Novel Cofactors and TFIIA Mediate Functional Core Promoter Selectivity by the Human TAF_II150-Containing TFIID Complex

Abstract

TATA-binding protein-associated factors (TAF II s) within TFIID control differential gene transcription through interactions with both activators and core promoter elements. In particular, TAF II 150 contributes to initiator-dependent transcription through an unknown mechanism. Here, we address whether TAF II s within TFIID are sufficient, in conjunction with highly purified general transcription factors (GTFs), for differential core promoter-dependent transcription by RNA polymerase II and whether additional cofactors are required. We identify the human homologue of Drosophila TAF II 150 through cognate cDNA cloning and show that it is a tightly associated component of human TFIID. More importantly, we demonstrate that the human TAF II 150-containing TFIID complex is not sufficient, in the context of all purified GTFs and RNA polymerase II, to mediate transcription synergism between TATA and initiator elements and initiator-directed transcription from a TAF II -dependent TATA-less promoter. Therefore, TAF II -promoter interactions are not sufficient for the productive core promoter-selective functions of TFIID. Consistent with this finding, we have partially purified novel cofactor activities (TICs) that potentiate the TAF II -mediated synergism between TATA and initiator elements (TIC-1) and TAF II -dependent transcription from TATA-less promoters (TIC-2 and -3). Furthermore, we demonstrate an essential function for TFIIA in TIC-and TAF II -dependent basal transcription from a TATA-less promoter. Our results reveal a parallel between the basal transcription activity of TAF II s through core promoter elements and TAF II -dependent activator function.The structure of the core promoter region (i.e., DNA sequences flanking the transcription start site and including the TATA, initiator, and downstream elements that interact with the general transcription machinery) of protein-coding genes has an important influence both on the efficiency of basal transcription and on the ability of the core promoter to respond to upstream promoter-bound activators in vivo and in vitro (reviewed in references 33, 38, and 46). Although the general transcription machinery has been well characterized, little is known about the factors and mechanisms that control its activity in a core promoter-specific manner. Whereas various factors like E2F, YY1, TFII-I, and USF may regulate transcription not only through upstream promoter elements but also through interactions with the core promoter regions of certain genes (for reviews, see references 38 and 50), several components of the basal transcription machinery have intrinsic and more general core promoter-selective functions. For instance, the differential affinity of TATA-binding protein (TBP) for different TATA box DNA sequences and conformations can influence the efficiency of transcription initiation (28, 37, 39; for a review, see reference 3). Previous studies have also demonstrated differential requirements for the general transcription factors (GTFs) TFIIE, TFIIF, and TFIIH for basal...

Taspase1 processing alters TFIIA cofactor properties in the regulation of TFIID

“…1 Importantly, TFIIA also promotes interactions between TFIID TAFs and core promoter DNA sequences downstream of the TATA region 13,19,20 and has been shown to be an essential cofactor for TAF-dependent INR function both in the absence and presence of a TATA element. 14 Consistent with these observations, results of site-specific DNA crosslinking studies provided first evidence that TFIIA affects the positioning of individual TFIID TAFs relative to downstream promoter DNA within TFIID nucleoprotein complexes. 20 Recent high-resolution single particle electron microscopy studies revealed 2 distinct TFIID conformations.…”

“…14,15 Positive cofactors for INR function include the Mediator complex and the architectural transcription factor HMGA1, which stimulate the activity of the basal transcription machinery in the presence of an INR element in a TFIID TAF-dependent manner and, at the same time, counteract repression of TATA-dependent transcription initiation by NC2. 15,16 Starting point of the work presented here was the surprising observation that our reconstituted in vitro transcription system composed of highly purified human GTFs and RNAP II can support significant levels of INR-selective core promoter activity in the presence of NC2 when supplied with excess amounts of highly purified f:TFIID (Fig.…”

“…27 Given the stringent conditions of the purification protocol, TFIIA in f:TFIID preparations must be tightly associated with the TFIID complex. Because TFIIA is required for synergistic binding of TFIID to TATA and INR core promoter elements 13 and for INR-dependent basal transcription in vitro, 14 we speculated that the subpopulation of TFIID complexes in our f:TFIID preparations that is resistant to NC2 repression in the presence of an INR element is stably associated with TFIIA. To further investigate this possibility, we carried out immunoblot analyses and identified two independent f:TFIID preparations (A) and (B), that differ significantly in TFIIA content.…”

“…However, TAF/promoter interactions are not sufficient to mediate the strong functional synergy of TATA and INR elements observed in crude nuclear extracts. 14,15 A strong response of TATA-containing core promoters to the presence of an INR element requires positive and negative TAF-and INR-dependent cofactors (TICs), 14 some of which were recently identified. 15, 16 We previously demonstrated that positive-acting TICs counteract repression of TATA-dependent basal transcription by the ubiquitous repressor NC2 in the presence of a correctly spaced INR element in a TFIID TAF-dependent manner.…”

The RNA polymerase II core promoter: a key component in the regulation of gene expression

“…For instance, NC2/Dr1-Drap1 represses TATA-dependent transcription and activates DPE-dependent transcription (Willy et al 2000). In addition, cofactors that specifically activate transcription from TATA-less core promoters have been partially purified (Martinez et al 1998).…”