טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentKhoury Hanan
SubjectCharacterizing New Players in Safeguarding Human
Genome Integrity
DepartmentDepartment of Biology
Supervisor Professor Nabieh Ayoub
Full Thesis textFull thesis text - English Version


Abstract

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Our genome is under constant assaults by DNA damaging agents. To maintain genomic integrity several major pathways have evolved including DNA damage response (DDR) and accurate regulation of chromosomes segregation during cell division. Defects in either of these pathways lead to genomic instabilities fueling carcinogenesis. Identifying new players in maintaining genome integrity is essential therefore to gain deep understanding of the mechanisms by which the cells preserve their genome. In my study, I provided evidence showing that KDM4D, KDM4C and RNase P complex are implicated in maintaining genome integrity.

KDM4D promotes double-strand break (DSB) repair: Various types of human cancers exhibit amplification or deletion of KDM4A-D members, which selectively demethylate H3K9 and H3K36, and thus implicating their activity in promoting carcinogenesis. Here, we revealed a previously unrecognized role of KDM4D in DDR. We showed that KDM4D is rapidly recruited to DNA damage sites in a PARP1-dependent manner. Further, this recruitment is mediated by the C-terminal region of KDM4D that binds poly(ADP-ribose) moieties and undergoes ADP-ribosylation in response to DNA damage. Also, KDM4D promotes DNA damage-induced phosphorylation of ATM substrates by regulating ATM localization on chromatin, and facilitates foci formation of Rad51 and 53BP1. Consequently, KDM4D depletion impairs homology-directed repair and non-homologous end-joining of DSBs.

KDM4C activity regulates mitotic chromosome segregation fidelity: We implicated KDM4C demethylase activity in regulating the fidelity of mitotic chromosomes segregation. We showed that unlike KDM4A-B, the Tudor domains of KDM4C mediates its association with chromatin during mitosis and promotes H3K9me3 demethylation. Interestingly, depletion or overexpression of KDM4C leads to over 3-fold increase in the frequency of abnormal mitotic chromosome segregation. Furthermore, overexpression of KDM4C demethylase-dead mutant has no detectable effect on mitotic chromosome segregation. Altogether, our findings implicate both KDM4D and KDM4C lysine demethylases in safeguarding human genome, furthering the links between the cancer-relevant networks of epigenetic regulation and genome stability.

RNase P complex involvement in DDR: RNase P is composed of 10 protein subunits and a catalytic H1-RNA that catalyzes the endonucleolytic 5’ cleavage of tRNA precursors and is involved in 3’ end maturation of noncoding RNAs. We revealed that RNase P protein subunits, Rpp21 and Rpp29, are recruited to DNA breakage sites in a PARP1-dependent, but in ATM independent-manner. Moreover, H1-RNA facilitates Rpp21 and Rpp29 recruitment. Noticeably, we demonstrate that DNA damage leads to a dramatic and transient increase in RNase P catalytic activity. On this basis, we speculate that RNase P is required for processing DNA damage-induced non-coding RNAs at DNA lesions. The emerging novel link between RNase P and DDR suggests that it may serve as a DDR biomarker that can be used for the development of new therapeutic strategies that exploit genomic instabilities of cancer cells in targeted cancer therapy.