TFIID Component TAF7 Functionally Interacts with Both TFIIH and P-Tefb

TFIID component TAF7 functionally interacts with both TFIIH and P-TEFb

Anne Gegonne*, Jocelyn D. Weissman*, Hanxin Lu*, Meisheng Zhou†, Arindam Dasgupta†, Robert Ribble†, John N. Brady†, and Dinah S. Singer*‡

*Experimental Immunology Branch and †Virus Tumor Biology Section, Basic Research Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892

Communicated by Gregory A. Petsko, Brandeis University, Waltham, MA, February 20, 2008 (received for review January 14, 2008)

Transcription consists of a series of highly regulated steps: assem- A - + TA 7F B - + FAT 7 C - + TA 7F bly of the preinitiation complex (PIC) at the promoter, initiation, ++ TFI HI ++ KAC ++ bFET-P ++ CTD + + DTC ++ DTC elongation, and termination. PIC assembly is nucleated by TFIID, a complex composed of the TATA-binding protein (TBP) and a series CTD DTC DTC of TBP-associated factors (TAFs). One component, TAF7, is incor- 7FAT porated in the PIC through its interaction with TFIID but is released T 7FA 7FAT 1 1 1 from TFIID upon transcription initiation. We now report that TAF7 )P-5reS( 5reS( - )P )P-2reS( interacts with the transcription factors, TFIIH and P-TEFb, resulting 8.0 0.8 8.0 in the inhibition of their Pol II CTD kinase activities. Importantly, in 0.6 6.0 6.0 in vitro transcription reactions, TAF7 inhibits steps after PIC assem- 4.0 4.0 4.0 bly and formation of the first phosphodiester bonds. Further, in vivo TAF7 coelongates with P-TEFb and Pol II downstream of the 0.2 2.0 0 2. 0 0 0 promoter. We propose a model in which TAF7 contributes to the Phosphorylation Relative CTD _ _ _ regulation of the transition from PIC assembly to initiation and 7FAT 7FAT 7FAT elongation. Fig. 1. TAF7 inhibits the kinase activities of TFIIH and P-TEFb. (A) TAF7 inhibits TFIIH-mediated phosphorylation of the Pol II CTD Ser-5. (Upper) MHC class I genes ͉ regulation ͉ transcription initiation Purified TFIIH and 100 ng of GST-tagged Pol II CTD were incubated in the presence or absence of recombinant Flag-tagged TAF7 (F-TAF7) in an in vitro 32P n eukaryotic cells, expression of protein-encoding genes de- kinase assay using [ ]ATP. Phosphorylation was determined by autoradiog- raphy after gel electrophoresis; positions of TAF7 and CTD are indicated based Ipends on the ordered recruitment of the general transcription on mobility markers (data not shown). (Lower) Purified TFIIH and 100 ng of factors (GTF) TFIID, TFIIB, and TFIIA to the promoter, GST-tagged Pol II CTD were incubated in the presence of increasing amount of followed by association of Pol II, the mediator, and the remain- recombinant F-TAF7 (125 and 250 ng), in an in vitro kinase assay. Ser-5 CTD ing GTFs, TFIIF, TFIIE, and TFIIH, to form a preinitiation phosphorylation was determined by Western blot analysis, using H14 anti- complex (PIC) (1). Once PIC assembly is complete, transcription phosphoSer5 antibody, and quantified by densitometry. (B) TAF7 inhibits the initiation ensues; Pol II with the elongation complex dissociate CAK-mediated phosphorylation of the Pol II CTD. (Upper) In vitro kinase assay from the PIC (2, 3). A required step during transcription with purified CAK, 100 ng of GST-tagged Pol II CTD, and F-TAF7 as in A Upper. initiation is the phosphorylation of serine 5 in the carboxy (Lower) In vitro kinase assay with purified CAK, 100 ng of GST-tagged Pol II CTD, and increasing F-TAF7 as in A Lower.(C) TAF7 inhibits P-TEFb-mediated terminal domain (CTD) heptad repeat of Pol II by the kinase phosphorylation of the Pol II CTD Ser-2. (Upper) In vitro kinase assay with 50 subunit of TFIIH, CDK7 (4, 5), after which Pol II pauses to ng of purified P-TEFb, 100 ng of GST-tagged Pol II CTD, and F-TAF7 as in A ensure proper pre-mRNA capping (6–9). The transition from Upper.(Lower) In vitro kinase assay with 50 ng of P-TEFb, 100 ng of GST- pausing to elongation is facilitated by the P-TEFb elongation tagged Pol II CTD, and increasing F-TAF7 as in A Lower. CTD Ser-2 phosphor- BIOCHEMISTRY complex, which also mediates efficient elongation (10). P-TEFb ylation was determined by Western blot analysis, using H5 anti-phosphoSer2 consists of two subunits, cyclin T1 and the kinase CDK9, which antibody. phosphorylates serine 2 of the CTD, required for productive elongation and the recruitment of complexes involved in mRNA processing (splicing and polyadenylation) (10–15). These studies were designed to determine the fate of TAF7 Although the general mechanics of transcription have been after its dissociation from the PIC. We report that TAF7 characterized, relatively little is known about how the transitions functionally interacts with both the general transcription factor from PIC assembly to initiation/pausing to elongation are reg- TFIIH and the elongation factor P-TEFb. Association of TAF7 ulated. Promoter recognition is largely mediated by TFIID, with TFIIH inhibits its CDK7 kinase thereby inhibiting TFIIH- mediated phosphorylation of the Pol II CTD Ser-5; binding of which is composed of the TATA binding protein (TBP) and over TAF7 to the P-TEFb elongation complex inhibits its CDK9- a dozen TBP associated factors (TAFs) (16, 17, 18). The largest mediated phosphorylation of Pol II CTD Ser-2. Importantly, we TFIID component, TAF1, has both acetyltransferase (AT) and show that TAF7 functions in vitro to inhibit transcription at steps kinase activities (19, 20). We demonstrated that TAF1 and its after PIC assembly and in vivo colocalizes with P-TEFb and Pol intrinsic acetyltransferase activity are essential for transcription II downstream of the promoter. Thus, in addition to its role in of an MHC class I gene (21). Importantly, MHC class I transcription is inhibited both in vitro and in vivo by the viral transactivator, HIV Tat, which binds to the TAF1 AT domain, Author contributions: A.G., J.N.B., and D.S.S. designed research; A.G., J.D.W., H.L., and M.Z. inhibiting its enzymatic activity (22, 23). TAF7, a cellular 55-kDa performed research; A.D. and R.R. contributed new reagents/analytic tools; A.G., J.D.W., TFIID component (24, 25), also binds to TAF1 inhibiting its AT and D.S.S. analyzed data; and A.G. and D.S.S. wrote the paper. activity and repressing MHC class I transcription (26) Signifi- The authors declare no conflict of interest. cantly, we have demonstrated that TAF7 remains bound to Freely available online through the PNAS open access option. TAF1/TFIID until PIC assembly is complete, whereupon it is ‡To whom correspondence should be addressed. E-mail: [email protected]. released enabling transcription initiation and elongation (27). This article contains supporting information online at www.pnas.org/cgi/content/full/ Thus, TAF7 is an intrinsic regulator of transcription. 0801637105/DCSupplemental.

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Fig. 2. TAF7 interacts with P-TEFb in vitro and in vivo and exists in a TFIID-independent complex. (A) TAF7 binds to P-TEFb through its central domain in in vitro pull-down assays. Puriﬁed recombinant P-TEFb (200 ng) was incubated with F-TAF7 or each of the F-TAF7 mutants immobilized on Sepharose beads. TAF7-bound fractions were assayed for retention of P-TEFb by immunoblotting, using anti-cyclin T1 antibody. (B) TAF7 binds to P-TEFb in vivo. Extracts from cells transiently transfected with either F-TAF7, pcDNA3, or Flag-SKD1, an irrelevant control protein, were immunoprecipitated with anti-cyclin T1 antibody. Immunoprecipitates were analyzed for cyclin T1 (Upper) or F-TAF7 (Lower) by Western blot. (Upper) Anti-cyclin T1. Total extract from cells transfected with vector (lane 1), Flag-SKD1 (lane 2), or F-TAF7 (lane 3) before immunoprecipitation and immunoprecipitated cyclin T1 (lanes 4–6). (Lower) Anti-Flag M2 antibody, detection of F-TAF7 coimmunoprecipitated from extracts of cells transfected with vector control (lane 4), Flag-SKD1 (lane 5), or F-TAF7 expression vector (lane 6). (C) TAF7 exists in a TFIID-independent form. C8166 whole cell extracts were fractionated on an FPLC column and fractions analyzed by Western blotting as indicated. Arrows refer to the indicated molecular sizes. Puriﬁed TFIID was included as a marker. TFIID consistently appears in the excluded volume in column fractionation of whole cell extracts. Fractions 16–28 and 30–36 were run in parallel but on two separate SDS/PAGEs.

transcription initiation as a TFIID component, TAF7 also competitive inhibitor. However, phosphorylation of SPT5, an- functions in the transition from PIC assembly to initiation and other P-TEFb substrate (30), is enhanced in the presence of elongation. We propose a model in which TAF7 regulates the TAF7 (Fig. S1A). Furthermore, TAF7 does not inhibit the orderly progression of events in transcription, preventing tran- activity of CDK2, another CDK family member (Fig. S1B). scription elongation until the steps of transcription initiation are Finally, neither CTD nor TAF7 alone has any kinase activity completed and the transcription elongation complex (TEC) is (Fig. S1C). Thus, TAF7 inhibition of TFIIH and P-TEFb CTD fully assembled. kinase activities is specific. Thus, TAF7 regulates TAF1 acetyl transferase activity and function and the CTD kinase activities Results of both TFIIH and P-TEFb. TAF7 Functionally Interacts with TFIIH and P-TEFb and Inhibits Their Phosphorylation of the Pol II CTD. We demonstrated that TAF7 is TAF7 Physically Interacts with P-TEFb. The experiments described released from the TFIID complex upon transcription initiation above demonstrate a functional association between TAF7 and (27). The present studies were designed to determine whether both TFIIH and P-TEFb. To examine the physical interactions, TAF7 associates with other components of the transcription we performed in vitro pull-down assays with recombinant Flag- complex after its dissociation from TFIID. Because TAF7 was tagged TAF7 and either purified P-TEFb or purified TFIIH. A released from a partially assembled preinitiation complex with weak association of TAF7 with the CAK subunit of TFIIH was TFIIB, TFIIF, and Pol II (27), none of these factors is likely to observed in the pull-down assay (data not shown). TAF7 effi- be a TAF7 interacting partner. Therefore, we considered the ciently recovered P-TEFb as revealed by anti-cyclin T1 immu- possibility that TAF7 functionally interacts with TFIIH and/or noblotting, indicating that TAF7 stably interacts with P-TEFb in P-TEFb (7). To assess the functional interaction of TAF7 with vitro (Fig. 2A; compare lanes1 and 2). Similar results were TFIIH or P-TEFb, its effect on their Pol II CTD kinase activities obtained in an anti-CDK9 immunoblot (data not shown). The was determined. TAF7 inhibited the CTD kinase activities of interaction domain of TAF7 with P-TEFb was mapped using a TFIIH (Fig. 1A). TFIIH consists of a complex of 10 subunits, 3 set of deletion mutants to the central 103- to 203-aa segment of of which including the CDK7 kinase form a subcomplex called TAF7 [MID (103–203)] (Fig. 2A, compare lanes 6 and 7), which CAK (28). TAF7 inhibited the phosphorylation of the Pol II is within the same domain that interacts with TAF1 (24). CTD by the CAK complex, demonstrating that TAF7 acts However, mutation of Thr-151 in the TAF1 binding domain (24) directly on CAK (Fig. 1B). Remarkably, TAF7 also inhibited has no effect on TAF7 binding to P-TEFb. Thus, TAF7 binds P-TEFb phosphorylation of the Pol II Ser-2 CTD (Fig. 1C). In P-TEFb in vitro. contrast, recombinant CREB has no effect on CDK9 kinase The in vivo interaction of TAF7 with P-TEFb was determined phosphorylation of the CTD (29) [supporting information (SI) by immunoprecipitation after transfection of Flag-TAF7 into Fig. S1D]. Thus, TAF7 functionally interacts with both TFIIH 293 cells. Consistent with the in vitro findings, immunoprecipi- and P-TEFb, inhibiting their respective CTD kinase activities. tation of endogenous cyclin T1 coimmunoprecipitated TAF7 Because both TFIIH and P-TEFb also phosphorylate TAF7 from Flag-TAF7-transfected 293 extracts but not control protein (Fig. 1), we considered the possibility that TAF7 acts as a from control extracts (Fig. 2B Lower, lanes 5 and 6).

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Fig. 3. TAF7 regulates multiple distinct steps in transcription initiation. (A) Scheme of experimental protocol. Basic IVT, TAF7, and the four NTPs are present during PIC assembly and initiation; InitiationϩPause, PIC preassembly occurs in the absence of NTPs or TAF7. TAF7 is added after transcription is initiated with AGC; UTP is added at 75 sec to permit clearance and elongation. Post-Initiation, PIC preassembly occurs in the absence of NTPs or TAF7, which is added after transcription is initiated with all four NTPs. (B) TAF7 regulates in vitro transcription. Lane 1, marker; lanes 2–3, HeLa nuclear extract in the presence or absence of purified TAF7 preincubated for 15 min in presence of rNTPs. Transcription was initiated by the addition of the MHC class I Ϫ313CAT plasmid. In lanes 4–7, Ϫ313CAT DNA and extracts were preincubated for 30 min in presence of 40 ␮M ATP to permit PIC assembly. Transcription was initiated by addition of ATP/CTP/GTP mix (lanes 4 and 5) or rNTP (lanes 6 and 7). Control TKEG buffer (Ϫ) or purified F-TAF7 (ϩ) were added 15 seconds after initiation of transcription and incubated for 30min. Analysis of Ϫ313CAT transcripts was by primer extension. [In previous studies (27), addition of TAF7 was after promoter clearance so did not affect transcription.] (C) Quantitation of results of three experiments, showing averages and SEM. Addition of TAF7 in each of these conditions results in significant inhibition of transcription: IVT, P Ͻ 0.003; pause, P Ͻ 0.03; initiation, P Ͻ 0.04.

The observed TAF7 inhibition of P-TEFb and TFIIH kinase tion initiation, or after initiation (schematized in Fig. 3A). The activities predicted that TAF7 exists in a TFIID-independent presence of TAF7 during the entire in vitro transcription assay is form in vivo. To test this prediction, native cell extracts were predicted to block transcription at all steps of transcription fractionated by FPLC gel filtration and assayed for the elution initiation: inhibition of TAF1 AT activity and of TFIIH and pattern of TAF7, TBP, and TAF1 (Fig. 2C). As expected, TFIID P-TEFb kinase activities (Basic IVT). Indeed, addition of TAF7

components (TBP, TAF1, and TAF7) were detected in high before PIC assembly significantly reduced transcription (Fig. 3B, BIOCHEMISTRY molecular weight fractions (fractions 16–18); a second peak of basic IVT, lanes 2 and 3). Transcriptional repression in the TBP was detected in fractions 26–30, corresponding to free presence of TAF7 is specific; addition of irrelevant proteins to TFIID. Importantly, TAF7 eluted in a distinct peak in fractions the in vitro transcription reaction had no effect (Fig. S3). corresponding to molecular mass between 230 and 440 kDa The effect of TAF7 on transcription after initiation was (fractions 30–32). Thus, TAF7 is found in a form independent determined as follows: After PIC assembly, transcription was of TFIID. Detecting the 55-kDa TAF7 in fractions correspond- initiated by the addition of ATP, GTP, and CTP to allow the ing to 230 kDa suggests that it is in a complex with other proteins. incorporation of the first three nucleotides in the nascent RNA and pausing of Pol II. (The sequence at the transcription start A TFIID-independent TAF7 fraction was also observed in site is gctcAGCTTCT; transcription initiates at A). Then, TAF7 MOLT4 whole cell extracts (Fig. S2). Whether TAF7 is associ- was added, followed by UTP to allow promoter clearance and ated with cyclin T1 or CDK9 or other proteins in the 230-kDa elongation (initiation plus pause). Consistent with a role for fraction remains to be determined. TAF7 after initiation of transcription, TAF7 significantly re- These experiments provide the first demonstration that TAF7 duced transcription even when added after phosphodiester bond exists in at least two distinct forms in vivo—a TFIID- formation (Fig. 3B, InitiationϩPause, lanes 4 and 5). Finally, to independent form and the well known TFIID complex. examine the effect of TAF7 after promoter clearance, transcription was initiated with the 4NTPs after PIC assembly, followed TAF7 Regulates Both PIC Assembly and Transcription Initiation in by addition of TAF7 (Fig. 3A, Post-Initiation). Even added after Vitro. TAF1, TFIIH and P-TEFb are critical for PIC assembly, PIC assembly and promoter clearance, TAF7 is still able to transcription initiation, and elongation. We have shown that significantly reduce transcription (Fig. 3B, lanes 6 and 7). The TAF7 functionally interacts with all three. Because TFIIH and results of three independent experiments are summarized in Fig. P-TEFb are involved in transcription initiation and elongation, 3C. The effects of TAF7 after transcription initiation, although we postulated that TAF7 functions in the regulation of tran- modest, are reproducible and significant. Thus, TAF7 function- scription at steps after PIC assembly. To address this question, ally targets components of the transcription machinery other in vitro transcription assays were performed in which TAF7 was than TAF1/TFIID, consistent with a functional role for its added at one of three times: before PIC assembly, at transcrip- inhibition of the kinase activities of TFIIH and P-TEFb.

Gegonne et al. PNAS ͉ April 8, 2008 ͉ vol. 105 ͉ no. 14 ͉ 5369 Downloaded by guest on September 29, 2021 Table 1. TAF7 deﬁciency compromises cell growth AB0.5 oP l I SI e 2r P- .0 4 Absence of colonies, T 7FA : - -+ + Construct % seeded wells Avg cells per well, no. 3.0 Ϯ 0-II 2.0 Control 4.2 21.8 1.9 Ϫ4 -II a (siRNA/ctrl) shRNA1 22.9 10.6 Ϯ 1.6, P ϭ 1.4 ϫ 10 .0 1 shRNA2 17.8 NT

2.01 2 1 0 2. 4 Relative RNA levels 0 evitaleR DTC sohP ohp lyr a it on n H F7 -B -A -C ti D A LA LA LA ac P Human 293 cells, stably transfected with two different TAF7 shRNA retro- T H H H β- GA viral vectors, shRNA1 or shRNA2, or the control vector, were seeded into 100 Fig. 4. TAF7 regulates promoter activity in vivo.(A) TAF7 inhibits Pol II CTD microtitre wells at an average density of ﬁve cells per well. After TAF7 shRNA phosphorylation in vivo. Nuclear extracts from either 293/F-TAF7 (ϩ) or con- induction, the number of wells with colonies and the size of each colony were trol 293T (Ϫ) cells were analyzed by Western blotting with either an antibody determined by microscopic examination. In the absence of induction of TAF7 speciﬁc for the Ser-2 CTD phosphorylated form of pol II (Right) or an antibody shRNA, transfectant growth was comparable to a control cell line transfected reactive with all forms of Pol II CTD (Left). (Quantitation) (Left) Phosphory- with an empty vector (data not shown). lated Pol II (II-0) is expressed relative to nonphosphorylated Pol II (II-a). (Right) The amount of Ser-2-P is expressed relative to the total amount of Pol II in the same sample (Left). (B) Depletion of TAF7 results in reduced levels of endog- considered the possibility that TAF7 levels may play a critical enous MHC class I RNA. Human 293T cells were transiently transfected with role in cell viability and growth. As assessed by limiting dilution TAF7 siRNA or scrambled siRNA,as described; levels of TAF7, HLA class I RNAs, analysis in HeLa tet-off cells stably transfected with an inducible actin, and GAPDH RNAs were determined by real time PCR. Results are TAF7 shRNA, TAF7 depletion reduced both cell viability and presented relative to control siRNA. growth (Table 1). Thus, TAF7 contributes to the regulation of sustained cell viability and growth. TAF7 Regulates Promoter Activity in Vivo. The biological implica- tions of TAF7 inhibition of Pol II CTD phosphorylation were TAF7 Is Associated with Both the PIC and Transcription Initiation/ examined in vivo in 293T cells stably transfected with TAF7 Elongation Complex in Vivo. Because TAF 7 interacts with P-TEFb, (293/F-TAF7). Both TAF7 protein and RNA levels were in- an elongation factor that moves with the elongation complex, we creased in the 293/F-TAF7 cells relative to control 293 cells (Fig. postulated that, in vivo, TAF7 would be associated with coding S4 A and B). Importantly, the fraction of total phosphorylated regions of the gene. To test this prediction, we examined the in Pol II species was markedly reduced in the 293/F-TAF7 cells situ localization of TAF7 by in vivo ChIP assays of the class I gene relative to controls (Fig. 4A; compare two left lanes); CTD Ser-2 in spleen and brain (Fig. 5). These two tissues differ in MHC Ͼ Pol II was also reduced by 75% in the TAF7-transfected cells class I expression by 2 orders of magnitude (32). In the (Fig. 4A; compare Ser-2 in Right to total Pol II in Left). These high-expressing spleen, TAF7 was associated with both the results clearly demonstrate that over-expression of TAF7 leads promoter and downstream sequences as far as exon 5 (Fig. 5A). to inhibition of CTD phosphorylation in vivo and in vitro. The extent of TAF7 association paralleled that of Pol II. In Because TAF7 regulates the functions of TAF1, TFIIH and contrast, the transcription factors CREB and c-jun were found P-TEFb and has a major effect on MHC class I expression in vitro only at the promoter (Fig. 5A and data not shown). Consistent (Fig. 3) (26, 27), we predicted that both overexpression and with the high level of transcription of the class I gene in spleen, underexpression of TAF7 would perturb transcription in vivo: acetylated H4 histone—a marker of active chromatin—was Overexpression would sustain inhibition of TAF1, TFIIH, and detected throughout the gene. In brain, where class I transcrip- P-TEFb functions, whereas under-expression would allow pre- tion is barely detectable, the levels of TAF7, Pol II, and mature initiation and promoter clearance, resulting in abortive acetylated H4 histone were correspondingly low. No TAF7 was transcripts. In either case, MHC class I expression would be detected either to sequences beyond the poly(A) addition site or reduced. Indeed, overexpression of TAF7 decreased MHC class to sequences 700 bp 5Ј to the promoter in either tissue (data not I promoter activity in a transient transfection assay (Fig. S5). To shown). Thus, association of TAF7 with the promoter and coding determine whether ablated expression of TAF7 affected tran- sequences is specific and dependent on active transcription. scription, human 293T cells were transiently transfected with To examine the relationship between TAF7 and the elonga- TAF7 siRNA, resulting in a reduction of TAF7 RNA to 19% of tion complex, the correlation between TAF7 and P-TEFb mi- the control level (Fig. 4B). As predicted, RNA levels of the gration along the gene was determined by ChIP analysis of endogenous MHC class I genes HLA-A,B,C were dramatically spleen cells with antibodies to TAF7, cyclin T1, and Pol II. reduced after TAF7 siRNA treatment (Fig. 4B); siRNA to a Because the abundance of the elongation complex varies along control transcript did not affect HLA-A RNA levels (Fig. S6B). the gene, the abundances of TAF7 and cyclin T1 were normal- A TAF7 shRNA construct stably introduced into HeLa cells ized to the level of Pol II association at each point along the gene. similarly decreased TAF7, HLA-A, and HLA-C RNA levels This normalization also corrects for differences between the (Fig. S6). Because the siRNA and shRNA sequences and the antibodies and efficiency of PCR primers at the various positions transfected cell lines were different, the observed effects of within the gene. As shown in Fig. 5B, the relative abundance of TAF7 knock-down on HLA class I RNA levels are unlikely due TAF7 paralleled that of cyclin T1 through the exon 2/intron 2 to off-target effects. Taken together, these findings demonstrate junction. Thus, the occurrence of TAF7 within the class I coding that TAF7 actively contributes to regulating transcription of sequences correlates with the abundance of Pol II and the level MHC class I genes in vivo. of transcription. From these experiments, we conclude that Interestingly, the depletion of TAF7 did not result in a global TAF7 is associated with initiation/elongation complexes in situ in inhibition of transcription, because the levels of actin and tissues. GAPDH were only modestly reduced, suggesting that TAF7 regulates a subset of mammalian promoters, consistent with the Discussion observation in yeast that loss of TAF7 affects only a subset of Transcription depends on the temporal and spatial integration of genes (31). preinitiation complex assembly, initiation, elongation, and termination. Regulation of the transitions from PIC assembly to TAF7 Plays a Critical Role in Cell Viability and Growth. Because initiation to elongation is necessary to assure proper rates of transcription of many cell cycle genes depends on TFIID, we productive transcription. Here, we report that the TFIID com-

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Fig. 5. TAF7 remains associated with the transcription elongation complex in vivo.(A) TAF7 is associated with MHC class I coding sequences in spleen but not brain. A representative ChIP assay out of four performed by using chromatin from spleen cells and brain of mice transgenic for the pig MHC class I gene, PD1 (B10.PD1). The fold enrichments of TAF7, Pol II, AcHistone H4, and CREB were calculated for different regions of the PD1 gene as described in Methods. The PD1 gene segments analyzed and primer sets in the ChIP assays are shown diagrammatically. Boxes indicate exons. PCR fragments are nonoverlapping. (B) TAF7 and Cyclin T1 remain associated with Pol II through exon 2 of the class I gene. ChIP assays were performed on B10.PD1 spleen cells. The amounts of TAF7 or cyclin T1 were determined relative to Pol II for different regions of the PD1 gene and are expressed as ‘‘relative abundance’’: the fold enrichment (Vϩ/VϪ) of TAF7 and cyclin T1 relative to the fold enrichment of Pol II, to normalize for variations in efﬁciency of PCR ampliﬁcation of primer sets across the gene. The PD1 gene segments analyzed and primer sets in the ChIP assays are shown diagrammatically. Gray boxes indicate exons. PCR fragments are nonoverlapping. The graph represents the composite of three independent experiments.

ponent, TAF7, functionally associates with other components of In vivo, TAF7 remains associated with the elongation complex the transcription machinery both in vitro and in vivo.We within MHC class I coding sequences, suggesting that, like demonstrate that TAF7 interacts with both the general tran- TFIIF, it may modulate the late pauses of transcription (2).The scription factor TFIIH and the elongation factor P-TEFb, in- extent of TAF7 association with the class I gene in different hibiting phosphorylation of the Pol II CTD. Importantly, as a tissues from the same animal parallels the extent of association presumed functional consequence of its modulation of the CTD of Pol II and the level of class I expression in those tissues. kinase activities of TFIIH/CDK7 and P-TEFb/CDK9, TAF7 Although the multiplicity of TAF7 interactions we have de- regulates transcription subsequent to PIC assembly and initia- scribed are unusual, they are not unique. A well documented tion (i.e., promoter clearance and elongation). Finally, we dem- example, with a strikingly similar pattern of interactions, is the HIV onstrate that TAF7 regulates MHC class I expression in vivo and transactivator protein Tat. Like TAF7, Tat (i) binds TAF1 and is associated with MHC class I DNA coding sequences. These inhibits its acetyl transferase activity (23), (ii) binds P-TEFb and studies demonstrate that TAF7 plays a critical role in regulating activates its autophosphorylation (34), and (iii) modulates the distinct steps in transcription initiation. phosphorylation of RNAP CTD by TFIIH and P-TEFb (35). We propose a model in which TAF7 contributes to the regulation Although there is no structural similarity between these two of early transitions in transcription (Fig. S7) as follows. As a proteins, the remarkable parallels between TAF7 and Tat function component of TFIID, TAF7 inhibits the TAF1 AT activity until suggest that Tat has subverted the cellular processes normally PIC assembly is complete, thereby preventing premature initiation regulated by TAF7. BIOCHEMISTRY (27). Upon completion of PIC assembly, TAF7 is released from In conclusion, the studies reported here define important new TAF1, allowing transcription to initiate with the first phosphodi- roles for TAF7 in regulating the transition from PIC assembly to ester bond formation. TAF7 inhibition of TFIIH phosphorylation productive elongation and demonstrate that transcription factor of Ser-5 CTD would delay 5Ј cap formation and the recruitment of complexes, such as TFIID, TFIIH, and P-TEFb, are not static CTD binding factors until formation of the first phosphodiester entities but rather dynamic ones with constituent proteins entering bonds and separation of the nascent elongation complex from the and leaving at various points during the transcription process. PIC, thus effectively pausing initiation. This model is consistent with the known pausing of Pol II Ϸ20–60 bp downstream of the Methods transcription start site. For genes, like the MHC class I gene, whose Kinase activities were measured with Flag-tagged TAF7 (125 or 250 ng) in 32 expression is dynamically regulated by extrinsic signaling events the presence of 100-ng GST-Pol II CTD, 10 ␮Ci of [ P] ␮ATP (6,000 Ci/mM) ␮ (i.e., hormonal or cytokine), Pol II pausing could provide a in 20 l of 50 mM Tris (pH 6.8 for P-TEFb, pH 7.9 for TFIIH), 5 mM DTT, 10 ␮M ZnSO4, 5 mM MnCl, 4 mM MgCl2 and 1 mM ATP. In vitro transcription mechanism to regulate the rate of transcription, either by modu- assays, immunoprecipitations, and Western blot anlyses were performed as lating the length of pausing or by maintaining the promoter in an described in ref. 27. Column fractionation of C8166 T whole cells extracts open conformation that is ‘‘poised’’ for rapid response but not was on a Superose 6 column in 150 mM Hepes (pH 7.9), 1.5 mM MgCl2, 1 mM actively transcribed (33). EGTA (pH 8), 10% glycerol, 150 mM NaCl, 10 mM B-glycerophosphate, 1 mM Release of TAF7 from TFIIH/CDK7 would overcome pausing, DTT, 50 mM NaF, 0.2 mM sodium vanadate, and protease inhibitors. TAF7 whereas its inhibition of P-TEFb phosphorylation of Ser-2 CTD siRNA transient transfections were performed with Hiperfect (Qiagen) would delay elongation to allow recruitment of CTD-associated according to the manufacturer’s protocol. siRNA duplexes specifically tar- complexes necessary for productive elongation. Overcoming paus- geting TAF7 expression (Dharmacon), a control target (AP2 RNA sequence ing and resumption of elongation are known to require activation GUGGAUGCCUUUCGGGUCA), and a scrambled duplex control (Dharma- con; catalog no. D-001810-10-05) were transfected in parallel experiments by additional factors, such as NELF, TFIIF, Elongins, and members to a final concentration of 25 nM. ChIP assays were performed using a of the ELL family (7). The finding that TAF7 does not affect modification of the method of Oberley and Farnham (36) on tissues from P-TEFb phosphorylation of Spt5 raises the intriguing possibility the B10.PD1 transgenic mouse strain (32). Further experimental details can that TAF7 functions to alter the substrate specificity of P-TEFb. be found in SI Methods.

Gegonne et al. PNAS ͉ April 8, 2008 ͉ vol. 105 ͉ no. 14 ͉ 5371 Downloaded by guest on September 29, 2021 ACKNOWLEDGMENTS. We thank Dr. Greg Hannon (Cold Spring Harbor Lab- MD), and Hui Ge (Protein One, College Park, MD) for providing TFIIH and CAK; oratory, Cold Spring Harbor, NY) for providing TAF7 shRNA constructs and Drs. Kevin Howcroft, Helit Cohen, Aparna Kotekar, and Steve Shaw for helpful helpful discussions; Drs. Danny Reinberg (New York University School of discussions; and Drs. Brian Lewis, David Levens, Keji Zhao for helpful discus- Medicine, New York, NY), David Levens (National Cancer Institute, Bethesda, sions and critical review of the manuscript.

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