Accessibility of α2-Repressed Promoters to the Activator Gal4

Abstract

It has been proposed that eukaryotic repressors of transcription can act by organizing chromatin, thereby preventing the accessibility of nearby DNA to activator proteins required for transcription initiation. In this study, we test this idea for the yeast ␣2 repressor using a simple, artificial promoter that contains a single binding site for the activator protein Gal4 and a single binding site for the repressor ␣2. When both the repressor and the activator are expressed in the same cell, the artificial promoter is efficiently repressed. In vivo footprinting experiments demonstrate that Gal4 can occupy its binding site even when the promoter is repressed. This result indicates that ␣2-directed repression must result from interference with some stage in transcription initiation other than activator binding to DNA.Negative regulation of transcription in eukaryotes occurs by a variety of mechanisms. Some repressors act by preventing the DNA binding of activators, some bind DNA and interact with nearby activators, ''quenching'' their activation surface, and some communicate directly with the general transcription machinery, blocking its function or assembly (for reviews, see references 14, 16, 18, and 26). Still other repressors appear to organize repressive forms of chromatin that block the accessibility of proteins to DNA (for reviews, see references 31, 33, and 45). For some repressors, more than one of these mechanisms is thought to function simultaneously, resulting in a very low level of gene expression under repressing conditions.One case in which two mechanisms of repression have been proposed is that of the yeast ␣2 protein. This protein is responsible for repressing the expression of two sets of cell-typespecific genes, a-specific genes and haploid-specific genes (for reviews, see references 7, 15, and 17). To repress a-specific genes, ␣2 binds cooperatively with the Mcm1 protein to a 34-bp DNA sequence called the a-specific gene operator. ␣2/ Mcm1 binds a second protein complex composed of the Tup1 and Ssn6 proteins. Tup1 and Ssn6 are required for the repression of at least five sets of yeast genes and have been proposed to function as a general repression machine in Saccharomyces cerevisiae, recruited to DNA by a variety of sequence-specific DNA-binding proteins (21,24,41,42).The a-specific gene operator will bring about repression when placed in many positions upstream of a target gene, and models for repression by ␣2/Mcm1/Ssn6/Tup1 (referred to as the ␣2 repression complex) must account for this action at a distance (20,32). One model proposes that the ␣2 repression complex interacts directly with the general transcription machinery at the promoter, blocking its assembly or maturation (13,20). A second model proposes that the ␣2 repression complex positions nucleosomes over promoter elements, blocking the accessibility of nearby DNA to proteins (23,34,35,37). In this work, we wished to determine whether an ␣2-repressed promoter is accessible to Gal4, a yeast activator protein that binds DNA. MATERIALS...

Amino Termini of Histones H3 and H4 Are Required for a1-α2 Repression in Yeast

“…Interestingly, the CYC1 promoter used here is the only RNA polymerase II-transcribed promoter described to date that escapes the positioning of nucleosomes adjacent to the ␣2-Mcm1 operator (23). These findings and our current data indicate that nucleosome positioning may play a secondary role in repression by ␣2-Mcm1.…”

The Cyc8–Tup1 complex inhibits transcription primarily by masking the activation domain of the recruiting protein

“…Importantly, these events occur long before redeposition of nucleosomes to promoters, indicating that Tup1 is able to block transcription before any repressive chromatin structure is established; such repression in the absence of nucleosomes has been previously demonstrated (Bryant et al 2008). Previous studies (Redd et al 1996;Papamichos-Chronakis et al 2002Proft and Struhl 2002;Zhang and Reese 2005) and our analysis of Gcn4 binding in the ''mechanistic strain'' indicate that Tup1 does not inhibit activator binding. As recruitment of all three coactivators is mediated by direct and independent interactions with activation domains of DNA-bound activator proteins, the kinetic profile of molecular events at target promoters indicates that Tup1 directly blocks activator-mediated recruitment of these coactivators.…”

“…In principle, Cyc8-Tup1 could block activator-mediated recruitment of coactivator complexes or block binding of the activator to target promoters, although previous studies suggest that activator binding is not inhibited (Redd et al 1996;Papamichos-Chronakis et al 2002Proft and Struhl 2002;Zhang and Reese 2005). As an independent approach to address the effect of Cyc8-Tup1 on activator binding, we generated a ''mechanistic'' (A) Pol II association with the indicated coding regions in the Tup1-anchor-away or Tup1/TBP-anchor-away strains that were or were not treated with rapamycin for 1 h. (B) H3 occupancy with the indicated promoters in the Tup1-anchor-away or Tup1/ TBP-anchor-away strains that were or were not treated with rapamycin for 1 h. (C) H3 occupancy with the indicated promoters in the Tup1-anchor-away and Tup1/Snf2-anchor-away strains that were or were not treated with rapamycin for 1 h. Averages and standard errors of three individual experiments are shown.…”

Gal4p-Mediated Chromatin Remodeling Depends on Binding Site Position in Nucleosomes but Does Not Require DNA Replication

“…In each plasmid, the ␣2 operator was intentionally placed at a relatively far distance from the promoter in order to abrogate both activation by Mcm1p (46) bound at the operator in a-cells (i.e., in the absence of MAT␣2p) (Fig. 6, YCpTALS4 data) and repression of RNA polymerase II transcription by MAT␣2p in ␣-cells (51). In addition, this excludes all CYC1 TATA elements, which are constitutively bound by TATA-binding protein (12), from the positioned nucleosome.…”