TOPOLOGICAL MECHANISMS OF DNA BREAK REPAIR IN LYMPHOCYTES

淋巴细胞 DNA 断裂修复的拓扑机制

• 批准号: 10305139
• 负责人: Eugene M Oltz
• 金额: $ 47.21万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10305139
• 关键词: 3-Dimensional; Address; Affect; Antigen Receptors; Architecture; Cell Cycle; Cells; Chromatin; Chromosomal translocation; Chromosome Deletion; Chromosomes; Complex; Coupled; DNA; DNA Damage; DNA Double Strand Break; DNA Sequence Alteration; DNA metabolism; DNA-Directed RNA Polymerase; Defect; Diffusion; Distant; Double Strand Break Repair; Elements; Enhancers; Environment; Epigenetic Process; Funding; G1 Arrest; G1 Phase; Gamma-H2AX; Gap Junctions; Gene Expression; Generations; Genes; Genetic Transcription; Genome; Genome Stability; Genomic Instability; Goals; Histones; Immunoglobulin Class Switching; Immunoglobulin Switch Recombination; Impairment; Knowledge; Lesion; Link; Location; Lymphocyte; Maintenance; Malignant Neoplasms; Mediating; Molecular Conformation; Nonhomologous DNA End Joining; Oncogenic; Outcome; Pattern; Phosphorylation; Physiological; Physiological Processes; Probability; Process; Receptor Gene; Repression; Resolution; Rest; Side; Site; Somatic Cell; Stretching; Structure; Surface; Testing; Topoisomerase; V(D)J Recombination; Variant; ataxia telangiectasia mutated protein; base; cohesin; defined contribution; gene repression; innovation; lymphoid neoplasm; mammalian genome; promoter; recombinational repair; recruit; repaired; response

Summary Mammalian genomes are subject to a constant barrage of damage from metabolites, external agents, or physiologic processes, including transcription and replication. Developing lymphocytes also target double- strand breaks (DSBs) to antigen receptor loci during V(D)J recombination. To maintain genomic stability, DSBs must be repaired with high fidelity, minimizing oncogenic alterations such as chromosomal deletions and translocations. The DSB response extensively revises flanking chromatin via ATM-mediated phosphorylation of the histone variant H2Ax, producing γH2Ax, which spreads for 100s of kb around a DSB. In somatic cells, most of which are non-cycling, γH2Ax domains serve as chromatin-based platforms to facilitate repair by the non- homologous end joining (NHEJ) and, likely, as adherent surfaces to hold broken chromosome ends together. Indeed, ends are destabilized in cells lacking ATM or H2Ax, which have elevated levels of translocations. Thus, a deeper understanding of mechanisms that coordinate DSB repair and sequester ends from the rest of the genome remains an important goal. In this regard, links between repair, transcription, and epigenetic landscapes around DSBs are emerging. A feature that bridges many of these processes is the 3D conformation of chromatin, which determines the range of chromosomal contacts made by a persistent DSB. The applicant has shown that the topological “environment” of a DSB in non-cycling lymphocytes determines the spread and contours of γH2Ax domains, paralleling chromosome contacts of the break site. In addition, transcription of genes within γH2Ax domains was repressed, perhaps minimizing introduction of new breaks associated with RNA polymerase readthrough. A key finding from the prior funding period was that DSBs near the border of topologically-associated domains (TADs) produce highly asymmetric γH2Ax platforms on each chromosome end – one of which is very short – which may enhance disassociation of chromosome ends when the break persists. Indeed, genomic alterations, including those associated with cancer, are enriched near topological borders. Launching from these discoveries, the applicant now proposes to define the functional relationships between chromosome topology and DSB repair outcomes. Overarching hypotheses for three aims of the project are: (i) persistent DSBs adjacent to TAD borders will generate distinct profiles of repair products due to unstable association of chromosome ends, promoting extensive deletions and translocations, (ii) the mechanism of TAD formation, called loop extrusion, is required for generation of DDR platforms; impairment of this process will deleteriously affect repair outcomes, and (iii) transcription within a γH2Ax domain harboring a persistent DSB will enhance the probability of its deletional repair to an expressed gene with which it contacts. Together, the proposed project will fill fundamental knowledge gaps about how DSB responses integrate spatial, transcriptional, and chromatin-based mechanisms to sequester chromosome ends for efficient repair, minimizing their oncogenic potential in somatic cells.

概括 哺乳动物基因组受到代谢物，外部药物或 生理过程，包括转录和复制。发展淋巴细胞也靶向双重 在V（d）J重组期间，链断裂（DSB）到抗原受体基因座。为了维持基因组稳定性，DSB 必须以高忠诚度修复，最大程度地减少染色体缺失和 易位。 DSB响应通过ATM介导的磷酸化广泛修订侧翼染色质 组蛋白变体H2AX产生γH2AX，在DSB周围扩散100 kb。在体细胞中，大多数 其中是非周期的，γH2AX结构域是基于染色质的平台，可促进非 - 同源端连接（NHEJ），并且很可能是将染色体末端固定在一起的坚固表面。 实际上，末端在缺乏ATM或H2AX的细胞中稳定，而ATM或H2AX的易位水平升高。 这是对协调DSB修复和隔离的机制的更深入的理解。 基因组仍然是一个重要目标。在这方面，修复，转录和表观遗传学之间的联系 DSB周围的景观正在出现。桥接许多这些过程的功能是3D 染色质的构象，它决定了持续的DSB进行的染色体接触范围。 申请人表明，确定的非周期淋巴细胞中DSB的拓扑“环境” γH2AX结构域的扩散和轮廓与断裂位点的染色体接触并行。此外， 反映了γH2AX结构域中基因的转录，也许最大程度地减少了新断裂的引入 与RNA聚合酶读取有关。以前的资金期间的一个关键发现是DSB附近 拓扑相关域（TAD）的边界在每个方面产生高度不对称的γH2AX平台 染色体末端 - 其中之一很短 - 当染色体结束时可能会增强染色体的分离 休息仍然存在。实际上，基因组改变，包括与癌症相关的基因组改变，富集在附近 拓扑边界。从这些发现启动，现在适用的提案来定义功能 染色体拓扑与DSB修复结果之间的关系。三个总体假设 该项目的目的是：（i）与tad边界相邻的持久性DSB将产生独特的维修概况 产品由于染色体末端不稳定的关联而引起的产品，促进了广泛的缺失和易位， （ii）生成DDR平台所必需的TAD形成的机理，称为循环扩展； 该过程的损害将对修复结果进行细微影响，并且（iii）转录在γH2AX中 带有持续DSB的域将提高其缺失修复的可能性 它与之接触。拟议的项目一起将填补有关DSB的基本知识差距 响应整合了空间，转录和基于染色质的机制，以隔离染色体末端 为了有效修复，将其在体细胞中的致癌潜力最小化。