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

基本信息

  • 批准号:
    10607414
  • 负责人:
  • 金额:
    $ 4.77万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 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

项目摘要

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: 共济失调毛细血管扩张症(AT)是一种以运动功能下降为特征的多系统神经退行性疾病, 协调,智力发育,免疫缺陷和眼睛的毛细血管扩张,影响高达1/40,000至 全球10万人。AT是一种隐性的儿童早期发病性疾病,由AT基因内的突变引起。 共济失调毛细血管扩张症突变(ATM)苏氨酸/丝氨酸激酶,在DNA损伤中起关键作用 响应(DDR)。然而,AT发病机制的精确分子机制以及ATM如何丢失- 功能缺失导致DDR缺陷仍然难以捉摸。R-loops,三股RNA-DNA结构, DNA-RNA杂合体和非模板DNA链已成为双链断裂的关键组分 (DSB)诱导的DDR。越来越多的证据证明了R环在引起和 回应DSB。由于DSB及其修复失败在AT的病理学中起主要作用, 调节异常可能有助于AT发病机制。一种最近鉴定的ATM激酶底物是 甲基转移酶样3(胃L3)蛋白,N6-甲基腺苷(m6 A)甲基转移酶。RNA上的m6 A R环链存在于细胞核内,并在DSB修复过程中影响R环的形成。的关系 ATM-L3磷酸化对DNA损伤的反应和R环形成的调节之间的关系, 可能在AT发病机制中起关键作用,尚未确定。我们的初步数据显示, 与健康对照相比,AT患者来源的神经元中R环减少的总体趋势。其中约20% 丢失的基因座在ATM突变已被纠正的等基因系中被挽救。我们假设, AT时,ATM引起的胃L3磷酸化的缺乏可能导致R环形成的全面失调,并导致AT的发生。 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。 在健康、AT衍生和同基因的神经元分化中, 神经元在目的2中,我们将定义ATM介导的胃L3磷酸化如何影响R- 循环我们将从年龄匹配的健康对照组、AT患者和他们的神经元中产生iPSC衍生的运动神经元。 具有通过基因组编辑校正的致病性突变的等基因系,以系统地鉴定关键的R- 与AT相关的环位点,并从机制上探索ATM截短在AT进展中的作用 通过胃L3依赖的R环调节。

项目成果

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