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

Activation of heterochromatic <i>hsp82-2001</i> occurs in the presence of minimal H2A.Z levels and is unimpaired by deletion of <i>HTZ1</i>.

<p>(a) ChIP analysis of H2A.Z at the indicated <i>hsp82</i> alleles under NHS (−) and 20 min HS (+) conditions conducted as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004202#pgen-1004202-g004" target="_blank">Figure 4B</a>, with H2A.Z content normalized per nucleosome (Myc-H4 abundance). Shown are means ± S.D. (N = 2). (b) <i>hsp82-2001</i> transcript abundance was determined in WT and <i>htz1Δ</i> cells (<i>sir4Δ</i> and <i>SIR⁺</i> as indicated) subjected to heat shock for the indicated periods of time. RNA was isolated, cDNA synthesized and <i>HSP82</i> transcripts were quantified using RT-qPCR and normalized to <i>SCR1</i> as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004202#pgen-1004202-g004" target="_blank">Figure 4A</a>. Depicted are means ± S.D. (N = 2; qPCR = 4).</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

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

Euchromatic <i>hsp82-ΔTATA</i> undergoes nucleosomal disassembly and H3 hyperacetylation within its 5′-regulatory region.

<p>(a) Expression analysis of isogenic strains bearing either <i>HSP82</i> or <i>hsp82-ΔTATA</i> (SLY101 background) and subjected to a heat shock time course for the indicated times. <i>hsp82</i> transcript levels were quantified and normalized as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004202#pgen-1004202-g004" target="_blank">Figure 4A</a> (N = 2; qPCR = 4). (b) Summary of Myc-H4 occupancy at the indicated regions within <i>HSP82</i> and <i>hsp82-ΔTATA</i>, normalized to its occupancy at <i>ARS504</i>. Quantification was done as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004202#pgen-1004202-g002" target="_blank">Figure 2C</a> (N = 2; qPCR = 4). (c) H3K18 acetylation analysis of <i>HSP82</i> over a 20 min heat shock time course as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004202#pgen-1004202-g005" target="_blank">Figure 5A</a> (means ± S.D.; N = 2; qPCR = 4). (d) H3K18ac analysis of <i>hsp82-ΔTATA</i>, performed as in C. (e) H3K18ac analysis of euchromatic <i>hsp82-2001</i> (<i>sir4Δ</i> cells). (f) H3K18ac analysis of heterochromatic <i>hsp82-2001</i> (<i>SIR⁺</i> cells) (note difference in scale).</p

Components of the transcriptional machinery are dynamically recruited to activated heterochromatic genes.

<p>(a) ChIP-qPCR analysis of Pol II occupancy at the promoter, ORF and 3′-UTR of the <i>hsp82-2001</i> gene in <i>sir4Δ</i> and <i>SIR⁺</i> cells heat shocked for the indicated times. All values represent net ChIP signals (immune – pre-immune serum) at <i>hsp82-2001</i> normalized to those at <i>ARS504</i>. Pol II occupancy at the euchromatic <i>hsp82-2001</i> promoter was set to 1.0; all other occupancies are scaled relative to this. (b) ChIP analysis of Cet1 occupancy of <i>hsp82-201</i>, <i>hsp82-1001</i> and <i>hsp82-2001</i> under NHS (−) and 20 min HS (+) conditions, conducted as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004202#pgen-1004202-g002" target="_blank">Figure 2A</a>. Left, gel analysis. Input sample (lane 1) represents 0.4% of soluble chromatin used in each IP. Mock, chromatin reacted with beads alone. Lanes 3–10, chromatin isolated from <i>SIR⁺</i> and <i>sir4Δ</i> strains as indicated (+ and −, respectively) and immunoprecipitated with a Cet1-specific antibody. Histogram represents net mean values (immune signal minus beads alone) of three independent experiments.</p

Transcriptional activation of the subtelomeric <i>YFR057w</i> gene is unlinked to covalent histone modification.

<p>(a) <i>YFR057w</i> mRNA levels in <i>SIR⁺</i> and <i>sir2Δ</i> cells (BY4741 background) exposed to 200 µg/ml cycloheximide (CX) for the indicated times. <i>YFR057w</i> transcripts were quantified by RT-qPCR, and normalized to those of <i>SCR1</i>. Depicted are means ± S.D. (N = 3; qPCR = 6). (b) ChIP-qPCR analysis of Pol II within the <i>YFR057w</i> promoter and ORF in <i>sir2Δ</i> and <i>SIR⁺</i> cells exposed to cycloheximide for the indicated times as in A. Pol II occupancy was determined as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004202#pgen-1004202-g003" target="_blank">Figure 3A</a>. Occupancy of the non-induced promoter (<i>sir2Δ</i>) was set to 1.0; all other occupancies (both promoter and ORF) are scaled relative to it. Depicted are means ± S.D. (N = 4; qPCR = 8). (c) ChIP-qPCR analysis of histone PTMs within the <i>YFR057w</i> promoter (H3K18ac, H4K16ac, H3K4me2,3) or ORF (H3K79me2) in <i>sir2Δ</i> and <i>SIR⁺</i> cells exposed for the indicated times to cycloheximide as in A. Shown are normalized PTM/histone H3 quotients. PTM-specific and H3 signals at <i>YFR057w</i> were normalized to those measured at <i>PMA1</i> and <i>ARS504</i>, respectively. The PTM/histone H3 quotient of the non-induced <i>sir2Δ</i> sample was set to 1.0 in each case. Depicted are means ± S.D. (N = 2; qPCR = 4). (d) ChIP-qPCR analysis of H3K36me3 enrichment within the ORF and Htz1 enrichment within the promoter as in C, for the indicated times following addition of cycloheximide (N = 2; qPCR = 4). (e) As in D, except H3K56ac enrichment over <i>YFR057w</i> promoter and ORF was assayed.</p

Activation of heterochromatic genes occurs in the context of negligible H4K12 acetylation.

<p>(a) H4K12ac enrichment at <i>hsp82-2001</i> subjected to heat shock for the indicated times. H4K12Ac and H3 ChIP signals were quantified at promoter, ORF and 3′-UTR using Real Time qPCR and normalized to those measured at <i>PMA1</i> and <i>ARS504</i> (for H4K12ac and H3, respectively). H4K12ac/H3 quotients were then determined and scaled to the non-induced promoter signal in <i>sir4Δ</i> cells, which was set to 1. Depicted are means ± S.D (N = 2; qPCR = 4). (b) H4K12ac enrichment at <i>YFR057W</i> promoter and ORF at the indicated times following addition of 200 µg/ml cycloheximide. Quantification was performed as in A.</p