Adr1 and Cat8 Mediate Coactivator Recruitment and Chromatin Remodeling at Glucose-Regulated Genes

Adr1 and Cat8 co-regulate numerous glucose-repressed genes in S. cerevisiae, presenting a unique opportunity to explore their individual roles in coactivator recruitment, chromatin remodeling, and transcription.We determined the individual contributions of Cat8 and Adr1 on the expression of a cohort of glucose-repressed genes and found three broad categories: genes that need both activators for full derepression, genes that rely mostly on Cat8 and genes that require only Adr1. Through combined expression and recruitment data, along with analysis of chromatin remodeling at two of these genes, ADH2 and FBP1, we clarified how these activators achieve this wide range of co-regulation. We find that Adr1 and Cat8 are not intrinsically different in their abilities to recruit coactivators but rather, promoter context appears to dictate which activator is responsible for recruitment to specific genes. These promoter-specific contributions are also apparent in the chromatin remodeling that accompanies derepression: ADH2 requires both Adr1 and Cat8, whereas, at FBP1, significant remodeling occurs with Cat8 alone. Although over-expression of Adr1 can compensate for loss of Cat8 at many genes in terms of both activation and chromatin remodeling, this over-expression cannot complement all of the cat8Delta phenotypes.Thus, at many of the glucose-repressed genes, Cat8 and Adr1 appear to have interchangeable roles and promoter architecture may dictate the roles of these activators

Expression in activator mutants^a

a<p>Values are the average of 3 biological samples, each one quantitated in duplicate and normalized to ACT1 values. The standard deviation is shown in parenthesis, except in cases where the samples were pooled prior to qPCR.</p>b<p>Genes are arranged in descending order of Cat8-dependence</p

Coactivators used in ChIP Analysis

a<p>Results in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001436#pone-0001436-g001" target="_blank">Figure 1</a></p>b<p>Results not shown</p>c<p>Results are averaged and shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001436#pone-0001436-g001" target="_blank">Figure 1f</a></p>d<p>Results shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001436#pone-0001436-t003" target="_blank">Table 3</a></p

Gene Expression Levels in Coactivator Deletion Strains^a

a<p>Values expressed as % wildtype after 4 hours of derepression</p

Differential roles for Adr1 and Cat8 in chromatin remodeling at <i>ADH2</i> and <i>FBP1.</i>

<p>NuSA results are displayed as the amount of relative protection, after normalization to the well-positioned nucleosome at <i>CEN3</i>. The position of each amplicon (referenced to the middle of each amplicon) within the promoter is shown on the x-axis, with approximate location of nucleosomes shown. (A) and (C) are the results at <i>ADH2,</i> (B) and (D) are at <i>FBP1</i>. (A) and (B) compare NuSA results of <i>Δadr1 (</i>pink) and <i>Δcat8</i> (green) in derepressed conditions to a wildtype strain either in repressed (dark blue) or derepressed (light blue) conditions. (C) and (D) compare NuSA results between over-expression of Adr1 with (red) or without Cat8 (blue) in derepressed conditions.</p

ChIP Analysis of Mediator Components^a

a<p>Values expressed as the %IP'ed divided by the %IP'ed at the <i>TEL</i></p

Expression in <i>cat8Δ</i> strains with single copy or multi-copy Adr1

a<p>Data taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001436#pone-0001436-t001" target="_blank">Table 1</a> (% WT strain)</p>b<p>Values are an average of biological duplicates after 4 hours of derepression, normalized to <i>ACT1</i> and expressed as the percent of activation in a multi-copy Adr1 <i>CAT8</i> strain, with the standard deviation shown in parenthesis.</p

Recruitment profiles under derepressed conditions.

<p>A–G: ChIP analysis for coactivators/general transcription machinery at the indicated promoter regions in a wildtype strain (black), <i>adr1Δ</i> (pink), <i>cat8Δ</i> (blue) and <i>adr1Δcat8Δ</i> (green). Binding is expressed as the percent of the wildtype derepressed value (set to 100%) after normalization to the <i>TEL</i> negative control locus. Error bars represent the standard deviation of biological replicates (two or more). Data is shown at 4 hours of derepression. H: ChIP analysis for Taf1 in repressing (grey bars) and derepressing (four hours, black bars) conditions. Error bars represent technical replicates. Inset: ChIP analysis by PCR at for Taf1 at <i>ACT1</i>. The protein(s) assayed for in each case is listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001436#pone-0001436-t002" target="_blank">Table 2</a>.</p