Genome-wide dysregulation of R-loops in Ataxia Telangiectasia neurological pathogenesis

共济失调毛细血管扩张症神经发病机制中 R 环的全基因组失调

• 批准号: 10607414
• 负责人: Katherine R. Westover
• 金额: $ 4.77万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-13 至 2026-01-12
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10607414
• 关键词: ATAC-seq; ATM Gene Mutation; ATM deficient; Affect; Age; Ataxia Telangiectasia; Ataxia Telangiectasia Patients; Autopsy; Cell Nucleus; Cell physiology; Cells; Central Nervous System; Chromatin; Clinical; Cytoplasm; DNA; DNA Damage; DNA Repair; DNA Structure; Data; Defect; Deposition; Disease; Double Strand Break Repair; Eye; Failure; Gene Expression; Genome; Goals; Hybrids; Immune; Impairment; Malignant Neoplasms; Maps; Mediating; Messenger RNA; Methyltransferase; Modeling; Modification; Molecular; Monitor; Motor; Motor Neurons; Mus; Mutation; Neurodegenerative Disorders; Neurologic; Neuronal Differentiation; Neurons; Onset of illness; Pathogenesis; Pathogenicity; Pathology; Pathway interactions; Patients; Persons; Phosphorylation; Phosphotransferases; Play; Post-Transcriptional Regulation; Protein-Serine-Threonine Kinases; Proteins; Purkinje Cells; RNA; RNA Splicing; RNA immunoprecipitation sequencing; Regulation; Role; Signal Pathway; Signal Transduction; Structure; Telangiectasis; Testing; Threonine; Transcriptional Regulation; Translating; ataxia telangiectasia mutated protein; autosome; brain tissue; cell type; clinically relevant; data integration; early childhood; genome editing; genome-wide; improved; induced pluripotent stem cell; loss of function; mRNA Translation; mental development; mimetics; motor neuron development; mutation correction; nervous system disorder; new therapeutic target; repaired; response; transcriptome sequencing; trend

Project Abstract: Ataxia Telangiectasia (AT), a multisystemic neurodegenerative disease characterized by decreasing motor coordination, mental development, immune defects, and telangiectasia of the eyes, affects up to 1 in 40,000 to 100,000 people worldwide. A recessive early childhood onset disorder, AT is caused by mutations within the ataxia telangiectasia mutated (ATM) threonine/serine kinase which plays crucial roles within the DNA damage response (DDR). However, the precise molecular mechanisms underlying AT pathogenesis and how ATM loss- of-function leads to deficient DDR remain elusive. R-loops, three stranded RNA-DNA structures composed of an DNA-RNA hybrid and a non-template DNA strand, have emerged as key components of double strand break (DSB)-induced DDR. Mounting evidence has documented critical roles of R-loops in both causing and responding to DSBs. As DSBs and the failure of their repair play major roles in the pathology of AT, R-loop dysregulation is likely to contribute to AT pathogenesis. One recently identified kinase substrate of ATM is methyltransferase like 3 (METTL3) protein, a N6-methyladenosine (m6A) methyltransferase. m6A on the RNA strand of R-loops is present inside nuclei and affects R-loop formation during DSB repair. The relationship between ATM-METTL3 phosphorylation in response to DNA damage and regulation of R-loop formation, which could play crucial roles in AT pathogenesis, has yet to be defined. Our preliminary data has demonstrated a global trend of R-loops decreasing in AT patient-derived neurons compared to healthy controls. ~20% of these lost loci were rescued in an isogenic line where the ATM mutation had been corrected. We hypothesize that in AT, the lack of METTL3 phosphorylation by ATM could globally dysregulate R-loop formation and underly AT progression. In Aim 1 we will investigate the global landscape of R-loops and analyze their effect on gene expression and chromatin accessibility throughout neuronal differentiation in healthy, AT-derived, and isogenic neurons. In Aim 2, we will define how ATM-mediated phosphorylation of METTL3 impacts the formation of R- loops. We will generate iPSC-derived motor neurons from age-matched healthy controls, AT patients, and their isogenic lines with the pathogenic mutations corrected by genome editing to systematically identify critical R- loop loci that are associated with AT and mechanistically explore the role of ATM truncations in AT progression through METTL3-dependent R-loop regulation.

项目摘要： 毛细血管扩张共济失调 (AT)，一种以运动能力下降为特征的多系统神经退行性疾病 协调性、智力发育、免疫缺陷和眼睛毛细血管扩张，影响多达四万分之一到 全球有 100,000 人。 AT 是一种隐性儿童早期发病疾病，由基因突变引起 共济失调毛细血管扩张突变 (ATM) 苏氨酸/丝氨酸激酶在 DNA 损伤中起着至关重要的作用 响应（DDR）。然而，AT 发病机制的精确分子机制以及 ATM 丢失的方式- 功能失效导致 DDR 缺陷仍然难以捉摸。 R 环，三链 RNA-DNA 结构，由 DNA-RNA 杂交体和非模板 DNA 链已成为双链断裂的关键组成部分 (DSB) 诱导的 DDR。越来越多的证据证明 R 环在导致和 回应 DSB。由于 DSB 及其修复失败在 AT 病理学中发挥着重要作用，R 环 失调可能导致 AT 发病机制。最近发现的一种 ATM 激酶底物是 甲基转移酶样 3 (METTL3) 蛋白，一种 N6-甲基腺苷 (m6A) 甲基转移酶。 RNA 上的 m6A R 环链存在于细胞核内部，并在 DSB 修复过程中影响 R 环的形成。关系 DNA 损伤引起的 ATM-METTL3 磷酸化与 R 环形成调节之间的关系， 可能在 AT 发病机制中发挥关键作用，但尚未明确。我们的初步数据表明 与健康对照相比，AT 患者来源的神经元 R 环减少的总体趋势。其中约 20% 丢失的基因座在 ATM 突变已被纠正的同基因系中被拯救。我们假设在 AT，ATM 缺乏 METTL3 磷酸化可能会全局失调 R 环形成和潜在的 AT 进展。在目标 1 中，我们将研究 R 环的全局景观并分析它们对基因的影响 健康、AT 衍生和同基因神经元分化过程中的表达和染色质可及性 神经元。在目标 2 中，我们将定义 ATM 介导的 METTL3 磷酸化如何影响 R-的形成 循环。我们将从年龄匹配的健康对照、AT 患者及其患者身上生成 iPSC 衍生的运动神经元。 通过基因组编辑纠正致病性突变的等基因系，以系统地识别关键的 R- 与 AT 相关的环基因座，并从机制上探讨 ATM 截短在 AT 进展中的作用 通过依赖 METTL3 的 R 环路调节。