also be involved in chromatin remodeling processes, either directly or indirectly. Studies on functional roles of general transcription initiation factors in remodeling of chromatin structure so far documented include only those related to TFIID and TFIID-interacting factors with histone acetyltransferase activity (Mizzen et al. 1996; Yamamoto & Horikoshi 1997). TFIIE is a GTF which functions at the later stage of transcription initiation; promoter opening and promoter clearance steps by recruiting TFIIH to the preinitiation complex and/or regulating the enzymatic activities of TFIIH, a ®nal component in preinitiation complex formation (Buratowski et al. 1989; Ohkuma et al. 1990, 1995; Goodrich & Tjian 1994; Ohkuma & Roeder 1994; Holstege et al. 1996; Okamoto et al. 1998; Yokomori et al. 1998). TFIIE may participate in key conformational switching occurring at the active centre upon RNA polymerase II±DNA interaction (Leuther et al. 1996). TFIIE may also participate in formation of the open complex, independent of TFIIH, which melts double-stranded DNA to single-stranded DNA (Tyree et al. 1993; Holstege et al. 1995), and it was reported that TFIIE is capable of binding to singlestranded DNA (Buratowski et al. 1991; Kuldell & Buratowski 1997). Formation of the open complex requires topological change of DNA, which could be largely affected by chromatin remodeling as disruption of the chromatin structure results in negative supercoiling of naked DNA. When the template is negatively supercoiled, immunoglobulin heavy chain (IgH) gene transcription in vitro can be reconstituted with a minimal reaction containing only TATA box-binding protein (TBP), TFIIB, and RNA polymerase II, without TFIIE (Parvin & Sharp 1993). In addition, it was reported that TFIIE may recruit TFIID with TFIIA at the ®rst stage of transcription initiation step (Yokomori et al. 1998). Moreover, yeast RNA polymerase II holoenzyme can support transcription, including TFIIA, TFIID, TFIIB forming the preinitiation complex, without TFIIE (Koleske & Young 1994), although a mammalian RNA polymerase II holoenzyme contains all components, including TFIIE (Ossipow et al. 1995). Interaction of KruÈppel, a transcriptional negative cofactor involved in chromatin transcription, with TFIIE results in transcriptional repression (Sauer et al. 1995). TFIIE could be acetylated by histone acetyltransferase p300 and PCAF (Imhof et al. 1997). In light of these ®ndings, we considered that TFIIE might have other functions not detectable by in vitro transcription assay, which makes use of naked DNA 252

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as a template. To consider the role of TFIIE in transcription initiation processes, its function on chromatin DNA template would be at the later stage of initiation or the transition step to elongation of transcription. Hence, interactions of TFIIE with chromatin factors which function on both initiation and early elongation steps of transcription, could be predicted. It was reported that human SPT16/CDC68 is a component of FACT, the chromatin-speci®c transcription elongation factor in vitro (LeRoy et al. 1998; Orphanides et al. 1999), speculating that this factor would be one candidate of TFIIE-interacting chromatin factors, for the reason given above. ySPT16/CDC68 is an essential nuclear protein required for transcription of various genes (Malone et al. 1991; Rowley et al. 1991) and shown to be antagonized by San1, a protein implicated in transcriptional silencing (Xu et al. 1993), suggesting its involvement of chromatin transcription (Brewster et al. 1998). In spt16 mutants, the cell cycle is arrested at the phase of START by a defect in SWI4 expression, which is required for expression of G1 cyclins (Lycan et al. 1994). Furthermore, SPT16/ CDC68 is reported to be required for DNA replication (Wittmeyer & Formosa 1997; Okuhara et al. 1999; Wittmeyer et al. 1999). Therefore, SPT16/CDC68 is a general chromatin factor which is commonly required for both transcription and DNA replication. Using the yeast two-hybrid system we screened TFIIE-interacting factors which have chromatinrelated functions (Durfee et al. 1993). Among the isolated TFIIE-interacting factors, the human homologue of ySPT16/CDC68 was identi®ed. We report here that TFIIE interacts with SPT16/CDC68 both biochemically and genetically. The human homologue of Spt16p was reported to be the subunit of FACT (Orphanides et al. 1999) which facilitates transcription elongation on chromatin templates. We also discuss another possibility that SPT16/CDC68 may also participate in transcription initiation via interaction with TFIIE, in addition to the transcription elongation process.

Results Isolation of human SPT16/CDC68 as a human TFIIE-b-interacting factor To investigate the functional role of TFIIE in transcription on the chromatin DNA template, we screened and obtained several TFIIE-interacting factors from a human peripheral lymphocyte cDNA library, q Blackwell Science Limited

Functional interaction of TFIIE and SPT16/CDC68

using the yeast two-hybrid system (Harper et al. 1993). We isolated two independent clones of the human homologue of Saccharomyces cerevisiae (y)Spt16p/Cdc68p as a TFIIE-b-interacting factor (Fig. 1A). Although the

deduced amino acid sequence of the isolated clone did not cover the full length of the protein, it was evolutionarily conserved (55% identity and 76% similarity, from 829 to 951 amino acids), compared to

Figure 1 Isolation of the cDNA encoding the human homologue of ySpt16p/Cdc68p as a hTFIIE-b-interacting factor. (A) Alignment of human and yeast SPT16/CDC68. The upper sequence is human SPT16/CDC68 and lower one is S. cerevisiae homologue. The middle one shows identical amino acids and () means similar amino acids grouped as (N,Q,D,E) (T,S,A) (L,I,V,F) (S,T) (Y,F) (E,Q) (G,S,D,N). The region from 829 to 951 amino acids is conserved and the region from 952 to 1002 includes an acidic amino acids stretch. (B) The b-galactosidase assay shows the speci®c interaction between hTFIIE and hSPT16/CDC68 in a yeast two-hybrid system. hTAFII250, hTAFII80, hRAP74, yTFIIB, hTFIIB, hTBP, SNF1 and hTFIIE-b were used as baits. CDC68 and SNF4 are preys. Blue colonies mean the interaction between the bait and the prey. (C) Physical interaction between hTFIIE-b and hSPT16/CDC68. The physical interaction between hTFIIE-b and hSPT16/CDC68 was detected, using GST pull-down assay. GSTor GST-hSPT16/CDC68 (100 ng) was immobilized on a glutathione-af®nity gel (Glutathione-Sepharose 4B, Pharmacia) and 6His-tagged TFIIE-b (50 ng) was added to the immobilized proteins. After the immobilized proteins were washed, as described under Experimental procedures, bound 6His-tagged hTFIIE-b was detected by Western blot assay using an antibody against hTFIIE-b (Santa Cruz). hTFIIE-b used in this assay (lane 1), GST-CDC68 hTFIIE-b (lane 2), GST hTFIIE-b (lane 3). The arrow indicates hTFIIE-b detected by anti-hTFIIE-b antibody. q Blackwell Science Limited

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Figure 2 Expression of hSPT16/CDC68 mRNA in human tissues. Northern blot analysis was performed with mRNA from human tissues. mRNA of hSPT16/CDC68 was detected in all tissues indicated in the ®gure, although the signal detected in the liver was weak. Size of hSPT16/CDC68 mRNA was 4.3 kb which was almost the same size as that of ySpt16/Cdc68 (Rowley et al. 1991). In testis, a smaller size mRNA was detected, suggesting existence of a special form and function of hSPT16/CDC68 and its speci®c role in testis-speci®c chromatin. Arrow shows mRNA of hSPT16/CDC68.

the primary structure of that of ySpt16p/Cdc68p (Fig. 1A). ySpt16p/Cdc68p is a general chromatin factor reported to be involved in the regulation of gene expression through modulation of the chromatin structure (Malone et al. 1991; Rowley et al. 1991; Brewster et al. 1998). Speci®city of the interaction between hTFIIE and the isolated hSPT16/CDC68 was examined against various types of transcription factors, using the yeast two-hybrid system (Fig. 1B). The interaction between hTFIIE-b and hSPT16/CDC68 was evident, while there was no interaction between

hSPT16/CDC68 and SNF1, hTBP, hTFIIB, yTFIIB, hRAP74, hTAFII80 or hTAFII250. Thus, the interaction between hSPT16/CDC68 and hTFIIE-b is speci®c. To determine if the interaction beween hTFIIE and hSPT16/CDC68 is direct, we carried out the in vitro binding assay by the GST pull-down method (Magnaghi-Jaulin et al. 1996). Recombinant GST or histidinetagged TFIIE or GST-Spt16/Cdc68 proteins were expressed in E. coli and puri®ed using af®nity chromatography. While hTFIIE-b did not bind to

Figure 3 Physical interaction between yTFIIE-b and ySpt16p/Cdc68p. Physical interaction between TFIIE-b and ySpt16p/Cdc68p. GST or GST-ySpt16/ Cdc68 immobilized on a glutathioneaf®nity gel (Glutathione-Sepharose 4B, Pharmacia). 6His-tagged yTFIIE-b was added to the immobilized proteins, and bound proteins were eluted and analysed by SDS-PAGE and Western blotting. 6His-yTFIIE-b (lane 1), GST (lane 2), GST 6His-yTFIIE-b (lane 3), GSTySpt16/Cdc68 (lane 4), GST-ySpt16/ Cdc68 6His-yTFIIE-b (lane 5). Arrow indicates 6His-yTFIIE-b.

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GST, the interaction between hTFIIE-b and GSThSPT16/CDC68 was evident (Fig. 1C), thereby indicating that this interaction is direct and speci®c. These results are interpreted to mean that hTFIIE-b physically binds to the C-terminal portion of hSPT16/ CDC68. The primary structure of Spt16p/Cdc68p is highly conserved from yeast to human (Fig. 1A), which suggests a general role for Spt16p/Cdc68p in interacting with the general transcription initiation factor TFIIE. This notion is supported by Northern blot analysis showing that hSPT16/CDC68 is expressed in all the human tissues so far examined (Fig. 2). Since it had to be determined if full-length SPT16/CDC68 could bind to TFIIE, we made use of yeast counterparts, in addition to testing the functional conservation of this interaction, as described in the next section.

Evolutionarily conserved interaction between TFIIE and Spt16p/Cdc68p from yeast to human Structure and function of TFIIE are highly conserved among eukaryotes (Ohkuma et al. 1991, 1995; Peterson et al. 1991; Sumimoto et al. 1991) and the primary structure of human and yeast SPT16/CDC68 is also well conserved. Based on the structural conservation of these two factors, conservation of the interaction between TFIIE and SPT16/CDC68 among species can be predicted. Therefore, we asked if interaction between yTFIIE and ySpt16p/Cdc68p could occur as in the case of human homologues. At ®rst, we examined the interaction between yTFIIE-b (Tfa2p) and ySpt16p/Cdc68p, using GST

Figure 4 Physical interaction between yTFIIE-a and ySpt16p/Cdc68p. (A) Complex formation of yTFIIE-a with yTFIIE-b in vitro. GST-yTFIIE-a immobilized on a glutathione-af®nity gel (Glutathione-Sepharose4B, Pharmacia) formed complex with 6His-tagged yTFIIE-b. Complex formation of GST-yTFIIE-a and 6His-tagged yTFIIE-b was con®rmed by SDS-PAGE. Size marker (lane 1), 6His-yTFIIE-b (lane 2), GST-yTFIIE-a 6His-yTFIIE-b (lane 3), GST-yTFIIE-a (lane 4). Arrows indicate GST-yTFIIE-a or HisyTFIIE-b. (B) Physical interaction between yTFIIE and ySpt16p/Cdc68p. Puri®ed 6His-tagged ySpt16p/Cdc68p bound on immobilized GST-yTFIIE-a/b (lane 1) and GST-yTFIIE-a (lane 2) after GST pull-down assay and was detected by Western blot assay, using an antibody against the 6His-tag (Santa Cruz). (lane 1) GST-yTFIIE-a 6His-yTFIIE-b 6His-ySpt16p/Cdc68p, (lane 2) GSTyTFIIE-a 6His-ySpt16p/Cdc68p, (lane 3) GST ySpt16p/Cdc68p, (lane 4) 6His-ySpt16p/Cdc68p. Arrow indicates His-tagged ySpt16p/Cdc68p. q Blackwell Science Limited

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Figure 5 Functional interaction between yTFIIE and ySpt16p/Cdc68p. (A) The mutated amino acids and their postions in yTFIIE-a mutants (cs) and (ts) show the growth defect at 11 8C and 37 8C, respectively. The mutants were gifts from Dr S. Buratowski (Kuldell & Buratowski 1997). (B) Suppression of yTFIIE-a cs mutant phenotype by over-expression of ySpt16p/ Cdc68p. Yeast strains were grown on SDhis, trp, leu for 3 days at 30 8C and 24 days at 11 8C. Strains YSB324(wt), YSB319(cs) and YSB335(ts) were transformed with multicopy type p68-Ba-1 (Cdc68) and YEp352 (control vector). Over-expression of ySpt16p/Cdc68p caused slightly growth defect of wild-type cells.

pull-down assay. While GST did not bind to yTFIIE-b protein, binding of GST-full length ySpt16p/Cdc68p to yTFIIE-b was evident (Fig. 3), thereby indicating that this TFIIE-b/CDC68 interaction was conserved from human to yeast. Since TFIIE forms a heterotetramer composed of TFIIE-a/b, next we asked if the TFIIE complex would bind to ySpt16p/Cdc68p. As the initial step, we immobilized the TFIIE complex by binding GST-yTFIIE-a (Tfa1p) on glutathione-af®nity chromatography and trapping yTFIIE-b (Fig. 4A). The amount of trapped yTFIIE-b was comparable to that of yTFIIE-a, thereby indicating formation of the TFIIE complex (Fig. 4A). The in vitro binding assay, using the GST pull-down method, revealed that this TFIIE complex can interact with ySpt16p/Cdc68p (Fig. 4B). We also found that yTFIIE-a alone can also interact with ySpt16p/Cdc68p (Fig. 4B). These results suggest that both subunits of TFIIE cooperatively bind to Spt16p/Cdc68p, which might strengthen the interaction between TFIIE complex and Spt16p/Cdc68p. Taking advantage of such characteristics, we examined in vivo functional interaction between TFIIE and Spt16p/Cdc68p, as described in the next section. Throughout these analyses, we found that multiple interactions between evolutionarily conserved TFIIE and SPT16/CDC68 are conserved and if so, the interactions between these factors have a crucial and fundamental function in vivo. 256

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Functional interaction between yTFIIE and ySpt16p/Cdc68p As interaction between TFIIE and SPT16/CDC68 are apparently evolutionarily conserved from yeast to human, we investigated the genetic interaction using established S. cerevisiae conditional mutant strains. To determine if the interaction between TFIIE and SPT16/CDC68 has any biological signi®cance, we examined the effect of over-expression of ySpt16p/ Cdc68p in two conditional yTFIIE-a (tfa1) mutant strains with mutations in distinct positions and represent two distinct functional domains (Kuldell & Buratowski 1997). The cold sensitive (cs) mutant strain encodes mutant yTFIIE-a protein in which lysine 222 is converted to a stop codon, producing yTFIIE-a mutant protein with a deleted C-terminal region (Fig. 5A) and the thermosensitive mutant strain has mutant yTFIIE-a protein in which cysteine 124 in the zinc ribbon motif is replaced by serine. Over-expression of ySpt16p/Cdc68p suppressed the yTFIIE-a-cs mutant phenotype at nonpermissive temperature (11 8C) (Fig. 5B). Growth of the yTFIIEa-cs mutant strain to over-express ySpt16p/Cdc68p at nonpermissive temperature was less than that at permissive temperature. This does not mean that the suppression activity was weak, because over-expression of ySpt16p/Cdc68p at nonpermissive temperature was toxic to even the wild-type strain (Fig. 5B). This ®nding q Blackwell Science Limited


binding af®nity to ySpt16p/Cdc68p, this would explain the phenotype caused by the yTFIIE-a-cs mutant strain.

Discussion Interaction of TFIIE with SPT16/CDC68

Figure 6 Physical interaction between yTFIIE-a(wt,cs) and ySpt16p/Cdc68p. GST-yTFIIE-a(wt) and GST-yTFIIE-a(cs) were immobilized on a glutathione-af®nity gel and GST pulldown assay was done using 6His-ySpt16p/Cdc68p (100 ng). Bound 6His-tagged ySpt16p/Cdc68p was detected by Western blot assay, using an antibody against the 6His-tag (Santa Cruz). GST-yTFIIE-a (wt) (lane 1), GST-yTFIIE-a (cs) (lane 2), GST (lane 3), 6His-ySpt16p/Cdc68p (lane 4). GST-yTFIIE-a (cs) bound to 6His-ySpt16/Cdc68 so less than GST-yTFIIE-a (wt) did. The amount of GST-TFIIE-a(cs) equals to wt GST-TFIIEa. Arrow indicates his-tagged ySpt16p/Cdc68p.

supports the full suppression by over-expression of ySpt16p/Cdc68p in the cs mutant strain (Fig. 5B). Over-expression of ySpt16p/Cdc68p in the yTFIIE-a-ts mutant strain had no evident effect at 37 8C (data not shown), indicating that the effect of over-expression of ySpt16p/Cdc68p was selective for the yTFIIE-a-cs mutant strain. Thus, the interaction between yTFIIE-a and ySpt16p/Cdc68p is functional and the deleted C-terminal region of yTFIIE-a is apparently important for actions of ySpt16p/Cdc68p, through interactions with TFIIE-a. To characterize the molecular mechanism underlying suppression against the phenotype of the yTFIIE-a-cs mutant strain by the over-expression of ySpt16p/ Cdc68p, we examined the relationship of the interaction between yTFIIE-a and ySpt16p/Cdc68p and suppression of the phenotype of yTFIIE-a-cs mutant strain. In an in vitro binding assay, the yTFIIE-a-cs mutant protein showed weak af®nity to ySpt16p/ Cdc68p even at permissive temperature (at 30 8C) (Fig. 6), an observation which implies that yTFIIE-a-cs mutant protein at a low temperature might lose the potential to bind to ySpt16p/Cdc68p. We detected no interaction between yTFIIE-a-cs protein and ySpt16p/ Cdc68p at low temperature (4 8C) (data not shown). Therefore, if yTFIIE-a-cs protein has diminished q Blackwell Science Limited

The importance of alterations in chromatin structure at transcription initiation have been elucidated (for review, Kornberg & Lorch 1999a). Three types of factors were found to participate in this reaction: histone chaperone (Laskey et al. 1978; Laskey & Earnshaw 1980; Ishimi et al. 1983; Stillman 1986; Dilworth et al. 1987; Matsumoto et al. 1993; Kawase et al. 1996; Ito et al. 1997), ATP-dependent nucleosome remodeling factor (Tsukiyama et al. 1994, 1995b; Tsukiyama & Wu 1995a; Cairns et al. 1996; Varga-Weisz & Becker 1998; Kornberg & Lorch 1999b) and histone modi®cation enzyme (Kleff et al. 1995; Bannister & Kouzarides 1996; Brownell et al. 1996; Kingston et al. 1996; Mizzen et al. 1996; Ogryzko et al. 1996; Yang et al. 1996; Chen et al. 1997; Spencer et al. 1997; Yamamoto & Horikoshi 1997; Kimura & Horikoshi 1998; Smith et al. 1998; Wolffe & Hayes 1999). These chromatin factors have been suggested to interact with DNA-binding transcription regulators, remodel chromatin and recruit transcription machinery onto chromatin DNA template (Neely et al. 1999; Suzuki et al. 2000). However, alterations in chromatin structure at the later stage of transcription are poorly understood. As shown in Fig. 1, SPT16/CDC68 binds to TFIIE-b but not to the other general transcription initiation factors examined: three subunits of TFIID: TBP, TAFII80 and TAFII250, TFIIB and RAP74: the largest subunit of TFIIF which functions at the early stage of transcription initiation (Buratowski et al. 1989). Figure 3 shows that TFIIE-a also binds to Spt16p/ Cdc68p. TFIIE participates at the later stage of transcription initiation (Zawel & Reinberg 1995; Roeder 1996). Since the FACT complex, including SPT16/CDC68, was reported to remodel nucleosome DNA in transcription elongation (Orphanides et al. 1998), TFIIE might recruit and stimulate SPT16/ CDC68 so as to remodel chromatin DNA template at the later stage of initiation and early stage of elongation in transcription, which might allow RNA polymerase II to pass through the nucleosome template. The interaction studies we have described provide new insight into both the mechanism of alteration of chromatin DNA at the later stage initiation and early stage of elongation of transcription, and the involvement and Genes to Cells (2000) 5, 251±263

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functional role of TFIIE at these stages of chromatin transcription.

Interaction between hTFIIE-b and the acidic C-terminal region of hSPT16/CDC68 Although hSPT16/CDC68 was shown to be the subunit of the FACT complex, related molecular mechanisms are poorly understood (Orphanides et al. 1999). The yeast protein complex FACT containing Cdc68 and Pob3 mediates core-promoter repression through the N-terminal domain of Cdc68 (Evans et al. 1998), but the role of the C-terminal acidic domain of Cdc68 remained to be analysed. hTFIIE-b binds to the C-terminal region rich in acidic amino acids of hSPT16/CDC68, as shown in Fig. 1. The acidic stretch region is the characteristic motif of chromatin factors, especially histone chaperones: e.g. NAP1, CAF1, TAF1, nucleoplasmin and N1/N2 (Ito et al. 1996, 1997; Kaufman 1996; Adams & Kamakaka 1999). In the case of TAF1, the acidic region was reported to be essential for template activation reaction (Okuwaki & Nagata 1998). These acidic regions have been investigated with regard to interactions with basic proteins, such as histones which are major components of chromatin (Kawase et al. 1996). SPT16/CDC68 was shown to bind to histones, although whether or not its C-terminal acidic region participated in this binding activity was not determined (Orphanides et al. 1999). Given that TFIIE interacts with the acidic region of SPT16/CDC68, TFIIE may bind to SPT16/CDC68 co-operatively or competitively, with histones. Hence, one of the regulatory mechanisms of action of SPT16/CDC68 controlled by TFIIE on the chromatin DNA template is to regulate the interaction between histones and Spt16/Cdc68p. Further investigation on interactions among histones, TFIIE and Spt16/Cdc68 are indeed required.

Genetic analysis of the interaction between TFIIE and Spt16/Cdc68 Since TFIIE binds to Spt16p/Cdc68p speci®cally and directly in vitro, this interaction may be functional. However, it is necessary to gain support for this prediction, using an in vivo system. We examined functional interactions in vivo between TFIIE and SPT16/CDC68 using two known TFIIE mutant strains. One of the two mutants of TFIIE showed functional interactions with Spt16/Cdc68. The cs mutant of yTFIIE-a used in this study lacks the C-terminal region. Over-expression of Spt16/Cdc68 258

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rescued this cs phenotype (Fig. 5) and in vitro binding studies showed that the interaction between TFIIE and Spt16p/Cdc68p was decreased but not lost (Fig. 6). This means that the C-terminal region of TFIIE-a likely participates in the interaction with Spt16p/ Cdc68p while the N-terminal region alone of TFIIE-a can support interactions with Spt16p/Cdc68p. Therefore, the C-terminal region of the TFIIE-a might be the internal regulator of the interaction between TFIIE and Spt16p/Cdc68p. Although we used the established strain of yTFIIE-a mutants, for detailed analysis of their functional interaction, random mutagenesis of TFIIE-a is required. Since not only TFIIE-a but also TFIIE-b binds to Spt16p/Cdc68p, in vivo functional interaction between yTFIIE-b and Spt16/Cdc68 remains to be investigated. For this, using random mutagenesis, isolation of mutants of yTFIIE-b which affect interactions between yTFIIE-b and ySpt16/Cdc68 will need to be done. Furthermore, functional interactions among TFIIE-a, TFIIE-b and Spt16/Cdc68 will need to be done to understand more detailed mechanisms of the action and regulation of these interactions.

Functional roles of TFIIE in the aspect of reaction centring on Spt16/Cdc68 TFIIE facilitates promoter melting on a negatively supercoiled template (Holstege et al. 1995) perhaps because the chromatin structure is disrupted. TFIIE may participate in formation of the open complex by creating a naked region in the chromatin template in addition to maintaining promoter opening by binding to single-stranded DNA (Kuldell & Buratowski 1997). It was suggested that TFIIE may be involved in the binding of TBP/TFIIA complex to the promoter, the ®rst critical step in the preinitiation complex assembly (Yokomori et al. 1998). Therefore, TFIIE might contact the chromatin template at the ®rst step of the transcription initiation reaction. Interaction between TFIIE and SPT16/CDC68 might play an important role in the ®rst contact with the promoter region of the chromatin template. This proposed mechanism is supported by ®ndings that ySpt16p/Cdc68p is highly abundant in the yeast nucleus and that over-expression of ySpt16p/Cdc68p obviates requirement of the Swi/Snf complex, a chromatin remodeling factor (Brewster et al. 1998). ySpt16p/Cdc68p was reported to interact with the DNA polymerase a catalytic subunit (Wittmeyer & Formosa 1997; Wittmeyer et al. 1999) and to have a DNA-unwinding activity as a component of DUF q Blackwell Science Limited


(Okuhara et al. 1999). These reports indicate that ySpt16p/Cdc68p is commonly required for several reactions regarding alterations in chromatin structure. As ySpt16p/Cdc68p is a component of DUF which has DNA-unwinding activity, our purported model concerning TFIIE and SPT16/CDC68 is supported, because unwinding of DNA may lead to alteration of the chromatin structure. We predict that there would be a TFIIE-like replication factor interacting with DUF or that TFIIE itself would function in DNA replication systems. Transcription factors such as CTF/NF-1 were reported to contribute to both transcription and DNA replication (Santoro et al. 1988) and zinc ®nger protein family (Sp1 and Replication Protein A) was seen to contribute to the both transcription (Dynan & Tjian 1983) and replication (Park et al. 1999). Reinberg and co-workers have reported that the human homologue of Spt16p/Cdc68p is a component of FACT which facilitates transcription elongation on chromatin templates (LeRoy et al. 1998; Orphanides et al. 1998, 1999). Thus, our results also imply that TFIIE can recruit FACT to facilitate establishment of an ef®cient elongation complex as well as TFIIH. There is also the report that successful traversal of the ®rst several nucleosomes in the transcription unit might be the most important step in establishing an ef®cient early elongation complex, because FACT functions at the early stage of transcription elongation (Brown et al. 1998). Since TFIIH is incorporated into the preinitiation complex after TFIIE in the process of transcription initiation, TFIIH might also associate with chromatin factors as TFIIE interacts with SPT16/CDC68. Furthermore, since TFIIH contributes to both transcription and DNA repair, TFIIH functions through elongation steps on chromatin DNA. We predict that TFIIH might be also involved in the regulation of chromatin transcription, as well as TFIIE. A recent report showed that TFIIE-a, TFIIE-b and TFIIHMAT1 remains tightly associated with the nuclear substructure (Kimura et al. 1999). Repression of basal transcription by HMG2, a non-histone component of chromatin, is counteracted by TFIIH-associated factors in an ATP-dependent process (Stelzer et al. 1994). Three subunits of TFIIH have ATPase domains (Schaeffer et al. 1993) and the DNA-dependent ATPase activity was reproted to associate with TFIIH, as ATP-dependent chromatin remodeling factors (Roy et al. 1994). Furthermore, TFIIH ATPase activitiy was reported to be regulated by TFIIE during active transcription initiation complex formation (Ohkuma & Roeder 1994). Hence, TFIIH should be a good target for investigations concerning interaction between q Blackwell Science Limited

general transcription initiation factor and chromatin factor. Our observations are the ®rst evidence that general chromatin factor SPT16/CDC68 functionally interacts with general transcription initiation factor TFIIE. If our thesis is valid, the functional role of TFIIE in chromatin transcription deserves ongoing attention.

Experimental procedures Yeast two-hybrid screening Full length open reading frame of human TFIIE was cloned in-frame with Gal4 DNA-binding domain into pAS1-CYH2 in order to construct pAS1-CYH2-TFIIE-b. A human peripheral lymphocyte cDNA library (a gift from Dr S.T. Elledge) was transformed with Y190 yeast cells carrying pAS1CYH2-TFIIE-b, resulting in 3.7 ´ 105 transformants. An interaction trap selection was done as described (Fields & Song 1989; Durfee et al. 1993; Bando et al. 1997). The transformants were selected based on resistance to the 3-aminotriazole (25 mM) and by b-galactosidase assay. Finally 246 positive clones were selected and plasmids from positive colonies were isolated by the zymolyase method (Kaiser et al. 1994). All the inserted cDNA was sequenced by the dideoxy method (Sanger et al. 1977) using DNA sequencer (ABI 377). Two independent clones showed the sequence similarities to the C-terminus of ySpt16/Cdc68 (Fig. 1A). The nucleotide sequence reported in this paper has been deposited in the GENBANK database under GENBANK Accession Number AF164924.

Construction, expression and puri®cation of recombinant proteins To obtain GST-fused proteins, cDNAs were subcloned into pGEX-4T-2 or pGEX-5X-2 (Pharmacia) with the NdeI site created by mutagenesis. To obtain histidine-tagged proteins, cDNAs were subcloned into 6HispET11d (Hoffmann & Roeder 1991). The histidine-tagged yTFIIE-a and -b construction vectors and histidine-tagged TFIIE-a-cs mutant construction vector were kindly gifted by Dr R. Kornberg (Feaver et al. 1994), and Dr S. Buratowski (Kuldell & Buratowski 1997), respectively. The constructed plasmids were introduced into the BL21 (DE3) strain of E. coli. Recombinant proteins were induced by IPTG and puri®ed using Ni-agarose or Glutathione-Sepharose for histidine-tagged or GST-fused proteins, respectively. GSTySpt16p/Cdc68p was further puri®ed by DEAE-Sepharose. The purities of these proteins were 60±90%.

In vitro binding assay A GST pull-down assay was used for detecting in vitro binding activity, performed as described (Hagemeier et al. 1993). GSThSPT16/CDC68 or GST-ySpt16/Cdc68 fusion proteins were immobilized on Glutathione-Sepharose beads. Histidine-tagged Genes to Cells (2000) 5, 251±263

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S-W Kang et al. hTFIIE or yTFIIE proteins were added to these immobilized fusion proteins in the binding buffer containing 25 mM HEPES, pH 7.5, 12.5 mM MgCl2, 20% glycerol, 0.1% IGEPAL CA630, 150 mM KCl, 20 mM ZnCl2, 0.2% 2-mercaptoethanol for 1 h or O/N at the indicated temperature. Bound fractions were washed by buffer containing 150 mM NaCl, 1 mM EDTA, 0.5% IGEPAL CA630, 25 mM HEPES, pH 7.5, 0.2% 2-mercaptoethanol three times and eluted with SDS-sample buffer, separated on SDSpolyacrylamide gel and transferred to a nitrocellulose ®lter by the electronic blotting (Burnette 1981). Bound proteins were detected with anti-hTFIIE-b (Santa Cruz) or anti-histidine tag antibodies (Santa Cruz) using ECL system (Amersham) and autoradiography.

Northern blot analysis Human Multiple Tissue Blot (Clontech) was used for hybridization with the isolated cDNA clone of hSPT16/CDC68 as probe. Procedure was previously described (Ausubel et al. 1995). Brie¯y, probe DNA was labelled with 32P-dCTP by random labelling method. Hybridization was performed at 42 8C with formamide buffer solution for overnight and the ®lter was washed stringently with buffer containing 0.1´ SSC, 0.1% SDS at 42 8C. Hybridized signals were detected by autoradiography using Kodak ®lm.

Genetic interaction assay TFIIE mutant strains were kindly given by Dr S. Buratowski (Kuldell & Buratowski 1997). For over-expression of ySpt16p/ Cdc68p, p68-Ba-1 was gifted from Dr G.E. Johnston. The control vector (YEp352) was prepared by deleting the insert region of p68-Ba-1. These plasmids were introduced to cs and wild-type strains by lithium chloride method (Ausubel et al. 1995). The resultant cells were cultured at 11 and 30 8C and suppression activity was analysed by the growth of their colonies.

Acknowledgements We thank Dr R. Kornberg for expression plasmid of yTFIIE, Dr S.T. Elledge for two-hybrid system, Dr S. Buratowski for TFIIE mutant strains and Dr G.E. Johnston for plasmid to over-express ySpt16p/Cdc68p. We also thank A. Kimura, T. Umehara, N. Yokoyama, T. Munakata and T. Sakuno for critical comments on the manuscript. This work was supported in part by a Grant-inAid for Scienti®c Research from the Ministry of Education, Science, Sports and Culture of Japan, and the Exploratory Research for Advanced Technology (ERATO) of the Japan Science and Technology Corporation ( JST).

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Received: 29 November 1999 Accepted: 24 December 1999

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