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TAF7: a Possible Transcription Initiation Check-Point Regulator

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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 30, 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.
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