Yeast Strains.

<p>Yeast Strains.</p

p53 Binds to Peptides that Mimic the Phosphorylated Pol II CTD.

<p>Biotinylated CTD peptides bearing four heptad repeats (28-mers) that were unphosphorylated (CTD), Ser5-phosphorylated (Ser5-P-CTD), or Ser2-phosphorylated (Ser2-P-CTD), were adsorbed to streptavidin-coated magnetic beads. Recombinant HA-p53, bearing an HA tag on its N-terminus, was incubated with beads alone or beads bearing peptides. Bound proteins (B) and those remaining in the supernatant (S) were subjected to SDS-PAGE and the presence of p53 was detected using the 1801 mAb. (B) Graphical summary of peptide-bound p53 relative to unbound p53. Depicted are means ± S.E.M. (N = 4). A two-sample <i>t</i>-test (two-tailed, equal variance) comparing p53 binding to the Ser5-phosphorylated CTD peptide <i>vs.</i> the unphosphorylated CTD peptide results in a <i>P</i> value of 0.058. A corresponding analysis of p53 binding to the Ser2-phosphorylated CTD peptide results in a <i>P</i> value of 0.11.</p

Core domain Mutations Abolish Synthetic Growth Phenotypes Observed in p53-Expressing Cells.

<p>(A) Fivefold serial dilutions of yeast cells (BY4741 background) expressing p53⁺, p53^R273H or p53^V143A behind the <i>GAL1</i> promoter or expressing no ectopic protein (p53⁻). Cells were spotted onto synthetic rich medium containing either 2% glucose (SDC) or 1.6% raffinose/0.4% galactose; 6-azauracil (6-AU) was added where indicated. Cells were incubated at 30°C for 3 to 6 days. (B) Immunoblot analysis of p53⁺ and p53 core domain mutant-expressing strains grown in liquid synthetic medium containing 1.6% raffinose/0.4% galactose. p53⁺ and its mutant derivatives were detected using the 1801 mAb. (C) As in A, except p53 expression was regulated by the <i>ADH1</i> promoter and BY4741 cells were grown on synthetic rich medium containing 2% glucose in the absence or presence of mycophenolic acid (MPA). (D) Spot dilution analysis of p53⁺ and p53 mutant-expressing cells as in A except grown on synthetic rich medium containing either 2% glucose or 1.5% raffinose/0.5% galactose in the absence or presence of MPA. (E) Fivefold serial dilutions of strain BY4741 (rows 1–3) or its isogenic <i>dst1Δ</i> counterpart (rows 4–6) transformed with p53⁺- or p53^R175H-expressing plasmids (or empty vector; p53⁻) as in C and spotted onto synthetic rich medium. The SDC panel, while non-selective, shows that roughly equivalent numbers of cells were spotted. Note that in replicate experiments, p53⁺ BY4741 cells exhibited a slow growth phenotype on SDC -Leu, -His similar to that seen in C. (F) Immunoblot analysis of p53⁺ and p53^R175H expression in the <i>dst1Δ</i> mutant grown in synthetic rich medium containing 2% glucose. (G) Spot dilution analysis of p53⁺ and p53^V143A-expressing BY4741 <i>ipk1Δ</i> cells grown on synthetic medium containing either 2% glucose (SDC) or 1.6% raffinose/0.4% galactose as indicated.</p

The Core Domain of p53 Mediates the p53-Pol II Interaction in Whole Cell Extracts.

<p>(A) Domain structures of p53⁺ and p53^coreΔ. TA1 and TA2, transactivation domains 1 and 2; Tetra., tetramerization domain; Reg., regulatory domain. (B) p53 immunoblot analysis of cells expressing p53⁺ (strain SY1000) and p53^coreΔ (strain SY1004) regulated by the <i>GAL1</i> promoter. Cells were grown in rich medium containing 1.8% raffinose and 0.2% galactose. p53 was detected through use of the DO-1 mAb; expression levels were internally normalized to those of Pgk1. (C) The Pol II large subunit co-immunoprecipitates with p53⁺, but not with p53^coreΔ, in yeast WCEs. Immunoprecipitates were obtained from WCEs isolated from strains SY1000 and SY1004 cultivated as above using Myc- or p53-specific antibodies (9E10 or DO-1, respectively) (lanes 1–4). Rpb1 was detected using anti-CTD antiserum. Lanes 1 and 3 serve as negative controls as cells do not express a Myc-tagged protein. (D) p53⁺, but not p53^coreΔ, co-IPs with Pol II. Immunoprecipitates were generated using either pre-immune or anti-Pol II (CTD) antiserum (lane 1–4) from extracts as above. p53 and p53^coreΔ were detected using DO-1.</p

p53 Increases Pol II Density at Constitutively Transcribed Genes.

<p>(A) Pol II ChIP analysis of the <i>PMA1</i> gene in p53⁺ and p53⁻ cells. Depicted is a multiplex PCR analysis (resolved on an 8% polyacrylamide gel) of DNA purified from immunoprecipitated chromatin samples isolated from SY1000 (<i>P_GAL1- p53</i>) and SLY101 (p53⁻) cells subjected to a 2% galactose induction for 0, 60, 120 and 180 min. Immunoprecipitations were conducted using pre-immune (lanes 1, 6) or anti-CTD antiserum (lane 2–5, 7–10). Lane 11, DNA isolated from chromatin used in the ChIPs of lanes 1 and 2. An ORF-free region on chromosome V (<i>ARS504</i>) was co-amplified with the <i>PMA1</i> loci, and serves as a non-specific IP control. (B) Summary of Pol II ChIP assays of the <i>PMA1</i> 5′-UTR, ORF, and 3′-UTR in p53⁺ and p53⁻ cells conducted as in panel A. Depicted is Pol II abundance at each locus (net signal, immune minus pre-immune) relative to <i>ARS504</i> (net signal, immune minus pre-immune). Shown are means ± S.D; N = 2. (C) As in panel B, except Pol II ChIP analysis of the <i>ACT1</i> gene. (D) Northern analysis of <i>PMA1</i> and <i>ACT1</i> in p53⁺ and p53⁻ cells following addition of galactose for the indicated times. Transcript levels were normalized to those of <i>SCR1</i> and are presented in arbitrary units (shown are means ±S.D; N = 2).</p

<i>SIR</i>-regulated heat shock transgene system.

<p>(a) Summary of <i>hsp82</i> transgenes used in this study with location, orientation and dosage of integrated <i>HMRE</i> silencers indicated by arrows (see Inset for location of silencer binding proteins ORC, Rap1 and Abf1). Transgenes occupy the native chromosomal <i>HSP82</i> locus (located ∼95 kb from TEL16L) and contain the indicated silencer insertions with no extraneous DNA sequence (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004202#s4" target="_blank">Materials and Methods</a>). Note that the transcription start site lies 60 bp upstream of the start codon and the 3′ integration site lies ∼50 bp 3′ of the mapped transcription termination site <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004202#pgen.1004202-Farrelly1" target="_blank">[74]</a>. The ORF is indicated as a black rectangle; coordinates are numbered relative to the ATG codon. (b) Transcriptional output of <i>hsp82</i> transgenes under non-inducing (30°C) and inducing conditions (20 min heat shock at 39°C). Depicted are bar graph summaries of Northern analyses of <i>SIR⁺</i> and <i>sir4Δ</i> cells bearing the indicated <i>hsp82</i> transgenes (arrows symbolize integrated silencers as in A); <i>HSP82</i>⁺ was analyzed in the parental strain. Transcript abundance was normalized to <i>ACT1</i> and is presented relative to the non-induced <i>HSP82</i>⁺ level set at 100 (depicted are means ± S.D.; N = 2).</p

Heterochromatic gene activation occurs in the context of minimal transcription-linked H3 methylation and is unimpaired by ablation of Dot1.

<p>(a) H3K56ac ChIP analysis of <i>hsp82-2001</i> in <i>sir4Δ</i> or <i>SIR⁺</i> cells subjected to an instantaneous 30° to 39°C thermal upshift for the times indicated. Quantification was done using Real Time qPCR. The acetylated H3K56/Myc-H4 quotient of the non-induced <i>sir4Δ</i> sample was set to 1.0 for each amplicon. PTM-specific and Myc-H4 signals at the heat shock transgene were normalized to those measured at <i>PMA1</i> and <i>ARS504</i>, respectively. Shown are means ± S.D. (N = 2; qPCR = 4). (b) H3K36me3 ChIP analysis of <i>hsp82-2001</i> conducted as in A. (c) H3K4me3 ChIP analysis of <i>hsp82-2001</i> in <i>sir4Δ</i> or <i>SIR⁺</i> cells subjected to an instantaneous 30° to 39°C thermal upshift for the times indicated. Quantification and scaling were done as in A, except H3K4me3/H3 quotients are depicted, and both PTM-specific and H3 signals were normalized to those measured at <i>ARS504</i>. Shown are means ± S.D. (N = 2; qPCR = 4). (d) H3K79me2 ChIP analysis of <i>hsp82-2001</i> in <i>sir4Δ</i> or <i>SIR⁺</i> cells as in C. (e) Pol II ChIP analysis of heterochromatic <i>hsp82-2001</i> in <i>DOT1⁺</i> and <i>dot1Δ</i> strains subjected to heat shock as above. Pol II occupancy was determined using ChIP-qPCR as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004202#pgen-1004202-g003" target="_blank">Figure 3A</a>. Shown are means ± S.D. (N = 2; qPCR = 4).</p

Uncoupling Transcription from Covalent Histone Modification

<div><p>It is widely accepted that transcriptional regulation of eukaryotic genes is intimately coupled to covalent modifications of the underlying chromatin template, and in certain cases the functional consequences of these modifications have been characterized. Here we present evidence that gene activation in the silent heterochromatin of the yeast <i>Saccharomyces cerevisiae</i> can occur in the context of little, if any, covalent histone modification. Using a SIR-regulated heat shock-inducible transgene, <i>hsp82-2001</i>, and a natural drug-inducible subtelomeric gene, <i>YFR057w</i>, as models we demonstrate that substantial transcriptional induction (>200-fold) can occur in the context of restricted histone loss and negligible levels of H3K4 trimethylation, H3K36 trimethylation and H3K79 dimethylation, modifications commonly linked to transcription initiation and elongation. Heterochromatic gene activation can also occur with minimal H3 and H4 lysine acetylation and without replacement of H2A with the transcription-linked variant H2A.Z. Importantly, absence of histone modification does not stem from reduced transcriptional output, since <i>hsp82-ΔTATA</i>, a euchromatic promoter mutant lacking a TATA box and with threefold lower induced transcription than heterochromatic <i>hsp82-2001</i>, is strongly hyperacetylated in response to heat shock. Consistent with negligible H3K79 dimethylation, <i>dot1Δ</i> cells lacking H3K79 methylase activity show unimpeded occupancy of RNA polymerase II within activated heterochromatic promoter and coding regions. Our results indicate that large increases in transcription can be observed in the virtual absence of histone modifications often thought necessary for gene activation.</p></div

Retention of the Sir protein complex and increased nucleosome density and stability at the heat shock-induced <i>hsp82-2001</i> transgene.

<p>(a) <i>In vivo</i> crosslinking analysis of Sir3 at the promoter, ORF and 3′-UTR of <i>hsp82-201</i>, <i>hsp82-1001</i> and <i>hsp82-2001</i>. Crosslinked chromatin, sheared to a mean size of ∼0.5 kb, was isolated from cells cultivated at 30°C and either maintained at that temperature or subjected to a 20 min 39°C heat shock (HS) (− and +, respectively). Following immunoprecipitation, crosslinks were reversed and purified DNA was subjected to quantitative multiplex PCR in the presence of [α-³²P] dATP using primers specific for the five loci indicated. A gel analysis of multiplex PCR products is presented on the left, while a summary of three independent experiments (means ± S.E.) is presented on the right. Input sample (lane 1), derived from strain EAS2011, represents 4% of soluble chromatin used in the corresponding IP (lane 2). In the histogram, Sir3 occupancy at each <i>hsp82</i> transgene was normalized to its occupancy at <i>HMRa1</i>. prom, promoter. (b) ChIP analysis of Sir3 at <i>hsp82-2001</i> as in A, except that cells were subjected to the indicated heat shock time course and quantification of Sir3 abundance was performed using Real Time qPCR. Sir3 abundance at the indicated regions was normalized to its occupancy at <i>HMRa2</i>; illustrated are means ± S.D. (N = 2; qPCR = 4). (c) Histone H3 abundance at the promoter, ORF and 3′-UTR of <i>hsp82-2001</i> in <i>sir4Δ</i> and <i>SIR⁺</i> contexts as indicated, normalized to its occupancy at <i>ARS504</i>. Cultures were maintained at 30°C (0 min) or subjected to an instantaneous 39°C upshift for the times indicated. Quantification performed as in B; depicted are means ± S.D.; N = 2, qPCR = 4.</p

Yeast strains.

<p>Yeast strains.</p