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

HTT 介导的 DNA 损伤修复和染色质重塑复合物的调节

• 批准号: 10800972
• 负责人: PARTHA S SARKAR
• 金额: $ 69.62万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-20 至 2028-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10800972
• 关键词: Age of Onset; Automobile Driving; Brain; CAG repeat; Cells; Central Nervous System; Chromatin; Chromatin Remodeling Factor; Code; Complex; Coupled; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Enzymes; DNA Single Strand Break; Data; Development; Disease Progression; Double Strand Break Repair; Drosophila genus; Enzymes; Gene Expression; Genes; Genetic Diseases; Genetic Transcription; Genome; Goals; Health; Human; Huntington Disease; Huntington gene; Impairment; Individual; Induced pluripotent stem cell derived neurons; Knowledge; Link; MJD1 protein; MSH3 gene; Maintenance; Mediating; Modeling; Modification; Monitor; Motor; Mus; Neurodegenerative Disorders; Neuroglia; Neuronal Differentiation; Neurons; Nonhomologous DNA End Joining; Outcome; Pathogenesis; Pathologic; Patients; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Play; Polynucleotide 5' -Hydroxyl-Kinase; Post-Translational Protein Processing; Process; Proteins; RNA Polymerase II; Regulation; Repair Complex; Reporting; Research; Role; Severity of illness; Single Strand Break Repair; Site; Sumoylation Pathway; System; Testing; Transcript; Transgenic Mice; Work; brahma; chromatin remodeling; functional restoration; gene repair; genome integrity; genome wide association study; improved; in vivo; induced pluripotent stem cell; mouse model; mutant; novel; polyglutamine; postmitotic; preclinical study; protein complex; protein inhibitor of activated STAT 1; response; therapeutic target; ubiquitin-protein ligase

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.

DNA双链断裂（DSB）的持续存在是对有丝分裂后细胞（如神经元）的存在威胁， DSB的持续积累与几种神经变性疾病的病理机制有关， 包括亨廷顿病（HD）。此外，完整的，和积极的DNA损伤修复是必需的， 中枢神经系统中神经元和神经胶质的正常发育和分化。HD是由 亨廷顿基因CAG重复序列的扩增与DNA损伤修复 基因是HD发病年龄的主要调节因子，如最近GWAS研究所报道的。我们最近发现， 野生型HTT与RNA形成转录偶联DNA单链断裂（TC-SSB）修复复合物 聚合酶II、共济失调蛋白-3和多核苷酸激酶3 '-磷酸酶（PNKP，一种必需的DNA修复酶）。在 反过来，突变HTT在HD小鼠模型和HD患者脑中的存在损害PNKP活性， 在转录基因中单链断裂的持久性。我们还证明了SUMO E3 激活STAT 1连接酶蛋白抑制剂（PIAS 1）调节HD患者PNKP活性和基因组完整性 转基因小鼠和患者iPSC衍生的神经元。此外，PIAS 1增强PNKP的SUMO修饰， 这表明DNA修复复合物组分的可逆翻译后修饰（PTM）可以 在调节DNA修复的功效和疾病严重程度中起重要作用。在一项重大进展中，我们 发现天然野生型HTT发挥着比以前在基因组中表征的更广泛的作用， 通过其在新型转录偶联非同源末端连接（TC-NHEJ）中的功能维持 复合物，由完善的NHEJ酶组成，包括修复双链断裂（DSB）的PNKP。 该复合物促进神经元中DSB的无错误修复。这些过程与监管密切相关 我们最近发现，染色质修饰剂BRG 1 （Brahma相关基因1）也是该NHEJ复合物的组分，并且在HD小鼠模型中失调。 基于这些研究，我们假设这种新的蛋白质复合物在转录过程中解决了DSB， 维持基因组的完整性和神经细胞的健康。我们的初步数据显示突变HTT抑制了 通过阻碍TC-NHEJ活性进行DSB修复，导致HD系统中DSB的持续存在。这个目标 建议的工作是了解活性TC-NHEJ复合物因存在而受损的机制。 CAG重复扩增，以及单个TCR蛋白组分的SUMO化和磷酸化 调节TCR复合物活性以响应DSB。此外，我们建议评估PIAS 1的贡献， PNKP和BRG 1至复员方案成果。目的1：确定mHTT如何改变HTT/BRG 1/TC-NHEJ的功能 复杂.目的2：确定TC-NHEJ和BRG 1的mHTT破坏的功能后果 染色质重塑复合物对DNA损伤反应的影响。目标3：确定调整 HTT/BRG 1/PIAS 1/TC-NHEJ复合物在mHTT存在下对复合物活性的影响。