Regulation of HTT-mediated DNA damage repair and chromatin remodeling Complexes

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Persistence of DNA double-strand breaks (DSBs) is an existential threat to postmitotic cells like neurons, and persistent accumulation of DSBs is implicated in the pathomechanism(s) of several neurodegenerative diseases, including Huntington's disease (HD). Furthermore, intact, and active DNA damage repair is required for the proper development and differentiation of neurons and glia in the central nervous system. HD is caused by the expansion of polyglutamine (polyQ)-coding CAG repeats in the gene huntingtin (HTT) and DNA damage repair genes are the major modifiers of HD age-of-onset as reported in recent GWAS studies. We recently showed that wild-type HTT forms a transcription-coupled DNA single strand-break (TC-SSB) repair complex with RNA polymerase II, Ataxin-3, and polynucleotide kinase 3'-phosphatase (PNKP), an essential DNA repair enzyme. In turn, the presence of mutant HTT in brains of HD mouse models and HD patients impairs PNKP activity, resulting in the persistence of single stranded breaks in transcribed genes. We also demonstrated that the SUMO E3 ligase Protein Inhibitor of Activated STAT 1 (PIAS1) modulates PNKP activity and genome integrity in HD transgenic mice and patient iPSC derived neurons. Further, PIAS1 enhances SUMO modification of PNKP, suggesting that reversible post-translational modification(s) (PTMs) of the DNA repair complex components may play a significant role in modulating efficacy of DNA repair and disease severity. In a major advance, we discovered that the native wildtype HTT plays a much broader role than previously characterized in genome maintenance through its function within a novel transcription-coupled-nonhomologous end-joining (TC-NHEJ) complex, comprised of well-established NHEJ enzymes including PNKP that repair double strand breaks (DSBs). This complex facilitates error-free repair of DSBs in neurons. These processes are tightly coupled with regulation of chromatin remodeling at sites of DNA damage, and we recently found that the chromatin modifier BRG1 (Brahma-related gene 1) is also a component of this NHEJ complex and is dysregulated in HD mouse models. Based on these studies, we hypothesize that this novel protein complex resolves DSBs during transcription to maintain genome integrity and the health of neural cells. Our preliminary data show that mutant HTT inhibits DSB repair by impeding TC-NHEJ activity, resulting in the persistence of DSBs in HD systems. The goal of this proposed work is to understand the mechanisms by which active TC-NHEJ complex is impaired by the presence of the CAG repeat expansion, and how SUMOylation and phosphorylation of individual TCR protein components modulates TCR complex activity in response to DSBs. Further, we propose to evaluate the contribution of PIAS1, PNKP and BRG1 to DDR outcomes. Aim 1: Identify how mHTT alters the function of the HTT/BRG1/TC-NHEJ complex. Aim 2: Determine the functional consequences of mHTT disruption of the TC-NHEJ and BRG1 chromatin remodeling complexes on DNA damage responses. Aim 3: Determine the impact of modulating the HTT/BRG1/PIAS1/TC-NHEJ complex on the complex activity in the presence of mHTT.