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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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Validation of the temporal sequence of changes in gene expression and H3K27me3 level induced by Ras signaling.(A) Time course of changes in H3K27me3 level at the Itgb5, Adcy7, and Smad6 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) Changes in gene expression (FPKM) and mean H3K27me3 level for Itgb5, Adcy7, and Smad6. The t-half values are indicated by the dashed lines. (C) RT-qPCR analysis of gene expression and ChIP-qPCR analysis of the ratio of H3K27me3 to total H3 for Itgb5, Adcy7, and Smad6 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 Itgb5 and i for Adcy7 shown in Figure S4. Data are representative of four independent experiments. (D) 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.
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pgen-1003698-g004: Validation of the temporal sequence of changes in gene expression and H3K27me3 level induced by Ras signaling.(A) Time course of changes in H3K27me3 level at the Itgb5, Adcy7, and Smad6 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) Changes in gene expression (FPKM) and mean H3K27me3 level for Itgb5, Adcy7, and Smad6. The t-half values are indicated by the dashed lines. (C) RT-qPCR analysis of gene expression and ChIP-qPCR analysis of the ratio of H3K27me3 to total H3 for Itgb5, Adcy7, and Smad6 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 Itgb5 and i for Adcy7 shown in Figure S4. Data are representative of four independent experiments. (D) 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.

Mentions: We selected three gene loci—Itgb5, Adcy7, and Smad6—for further study to confirm the results of our genome-wide RNA-seq and ChIP-seq analyses. Itgb5 and Adcy7 manifested Ras-induced changes in H3K27me3 level in the gene body (Figure 4A). The time courses of the ChIP-seq and RNA-seq data showed that Ras signaling initially affected transcription and then gradually changed the H3K27me3 content of the gene body for Itgb5 and Adcy7 (Figure 4B) as well as for four additional genes, Plekha4, Ephx1, Bpifc, and Sorcs2 (Figure S3). In the case of Smad6, the H3K27me3 level increased prominently in the region around the TSS but only slightly in the gene body as previously reported (Figure 4A) [30]. Ras signaling again affected transcription first and then gradually changing H3K27me3 content (Figure 4B). In addition to Smad6, we found other genes that showed a prominent increase in H3K27me3 level in the region around the TSS by visual inspection of the genome browser (data not shown). These results for Itgb5, Adcy7, and Smad6 were confirmed by RT-qPCR and ChIP-qPCR analyses (Figure 4C). We also confirmed that changes in gene expression precede those in H3K27me3 level with the use of NIH 3T3 cells that stably express Raf-ER, a fusion protein composed of the catalytic domain of Raf-1 and the ligand binding domain of the estrogen receptor. Treatment of these cells with 4-hydroxytamoxifen (4HT) activates Raf-ER and downstream MAPK pathways [31]. Activation of Raf-ER thus also affected mRNA abundance before H3K27me3 level for Itgb5, Adcy7, and Smad6 (Figure 4D) as well as for four additional genes, Plekha4, Ephx1, Bpifc, and Sorcs2 (Figure S7A).


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

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

Validation of the temporal sequence of changes in gene expression and H3K27me3 level induced by Ras signaling.(A) Time course of changes in H3K27me3 level at the Itgb5, Adcy7, and Smad6 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) Changes in gene expression (FPKM) and mean H3K27me3 level for Itgb5, Adcy7, and Smad6. The t-half values are indicated by the dashed lines. (C) RT-qPCR analysis of gene expression and ChIP-qPCR analysis of the ratio of H3K27me3 to total H3 for Itgb5, Adcy7, and Smad6 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 Itgb5 and i for Adcy7 shown in Figure S4. Data are representative of four independent experiments. (D) 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.
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Related In: Results  -  Collection

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

pgen-1003698-g004: Validation of the temporal sequence of changes in gene expression and H3K27me3 level induced by Ras signaling.(A) Time course of changes in H3K27me3 level at the Itgb5, Adcy7, and Smad6 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) Changes in gene expression (FPKM) and mean H3K27me3 level for Itgb5, Adcy7, and Smad6. The t-half values are indicated by the dashed lines. (C) RT-qPCR analysis of gene expression and ChIP-qPCR analysis of the ratio of H3K27me3 to total H3 for Itgb5, Adcy7, and Smad6 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 Itgb5 and i for Adcy7 shown in Figure S4. Data are representative of four independent experiments. (D) 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.
Mentions: We selected three gene loci—Itgb5, Adcy7, and Smad6—for further study to confirm the results of our genome-wide RNA-seq and ChIP-seq analyses. Itgb5 and Adcy7 manifested Ras-induced changes in H3K27me3 level in the gene body (Figure 4A). The time courses of the ChIP-seq and RNA-seq data showed that Ras signaling initially affected transcription and then gradually changed the H3K27me3 content of the gene body for Itgb5 and Adcy7 (Figure 4B) as well as for four additional genes, Plekha4, Ephx1, Bpifc, and Sorcs2 (Figure S3). In the case of Smad6, the H3K27me3 level increased prominently in the region around the TSS but only slightly in the gene body as previously reported (Figure 4A) [30]. Ras signaling again affected transcription first and then gradually changing H3K27me3 content (Figure 4B). In addition to Smad6, we found other genes that showed a prominent increase in H3K27me3 level in the region around the TSS by visual inspection of the genome browser (data not shown). These results for Itgb5, Adcy7, and Smad6 were confirmed by RT-qPCR and ChIP-qPCR analyses (Figure 4C). We also confirmed that changes in gene expression precede those in H3K27me3 level with the use of NIH 3T3 cells that stably express Raf-ER, a fusion protein composed of the catalytic domain of Raf-1 and the ligand binding domain of the estrogen receptor. Treatment of these cells with 4-hydroxytamoxifen (4HT) activates Raf-ER and downstream MAPK pathways [31]. Activation of Raf-ER thus also affected mRNA abundance before H3K27me3 level for Itgb5, Adcy7, and Smad6 (Figure 4D) as well as for four additional genes, Plekha4, Ephx1, Bpifc, and Sorcs2 (Figure S7A).

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