|M.Sc Student||Kupershmit Ilana|
|Subject||Sub-Cellular Localization and Post-Translational|
Regulation of KDM4A-D Histone Demethylases
|Department||Department of Biology||Supervisor||Professor Nabieh Ayoub|
|Full Thesis text|
In eukaryotic cells the DNA is tightly packed into a highly organized dynamic structure called chromatin. Chromatin dynamics can be achieved by various processes including histones post-translational modifications (PTMs). One of the most common PTMs is lysine methylation, which is implicated in diverse cellular processes such as transcriptional regulation, heterochromatin formation, DNA replication and DNA repair. Lysine methylation is a reversible process and its dynamicity is regulated by histone methyltransferases and histone demethylases. The Lysine-specific demethylase 4 (KDM4A-D) are JmjC catalytic domain-containing enzymes, which catalyze the demethylation of H3K9me2/me3, H3K36me2/me3 and H1.4K26me2/me3 marks. Interestingly, over-activity of the different KDM4A-D members was found in several types of human cancer. Moreover, KDM4 depletion impairs cancer cell proliferation and tumor formation. These findings suggest that KDM4 proteins play a causative role in promoting carcinogenesis. The mechanism that regulates the demethylase activity of KDM4 proteins is largely unknown. My research project focuses on studying the biology of KDM4 proteins and includes two parts .
The first part focuses on mapping PTMs of KDM4A-D proteins and assessing their effect on the demethylase activity. To do so, each of EGFP-KDM4A-D fusions was conditionally expressed in U2OS-Tet-ON cells upon the induction of Doxycycline, then pulled-down using GFP-Trap methodology and sent to mass spectrometry analysis to map the post-translationally modified residues. In addition, a structure-based approach was devised to identify residues whose modification can potentially influence the demethylase activity of KDM4A-D proteins. Altogether, I have mapped 31 novel PTMs of KDM4 enzymes. To determine their effect on the demethylase activity of KDM4A-D isoforms, I mutated these residues and overexpressed them in U2OS cells. The catalytic activity of these mutants was assessed by monitoring the levels of H3K9me3 mark using immunofluorescence technique. My results revealed three mutants that lack their demethylase activity: KDM4B-T351A-S352A-S247A-S259A, KDM4B-R240F and KDM4A-T167D .
In the second part I have determined KDM4A-C sub-cellular localization during the cell cycle. Although KDM4A-C isoforms share the same domain architecture and erase the same methyl marks, our results revealed different localization patterns during mitosis. While both KDM4A and KDM4B isoforms are excluded from mitotic chromatin, KDM4C remains associated with chromatin. Next, domain-swapping analysis reveals that the Tudor domains are essential and sufficient for KDM4C localization at mitotic chromatin. These results suggest that KDM4C protein is likely to play a role in chromosomes segregation. If so, these observations may help us to better understand the mechanism by which KDM4C misregulation promotes chromosome instability and carcinogenesis .