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)的持续存在是对神经元等有丝分裂后细胞的生存威胁 DSB的持续堆积与几种神经退行性疾病的病理机制有关(S)， 包括亨廷顿氏病(HD)。此外，完整的和活跃的DNA损伤修复是必需的 中枢神经系统中神经元和神经胶质细胞的正常发育和分化。高血压病是由 多聚谷氨酰胺(PolyQ)编码CAG重复序列在亨廷顿蛋白(HTT)基因中的扩增与DNA损伤修复 基因是高血压病发病年龄的主要修饰因素，最近的GWAS研究报告了这一点。我们最近展示了 野生型HTT与RNA形成转录偶联DNA单链断裂(TC-SSB)修复复合体 聚合酶II，Aaxin-3，以及DNA修复所必需的多核苷酸磷酸酶(PNKP)。在……里面 反过来，HD小鼠模型和HD患者大脑中突变HTT的存在会削弱PNKP活性，从而导致 在转录的基因中持续存在单链断裂。我们还演示了相扑E3 活化STAT 1连接酶抑制因子1(PIAS1)对HD患者PNKP活性和基因组完整性的调节 转基因小鼠和患者IPSC来源的神经元。此外，PIAS1增强了PNKP的相扑修饰， 提示DNA修复复合体成分的可逆翻译后修饰(S)(PTMS)可能 在调节DNA修复效果和疾病严重程度方面发挥重要作用。在一个重大的进步中，我们 发现原生野生型HTT在基因组中发挥的作用比以前描述的要广泛得多 一种新的转录偶联非同源末端连接(TC-NHEJ)中功能的维持 复合体，由成熟的NHEJ酶组成，包括修复双链断裂(DSB)的PNKP。 这种复合体有助于神经元中DSB的无错误修复。这些过程与监管紧密相连。 DNA损伤部位的染色质重塑，我们最近发现染色质修饰物BRG1 (梵天相关基因1)也是这个NHEJ复合体的一个组成部分，在HD小鼠模型中调节失调。 在这些研究的基础上，我们假设这种新的蛋白质复合体在转录过程中将DSB分解为 保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，突变的HTT抑制了 DSB通过抑制TC-NHEJ活性进行修复，导致DSB在HD系统中持续存在。这样做的目的是 建议的工作是了解活跃的TC-NHEJ复合体因存在而受损的机制 CAG重复序列的扩展，以及单个TCR蛋白组分的SUMO化和磷酸化 调节TCR复合体活性以响应DSB。此外，我们建议评估PIAS1的贡献， PNKP和BRG1到解甲返乡的结果。目标1：确定mHTT如何改变HTT/BRG1/TC-NHEJ的功能 很复杂。目的2：确定mHTT干扰TC-NHEJ和BRG1的功能后果 染色质重塑复合体对DNA损伤反应的影响。目标3：确定调整 HTT/BRG1/PIAS1/TC-NHEJ络合物在mHTT存在下的络合活性。