Limits...
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.

Show MeSH

Related in: MedlinePlus

Ras-induced H3K27me3 accumulation and transcriptional changes are reversed by inactivation of Ras signaling.(A) Time line for exposure to and removal of 4HT as well as sample analysis (arrows) for NIH 3T3–ER-Ras cells studied in (B) through (D). (B) Immunoblot analysis of ER-Ras (arrow) and α-tubulin in the cytosolic fraction of the cells. (C) RT-qPCR analysis of relative Itgb5, Adcy7, and Smad6 expression. Data are means ± SE from two independent experiments. (D) ChIP-qPCR analysis of H3K27me3 and total H3 levels for the regions of Itgb5, Adcy7, and Smad6 indicated in Figure 4A. Data are means ± SE from two independent experiments. (E) Time line for exposure to and removal of 4HT as well as sample analysis (arrows) for NIH 3T3–Raf-ER cells studied in (F) and (G). (F) RT-qPCR analysis of relative Itgb5, Adcy7, and Smad6 expression. Data are means ± SE from two independent experiments. (G) ChIP-qPCR analysis of H3K27me3 and total H3 at Itgb5, Adcy7, and Smad6. Data are means ± SE from two independent experiments. The position of PCR primers of Itgb5 correspond to positions c in Figure S4A.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3757056&req=5

pgen-1003698-g006: Ras-induced H3K27me3 accumulation and transcriptional changes are reversed by inactivation of Ras signaling.(A) Time line for exposure to and removal of 4HT as well as sample analysis (arrows) for NIH 3T3–ER-Ras cells studied in (B) through (D). (B) Immunoblot analysis of ER-Ras (arrow) and α-tubulin in the cytosolic fraction of the cells. (C) RT-qPCR analysis of relative Itgb5, Adcy7, and Smad6 expression. Data are means ± SE from two independent experiments. (D) ChIP-qPCR analysis of H3K27me3 and total H3 levels for the regions of Itgb5, Adcy7, and Smad6 indicated in Figure 4A. Data are means ± SE from two independent experiments. (E) Time line for exposure to and removal of 4HT as well as sample analysis (arrows) for NIH 3T3–Raf-ER cells studied in (F) and (G). (F) RT-qPCR analysis of relative Itgb5, Adcy7, and Smad6 expression. Data are means ± SE from two independent experiments. (G) ChIP-qPCR analysis of H3K27me3 and total H3 at Itgb5, Adcy7, and Smad6. Data are means ± SE from two independent experiments. The position of PCR primers of Itgb5 correspond to positions c in Figure S4A.

Mentions: The presence of H3K27me3 at an exogenous transgene was previously shown to maintain the repressed state [33], suggesting the possibility that an increase in H3K27me3 level induced by Ras signaling might be able to maintain repression of gene expression after signaling is inactivated. To test this possibility, we introduced ER-Ras [a fusion protein of human H-Ras(G12V) and the estrogen receptor] into NIH 3T3 cells, exposed the cells to 4HT for 9 days in order to induce changes in both H3K27me3 level and transcription, and then removed 4HT to inactivate Ras signaling (Figure 6A). Immunoblot analysis revealed that ER-Ras was induced by 4HT and that its abundance decreased rapidly after removal of 4HT (Figure 6B), the latter indicative of inactivation of the Ras signal. Changes in the transcription of Itgb5, Adcy7, and Smad6 were also apparent after exposure of the cells to 4HT for 9 days, whereas these changes were completely reversed after 4HT removal (Figure 6C). Moreover, an increase in H3K27me3 content at Itgb5 and Smad6 was observed in the presence of 4HT, whereas H3K27me3 abundance at these genes returned essentially to basal levels after signal inactivation (Figure 6D). The H3K27me3 level at Adcy7 was reduced by exposure of the cells to 4HT and remained low after 4HT removal, suggesting that the dynamics of H3K27 methylation and demethylation might differ. We obtained similar results with four additional genes—Plekha4, Ephx1, Bpifc, and Sorcs2 (Figure S8)—as well as with cells expressing Raf-ER (Figure 6E–6G). From these data, we concluded that changes in H3K27me3 level are dependent on Ras signaling, and that H3K27me3 enrichment is not maintained after inactivation of such signaling, resulting in reactivation of transcription.


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

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

Ras-induced H3K27me3 accumulation and transcriptional changes are reversed by inactivation of Ras signaling.(A) Time line for exposure to and removal of 4HT as well as sample analysis (arrows) for NIH 3T3–ER-Ras cells studied in (B) through (D). (B) Immunoblot analysis of ER-Ras (arrow) and α-tubulin in the cytosolic fraction of the cells. (C) RT-qPCR analysis of relative Itgb5, Adcy7, and Smad6 expression. Data are means ± SE from two independent experiments. (D) ChIP-qPCR analysis of H3K27me3 and total H3 levels for the regions of Itgb5, Adcy7, and Smad6 indicated in Figure 4A. Data are means ± SE from two independent experiments. (E) Time line for exposure to and removal of 4HT as well as sample analysis (arrows) for NIH 3T3–Raf-ER cells studied in (F) and (G). (F) RT-qPCR analysis of relative Itgb5, Adcy7, and Smad6 expression. Data are means ± SE from two independent experiments. (G) ChIP-qPCR analysis of H3K27me3 and total H3 at Itgb5, Adcy7, and Smad6. Data are means ± SE from two independent experiments. The position of PCR primers of Itgb5 correspond to positions c in Figure S4A.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003698-g006: Ras-induced H3K27me3 accumulation and transcriptional changes are reversed by inactivation of Ras signaling.(A) Time line for exposure to and removal of 4HT as well as sample analysis (arrows) for NIH 3T3–ER-Ras cells studied in (B) through (D). (B) Immunoblot analysis of ER-Ras (arrow) and α-tubulin in the cytosolic fraction of the cells. (C) RT-qPCR analysis of relative Itgb5, Adcy7, and Smad6 expression. Data are means ± SE from two independent experiments. (D) ChIP-qPCR analysis of H3K27me3 and total H3 levels for the regions of Itgb5, Adcy7, and Smad6 indicated in Figure 4A. Data are means ± SE from two independent experiments. (E) Time line for exposure to and removal of 4HT as well as sample analysis (arrows) for NIH 3T3–Raf-ER cells studied in (F) and (G). (F) RT-qPCR analysis of relative Itgb5, Adcy7, and Smad6 expression. Data are means ± SE from two independent experiments. (G) ChIP-qPCR analysis of H3K27me3 and total H3 at Itgb5, Adcy7, and Smad6. Data are means ± SE from two independent experiments. The position of PCR primers of Itgb5 correspond to positions c in Figure S4A.
Mentions: The presence of H3K27me3 at an exogenous transgene was previously shown to maintain the repressed state [33], suggesting the possibility that an increase in H3K27me3 level induced by Ras signaling might be able to maintain repression of gene expression after signaling is inactivated. To test this possibility, we introduced ER-Ras [a fusion protein of human H-Ras(G12V) and the estrogen receptor] into NIH 3T3 cells, exposed the cells to 4HT for 9 days in order to induce changes in both H3K27me3 level and transcription, and then removed 4HT to inactivate Ras signaling (Figure 6A). Immunoblot analysis revealed that ER-Ras was induced by 4HT and that its abundance decreased rapidly after removal of 4HT (Figure 6B), the latter indicative of inactivation of the Ras signal. Changes in the transcription of Itgb5, Adcy7, and Smad6 were also apparent after exposure of the cells to 4HT for 9 days, whereas these changes were completely reversed after 4HT removal (Figure 6C). Moreover, an increase in H3K27me3 content at Itgb5 and Smad6 was observed in the presence of 4HT, whereas H3K27me3 abundance at these genes returned essentially to basal levels after signal inactivation (Figure 6D). The H3K27me3 level at Adcy7 was reduced by exposure of the cells to 4HT and remained low after 4HT removal, suggesting that the dynamics of H3K27 methylation and demethylation might differ. We obtained similar results with four additional genes—Plekha4, Ephx1, Bpifc, and Sorcs2 (Figure S8)—as well as with cells expressing Raf-ER (Figure 6E–6G). From these data, we concluded that changes in H3K27me3 level are dependent on Ras signaling, and that H3K27me3 enrichment is not maintained after inactivation of such signaling, resulting in reactivation of transcription.

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