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A NOVEL ROLE OF POLYCOMB PROTEINS IN DNA DAMAGE RESPONSE

by
Jay Hrushikesh Oza
B.S., Rutgers University 2006


Thesis Advisor: Shridar Ganesan, M.D., Ph.D.

Graduate Program in Cellular & Molecular Pharmacology


CINJ, Auditorium A
195 Little Albany Street
New Brunswick

Friday, April 13, 2012
10:00 a.m.


Abstract

Genomic homeostasis is a dynamic process involving the action of both DNA repair factors and epigenetic regulators. In order for DNA repair to initiate the repair factors must gain access to DNA lesions; for this chromatin alternations must occur. Furthermore, once the repair is complete the chromatin must be restored to its original configuration to preserve the gene expression profile.
We have identified that a novel group of proteins the CBX proteins, known primarily for their roles in epigenetic regulation are rapidly recruited to the sites of DNA damage. We use one such protein CBX8, as our prototype and show using state-of-the-art live-cell imaging technique that it starts to accumulate at sites of DNA damage within 30 seconds and progresses to accumulate until 4 minutes. Thereafter, CBX8 gradually disperses back to its pre-damage distribution by 15 minutes. We cloned a series of CBX8 deletion constructs fused to GFP to identify the structural features responsible for its recruitment and found that the N-terminal chromodomain, as well as the adjacent ATHL1 and CBX8 specific Cx8.1 domains are essential and sufficient for its recruitment to laser-scissor induced DNA damage.

To identify the signals responsible for CBX8 recruitment to sites of DNA damage we used a combination of knock-out cell lines and small molecule inhibitors and found that CBX8 recruitment is independent of other polycomb group proteins such as EZH2 or BMI1 or components of PI3-like kinase pathways such as ATM or H2ax. Given the extremely rapid recruitment of CBX8 to the sites of DNA damage, similar to that previously reported for PARP1, we interestingly found that CBX8 recruitment to the sites of DNA damage is downstream of PARP1 signaling. Furthermore, CBX8 recruitment is also downstream of TRIM33-another DNA repair protein identified by our lab to be downstream of the PARP1 mediated DNA damage response. The results of our biochemical analysis show that both PARP1 and TRIM33 co-immunoprecipitate with CBX8.

In order to identify the functional role of CBX8 at sites of DNA damage, we performed an assay for homologous recombination and found that the knock-down of CBX8 using siRNA reduces the efficiency of homologous recombination. Furthermore, we also found that the knock-down of CBX8 significantly reduces the ability of U2OS cells to form colonies after exposure to ionizing radiation. These findings suggest a functional role for CBX8 and similar CBX proteins in maintaining genomic integrity.


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