Project 3: Chromatin modifiers of BRCA-related DNA repair Pathways

PROJECT SUMMARY/ABSTRACT: Induction of a DNA break initiates DNA damage signaling pathways that lead to coordinated and dynamic modification of the local chromatin landscape, including regulated changes in post-translational modification of histones flanking the site of DNA damage. These chromatin changes are required to recruit repair complexes to the site of damage, and repress transcription of genes flanking the break during repair. We recently found that euchromatic histone methyl transferase 2 (EHMT2, also known as G9a) plays a crucial role in regulating DNA repair. EHMT2 is rapidly recruited to sites of DNA breaks in a manner dependent on its phosphorylation by ATM at serine 569. Interestingly, the methyl-transferase activity of EHMT2 is required for the early, H2AX- independent recruitment of DNA repair factors including RNF8, 53BP1 and BRCA1; We have also recently found that EHMT2 activity is required for normal regulation of key features of ATM-mediated induction of transcriptional silencing near a DNA break. This includes regulation and recruitment of key silencing factors such as BMI1 and CDYL. These findings suggest that EHMT2 has a key role in regulating both DNA repair efficiency and modulating transcriptional regulation near a DNA break. As small molecule inhibitors of EHMT2 are now being developed for clinical use, EHMT2 may be one of the components of the DNA repair pathway that may be therapeutically targetable. We hypothesize that EHMT2 activity during the DNA damage response is 1) required to mediate local transcriptional silencing by regulating both histone and non-histone substrates 2) enables the early, H2AX independent, recruitment of DNA repair factors to sites of DNA damage through the action of key binding partners and substrates and 3) is critical for cell survival in the presence of specific cancer-associated DNA repair defects. These hypotheses will be investigated through the following set of specific aims. Aim 1. Determine the role of EHMT2 in DNA-damage-induced transcriptional silencing. Aim 2: Determine role of EHMT2 in H2AX-indpendent recruitment of DNA repair factors. Aim 3: Define synthetic lethal interactions of EHMT2 in DNA repair and chromatin regulation. To address these aims we will use carefully interrogate the effect of EHMT2 loss and inhibition on multiple aspects of DNA repair and local chromatin landscape near a DNA break. This will be done in cell and mouse systems where induction of DNA damage is engineered at specific genomic locations, and the effect of DNA damage on local chromatin landscape and recruitment of DNA repair factors can be systematically interrogated. We will also determine how EHMT2 inhibition using small molecule catalytic inhibitors perturbs repair pathway and survival in both wt cells and cells harboring specific, clinically relevant mutations in DNA repair pathways, including ATM, BRCA1, PALB2 and BRCA2. By this approach we will gain new insight into the fundamental role of EHMT2 in regulating DNA repair, and develop synthetic lethal strategies to target cancers harboring specific defects in DNA repair or chromatin regulation with EHMT2 inhibitors.