Investigation of Huntingtin’s Role in DNA Repair and Transcription

Huntington’s disease (HD) is a devastating and fatal neurodegenerative disease, caused by the expansion of a CAG repeat tract in the mutant Huntingtin gene (mHTT). The mechanism of toxicity imparted by mHTT has yet to be fully elucidated, despite decades of research since its description. Research into the normal cellular function of wild type HTT has also been hindered due to its size, complexity, and promiscuity. This dissertation presents a novel function of HTT in the cell as a member of the nuclear Transcription-Coupled DNA Repair (TCR) complex. Both wild type and mHTT were shown to interact with several nuclear DNA repair and transcription proteins, including PNKP, RNA Polymerase, DNA ligase III, CREB, and Ataxin3 (Atxn3). HTT is directly implicated in regulating the DNA repair activities of PNKP, and deubiquitinase activity of Atxn3. This finding is supported by accumulation of DNA damage in transcriptionally active regions of the genome, compared to silenced regions, in HD cell and animal models. Furthermore, mHTT induced cell toxicity is causally linked to prolonged DNA damage response and ATM activation, and inhibition of ATM activity can ameliorate this deleterious response. This dissertation presents additional functions for HTT in mitochondria which parallels its role in the nucleus, as a part of the mitochondrial transcription and DNA repair pathways. HTT interacts with mitochondrial RNA polymerase, DNA polymerase gamma, mitochondrial transcription factors, PNKP, and Atxn3. Chromatin immunoprecipitation confirms binding of HTT with the mitochondrial DNA. As observed in the nucleus, mHTT impairs the activity of mitochondrial PNKP and Atxn3, leading to accumulated mitochondrial DNA damage. Collectively, these findings provide new insights into how mHTT-mediated abrogation of PNKP and Atxn3 activities simultaneously impairs both nuclear and mitochondrial DNA repair; disrupting transcription, inducing DNA strand breaks, and activating the pro-apoptotic DDR-ATM→p53 signaling pathway to trigger neurotoxicity. Our results also suggest potential targets for developing therapeutic modalities to combat neurodegeneration and neuronal dysfunction in HD.