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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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Activation of Ras signaling increases H3K27me3 abundance at the Fas locus in NIH 3T3 cells.(A) 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) Phase-contrast images of Ras and Vec cells. Scale bars, 100 µm. (C) RT-qPCR analysis of Fas, Acta2, and Stambpl1 expression in Ras cells relative to that in Vec cells. Data are means ± SE from five independent experiments. (D) ChIP-qPCR analysis of H3K9me2, H3K9me3, and H3K27me3 at the Fas 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.
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pgen-1003698-g001: Activation of Ras signaling increases H3K27me3 abundance at the Fas locus in NIH 3T3 cells.(A) 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) Phase-contrast images of Ras and Vec cells. Scale bars, 100 µm. (C) RT-qPCR analysis of Fas, Acta2, and Stambpl1 expression in Ras cells relative to that in Vec cells. Data are means ± SE from five independent experiments. (D) ChIP-qPCR analysis of H3K9me2, H3K9me3, and H3K27me3 at the Fas 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.

Mentions: We established mouse NIH 3T3 cells that express a constitutively active mutant (G12V) of human H-Ras or that were infected with the corresponding empty retroviral vector (referred to hereafter as Ras cells and Vec cells, respectively). Expression of the Ras transgene resulted in increased phosphorylation of the MAPK isoforms Erk1 and Erk2 (Figure 1A) as well as in morphological transformation of the cells (Figure 1B). Moreover, reverse transcription (RT) and quantitative polymerase chain reaction (qPCR) analysis revealed that the Ras cells exhibited transcriptional repression of Fas locus genes including Fas, Acta2, and Stambpl1 (Figure 1C), consistent with previous observations [26], [28].


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

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

Activation of Ras signaling increases H3K27me3 abundance at the Fas locus in NIH 3T3 cells.(A) 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) Phase-contrast images of Ras and Vec cells. Scale bars, 100 µm. (C) RT-qPCR analysis of Fas, Acta2, and Stambpl1 expression in Ras cells relative to that in Vec cells. Data are means ± SE from five independent experiments. (D) ChIP-qPCR analysis of H3K9me2, H3K9me3, and H3K27me3 at the Fas 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.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003698-g001: Activation of Ras signaling increases H3K27me3 abundance at the Fas locus in NIH 3T3 cells.(A) 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) Phase-contrast images of Ras and Vec cells. Scale bars, 100 µm. (C) RT-qPCR analysis of Fas, Acta2, and Stambpl1 expression in Ras cells relative to that in Vec cells. Data are means ± SE from five independent experiments. (D) ChIP-qPCR analysis of H3K9me2, H3K9me3, and H3K27me3 at the Fas 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.
Mentions: We established mouse NIH 3T3 cells that express a constitutively active mutant (G12V) of human H-Ras or that were infected with the corresponding empty retroviral vector (referred to hereafter as Ras cells and Vec cells, respectively). Expression of the Ras transgene resulted in increased phosphorylation of the MAPK isoforms Erk1 and Erk2 (Figure 1A) as well as in morphological transformation of the cells (Figure 1B). Moreover, reverse transcription (RT) and quantitative polymerase chain reaction (qPCR) analysis revealed that the Ras cells exhibited transcriptional repression of Fas locus genes including Fas, Acta2, and Stambpl1 (Figure 1C), consistent with previous observations [26], [28].

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