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Activation of Ras signaling increases H3K27me3 abundance at the <i>Fas</i> locus in NIH 3T3 cells.

<p>(<b>A</b>) Immunoblot analysis of H-Ras, phosphorylated (p-) and total forms of Erk1/2, and α-tubulin (loading control) in the cytosolic fraction of NIH 3T3 cells expressing human H-Ras(G12V) (Ras cells) and control (Vec) cells. (<b>B</b>) Phase-contrast images of Ras and Vec cells. Scale bars, 100 µm. (<b>C</b>) RT-qPCR analysis of <i>Fas</i>, <i>Acta2</i>, and <i>Stambpl1</i> expression in Ras cells relative to that in Vec cells. Data are means ± SE from five independent experiments. (<b>D</b>) ChIP-qPCR analysis of H3K9me2, H3K9me3, and H3K27me3 at the <i>Fas</i> locus in Ras and Vec cells. The positions of genes on the chromosome and their transcriptional orientation are indicated at the bottom of the panel. Data are expressed as fold enrichment relative to the value for Vec cells at each position, and are means ± SE from at least two independent experiments.</p

Ras-Induced Changes in H3K27me3 Occur after Those in Transcriptional Activity

<div><p>Oncogenic signaling pathways regulate gene expression in part through epigenetic modification of chromatin including DNA methylation and histone modification. Trimethylation of histone H3 at lysine-27 (H3K27), which correlates with transcriptional repression, is regulated by an oncogenic form of the small GTPase Ras. Although accumulation of trimethylated H3K27 (H3K27me3) has been implicated in transcriptional regulation, it remains unclear whether Ras-induced changes in H3K27me3 are a trigger for or a consequence of changes in transcriptional activity. We have now examined the relation between H3K27 trimethylation and transcriptional regulation by Ras. Genome-wide analysis of H3K27me3 distribution and transcription at various times after expression of oncogenic Ras in mouse NIH 3T3 cells identified 115 genes for which H3K27me3 level at the gene body and transcription were both regulated by Ras. Similarly, 196 genes showed Ras-induced changes in transcription and H3K27me3 level in the region around the transcription start site. The Ras-induced changes in transcription occurred before those in H3K27me3 at the genome-wide level, a finding that was validated by analysis of individual genes. Depletion of H3K27me3 either before or after activation of Ras signaling did not affect the transcriptional regulation of these genes. Furthermore, given that H3K27me3 enrichment was dependent on Ras signaling, neither it nor transcriptional repression was maintained after inactivation of such signaling. Unexpectedly, we detected unannotated transcripts derived from intergenic regions at which the H3K27me3 level is regulated by Ras, with the changes in transcript abundance again preceding those in H3K27me3. Our results thus indicate that changes in H3K27me3 level in the gene body or in the region around the transcription start site are not a trigger for, but rather a consequence of, changes in transcriptional activity.</p></div

Signaling-induced changes in H3K27me3 level are not required for those in transcriptional activity.

<p>(<b>A</b>) Time line for transfection with control (Ctrl) or Suz12 siRNAs, treatment with 4HT or ethanol (EtOH) vehicle, and sample analysis (arrow) for NIH 3T3–Raf-ER cells studied in (B) through (D). (<b>B</b>) Immunoblot analysis of Suz12 (arrow) and α-tubulin in the cytosolic fraction as well as of H3K27me3 and total H3 in the chromatin fraction. (<b>C</b>) ChIP-qPCR analysis of H3K27me3 normalized by total H3 for the regions of <i>Itgb5</i>, <i>Adcy7</i>, and <i>Smad6</i> indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003698#pgen-1003698-g004" target="_blank">Figure 4A</a>. Data are means ± SE from two independent experiments. (<b>D</b>) RT-qPCR analysis of relative <i>Itgb5</i>, <i>Adcy7</i>, and <i>Smad6</i> expression. Data are means ± SE from three independent experiments. (<b>E</b>) Time line for transfection with control or Suz12 siRNAs, treatment with 4HT or ethanol vehicle, and sample analysis (arrow) for NIH 3T3–Raf-ER cells studied in (F) and (G). (<b>F</b>) ChIP-qPCR analysis of H3K27me3 normalized by total H3 at <i>Itgb5</i>, <i>Adcy7</i>, and <i>Smad6</i>. Data are means ± SE from two independent experiments. (<b>G</b>) RT-qPCR analysis of relative <i>Suz12</i>, <i>Itgb5</i>, <i>Adcy7</i>, and <i>Smad6</i> expression. Data are means ± SE from two independent experiments.</p

Validation of the temporal sequence of changes in gene expression and H3K27me3 level induced by Ras signaling.

<p>(<b>A</b>) Time course of changes in H3K27me3 level at the <i>Itgb5</i>, <i>Adcy7</i>, and <i>Smad6</i> loci as determined by ChIP-seq analysis of Ras0 cells and cells infected with the retroviral vector for H-Ras(G12V) for 2, 4, 7, or 12 days. The regions for which the mean H3K27me3 level and corresponding t-half were calculated are highlighted in pink. (<b>B</b>) Changes in gene expression (FPKM) and mean H3K27me3 level for <i>Itgb5</i>, <i>Adcy7</i>, and <i>Smad6</i>. The t-half values are indicated by the dashed lines. (<b>C</b>) RT-qPCR analysis of gene expression and ChIP-qPCR analysis of the ratio of H3K27me3 to total H3 for <i>Itgb5</i>, <i>Adcy7</i>, and <i>Smad6</i> at the indicated times after introduction of the retroviral vector for H-Ras(G12V). Data are expressed relative to the corresponding value for time 0. The positions of PCR primers are indicated by arrowheads in (A), and correspond to positions e for <i>Itgb5</i> and i for <i>Adcy7</i> shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003698#pgen.1003698.s004" target="_blank">Figure S4</a>. Data are representative of four independent experiments. (<b>D</b>) Gene expression (RT-qPCR) and the ratio of H3K27me3 to total H3 (ChIP-qPCR) at the indicated times after exposure of NIH 3T3 cells expressing Raf-ER to 4HT. Data are representative of four independent experiments.</p

Signaling-induced changes in the production of novel transcripts from intergenic regions occur before changes in H3K27me3 level.

<p>(<b>A</b>) ChIP-seq analysis of H3K27me3 level as well as strand-specific assignment of sequencing reads from RNA-seq analysis by SOLiD sequencing for <i>Col1a1</i> and <i>Mink1</i> loci in Ras and Vec cells. Antisense transcription from the region upstream of each gene was detected predominantly in the vicinity of regions that showed changes in H3K27me3 level, with the predicted transcribed region being denoted schematically by the magenta box. (<b>B</b>) RT-qPCR analysis of transcripts derived from the regions upstream of <i>Col1a1</i> (<i>uCol1a1</i>) and <i>Mink1</i> (<i>uMink1</i>) in Ras and Vec cells. The analysis was performed with or without the RT reaction. Primers (red arrows) were targeted to the intergenic regions sensitive to Ras-induced modulation of H3K27me3 content. Data are means ± SE from four independent experiments. (<b>C</b>) RT-qPCR analysis of expression as well as ChIP-qPCR analysis of H3K27me3 normalized by total H3 for <i>uCol1a1</i> and <i>uMink1</i> at the indicated times after exposure of NIH 3T3 cells expressing Raf-ER to 4HT. Data are expressed relative to the values for time 0 and are representative of four independent experiments. (<b>D</b>) RT-qPCR analysis of the relative abundance of transcripts derived from <i>uCol1a1</i> and <i>uMink1</i> in NIH 3T3–Raf-ER cells transfected with Suz12 or control siRNAs and exposed to 4HT or ethanol as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003698#pgen-1003698-g005" target="_blank">Figure 5A</a>. Data are means ± SE from two independent experiments.</p

Comprehensive analysis of Ras-induced changes in gene transcription and H3K27me3 content in the gene body.

<p>(<b>A</b>) Venn diagram indicating the number of genes showing Ras-induced changes in expression and in the mean H3K27me3 level of the gene body. (<b>B</b>) Clustering of the temporal profiles of mean H3K27me3 level in the gene body. Each line represents one of 115 genes whose H3K27me3 level in the gene body and expression changed in NIH 3T3 cells during expression of H-Ras(G12V) for the indicated times. Results of hierarchical clustering are depicted on the left with colors of purple, gray, and brown. Changes in expression level (FPKM) of individual genes (as determined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003698#pgen.1003698.s001" target="_blank">Figure S1B</a>) are depicted on the right with colors of red (increase) or blue (decrease). (<b>C</b>) Averaged changes in expression and H3K27me3 level for the purple cluster (upper) and the gray cluster (lower) of genes shown in (B). Dashed lines represent “t-half,” the time corresponding to half of the difference between the values for Ras0 cells and cells expressing H-Ras(G12V) for 12 days. (<b>D</b>) The t-half values for expression and mean H3K27me3 level in the gene body for the purple and the gray clusters in (B).</p

Genome-wide identification of genomic regions at which H3K27me3 enrichment is associated with transcriptional repression.

<p>(<b>A</b>) Representative distribution of gene expression level and H3K27me3 abundance (as determined by RNA-seq and ChIP-seq, respectively) in control (Ras0) cells. Both parameters are normalized by total read counts. (<b>B</b>) Clustering of 2000 randomly selected genes of control cells based on H3K27me3 level. Each line represents an individual gene, including the upstream region, gene body, and downstream region. The length of the gene body is defined as 100% (consisting of 200 data points), and the flanking regions are ±100% of the gene body. Results of hierarchical clustering are depicted on the left with colors of brown, gray, and purple. The expression level (FPKM) for individual genes is depicted on the right with colors from blue (low FPKM) to red (high FPKM). (<b>C</b>) Definition of the gene body and the region around the TSS for the purposes of this study. (<b>D</b>) Relation between gene expression (FPKM) and H3K27me3 level both in and around the gene body (left) and in the region around the TSS (right) for all RefSeq genes. (<b>E</b>) Relation between gene expression (FPKM) and mean H3K27me3 level either in the gene body (left) or in the region around the TSS (right) for all RefSeq genes. The plots show the median, 25th and 75th percentiles, and range.</p