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: 9899620
• 负责人: Eugene M Oltz
• 金额: $ 39万
• 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 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9899620
• 关键词: 3-Dimensional; Address; Affect; Antigen Receptors; Architecture; Area; Automobile Driving; B-Cell Development; Cancer Biology; Cells; Chromatin; Chromosomal translocation; Chromosome Deletion; Chromosome Structures; Chromosomes; Complex; Consensus; Coupled; DNA; DNA Double Strand Break; DNA Repair; Dimensions; Distant; Double Strand Break Repair; Elements; Epigenetic Process; Exposure to; Gene Expression; Genetic Recombination; Genetic Transcription; Genome; Genome Stability; Genomics; Goals; Histones; IgK; Immunoglobulin Class Switching; Immunoglobulin Switch Recombination; Immunoglobulins; Interphase Cell; Introns; Knowledge; Lesion; Link; Location; Lymphocyte; Maintenance; Malignant Neoplasms; Maps; Mediating; Metabolism; Modeling; Molecular; Molecular Conformation; Mutagens; Nonhomologous DNA End Joining; Oncogenic; Pathway interactions; Phosphorylation; Phosphotransferases; Physiological; Physiological Processes; Population; Process; Receptor Gene; Repair Complex; Reporting; Resolution; Rest; Risk; Role; Secure; Side; Site; Structure; Surface; Testing; V(D)J Recombination; Variant; ataxia telangiectasia mutated protein; base; cellular targeting; chromatin modification; density; environmental agent; experience; experimental study; innovation; insight; lymphoid neoplasm; nuclease; prevent; programs; public health relevance; recombinational repair; recruit; repaired; response

 DESCRIPTION (provided by applicant): Our genomes are subject to a constant barrage of damage from reactive metabolites, environmental agents, or physiologic processes. A major form of physiologic damage is DNA double-strand breaks (DSBs) arising from transcription and replication. Developing lymphocytes also target DSBs to antigen receptor (AgR) loci as part of their assembly by 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 leads to extensive revision of flanking chromatin, including phosphorylation of the histone variant H2AX by the damage-sensing kinase ATM. Phosphorylated H2AX (-H2AX) spreads for 100s of kb from a DSB. In non-cycling cells, the -H2AX domain serves as a chromatin-based platform to facilitate repair by the non-homologous end joining (NHEJ) machinery and, perhaps, as an adherent surface to hold broken chromosome ends together. Indeed, broken chromosomes are destabilized in cells deficient for ATM or H2AX, which have elevated levels of translocations. Thus, a deeper understanding of mechanisms that coordinate DSB repair and sequester lesions from other parts of the genome remains an important goal in cancer biology. In this regard, mechanistic links between DNA repair, transcription, and epigenetic landscapes around DSBs are beginning to emerge. A feature that may bridge these processes is the 3D conformation of chromatin flanking a DSB. However, the impact of DSBs on genome conformation and, conversely, the role of its reconfiguration in stabilizing DNA ends for repair, remain unexplored. Conformational mechanisms are likely important to generate compact platforms for repair complexes and to spatially restrict DSBs from other regions of the genome. A breakdown in these processes may destabilize unrepaired chromosome ends, allowing them to drift apart or to participate in translocations. The applicant has discovered that DSBs in precursor lymphocytes induce compaction of chromatin over 100s of kb flanking DSB sites in AgR loci, paralleling the spread of -H2AX. Borders of compacted -H2AX domains correspond with those of topologically associated domains (TADs), the architectural building blocks of chromosome structure. Launching from these discoveries, the overarching hypothesis of the project is that -H2AX domains are limited by inherent topological features around the DSB site, forming a spatially compact platform to stabilize association of chromosome ends and focus repair. Three aspects of the hypothesis will be studied: (i) how DSB location within a TAD affects the intensity and breadth of -H2AX domains, linking these features to translocation potential, (ii) how damage response factors mediate DSB-induced conformational changes and end stabilization, and (iii) how DSBs impact structural and regulatory loops that drive gene expression. Findings from this project will advance the field, providing new insights into how DSB responses integrate spatial, transcriptional, and chromatin-based mechanisms to sequester chromosome ends for efficient repair, minimizing their oncogenic potential.

 描述(由申请人提供)：我们的基因组不断受到反应性代谢物、环境因素或生理过程的破坏。生理损伤的一种主要形式是由转录和复制引起的DNA双链断裂。发育中的淋巴细胞也通过V(D)J重组将DSB靶向抗原受体(AGR)基因座作为其组装的一部分。为了保持基因组的稳定性，必须高保真地修复DSB，将染色体缺失和易位等致癌改变降至最低。DSB反应导致侧翼染色质的广泛修改，包括组蛋白变异体H2AX被损伤敏感激酶ATM磷酸化。磷酸化的H_2AX(-H_2AX)从DSB扩散到100s kb。在非周期细胞中，-H2AX结构域作为一个以染色质为基础的平台，促进非同源末端连接(NHEJ)机制的修复，也可能作为一个黏附表面将断裂的染色体末端结合在一起。事实上，在缺乏ATM或H2AX的细胞中，断裂的染色体会变得不稳定，因为这些细胞的易位水平较高。因此，更深入地了解协调DSB修复和隔离基因组其他部分损伤的机制仍然是癌症生物学的一个重要目标。在这方面，DNA修复、转录和DSB周围的表观遗传景观之间的机械联系开始出现。一个可能连接这些过程的特征是染色质在DSB两侧的3D构象。然而，DSB对基因组构象的影响，以及相反，其重组在稳定DNA末端以供修复方面的作用仍未被探索。构象机制对于生成修复复合体的紧凑平台和在空间上限制来自基因组其他区域的DSB可能是重要的。这些过程的中断可能会破坏未修复的染色体末端的稳定性，使它们漂移或参与易位。申请人已经发现，前体淋巴细胞中的DSB导致AGR基因座DSB位点两侧超过100kb的染色质紧凑，与-H_2AX的扩散平行。致密的-H2AX结构域的边界与拓扑相关结构域(TADS)的边界相对应，拓扑相关结构域是染色体结构的构建块。从这些发现出发，该项目的总体假设是，-H2 AX结构域受到DSB位点周围固有拓扑特征的限制，形成了一个空间紧凑的平台，以稳定染色体末端的关联和焦点修复。该假说的三个方面将被研究：(I)双链断裂在TAD内的位置如何影响-H_2AX结构域的强度和宽度，将这些特征与易位潜能联系起来；(Ii)损伤反应因子如何介导双链断裂诱导的构象变化和末端稳定；以及(Iii)双链断裂如何影响驱动基因表达的结构和调控环。该项目的发现将推动该领域的发展，为DSB反应如何整合空间、转录和染色质机制以隔离染色体末端以实现有效修复提供新的见解，从而将其致癌潜力降至最低。