TOPOLOGICAL MECHANISMS OF DNA BREAK REPAIR IN LYMPHOCYTES

总结 哺乳动物的基因组经常受到代谢物、外部因子或 生理过程，包括转录和复制。发育中的淋巴细胞也靶向双- 在V（D）J重组过程中抗原受体基因座的链断裂（DSB）。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， 易位DSB反应通过ATM介导的磷酸化广泛地修改侧翼染色质， 组蛋白变体H2 Ax，产生γ H2 Ax，其在DSB周围扩散100 kb。在体细胞中，大多数 其中非循环，γ H2 Ax结构域作为基于染色质的平台，以促进非循环的修复。 同源末端连接（NHEJ），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，末端在缺乏ATM或H2 Ax的细胞中是不稳定的，其具有升高的易位水平。 因此，更深入地了解协调DSB修复和隔离的机制， 基因组仍然是一个重要的目标。在这方面，修复，转录和表观遗传之间的联系 DSB周围的景观正在出现。连接许多这些过程的功能是3D 染色质的构象，这决定了由持久性DSB进行的染色体接触的范围。 申请人已经表明，非循环淋巴细胞中DSB的拓扑“环境”决定了 γ H2 Ax结构域的扩展和轮廓，平行于断裂位点的染色体接触。此外，本发明还提供了一种方法， γ H2 Ax结构域内的基因转录受到抑制，可能使新断裂的引入最小化 与RNA聚合酶通读相关。上一个供资期的一个关键发现是， 拓扑相关结构域（TADs）的边界在每个上产生高度不对称的γ H2 Ax平台， 染色体末端-其中一个非常短-这可能会增强染色体末端的解离， 中断持续存在。事实上，基因组改变，包括与癌症相关的基因组改变， 拓扑边界从这些发现出发，申请人现在提出定义功能性的 染色体拓扑结构和DSB修复结果之间的关系。关于三个的过度假设 该项目的目标是：（i）邻近于欧洲联盟边界的持久性DSB将产生不同的修复轮廓 由于染色体末端的不稳定结合而产生的产物，促进广泛的缺失和易位， (ii)DDR平台的生成需要称为环挤出的微结构形成机制; 这一过程的损害将有害地影响修复结果，和（iii）γ H2 Ax内的转录 一个含有持久性DSB的结构域将增加其对表达基因的缺失修复的可能性 与之接触。总之，拟议的项目将填补有关DSB如何 反应整合了空间、转录和基于染色质的机制来隔离染色体末端 以进行有效的修复，最大限度地减少它们在体细胞中的致癌潜力。

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.