UBC Theses and Dissertations
Novel functions of MCL-1 in ATM signalling, homologous recombination, and heterochromatin dynamics Pesarchuk, Eric
MCL‑1 is a pro-survival member of the BCL-2 family of proteins that is over-expressed in a wide variety of human cancers. Beyond its canonical role regulating apoptosis, our lab was the first to describe an additional function of MCL‑1 in the nucleus where it is chromatin-bound at sites of DNA double strand breaks and potentiates DNA damage response signalling. This thesis further characterizes the nuclear functions of MCL‑1 and shows that it has a shorter protein half life in the nucleus compared to the cytosol. In response to low dose etoposide, MCL‑1-/- MEFs had diminished H2AX and pS824 KAP-1, while pS1981 ATM and pT68 Chk2 were lower across a 100-fold range of etoposide concentrations. Analysis of a panel of pathway-specific U2OS reporter cell lines determined that MCL‑1 participates in homologous recombination. Analysis of H2AX and RPA foci by immunofluorescence revealed that MCL‑1-depleted cells had elevated levels of RPA at 8 hours post-ionizing radiation but exhibited no difference in H2AX kinetics. A LC-MS/MS screen of endogenous MCL‑1 immunoprecipitations from etoposide-treated HeLa-S3 cells identified HP1 and p150CAF-1 as putative binding partners, which were validated by immunoblot and suggested a role for MCL‑1 in heterochromatin dynamics. MCL‑1 was found to bind HP1 and p150CAF-1 in the presence or absence of exogenous DNA damage, and was dispensable for the established interaction of HP1 and p150CAF-1. MCL‑1 was also not required for the localization of HP1, p150CAF-1, or KAP-1 to sites of DNA damage. MCL‑1 nevertheless had a functional role in promoting heterochromatin compaction as determined using the U2OS 2-6-3 cell line that has a quantifiable heterochromatin array. Furthermore, cells lacking MCL‑1 had reduced basal levels of H3K9me2/3. Taken together, I propose that MCL‑1 promotes heterochromatin compaction and H3K9me3 via novel interactions with HP1 and p150CAF-1. Other reports indicate that H3Kme3 leads to Tip60-mediated ATM activation following DNA damage, and that heterochromatic factors enhance downstream processes in homologous recombination. Therefore, MCL‑1 is capable of regulating numerous double strand break repair processes by influencing chromatin architecture.
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