TAF7: a Possible Transcription Initiation Check-Point Regulator

TAF7: A possible transcription initiation check-point regulator

Anne Gegonne*, Jocelyn D. Weissman*, Meisheng Zhou†, 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 Laurie H. Glimcher, Harvard Medical School, Boston, MA, November 23, 2005 (received for review November 4, 2005) Transcription consists of a series of highly regulated steps: assem- restricted TAF, TAF4b is found in the TFIID complexes purified bly of a preinitiation complex (PIC) at the promoter nucleated by from a mammalian mature B cell line (18). TFIID, followed by initiation, elongation, and termination. The Four major roles have been ascribed to the individual TAFs. present study has focused on the role of the TFIID component, First, they may act as specific coactivators interacting with TAF7, in regulating transcription initiation. In TFIID, TAF7 binds to transactivators (19–22). Second, some are critical for the stabil- TAF1 and inhibits its intrinsic acetyl transferase activity. We now ity of either the TFIID or SAGA complexes (e.g., TAF10 which report that although TAF7 remains bound to TAF1 and associated contains a histone folding motif; ref. 23). Third, some TAFs are with TFIID during the formation of the PIC, TAF7 dissociates from involved in promoter recognition through direct contact with the PIC upon transcription initiation. Entry of polymerase II into the different core promoter elements (24–27). Finally, TAFs may be assembling PIC is associated with TAF1 and TAF7 phosphorylation, selectively required by subsets of genes (28, 29). For example, the coincident with TAF7 release. We propose that the TFIID compo- largest TAF, TAF1, has been reported to be absolutely indis- sition is dynamic and that TAF7 functions as a check-point regulator pensable for expression of 18% of the mammalian genes (30). suppressing premature transcription initiation until PIC assembly is TAF1 is known to have kinase activity and acetyltransferase complete. activity (31–33), but the roles of these activities and the function of TAF1 are still not understood. Although relatively little is MHC class I ͉ regulation ͉ preinitiation complex ͉ TFIID known about the functions of any of the TAFs, TFIID structure and composition are thought to be relatively invariant on the assembled PIC. n eukaryotic cells, expression of most protein-encoding genes We have previously demonstrated that TAF1 is necessary for Idepends on the RNA polymerase II (Pol II)-dependent tran- the transcription of an MHC class I gene, and that its intrinsic scription machinery. The RNA Pol II machinery assembled on acetyltransferase activity is essential for both in vivo and in vitro the promoter is composed of distinct complexes that are respon- transcription of naked DNA (34). Thus, in ts13 cells with a sible for effecting the sequential steps of transcription initiation temperature-sensitive mutation in TAF1, MHC class I expres- and elongation (1–5). The first step in transcription is the sion is abrogated at the nonpermissive temperature. Similarly, recognition of the core promoter by the TFIID complex and the MHC class I transcription is inhibited, both in vitro and in vivo, assembly of a preinitiation complex (PIC) through an ordered by the transactivator, HIV Tat, which binds to the TAF1 AT recruitment of the general transcription factors (GTFs) TFIIB domain and inhibits its enzymatic activity (35). Of particular and TFIIA, followed by the RNA Pol II holoenzyme, the interest, we recently reported that TAF7, a TFIID component, mediator and the remaining GTFs, TFIIF, TFIIE, and TFIIH. also binds to TAF1 and inhibits its AT activity, resulting in Once the PIC has been assembled, transcription initiation repression of MHC class I transcription (36). Thus, TAF7 is an ensues: local melting of the promoter DNA, formation of the intrinsic regulator of MHC class I transcription. first phosphodiester bond, followed by the synthesis of a short These findings lead to the hypothesis that repression of TAF1 nascent RNA at which point the polymerase pauses. The initi- AT activity by TAF7 must be relieved upon completion of PIC ation of transcription is accompanied by the phosphorylation of assembly to allow the transition to transcription initiation. The serine 5 in the C-terminal domain (CTD) heptad repeat of RNA present studies were designed to determine the fate of TAF7 Pol II (CTD) by the kinase subunit of TFIIH, CDK7 (6–9). during transcription. We report that TAF7, which is initially Despite the extensive understanding of the general require- associated with TFIID in the PIC, is released upon initiation of ments of transcription, many details remain unresolved and transcription. Release of TAF7 from the complex is coincident there is considerable variation among different promoters and with the phosphorylation of TAF1 in the complex; TAF1 cell types. Promoter recognition is mediated by members of phosphorylation reduces the binding of TAF7. We propose a either the TFIID complex family (TFIID, TFIID-like) or the model in which TAF7 is a transcription check-point regulator, that prevents transcription initiation until the PIC is fully SAGA complex family (e.g., TFTC, PCAF, SAGA) (10–12). In assembled. yeast, 90% of gene expression is TFIID-dependent transcription; the remaining 10% is largely dependent on the SAGA complex Results (13). The TFIID complexes are composed of either the TATA- TAF7 Inhibits Transcription in Vivo and in Vitro. TAF7 is a compo- binding protein (TBP) or a TBP related protein (TRF1, TRF2) nent of the general transcription factor, TFIID. We have shown and several TBP-associated factors (TAFs) (14–17). The SAGA that TAF7 binds to TAF1, inhibiting its acetyl transferase activity family complexes do not contain TBP or TBP-related proteins; which is required for basal MHC class I transcription (36). This rather, they contain a GCN5-related histone acetyl transferase finding leads to the surprising prediction that TAF7 is a tran- (AT) subunit, several adapter and Spt proteins and a subset of TAFs. The composition of the TAFs present in these different complexes varies depending on the structure of the promoter and Conﬂict of interest statement: No conﬂicts declared. the cell cycle and tissue-specific gene expression requirement. Freely available online through the PNAS open access option. For example, in yeast, the SAGA complex associated with Abbreviations: Pol II, polymerase II; PIC, preinitiation complex; GTF, general transcription stress-induced gene expression contains five of the TAFs de- factor; TBP, TATA-binding protein; TAF, TBP-associated factor; AT, acetyl transferase. scribed in the TFIID complex (13, 10–12). Conversely, a tissue- ‡To whom correspondence should be addressed. E-mail: [email protected].

602–607 ͉ PNAS ͉ January 17, 2006 ͉ vol. 103 ͉ no. 3 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0510031103 Downloaded by guest on September 29, 2021 Fig. 1. TAF7 inhibits transcription from an MHC class I promoter. An MHC class I promoter construct (1) consisting of 516 bp of extended promoter sequences ligated to the CAT reporter (5 ␮g) was cotransfected with either the TAF7 expression vector (2 or 5 ␮g) or the empty vector (5 ␮g) into CHO cells. After 48 h, CAT activity was measured and corrected relative to protein concentration.

scriptional inhibitor. To test this possibility, CHO cells were cotransfected with an MHC class I promoter construct and a TAF7 expression vector (Fig. 1). As predicted, TAF7 overex- pression resulted in a significant inhibition of class I promoter activity. This inhibition was a direct effect of TAF7 on tran- Fig. 2. TAF7 is associated with a transcriptionally active PIC assembled on a scription, because addition of TAF7 to an in vitro transcription class I promoter. (A) PICs were assembled as described (Supporting Text and Fig. 9) on either the MHC class I promoter or a control promoter-less fragment assay using HeLa nuclear extract similarly inhibited transcription PSV0. Associated proteins were revealed by Western blotting with antibodies (Fig. 8, which is published as supporting information on the to the indicated proteins. The extents of nonspeciﬁc binding of Pol II and TAF7 PNAS web site). Furthermore, TAF7 inhibition is not unique to to the PSV0 control were comparable, as assessed by densitometry (data not the MHC class I promoter: it similarly inhibited a number of shown). (B) Plasmid DNA containing the MHC class I promoter region (lane 1) other promoters (data not shown). This is consistent with the or naked beads (lane 2) or two different concentrations of PIC assembled on widespread (Ϸ24%) dependence of yeast genes on TAF7, which the immobilized promoter DNA template (420 and 600 ng, lanes 3 and 4) were contrasts with TAF1, which is required for only Ϸ10% of genes used as templates in in vitro transcription reactions. The synthesized RNA (37). These findings raise the question of how TAF7 inhibition products were revealed by primer extension using an oligonucleotide primer corresponding to the cat gene region of the template. The two major MHC is relieved in order for transcription to proceed. We considered class I start sites are indicated. two possibilities: (i) TAF7 dissociates from TFIID during PIC assembly, or (ii) TAF7 remains associated with TFIID during ϩ PIC assembly, but is released upon initiation of transcription. synthesized, corresponding to the dominant initiation sites at 1 and ϩ12 bp, observed both in vivo (38) and on naked DNA

TAF7 Is Associated with the DNA-Bound PIC. To determine whether templates (Fig. 2B lane 1 and ref. 35). Thus, TAF7 remains CELL BIOLOGY TAF7 remains associated with TFIID on the PIC, we assembled associated with TFIID during active PIC formation. Addition of TAF7 to an in vitro transcription reaction de- PICs on an immobilized MHC class I promoter template. The creases MHC class I transcription (Fig. 9 and ref. 36). To MHC class I DNA fragment used to nucleate the preinitiation determine whether the TAF7 acts directly on the PIC, we complex was a 731-bp segment, encompassing the MHC class I determined the effect of adding exogenous TAF7 on the activity extended promoter (core and promoter-proximal regulatory of the purified, assembled PIC. Addition of excess TAF7 to the elements) and part of the first exon (Ϫ516͞ϩ44) linked to the 67 PIC assembly reaction before transcription initiation results in first base pairs of the cat reporter gene (Fig. 9, which is published decreased transcription (Fig. 3, compare lanes 1 and 2). How- as supporting information on the PNAS web site). The PICs were ever, addition of TAF7 30 sec after initiation of transcription has assembled with HeLa nuclear extract in the absence of nucleo- no effect, suggesting that TAF7 targets the PIC during initiation tides to prevent transcription initiation. The assembled PIC was (Fig. 3, compare lanes 1 and 3). To directly determine whether purified and associated proteins analyzed by SDS͞PAGE and TAF7 inhibits at initiation or at a subsequent stage in transcrip- Western blotting. As shown in Fig. 2A, the PIC contained major tion, reinitiation of PIC transcription was inhibited by the TFIID components, TAF1 and TAF4, and RNA Pol II, as well addition of sarkosyl at various times after the addition of NTPs. as other GTFs (data not shown). Of note, TAF7 was also a Addition of low concentrations of sarkosyl prevents reinitiation component of the isolated PIC, indicating that TAF7 is still but allows completion of elongating transcripts (Fig. 3, lanes 4 and 6). TAF7 decreased transcription when added before initi- associated with TFIID. Although some background association ation (Fig. 3, lane 4 and 5). In contrast, in the presence of of Pol II and TAF7 was observed to the promoterless control, sarkosyl, TAF7 had no effect when added 30 sec after transcrip- this binding is probably due to some nonspecific association of tion initiation, (Fig. 3, lanes 6 and 7). Thus, excess TAF7 blocks Pol II with DNA and consistently was significantly below the transcription initiation from the assembled PIC, consistent with extent of binding to the class I promoter (see below). its inhibition of the TAF1 AT activity. The assembled, purified PICs were active in transcription: addition of the four NTPs after PIC assembly and isolation Endogenous TAF7 Is Released from the PIC During Transcription. resulted in correct transcription initiation and elongation (Fig. Because endogenous TAF7 remains associated with the assem- 2B, lanes 3 and 4). Two transcripts of 67 and 55 bp were bled PIC but is a negative regulator of the TAF1 AT activity

Gegonne et al. PNAS ͉ January 17, 2006 ͉ vol. 103 ͉ no. 3 ͉ 603 Downloaded by guest on September 29, 2021 ably reflects the dissociation of Pol II as a result of reaching the end of the template and completion of transcription. The Pol II that remained bound to the template is presumed to be associated with inactive PICs, because it was largely unphosphorylated (Fig. 4). Importantly, a significant fraction of TAF7 was released into the supernatant concomitant with transcription initiation and elongation (Fig. 4 and Fig. 10A, which is published as supporting information on the PNAS web site). In eight independent experiments, an average of 18% of the TAF7 was released into the supernatant after transcription. In contrast to TAF7, only a very small amount (8%) of the TFIID component TAF4 was recovered in the supernatant (Figs. 4 and 10A). When normalized to the amount of Pol II recovered in the supernatant after transcription, the amount of TAF7 released into Fig. 3. TAF7 inhibits transcription initiation on an assembled PIC. TAF7 the supernatant was significantly greater (P Ͻ 0.0025) than and͞or sarkosyl at a concentration of either 0.05% or 0.5%, were added either that of TAF4. To control for the possibility that the simple during PIC assembly (0 sec) or 30 sec after the start of transcription, as addition of rNTPs to the PIC nonspecifically released TAF7, indicated. In all cases, the reactions were incubated for 30 min to allow purified PICs were incubated with 20 mM rATP. No prema- completion of transcription. The positions of the two major MHC class I start ture dissociation of either TAF7 or Pol II from the PIC was ϩ ϩ sites ( 1 and 12) are indicated with arrowheads (lane M); the relative level observed in the presence of rATP alone (data not shown). of transcription at each site was determined by densitometry, normalized to Furthermore, TAF7 release requires transcription, because it the level of transcription observed in the absence of either TAF7 or sarkosyl, ␣ and indicated under the appropriate lane. was not released in the presence of the inhibitor -amanitin (Fig. 10B). Therefore, TAF7 is specifically released from the TFIID complex during transcription. necessary for transcription, its inhibition must be relieved to allow transcription to initiate normally. Therefore, we consid- TAF1 Phosphorylation Results in the Dissociation of the TAF1͞TAF7 ered the possibility that TAF7 dissociated from the TFIID Complex. We next examined the mechanism by which TAF7 is complex during transcription initiation. To address this question, released from the PIC during transcription. TAF1 is known to have PICs were assembled on the immobilized class I promoter both a kinase activity and an AT activity. Because TAF1 does not template (Fig. 9). After PIC assembly and isolation, transcription acetylate TAF7 (36), we asked whether TAF7 was phosphorylated was initiated by addition of nucleotides. After incubation to by TAF1 and, if so, whether it affected the interaction. We allow completion of transcription, proteins that remained asso- performed in vitro kinase assays using purified recombinant TAF7 ciated with the immobilized template were removed by magnetic and TAF1 proteins, either alone or in combination. TAF1 alone bead separation. Proteins released into the supernatant during autophosphorylated through its intrinsic kinase activity (Fig. 5A, transcription (as well as those remaining associated with the lane 2). (Coomassie blue staining of purified recombinant TAF1 template and recovered on beads), were assessed by SDS͞PAGE revealed only a single band; data not shown). In contrast, TAF7 did and Western blotting. Before transcription initiation (in the not autophosphorylate (Fig. 5A, lanes 3–5). In the presence of absence of rNTPs), the assembled and purified PIC contained increasing amounts of TAF1, TAF7 was increasingly phosphory- TAF7, TAF4, and Pol II (Fig. 4). After transcription, an average lated (Fig. 5A, lanes 6–8). Thus, the interaction of TAF7 with TAF1 resulted in its phosphorylation. of 40% of the PIC-associated Pol II was recovered in the The TAF7 phosphorylation site(s) were mapped by using a supernatant (based on eight independent experiments). A slow- series of TAF7 deletion mutants (Fig. 5B). The N terminus of mobility form of Pol II, corresponding to a phosphorylated form TAF7 (amino acids 1–109) contains the domain responsible for as shown by immunoblotting with a phospho-Ser-2-specific inhibition of TAF1 AT activity. The central region of the antibody (data not shown), was consistently detected in post- molecule is a serine-rich region that contains the TAF1 inter- transcriptional supernatants and represented an average of Ϯ acting domain (39). As shown in Fig. 5B, TAF7 peptides derived 42.5 2.3% (over six experiments) of the total Pol II in the from the central TAF1-interacting domain and the C-terminal supernatant. The presence of Pol II in the supernatant presum- domain are efficiently phosphorylated by TAF1, whereas the N terminal domain is not a substrate for phosphorylation. These results identify TAF7 as a substrate of the TAF1 kinase activity. The finding that the TAF1-interacting domain of TAF7 is phosphorylated by TAF1 suggested that phosphorylation might affect the interaction between TAF7 and TAF1. To test this possibility, recombinant GST-TAF7 was incubated with Flag-tagged TAF1 in the presence of increasing amounts of [␥-32P]ATP. After the kinase reaction, Flag-tagged TAF1 was recovered on anti-Flag M2 beads, and the amount of TAF7 that copurified with TAF1 was determined by SDS͞PAGE and Western blotting. The extent of phosphorylation of both TAF1 Fig. 4. TAF7, but not TAF4, is released from the MHC class I PIC after and TAF7 (as assessed by extent of 32P incorporation) in- transcription initiation. In vitro transcription reactions of PICs assembled on creased as a function of ATP concentration (data not shown). the MHC class I promoter were done in absence or presence of rNTP. Proteins The amount of TAF7 protein bound to TAF1 (as determined remaining associated with the promoter (beads) or released from the promoter (sup) during transcription were analyzed by Western blot using anti- by Western blotting) was calculated as a function of ATP bodies to the indicated proteins. Two forms of RNA Pol II polymerase were concentration in the kinase reaction. As shown in Fig. 6A, the detected: the hyperphosphorylated upper band and the hypophosphorylated binding of TAF1 to TAF7 decreased with increasing phos- lower band. phorylation of the TAF1 and TAF7 proteins. Thus, the

604 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0510031103 Gegonne et al. Downloaded by guest on September 29, 2021 Fig. 6. TAF1 autophosphorylation results in the dissociation of TAF7. (A) Kinase reactions were performed by combining recombinant Flag-tagged TAF1 and recombinant GST-tagged TAF7 in presence of increasing amounts of ATP. Flag-tagged TAF1 was immobilized on anti-Flag M2 agarose beads, and the amount of both TAF1 and associated TAF7 recovered were determined by Western blot analysis using the anti-Flag M2 antibody and a GST antibody, respectively. The graph represents the amount of TAF7 associated with TAF1 (corrected to the amount of TAF1 present on the beads) as a function of the ATP added to the kinase reaction. Phosphorylation of the TAF1 and TAF7 was monitored by including [␥-32P]ATP in the assay. (B) Recombinant Flag-tagged TAF1 was phosphorylated in presence of increasing amount of ATP, immobilized on anti-Flag M2 agarose beads; after extensive washing, GST-tagged Fig. 5. TAF1 phosphorylates itself and TAF7. (A) Kinase reactions were TAF7 was added. The amount of TAF1 and associated TAF7 recovered was performed with recombinant Flag-tagged TAF1 alone (200 ng; lane 2), recom- determined by Western blot analysis using the anti-Flag M2 antibody and a binant Flag-tagged TAF7 with increasing amounts of control extract (lanes GST antibody, respectively. The graph represents the amount of TAF7 associ- 3–5), and Flag-tagged TAF7 in presence of increasing amounts of Flag-tagged ated with TAF1 (corrected to the amount of TAF1 present on the beads) as a TAF1 (lanes 6–8, 40, 80, and 200 ng, respectively). The control was extract function of the ATP added to the kinase reaction. puriﬁed from uninfected SF9 cells. (B) Kinase reactions were performed as in

A, but using recombinant Flag-TAF7 peptide fragments, as diagrammed, CELL BIOLOGY derived from the N terminus, central region and C terminus of TAF7. The TAF1 and TAF7 were phosphorylated, suggesting that TAF1 additional bands observed in lane 8 result from minor degradations observed phosphorylation is the primary determinant in the extent of in this fragment. interaction. These results indicate that although TAF1 phosphorylates TAF7, TAF1 autophosphorylation is sufficient to interaction of TAF7 and TAF1 is negatively regulated by reduce the binding of TAF7 to TAF1. The functional conse- phosphorylation. quences of TAF7 phosphorylation remain to be determined; the Under the conditions of this experiment, it was not possible to possibility that TAF7 phosphorylation further reduces the in- distinguish whether the transphosphorylation of TAF7 or auto- teraction with TAF1 cannot be excluded. phosphorylation of TAF1, or both, was responsible for the loss Although recombinant TAF1 undergoes autophosphorylation of binding. To address this question, the following experiment and transphosphorylates TAF7, the phosphorylation states of was performed (see Fig. 6B Inset): Flag-tagged TAF1 alone was TAF1 and TAF7 in the context of TFIID has not been examined incubated in the presence of increasing concentrations of previously. Therefore, we determined whether (and when) [␥-32P]ATP to permit autophosphorylation. The phosphorylated TAF1 and͞or TAF7 are phosphorylated in TFIID by sequential Flag-tagged TAF 1 was bound to anti-Flag M2 agarose beads and addition of purified GTFs in the presence of [␥-32P]ATP to washed to remove unincorporated ATP. Unphosphorylated assemble PICs on the class I promoter fragment. Phosphoryla- GST-TAF7 was then incubated with the phosphorylated TAF1 tion of TAF1 and TAF7 were monitored at each step in the immobilized on the beads. The ability of TAF7 to bind to TAF1 assembly (Fig. 7 Left). Neither TAF1 nor TAF7 were phosphor- was determined by recovering the beads and analyzing the ylated in the presence of only TFIID and the GTFs. Phosphor- amount of TAF7 bound to TAF1 by SDS͞PAGE and Western ylation of both TAF1 and TAF7 depended on the addition of Pol blotting. Surprisingly, with increasing ATP concentration and II to the PIC assembly reaction. Thus, TAF1 and TAF7 in TFIID phosphorylation of TAF1, the subsequent binding of TAF7 to are phosphorylated, but only after the entry of Pol II to the TAF1 decreased, reaching a plateau at Ϸ50% of the initial assembled PIC. It should be noted that these experiments do not binding to unphosphorylated TAF1 (Fig. 6B). The extent of the distinguish TAF1-dependent phosphorylation from phosphory- decrease in binding approximated that observed when both lation by another kinase. Further, and importantly, TAF7 was

Gegonne et al. PNAS ͉ January 17, 2006 ͉ vol. 103 ͉ no. 3 ͉ 605 Downloaded by guest on September 29, 2021 Even less common are phosphorylation-mediated dissociations that lead to activation. Perhaps the best characterized example is that of the ATM molecule involved in repair of double-strand breaks in DNA. In the absence of DNA damage, ATM exists as an inactive dimer; phosphorylation of the ATM dimer triggers its dissociation and activation (40). The interaction between TAF7 and TAF1 provides another example of phosphorylation- mediated dissociation, in this case resulting in activation of TAF1 AT activity (36). The present findings also raise the question of the role of TAF1 in PIC assembly and transcription initiation. As the largest component of the TFIID complex, TAF1 was originally thought to function primarily as a scaffold for the other TFIID constit- uents. It is now clear that TAF1 plays an active role in tran- Fig. 7. TAF1 phosphorylation in TFIID occurs upon entry of Pol II into the PIC scription. The AT activity of TAF1 is necessary for transcription, and is coincident with TAF7 release. (A) Transcription complexes were assem- although its exact role is still not known (35). The TAF1 AT bled from various combinations of purified transcription factors on the im- activity is inhibited by the binding of TAF7 (36). The present mobilized MHC class I promoter in presence of [␥-32P]ATP, as indicated above each lane. Phosphorylated proteins were analyzed by gel electrophoresis and studies suggest that the release of TAF7 after completion of PIC autoradiography. The position of RAP74, which is known to be phosphory- assembly relieves the inhibition of TAF1 AT activity, allowing lated by TAF1, is shown as a control and indicated based on mobility. The transcription to proceed. Indeed, excess TAF7, both in vitro and positions of TAF1 and of TAF7 are indicated based on both mobility and in vivo, blocks initiation, presumably by maintaining the associ- Western blot analysis of the same gel; it is the lower band of the doublet in ation with TAF1. lanes 3, 6, and 7. The purified transcription factors were TFIID, D; TFIIB, B; TFIIE, The extent of TAF7 release from the PIC parallels the extent E; TFIIF, F; TFIIH, H; and RNA polymerase II. (B) Purified transcription complexes of productive in vitro transcription, suggesting that the two were assembled as in A. Phosphorylation of proteins that either remained processes are closely linked. Transcription from an immobilized associated with the PIC (B) or were released into the supernatant (S) was identified by separation of the assembled PIC from the supernatant and MHC class I promoter DNA fragment resulted in the release of analysis on PAGE, followed by autoradiography. The position of TAF7 is 20% of the TAF7 initially associated with the PIC. This amount indicated based on both mobility and Western blot analysis of the same gel. corresponds approximately to the fraction of hyperphosphorylated Pol II that mediated productive transcription. Thus, productive transcription is accompanied by the release of TAF7 only released from the complex upon addition of Pol II (Fig. 7 from TFIID and the hyperphosphorylation of Pol II. In similar Right). From these experiments, we conclude that completion of experiments in yeast, using an immobilized HIS4 promoter and PIC assembly signals TAF1 and TAF7 phosphorylation and yeast extracts to reconstitute the PIC, Yudkovsky et al. found release of TAF7 allowing transcription to initiate. that TFIID (and particularly TBP, TAF5, TAF7) and TFIIA remained associated with the promoter (41) during transcription Discussion elongation. They did not observe the release of TAF7. One Transcription depends on the temporal and spatial integration of possible explanation for the discrepancy between their results preinitiation complex assembly, initiation, elongation, and ter- and ours may relate to differences in the TFIID dependency of mination. Proper regulation of the transition from PIC assembly the promoters examined. The release of TAF7 from TFIID may to initiation is necessary to prevent aberrant or abortive tran- only be observed on promoters such as the MHC class I scription. Here, we have demonstrated that TAF7, which inhibits promoter whose transcription requires TAF1 and its AT activity the AT activity of TAF1 required for TFIID-dependent tran- (35). The TAF1-dependence of the HIS4 promoter was not scription, is associated with TFIID during PIC formation. Upon examined. addition of Pol II to the assembling PIC, TAF1 and TAF7 In conclusion, we propose that TAF7 acts as a checkpoint undergo phosphorylation, releasing TAF7 from the PIC. We regulator in the transition from PIC assembly to transcription propose that the association of TAF7 with TFIID during PIC initiation. We propose that, during PIC assembly, TAF7 remains assembly prevents transcription initiation before the successful bound to TFIID, thereby inhibiting the AT activity of TAF1 and completion of the recruitment of the other GTFs and Pol II. The preventing premature transcription initiation. Once PIC assem- release of TAF7 from TFIID relieves the inhibition of TAF1 AT bly is completed, TAF1 undergoes phosphorylation, resulting in activity and allows transcription to initiate. Thus, we propose the release of TAF7 and activation of its AT activity, which that TAF7 serves as a checkpoint regulator in the transition from allows transcription to initiate. Further studies are needed to test PIC assembly to initiation. this model. The release of TAF7 from TFIID in an assembled PIC is coincident with TAF1 phosphorylation. In vitro, autophosphor- Materials and Methods ylation of recombinant TAF1 reduces its binding to TAF7. This For further details, see Supporting Text, which is published as finding, which reports a role for TAF1 autophosphorylation, supporting information on the PNAS web site. suggests that autophosphorylation may regulate the release of TAF7 from TFIID during the transition from PIC assembly to Assembly and Purification of PICs. Biotinylated PCR product (600 initiation. What regulates TAF1 phosphorylation in the assem- ng), dIdC (600 ng), and HeLa nuclear extract (150 ␮g, Promega) bled PIC and whether it is by the TAF1 kinase or another kinase were incubated for 40 min at 23°C in in vitro transcription buffer remain to be determined. The mechanism by which phosphor- (20 mM Hepes, pH 7.9͞50 mM KCl͞6.24 mM MgCl2͞0.5 mM ylation of TAF1 alters the avidity of TAF7 binding also remains EDTA͞2mMDTT͞10 ␮M ZnSO4͞10 mM creatine phosphate͞ to be determined. It is possible that a conformational change in 100 ␮g/ml creatine kinase͞8.5% glycerol). The assembled PIC autophosphorylated TAF1 obscures or destroys the TAF7- was captured on streptavidin-coated magnetic beads that had binding site. Although phosphorylation-mediated changes in been preequilibrated in binding buffer (20 mM Hepes, pH molecular interactions are well known, in most cases, phosphor- 7.9͞80 mM KCl͞10 mM MgCl2͞2mMDTT͞10 ␮M ZnSO4͞100 ylation results in intermolecular associations, not dissociations. ␮gofBSA͞0.5% Nonidet P-40, and 10% glycerol) (BB) by

606 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0510031103 Gegonne et al. Downloaded by guest on September 29, 2021 incubation for an additional 30 min. The captured DNA͞PIC GST antibody to detect TAF7. The amount of TAF7 bound to complex was washed five times in BB. TAF1 was normalized according to the amount of TAF1 on the agarose beads. In Vitro Transcription (IVT) with Purified PIC. IVT reactions were performed by resuspending purified PICs in 25 ␮l of IVT buffer TAF7 Binding to Autophosphorylated TAF1. Flag-TAF1 (250 ng) was 32 (8 mM Hepes, pH 7.9͞40 mM KCl͞6 mM MgCl2͞0.2 mM incubated in presence of increasing amount of [␥- P]ATP as DTT͞80 ␮M EDTA͞8% glycerol) in presence of 1.6 mM of each described above. One fifth of the reaction was loaded on an rNTP and incubating for 30 min at 20°C. Transcripts were SDS͞PAGE to test the efficiency of the kinase reaction. The revealed by primer extension (2). Proteins either released into remainder was captured on anti-Flag M2 agarose in kinase the supernatant or remaining on the beads after IVT were buffer supplemented with 17% glycerol and a mixture of phos- separated by SDS͞PAGE gel and analyzed by Western blotting. phatase inhibitors. Agarose-bound [32P]TAF1 was washed four When added, 2 ␮gof␣-amanitin was presented during PIC times and then mixed with 200 ng of GST-TAF7 the same buffer. assembly and IVT. Agarose-bound complexes were separated on SDS͞PAGE. TAF1 and TAF7 were revealed by Western blotting using TAF1 Kinase Assays. Flag-tagged TAF7 (250 ng) was incubated for anti-Flag M2 or GST antibodies, respectively. The amount of 30 min at 30°C with increasing amounts of Flag-tagged TAF1 TAF7 bound to TAF1 was normalized according to the amount (50, 100, 250 ng) or control extract immobilized on anti-Flag M2 of TAF1 on the agarose beads. agarose in 20 ␮l of kinase buffer (25 mM Hepes, pH 7.9͞100 mM ͞ ͞ ͞ KCl 12.5 mM MgCl2 0.1 mM EDTA 0.1% Nonidet P-40) in the PIC Assembly and in Vitro Kinase Assay. Biotinylated PCR product ␮ ␥ 32 ͞ presence of 10 Ci of [ - P]ATP (6,000 Ci mM); the proteins (30 ng) was immobilized on streptavidin magnetic beads and ͞ were resolved by SDS PAGE gel, and labeling was quantitated then incubated in presence of 10 ␮Ci of [␥-32P]ATP (6,000 by PhosphorImager. All kinase assays of TAF7 or different Ci͞mM) with combinations 100 ng of TFIID, 20 ng of TFIIB, 10 TAF7 mutants were performed with the same molar amount of ng of TFIIE, 10 ng of TFIIF, 50 ng of TFIIH, and 50 ng of Pol proteins. II as indicated. Purified GTFs were obtained from Protein One, Bethesda, MD. The reactions were incubated for1hin20␮lof TAF1 Phosphorylation and Binding to TAF7. Flag-TAF1 (250 ng) and kinase buffer (50 mM Tris, pH 7.9͞5mMDTT͞10 ␮M ZnSO4͞5 GST-TAF7 (200 ng) were mixed together in presence of increas- mM MnCl͞4 mM MgCl2͞1 mM ATP). The proteins were ing amounts of [␥-32P]ATP in the same conditions as described ͞ ͞ resolved by SDS PAGE gel, and labeling was revealed by above. One-fifth of the reaction was loaded on an SDS PAGE PhosphorImager. to test the efficiency of the kinase reaction. The remainder was ϫ subject to anti-Flag M2 agarose in 1 kinase buffer supple- We thank Drs. David Levens, Keji Zhao, Alfred Singer, and Bob Tjian mented with 17% glycerol and a mixture of phosphatase inhib- for helpful discussions and critical reading of the manuscript. This itors. Agarose-bound complexes were washed twice, and pro- research was supported by the Intramural Research Program of the teins were separated on SDS͞PAGE and revealed by Western National Institutes of Health, National Cancer Institute Center for blotting using either anti-Flag M2 antibody to detect TAF1 or an Cancer Research.

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