TOPOLOGICAL MECHANISMS OF DNA BREAK REPAIR IN LYMPHOCYTES

摘要 哺乳动物基因组受到代谢物、外部因素或 生理过程，包括转录和复制。发育中的淋巴细胞也以双重目标为目标 在V(D)J重组过程中，链断裂(DSB)到抗原受体基因座。为了保持基因组的稳定性，DSB 必须高保真地修复，最大限度地减少致癌改变，如染色体缺失和 易位。DSB反应通过ATM介导的磷酸化广泛地修正侧翼染色质 组蛋白变异体H_2AX，产生γH_2AX，它围绕DSB扩散100s kb。在体细胞中，大多数 其中非循环的，γ的H2 AX结构域作为以染色质为基础的平台，促进非 同源末端连接(NHEJ)，并可能作为粘附面将断裂的染色体末端结合在一起。 事实上，在缺乏ATM或H_2AX的细胞中，末端是不稳定的，这些细胞的易位水平升高。 因此，对协调DSB修复和隔离的机制的更深入理解结束于 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周围的景观正在浮现。3D是连接这些过程中许多过程的一个功能 染色质的构象，它决定了持续的DSB形成的染色体接触的范围。 申请人已经证明，非循环淋巴细胞中DSB的拓扑环境决定了 γH_2AX结构域的扩展和轮廓，平行于断裂部位的染色体接触。此外, γH2 AX结构域中的基因转录受到抑制，可能会最大限度地减少新断裂的引入 与RNA聚合酶通读相关。上一个资金期的一个关键发现是，DSB接近 拓扑相关结构域(TAD)的边界在每个结构域上产生高度不对称的γH_2AX平台 染色体末端-其中一个很短-这可能会加强染色体末端的解离，当 这种裂痕依然存在。事实上，基因组的改变，包括那些与癌症相关的改变，几乎是浓缩的 拓扑边界。从这些发现出发，申请人现在建议定义功能 染色体拓扑结构与DSB修复结果之间的关系。三个最重要的假设 该项目的目标是：(1)与TAD边界相邻的永久性DSB将产生不同的修复轮廓 由于染色体末端的不稳定关联，促进了广泛的缺失和易位， (2)生成DDR平台需要TAD形成机制，称为环状挤压； 这一过程的损伤将有害地影响修复结果，以及(Iii)γH2AX内的转录 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 它与之接触的。总之，拟议的项目将填补有关DSB如何实现的基本知识空白 响应整合了空间、转录和染色质的机制，以隔离染色体末端 为了有效的修复，将它们在体细胞中的致癌潜力降至最低。

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.