The Mechanism Of The Function Of Demethylase JMJD3 In SAHF Formation

Cellular senescence is an irreversible process of cell cycle arrest. The senescent cells remain metabolically active, but are unable to express genes required for cell proliferation. Primary human fibroblasts undergoing oncogene-induced or replicative senescence are known to form senescence-associated heterochromatin foci(SAHF), which can stabilize the state of senescence. SAHF are condensed regions of DNA that correlate with transcriptionally inactive sites, each chromosome forms a heterochromatin focus, which can be identified by DAPI. These heterochromatin foci are hallmarked by the histone modification of H3K9me3, as well as the incorporation of heterochromatin protein HP1, HMGA1, PML and macroH2 A, repress the expression of E2 F target genes. It is reported that the formation of SAHF repress the expression of genes required for cell proliferation, and some tumor cells can also form SAHF, which provide a new method for therapeutic strategy.The retinoblastoma(RB) protein plays important roles in cell activity; the mutant of RB was reported in many cancers. We found in our earlier study that the activation of RB pathway was necessary for SAHF formation in cancer cells, and cell lines in which the RB pathway could not be activated failed to form SAHF. It has been known that the post-translational modifications of RB, e.g., acetylation, ubiquitination, phosphorylation, methylation etc, can regulate its function. For instance, the phosphorylation of RB Ser807/811 regulates cell life activity, and methylation of RB affects the phosphorylation of Ser807/811. To date, how RB regulates the formation of SAHF, and whether methylation of RB impacts on the SAHF formation is unknown.JMJD3 is a member of the JMJD family, all of which contain a JmjC domain. JMJD3 can demethylates histone on H3K27me3/me2, enhances gene expression by demethylating the repressive H3K27me3 at promoters and gene bodies. JMJD3 has also been linked to the regulation of other biological processes, such as differentiation of embryonic stem cells and neural cell, inflammatory responses in macrophages and cell senescence. Abnormal expression of JMJD3 is related to many diseases. It is reported that JMJD3 acts as a tumor suppressor through demethylating the H3K27me3 at INK4a/ARF locus, thus increasing the expression of p16 in human and mouse fibroblasts. However, in our preparatory experiments of this study, we ectopic expressed JMJD3 in MDA-MB-231 cells, which is p16 deficient, and we found it could also induce cell senescence and SAHF formation. Therefore, we speculate that JMJD3 can also induce cell senescence in a p16-independent pathway.In this thesis, we report that knockdown of the expression of JMJD3 repressed the formation of SAHF, and JMJD3 was necessary for SAHF formation. We detected a significant upregulation of JMJD3 during cellular senescence and SAHF formation in WI38 cells induced by H-RasV12, and we found that ectopic expression of JMJD3 promoted cellular senescence and SAHF formation in WI38 cells, and this process required the demethylate activity of JMJD3. Furthermore, during the process of SAHF assembly, JMJD3 was transported to the cytoplasm and interacted with the RB 659-840 domain through its demethylase domain JmjC. JMJD3 demethylated RB at a site which is methylated by Set7/9, and we defined that the site was K810 of RB. Significantly, our data demonstrated that the JMJD3-mediated demethylation of RB at K810 impeded the interaction of RB with the protein kinase CDK4, resulted in reduced level of phosphorylation of RB at Serine807/811(S807/811), repressed the expression of the target genes of E2 F, promoted formation of SAHF, implicating an important role of the interplay between the demethylation and phosphorylation of RB in SAHF assembly.This study highlights the role of JMJD3 as a novel inducer of SAHF formation, and it can demethylate non-histone protein RB at K810, to promote SAHF formation. We demonstrate that the posttranslational modifications play a role in cell senescence and SAHF formation, which provide new insights into the mechanisms of cellular senescence and SAHF assembly.