Exploring a Functional Role of Chromosome Loop Extrusion Direction on Regulating Genome Biology

摘要/项目总结 阐明控制染色体拓扑结构的机制是非常感兴趣的，因为约10%的人类疾病发生 基因组结构的变化。Cohesin和CTCF蛋白建立和修改染色体环 引导启动子/增强子接触，介导组织和发育阶段特异性基因转录。 在细胞群中，环可以通过CTCF之间的单向或双向粘附素介导的环挤出形成。 结合元件（CBE）被CTCF结合。然而，任何潜在的作用，方向性的循环形成还没有 被考虑。抗原受体（AgR）基因座是研究染色体拓扑结构功能的重要模型 因为淋巴细胞发育过程中其结构的变化有助于建立至关重要的AgR库， 免疫力AgR基因座可变（V）、多样性（D）和连接（J）基因片段的重组产生了 AgR多样性。因此，研究AgR基因座也产生了关于染色体拓扑结构如何控制的机制的见解 启动V（D）J重组和细胞对抑制转化的DNA损伤的反应 所有的细胞。AgR基因座包含许多V片段和CBE，它们跨越位于遥远上游的巨大基因组距离 （D）J簇两侧的CBE收敛方向与V CBE。当AgR位点激活时，RAG 核酸内切酶结合（D）J片段以建立重组中心（RC），CTCF和粘附素形成环 将所有V段重新定位在与RC相似的空间接近度内。早期的研究建立了一个模型， V和RC CBE之间的粘附素/CTCF介导的环产生紧凑的基因座结构，其驱动长- 通过增加V段之间基于扩散的碰撞的机会来实现V到RC的距离复合 RC（Structural Synapsis）这种机制将支持通过缺失或倒位的重组。更 最近对Igh和Tcra/d的下一代测序（NGS）研究表明， 通过允许RAG单向扫描该基因座并捕获V RSS（扫描突触）。这一机制决定了重组仅通过缺失发生，并使用V CBE 以阻止环挤出。虽然扫描突触可能通过在所有位点缺失来介导V到RC重排， 它不能指导通过Tcrb和Igk基因座倒位发生的长距离V-到-RC重排。基于 鉴于申请人已发表的Igh研究和未发表的Tcrb数据之间的差异，他假设 粘着蛋白介导的环挤出穿过一个位点的方向决定了扫描或结构突触 介导长程重排。为了检验这一假设，申请人提出： 扫描和结构突触在Tcrb和Aim中的作用程度2）阐明了Vb CBE的影响 每种机制对长距离突触的修饰。预期的结果将产生重大进展 通过提供强有力的证据表明，染色体环挤出的方向决定了潜在的机制， 长距离V到RC重组这将作为实验的基础，以确定功能 在调控基因表达、修复、复制和基因组分离等方面具有重要意义。

ABSTRACT/PROJECT SUMMARY Elucidating mechanisms that control chromosome topology is of much interest as ~10% of human diseases arise from changes in genomic architecture. The Cohesin and CTCF proteins establish and revise chromosome loops to direct promoter/enhancer contacts that mediate tissue- and developmental stage-specific gene transcription. In a cell population, loops can form through uni- or bi-directional cohesin-mediated loop extrusion between CTCF binding elements (CBEs) bound by CTCF. Yet, any potential role for the directionality of loop formation has not been considered. Antigen receptor (AgR) loci are great models for studying functions of chromosome topology because changes in their architectures during lymphocyte development help establish AgR repertoires vital for immunity. The recombination of AgR locus variable (V), diversity (D), and joining (J) gene segments produces AgR diversity. Thus, studying AgR loci also yields mechanistic insights into how chromosome topology controls the initiation of V(D)J recombination and the cellular response to DNA damage that suppresses transformation of all cells. AgR loci contain many V segments and CBEs spanning vast genomic distances located far upstream of (D)J clusters flanked by CBEs of convergent orientation with V CBEs. When an AgR locus activates, the RAG endonuclease binds (D)J segments to establish a recombination center (RC) and CTCF and Cohesin form loops that reposition all V segments within similar spatial proximity to the RC. Early studies formulated a model wherein Cohesin/CTCF-mediated loops between V and RC CBEs generate compacted locus structures that drive long- distance V-to-RC recombination by increasing the chance for diffusion-based collisions between V segments and the RC (structural synapsis). This mechanism would support recombination by deletion or inversion. More recent next generation sequencing (NGS) studies of Igh and Tcra/d show that cohesin-directed loop extrusion from the RC directs V-to-RC recombination by allowing RAG to unidirectionally scan the locus and capture a V RSS (scanning synapsis). This mechanism dictates that recombination occurs only by deletion and uses V CBEs to impede loop extrusion. While scanning synapsis might mediate V-to-RC rearrangements by deletion at all loci, it cannot direct long-range V-to-RC rearrangements that occur through inversion in Tcrb and Igk loci. Based on differences between published Igh studies and unpublished Tcrb data of the applicant, he hypothesizes that the direction of cohesin-mediated loop extrusion across a locus determines whether scanning or structural synapsis mediates long-range rearrangement. To test this hypothesis, the applicant proposes to: Aim 1) determine the extents that scanning and structural synapsis operate within Tcrb and Aim 2) elucidate the impacts of Vb CBE modifications on long-range synapsis by each mechanism. The expected outcomes would yield a major advance by providing strong evidence that the direction of chromosome loop extrusion dictates the underlying mechanism of long-range V-to-RC recombination. This would serve as a foundation for experiments to determine the function of loop extrusion direction in regulating gene expression and repair, replication, and segregation of genomes.