Mechanism of transcriptional antirepression by GAL4-VP16.

Abstract

Promoter-and enhancer-binding factors appear to function by facilitating the transcription reaction as well as by counteracting chromatin-mediated repression (antirepression). We have examined the mechanism by which a hybrid activator, GAL4-VP16, is able to counteract histone HI-mediated repression by using both H1-DNA complexes and reconstituted HI-containing chromatin templates. The GAL4 DNA bind{ng domain alone was sufficient to disrupt local H1-DNA interactions, but a transcriptional activation region was additionally necessary for antirepression. GAL4--VP16-mediated antirepression required an auxiliary factor, denoted as a co-antirepressor, which was partially purified from Drosophila embryos. We have found that the co-antirepressor activity was sensitive to digestion with RNase A. Moreover, total RNA from Drosophila embryos could partially substitute for the co-antirepressor fraction, which indicated that the co-antirepressor may function as a histone acceptor ("histone sink"}. These findings suggest a model for gene activation in which sequence-specific transcription factors disrupt H1-DNA interactions at the promoter to facilitate transfer of HI to a histone acceptor, which then allows access of the basal transcription factors to the DNA template.[Key Words: Transcriptional regulation; histone H1; chromatin; RNA polymerase II; in vitro transcription] Received July 16, 1992; revised version accepted September 29, 1992.The proper control of gene expression is essential for the development, growth, and sustenance of eukaryotic organisms, yet the strategies and mechanisms by which genes are regulated remain to be clarified. An early step in the pathway leading to gene expression is initiation of transcription. Synthesis of mRNA is carried out by the RNA polymerase II transcriptional machinery, which comprises RNA polymerase II and several auxiliary factors that are commonly referred to as general factors (for recent reviews, see Saltzman and Weinmann 1989;Sawadogo and Sentenac 1990;Conaway and Conaway 1991; Zawel and Reinberg 1992). Transcription by the basal transcriptional apparatus is regulated by sequencespecific DNA-binding factors that interact with promoter and enhancer elements (for review, see Johnson and McKnight 1989;Mitchell and Tjian 1989), and it presently appears that many of these promoter-and enhancer-binding proteins may stimulate transcription by acting in conjunction with another class of factors that are referred to as coactivators, mediators, adaptors, or intermediary factors {for review, see Lewin 1990; Ptashne and Gann 1990; Pugh and Tjian 1992}. Transcriptional activity is also affected by chromatin structure {for review, see Weintraub 1985;Elgin 1988;Gross and Garrard 1988; van Holde 1989;Grunstein 1990;Wolffe 1990Wolffe , 1992 Felsenfeld 1992}; thus, it is important to consider the function of the general transcriptional machinery, the promoter-and enhancer-binding factors, and the coactivators with the chromatin template. 1991;To study the relationship between chromatin structure ...

Promoter-proximal pausing of RNA polymerase II defines a general rate-limiting step after transcription initiation.

“…The mechanism could be attributable to cooperativity in binding of trans-activators. Cooperative binding is not always observed on naked DNA templates and in some cases requires DNA packaged into chromatin (Workman et al 1991;Croston et al 1992;Chang and Gralla 1994). However, synergistic activation has been observed under conditions where the binding sites of the activator are saturated Lin et al 1990}.…”

Role of the histone amino termini in facilitated binding of a transcription factor, GAL4-AH, to nucleosome cores.

“…For example, nucleosome cores repress basal transcription both in vivo and in vitro, which increases the dependence of promoter function on the action of upstream regulatory factors (16,23,24,34,60,63,64). Similarly, subsequent binding of the linker histone Hi further represses promoter function and increases the dependence of transcription on regulatory factors in vitro (15,30). These studies implicate a crucial role of upstream activators in histone displacement as well as transcription complex formation at the core promoter (i.e., the TATA box and transcription start site).…”

“…These studies implicate a crucial role of upstream activators in histone displacement as well as transcription complex formation at the core promoter (i.e., the TATA box and transcription start site). This function of upstream activators is dependent on their activation domains (5,15,34,43,64) and is affected in vivo by the stability of nucleosomes located over core promoter sequences (50) and mutations in the N termini of histone H4 (17).…”

“…The binding of GAL4-AH (which contains the DNA-binding and dimerization domains of GALA) to (5,15,34,43,64) and is affected in vivo by the stability of nucleosomes located over core promoter sequences (50) and mutations in the N termini of histone H4 (17).Before upstream activators can act on core promoters, they must first gain access to their respective upstream binding elements (reviewed in reference 1). Studies thus far implicate at least three criteria which govern the ability of factors to access their binding sites on nucleosomes.…”

“…The binding of GAL4-AH (which contains the DNA-binding and dimerization domains of GALA) to (5,15,34,43,64) and is affected in vivo by the stability of nucleosomes located over core promoter sequences (50) and mutations in the N termini of histone H4 (17).…”