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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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Comprehensive analysis of Ras-induced changes in gene transcription and H3K27me3 content in the gene body.(A) Venn diagram indicating the number of genes showing Ras-induced changes in expression and in the mean H3K27me3 level of the gene body. (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 Figure S1B) are depicted on the right with colors of red (increase) or blue (decrease). (C) 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. (D) The t-half values for expression and mean H3K27me3 level in the gene body for the purple and the gray clusters in (B).
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pgen-1003698-g003: Comprehensive analysis of Ras-induced changes in gene transcription and H3K27me3 content in the gene body.(A) Venn diagram indicating the number of genes showing Ras-induced changes in expression and in the mean H3K27me3 level of the gene body. (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 Figure S1B) are depicted on the right with colors of red (increase) or blue (decrease). (C) 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. (D) The t-half values for expression and mean H3K27me3 level in the gene body for the purple and the gray clusters in (B).

Mentions: We next identified genes whose transcription and H3K27me3 level are both regulated by Ras. We calculated the fold change in mean H3K27me3 level over the gene body for individual genes in cells infected with the Ras retroviral vector for 2, 4, 7, or 12 days relative to that in Ras0 cells. Among a total of 23,232 RefSeq genes, 1027 genes showed at least a twofold change in H3K27me3 level at least one time point (Figure 3A). A total of 933 genes showed a significant change in expression level at at least one time point after Ras introduction (see Materials and Methods). We then subjected the 115 genes whose H3K27me3 level and expression were both regulated by Ras to hierarchical clustering based on the time course of the change in H3K27me3 abundance (Figure 3B). This analysis revealed three distinct patterns of H3K27me3 dynamics induced by Ras: A purple cluster of genes in which the H3K27me3 level increased after Ras activation, and gray and brown clusters in which the H3K27me3 level decreased. Whereas changes in H3K27me3 abundance in the brown cluster were not associated with a characteristic transcriptional trend, those in the purple and gray clusters were inversely correlated with changes in transcription (Figure 3B, Figure S1A and S1B). Moreover, in these transcription-correlated clusters, changes in transcription were apparent within 2 days after Ras activation, whereas the mean H3K27me3 level remained essentially unchanged at this time point (Figure 3C). We calculated “t-half” to evaluate the timing of these two events (Figure S1C). In the purple cluster, the median t-half for mRNA abundance occurred at 1.1 days and that for H3K27me3 level occurred at 6.9 days (Figure 3D). In the gray cluster, the median t-half for mRNA abundance occurred at 3.3 days and that for H3K27me3 level occurred at 4.8 days. These results thus indicated that changes in transcription precede those in H3K27me3 level in the gene body.


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

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

Comprehensive analysis of Ras-induced changes in gene transcription and H3K27me3 content in the gene body.(A) Venn diagram indicating the number of genes showing Ras-induced changes in expression and in the mean H3K27me3 level of the gene body. (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 Figure S1B) are depicted on the right with colors of red (increase) or blue (decrease). (C) 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. (D) The t-half values for expression and mean H3K27me3 level in the gene body for the purple and the gray clusters in (B).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3757056&req=5

pgen-1003698-g003: Comprehensive analysis of Ras-induced changes in gene transcription and H3K27me3 content in the gene body.(A) Venn diagram indicating the number of genes showing Ras-induced changes in expression and in the mean H3K27me3 level of the gene body. (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 Figure S1B) are depicted on the right with colors of red (increase) or blue (decrease). (C) 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. (D) The t-half values for expression and mean H3K27me3 level in the gene body for the purple and the gray clusters in (B).
Mentions: We next identified genes whose transcription and H3K27me3 level are both regulated by Ras. We calculated the fold change in mean H3K27me3 level over the gene body for individual genes in cells infected with the Ras retroviral vector for 2, 4, 7, or 12 days relative to that in Ras0 cells. Among a total of 23,232 RefSeq genes, 1027 genes showed at least a twofold change in H3K27me3 level at least one time point (Figure 3A). A total of 933 genes showed a significant change in expression level at at least one time point after Ras introduction (see Materials and Methods). We then subjected the 115 genes whose H3K27me3 level and expression were both regulated by Ras to hierarchical clustering based on the time course of the change in H3K27me3 abundance (Figure 3B). This analysis revealed three distinct patterns of H3K27me3 dynamics induced by Ras: A purple cluster of genes in which the H3K27me3 level increased after Ras activation, and gray and brown clusters in which the H3K27me3 level decreased. Whereas changes in H3K27me3 abundance in the brown cluster were not associated with a characteristic transcriptional trend, those in the purple and gray clusters were inversely correlated with changes in transcription (Figure 3B, Figure S1A and S1B). Moreover, in these transcription-correlated clusters, changes in transcription were apparent within 2 days after Ras activation, whereas the mean H3K27me3 level remained essentially unchanged at this time point (Figure 3C). We calculated “t-half” to evaluate the timing of these two events (Figure S1C). In the purple cluster, the median t-half for mRNA abundance occurred at 1.1 days and that for H3K27me3 level occurred at 6.9 days (Figure 3D). In the gray cluster, the median t-half for mRNA abundance occurred at 3.3 days and that for H3K27me3 level occurred at 4.8 days. These results thus indicated that changes in transcription precede those in H3K27me3 level in the gene body.

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