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.