Yeast NC2 associates with the RNA polymerase II preinitiation complex and selectively affects transcription in vivo.

NC2 (Dr1-Drap1 or Bur6-Ydr1) has been characterized in vitro as a general negative regulator of RNA polymerase II (Pol II) transcription that interacts with TATA-binding protein (TBP) and inhibits its function. Here, we show that NC2 associates with promoters in vivo in a manner that correlates with transcriptional activity and with occupancy by basal transcription factors. NC2 rapidly associates with promoters in response to transcriptional activation, and it remains associated under conditions in which transcription is blocked after assembly of the Pol II preinitiation complex. NC2 positively and negatively affects approximately 17% of Saccharomyces cerevisiae genes in a pattern that resembles the response to general environmental stress. Relative to TBP, NC2 occupancy is high at promoters where NC2 is positively required for normal levels of transcription. Thus, NC2 is associated with the Pol II preinitiation complex, and it can play a direct and positive role at certain promoters in vivo.

TATA-binding protein mutants that increase transcription from enhancerless and repressed promoters in vivo.

Using a genetic screen, we isolated three TATA-binding protein (TBP) mutants that increase transcription from promoters that are repressed by the Cyc8-Tup1 or Sin3-Rpd3 corepressors or that lack an enhancer element, but not from an equivalently weak promoter with a mutated TATA element. Increased transcription is observed when the TBP mutants are expressed at low levels in the presence of wild-type TBP. These TBP mutants are unable to support cell viability, and they are toxic in strains lacking Rpd3 histone deacetylase or when expressed at higher levels. Although these mutants do not detectably bind TATA elements in vitro, genetic and chromatin immunoprecipitation experiments indicate that they act directly at promoters and do not increase transcription by titration of a negative regulatory factor(s). The TBP mutants are mildly defective for associating with promoters responding to moderate or strong activators; in addition, they are severely defective for RNA polymerase (Pol) III but not Pol I transcription. These results suggest that, with respect to Pol II transcription, the TBP mutants specifically increase expression from core promoters. Biochemical analysis indicates that the TBP mutants are unaffected for TFIID complex formation, dimerization, and interactions with either the general negative regulator NC2 or the N-terminal inhibitory domain of TAF130. We speculate that these TBP mutants have an unusual structure that allows them to preferentially access TATA elements in chromatin templates. These TBP mutants define a criterion by which promoters repressed by Cyc8-Tup1 or Sin3-Rpd3 resemble enhancerless, but not TATA-defective, promoters; hence, they support the idea that these corepressors inhibit the function of activator proteins rather than the Pol II machinery.

Subregions of the adenovirus E1A transactivation domain target multiple components of the TFIID complex.

Transcriptional activation by the adenovirus E1A 289R protein requires direct contacts with the TATA box-binding protein (TBP) and also displays a critical requirement for TBP-associated factors (TAFs) (T.G. Boyer and A. J. Berk, Genes Dev. 7:1810-1823, 1993; J. V. Geisberg, W. S. Lee, A. J. Berk, and R. P. Ricciardi, Proc. Natl. Acad. Sci. USA 91:2488-2492, 1994; W. S. Lee, C. C. Kao, G. O. Bryant, X. Liu, and A. J. Berk, Cell 67:365-376, 1991; and Q. Zhou, P. M. Lieberman, T. G. Boyer, and A. J. Berk, Genes Dev. 6:1964-1974, 1992). In this report, we demonstrate that the activation domain of E1A (CR3) specifically binds to two TAFs, human TAFII250 (hTAFII250) and Drosophila TAFII110 (dTAFII110). These interactions can take place both in vivo and in vitro and require the carboxy-terminal region of CR3; the zinc finger region of CR3, which binds TBP, is not needed to bind these TAFs. We mapped the E1A-binding sites on hTAFII250 to an internal region that contains a number of structural motifs, including an HMG box, a bromodomain, and direct repeats. This represents the first demonstration that hTAFII250 may serve as a target of a transcriptional activator. We also mapped the E1A binding on dTAFII110 to its C-terminal region. This is of significance since, by contrast, Sp1-mediated activation requires binding to the N-terminal domain of dTAFII110. Thus, distinct surfaces of dTAFII110 can serve as target sites for different activators. Our results indicate that E1A may activate transcription, in part, through direct contacts of the CR3 subdomains with selected components of the TFIID complex.

The viral oncoproteins Ad5 E1A, HPV16 E7 and SV40 TAg bind a common region of the TBP-associated factor-110.

A function shared by the adenovirus E1A, papillomavirus E7 and SV40 TAg oncoproteins is their ability to interfere with normal cell growth by interacting with members of the retinoblastoma protein family. In this study, we show that each of these oncoproteins can also bind to the 921 amino acid TBP-associated factor-110 (TAF-110). The significance of the binding is underscored by the observation that each oncoprotein binds to the same 77 amino acid carboxyl region of TAF-110. In the case of E1A and TAg, this finding is consistent with their abilities to stimulate transcription initiation, in part, through their known interactions with TBP. While it is not clear whether E7 can also activate promoters through protein:protein interactions with components of the transcription initiation complex, our demonstration that E7 can bind to TAF-110, as well as TBP, suggests that E7 may modulate the expression of specific promoters which could contribute to the pathogenesis of human papillomavirus.

The zinc finger region of the adenovirus E1A transactivating domain complexes with the TATA box binding protein.

The 289R E1A protein of adenovirus transactivates a variety of viral and cellular promoters through protein-protein interactions. In earlier studies, mutational analyses of the E1A transactivating domain identified residues that are critical for transactivation and implied that the zinc finger region of the transactivating domain binds a transcription factor. Also, the E1A activation domain was found to bind to the TATA box binding protein (TBP) in vitro. Here, we tested the significance of the E1A-TBP interaction for E1A transactivation by analyzing the effects of conservative substitutions at each of the 49 residues of the E1A activation domain. Seven of the substitutions significantly diminished TBP binding in vitro. All of these were in the zinc finger region and were defective for transactivation in vivo. The perfect correlation between reduced TBP binding and transactivation argues strongly that a direct interaction between the E1A activation domain and TBP is critical to the mechanism of E1A activation. This genetic analysis leads us to further suggest that another factor, which is limiting, is also necessary for E1A-mediated transactivation.