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Ras-induced changes in H3K27me3 occur after those in transcriptional activity.

Hosogane M, Funayama R, Nishida Y, Nagashima T, Nakayama K - PLoS Genet. (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Proliferation, United Center for Advanced Research and Translational Medicine, Graduate School of Medicine, Tohoku University, Seiryo-machi, Aoba-ku, Sendai, Japan.

ABSTRACT
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.

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Signaling-induced changes in the production of novel transcripts from intergenic regions occur before changes in H3K27me3 level.(A) ChIP-seq analysis of H3K27me3 level as well as strand-specific assignment of sequencing reads from RNA-seq analysis by SOLiD sequencing for Col1a1 and Mink1 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) RT-qPCR analysis of transcripts derived from the regions upstream of Col1a1 (uCol1a1) and Mink1 (uMink1) 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. (C) RT-qPCR analysis of expression as well as ChIP-qPCR analysis of H3K27me3 normalized by total H3 for uCol1a1 and uMink1 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. (D) RT-qPCR analysis of the relative abundance of transcripts derived from uCol1a1 and uMink1 in NIH 3T3–Raf-ER cells transfected with Suz12 or control siRNAs and exposed to 4HT or ethanol as in Figure 5A. Data are means ± SE from two independent experiments.
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pgen-1003698-g007: Signaling-induced changes in the production of novel transcripts from intergenic regions occur before changes in H3K27me3 level.(A) ChIP-seq analysis of H3K27me3 level as well as strand-specific assignment of sequencing reads from RNA-seq analysis by SOLiD sequencing for Col1a1 and Mink1 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) RT-qPCR analysis of transcripts derived from the regions upstream of Col1a1 (uCol1a1) and Mink1 (uMink1) 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. (C) RT-qPCR analysis of expression as well as ChIP-qPCR analysis of H3K27me3 normalized by total H3 for uCol1a1 and uMink1 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. (D) RT-qPCR analysis of the relative abundance of transcripts derived from uCol1a1 and uMink1 in NIH 3T3–Raf-ER cells transfected with Suz12 or control siRNAs and exposed to 4HT or ethanol as in Figure 5A. Data are means ± SE from two independent experiments.

Mentions: Visual inspection of H3K27me3 distribution revealed that Ras signaling alters H3K27me3 levels in intergenic regions located several kilobases distant from known gene bodies. Two representative loci, Col1a1 and Mink1, are shown in Figure 7A. H3K27me3 was enriched in the region upstream of Col1a1 but was depleted in the region upstream of Mink1 in Ras cells.


Ras-induced changes in H3K27me3 occur after those in transcriptional activity.

Hosogane M, Funayama R, Nishida Y, Nagashima T, Nakayama K - PLoS Genet. (2013)

Signaling-induced changes in the production of novel transcripts from intergenic regions occur before changes in H3K27me3 level.(A) ChIP-seq analysis of H3K27me3 level as well as strand-specific assignment of sequencing reads from RNA-seq analysis by SOLiD sequencing for Col1a1 and Mink1 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) RT-qPCR analysis of transcripts derived from the regions upstream of Col1a1 (uCol1a1) and Mink1 (uMink1) 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. (C) RT-qPCR analysis of expression as well as ChIP-qPCR analysis of H3K27me3 normalized by total H3 for uCol1a1 and uMink1 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. (D) RT-qPCR analysis of the relative abundance of transcripts derived from uCol1a1 and uMink1 in NIH 3T3–Raf-ER cells transfected with Suz12 or control siRNAs and exposed to 4HT or ethanol as in Figure 5A. Data are means ± SE from two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3757056&req=5

pgen-1003698-g007: Signaling-induced changes in the production of novel transcripts from intergenic regions occur before changes in H3K27me3 level.(A) ChIP-seq analysis of H3K27me3 level as well as strand-specific assignment of sequencing reads from RNA-seq analysis by SOLiD sequencing for Col1a1 and Mink1 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) RT-qPCR analysis of transcripts derived from the regions upstream of Col1a1 (uCol1a1) and Mink1 (uMink1) 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. (C) RT-qPCR analysis of expression as well as ChIP-qPCR analysis of H3K27me3 normalized by total H3 for uCol1a1 and uMink1 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. (D) RT-qPCR analysis of the relative abundance of transcripts derived from uCol1a1 and uMink1 in NIH 3T3–Raf-ER cells transfected with Suz12 or control siRNAs and exposed to 4HT or ethanol as in Figure 5A. Data are means ± SE from two independent experiments.
Mentions: Visual inspection of H3K27me3 distribution revealed that Ras signaling alters H3K27me3 levels in intergenic regions located several kilobases distant from known gene bodies. Two representative loci, Col1a1 and Mink1, are shown in Figure 7A. H3K27me3 was enriched in the region upstream of Col1a1 but was depleted in the region upstream of Mink1 in Ras cells.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Proliferation, United Center for Advanced Research and Translational Medicine, Graduate School of Medicine, Tohoku University, Seiryo-machi, Aoba-ku, Sendai, Japan.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus