Mechanisms of R loop-mediated genome instability in Wiskott-Aldrich syndrome

摘要 DNA损伤介导的基因组不稳定是导致人类疾病的一个促成因素，包括 神经退行性变、免疫紊乱和癌症。了解细胞如何预防和管理DNA 破坏是非常严重的。R环，一种转录连接的3核苷酸结构，由RNA：DNA组成 杂交种和移位的单链DNA(SsDNA)，当装饰上组蛋白H3S10p标记时，会引起基因组 在其裂解成DNA双链断裂(DSB)时的不稳定性。目前，我们对R循环一无所知 人类原发免疫缺陷疾病(PIDD)的病因中的功能障碍，又称先天缺陷 豁免权。使用人类Wiskott-Aldrich综合征(Was)的PID疾病模型，我们最近发现了一种 黄蜂在限制R环介导的CD4+细胞DNA损伤中的重要核作用 辅助性T细胞1(Th1)。这一发现开辟了一条研究黄蜂是如何 核质蛋白在F-肌动蛋白聚合和基因转录中的双重作用，确保R环连接 基因组的稳定性。目前的提案试图定义所涉及的核信号通路和机制 在人类Th细胞中确保健康的R环平衡，从而确保稳定的基因组，以及它们如何 As基因突变的破坏与免疫缺陷和临床的发展有因果关系 AS的表型。目标1侧重于定义基于染色质的机制，黄蜂通过这些机制影响 有益的(好的)R环和有害的(坏的)R环之间的平衡以及它们在因果关系中的影响 Th1细胞表型和Th2细胞表型。目标2将阐明新发现的信使核糖核酸的机制。 Was Th细胞剪接缺陷与R环的形成和基因组的不稳定性有关。目标3将利用主要 来自不同临床严重程度的多发性AS患者的T细胞在T细胞中建立R环负荷作为 “动态”疾病生物标志物，并确定黄蜂参与核-F-肌动蛋白的作用。 表型发育。从长远来看，从这些研究中获得的知识将促进发展 对此PID和其他R环介导的免疫疾病的新的预后、诊断和治疗方法 精神错乱。

ABSTRACT DNA damage-mediated genome instability is a contributing factor in the causation of human diseases, including neurodegeneration, immunological disorders, and cancer. Understanding how cells prevent and manage DNA damage is highly significant. R loop, a transcription-linked 3 nucleic-acid structure consisting of a RNA:DNA hybrid and a displaced single-strand DNA (ssDNA), when decorated with histone H3S10p mark causes genomic instability upon its cleavage into DNA double strand breaks (DSBs). Currently, nothing is known about R loop dysfunction in the causation of human primary immunodeficiency disorders (PIDDs), aka, inborn errors of immunity. Using a PID disease model of human Wiskott-Aldrich syndrome (WAS), we recently discovered an essential nuclear role of WASp, the protein deficient in WAS, in limiting R loop-mediated DNA damage in CD4+ T helper 1 (Th1) lymphocytes. This discovery has opened up a new avenue of research into how WASp, a nucleocytoplasmic protein with dual-roles in F-actin polymerization and gene transcription, ensures R loop-linked genome stability. The current proposal seeks to define the nuclear signaling pathways and mechanisms involved in ensuring a healthy R loop balance, and therefore a stable genome, in human Th cells, and how their disruptions by WAS gene mutations is causally-linked to the development of immune deficiency and clinical phenotypes in WAS. Aim 1 focuses on defining chromatin-based mechanisms by which WASp influences the balance between beneficial (“good”) R loops and deleterious (“bad”) R loops, and their effects in the causation of WAS Th1 and WAS Th2 cellular phenotypes. Aim 2 will clarify the mechanism of the newly identified mRNA splicing defect in WAS Th cells as it relates to R loop formation and genome instability. Aim 3 will utilize primary T cells from multiple WAS patients of differing clinical severities to establish R loop load in the T cells as a “dynamic” disease biomarker, and to define the involvement of nuclear-F-actin effects of WASp in WAS phenotype development. In the long-term, the knowledge gained from these studies will foster the development of novel prognostics, diagnostics, and therapeutics for this PID and other R loop-mediated immunological disorders.