Published online Feb 26, 2016. doi: 10.4331/wjbc.v7.i1.188
Peer-review started: July 16, 2015
First decision: September 17, 2015
Revised: October 30, 2015
Accepted: December 3, 2015
Article in press: December 4, 2015
Published online: February 26, 2016
AIM: To investigate epigenomic and gene expression alterations during cellular senescence induced by oncogenic Raf.
METHODS: Cellular senescence was induced into mouse embryonic fibroblasts (MEFs) by infecting retrovirus to express oncogenic Raf (RafV600E). RNA was collected from RafV600E cells as well as MEFs without infection and MEFs with mock infection, and a genome-wide gene expression analysis was performed using microarray. The epigenomic status for active H3K4me3 and repressive H3K27me3 histone marks was analyzed by chromatin immunoprecipitation-sequencing for RafV600E cells on day 7 and for MEFs without infection. These data for Raf-induced senescence were compared with data for Ras-induced senescence that were obtained in our previous study. Gene knockdown and overexpression were done by retrovirus infection.
RESULTS: Although the expression of some genes including secreted factors was specifically altered in either Ras- or Raf-induced senescence, many genes showed similar alteration pattern in Raf- and Ras-induced senescence. A total of 841 commonly upregulated 841 genes and 573 commonly downregulated genes showed a significant enrichment of genes related to signal and secreted proteins, suggesting the importance of alterations in secreted factors. Bmp2, a secreted protein to activate Bmp2-Smad signaling, was highly upregulated with gain of H3K4me3 and loss of H3K27me3 during Raf-induced senescence, as previously detected in Ras-induced senescence, and the knockdown of Bmp2 by shRNA lead to escape from Raf-induced senescence. Bmp2-Smad inhibitor Smad6 was strongly repressed with H3K4me3 loss in Raf-induced senescence, as detected in Ras-induced senescence, and senescence was also bypassed by Smad6 induction in Raf-activated cells. Different from Ras-induced senescence, however, gain of H3K27me3 did not occur in the Smad6 promoter region during Raf-induced senescence. When comparing genome-wide alteration between Ras- and Raf-induced senescence, genes showing loss of H3K27me3 during senescence significantly overlapped; genes showing H3K4me3 gain, or those showing H3K4me3 loss, also well-overlapped between Ras- and Raf-induced senescence. However, genes with gain of H3K27me3 overlapped significantly rarely, compared with those with H3K27me3 loss, with H3K4me3 gain, or with H3K4me3 loss.
CONCLUSION: Although epigenetic alterations are partly different, Bmp2 upregulation and Smad6 repression occur and contribute to Raf-induced senescence, as detected in Ras-induced senescence.
Core tip: By examining gene expressions and active and repressive histone marks on genome-wide scale, many genes were found to show common expression changes during Ras- and Raf-induced senescence, including genes related to secreted factors and cell cycle. Upregulation of Bmp2 and downregulation of Smad6 were detected and played a role in Raf-induced senescence as well as in Ras-induced senescence. Epigenetic alterations to regulate gene expression were also similar, including gain and loss of active histone mark H3K4me3 and loss of H3K27me3. But epigenetic changes are different in part, e.g., in gain of H3K27me3 in Smad6 promoter region.