Topological Mechanisms of DNA Break Repair in Lymphocytes

 描述（由申请人提供）：我们的基因组受到来自活性代谢物、环境因子或生理过程的持续破坏。生理损伤的主要形式是由转录和复制引起的DNA双链断裂（DSB）。发育中的淋巴细胞也将DSB靶向抗原受体（AgR）基因座，作为它们通过V（D）J重组组装的一部分。为了保持基因组的稳定性，DSB必须以高保真度修复，最大限度地减少致癌改变，如染色体缺失和易位。DSB反应导致侧翼染色质的广泛修改，包括损伤敏感激酶ATM对组蛋白变体H2 AX的磷酸化。磷酸化的H2 AX（H2 AX-H2 AX）从DSB扩散100 kb。在非循环细胞中，NH-H2 AX结构域作为基于染色质的平台，以促进通过非同源末端连接（NHEJ）机制的修复，并且可能作为粘附表面将断裂的染色体末端保持在一起。事实上，在缺乏ATM或H2 AX的细胞中，断裂的染色体是不稳定的，其具有升高的易位水平。因此，更深入地了解协调DSB修复和隔离来自基因组其他部分的病变的机制仍然是癌症生物学的重要目标。 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。可以桥接这些过程的特征是DSB侧翼的染色质的3D构象。然而，DSB对基因组构象的影响，以及相反地，其重构在稳定DNA末端以进行修复中的作用，仍然未被探索。构象机制对于产生用于修复复合物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，使它们漂离或参与易位。申请人已经发现，前体淋巴细胞中的DSB诱导AgR基因座中DSB位点侧翼超过100 kb的染色质的压实，与β-H2 AX的扩散平行。紧凑的H2 AX结构域的边界对应于拓扑相关结构域（TADs）的边界，TADs是染色体结构的建筑构件。从这些发现开始，该项目的总体假设是，DSB位点周围的固有拓扑特征限制了H2 AX结构域，形成了空间紧凑的平台，以稳定染色体末端的缔合和聚焦修复。将研究该假设的三个方面：（i）DSB在DNA中的位置如何影响DSB-H2 AX结构域的强度和宽度，将这些特征与易位潜力联系起来，（ii）损伤应答因子如何介导DSB诱导的构象变化和末端稳定化，以及（iii）DSB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，转录和基于染色质的机制以隔离染色体末端进行有效修复提供新的见解，最大限度地减少其致癌潜力。

 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.