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

探索染色体环挤出方向在调节基因组生物学中的功能作用

• 批准号: 10606672
• 负责人: CRAIG H BASSING
• 金额: $ 22.25万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-11-08 至 2024-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10606672
• 关键词: Antigen Receptors; Architecture; Binding; Biology; CCCTC-binding factor; Cells; Chromosome Pairing; Chromosome Structures; Chromosomes; DNA Damage; Data; Development; Diffusion; Elements; Enhancers; Foundations; Frequencies; Gene Expression; Genes; Genetic Recombination; Genetic Transcription; Genome; Genomics; IgK; Immunity; Lymphocyte; Mammals; Mediating; Modeling; Modification; Mus; Mutation; Outcome; Peptide Signal Sequences; Population; Positioning Attribute; Publishing; Receptor Gene; Role; Scanning; Structure; Study models; Testing; Tissues; V(D)J Recombination; adaptive immunity; chromosome conformation capture; cohesin; endonuclease; experimental study; gene repair; genomic locus; human disease; innovation; insight; interest; next generation sequencing; prevent; promoter; protein complex; repaired; response; segregation; thymocyte; transcription factor

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.

摘要/项目摘要 随着约10%的人类疾病的出现，阐明控制染色体拓扑的机制非常有意义 基因组结构的变化。粘附素和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的基因转录。 在细胞群体中，环可以通过CTCF之间的单向或双向粘附素介导的环挤出而形成 CTCF结合的结合元件(CBE)。然而，环路形成的方向性的任何潜在作用都没有 被考虑过了。抗原受体(AGR)基因座是研究染色体拓扑功能的重要模型 因为在淋巴细胞发育过程中它们结构的变化有助于建立对 豁免权。AGR基因座变量(V)、多样性(D)和连接(J)基因片段的重组产生 AGR多样性。因此，对AGR基因座的研究也产生了对染色体拓扑如何控制的机械性见解。 V(D)J重组的启动和细胞对抑制转化的DNA损伤的反应 在所有的细胞中。AGR基因座包含许多跨越遥远基因组距离的V片段和CBE，位于遥远的上游 在(D)J团簇的两侧是具有V CBE的收敛取向的CBE。当AGR基因座被激活时，碎屑 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线段重新定位在与RC相似的空间邻近度内。早期的研究形成了一个模型，其中 粘附素/CTCF介导的V和RC CBE之间的环产生致密的轨迹结构，从而驱动长- 通过增加V段之间基于扩散的碰撞的机会来实现V到RC的距离重组 和RC(结构突触)。这一机制将支持通过缺失或倒置进行重组。更多 最近对IgH和TCRA/d的下一代测序(NGS)研究表明，粘附素导向的环挤出 通过允许RAG单向扫描轨迹并捕获V-to-RC来引导V-to-RC重组 RSS(扫描突触)。这一机制规定重组仅通过删除发生，并使用VCBE 以阻止弧形挤出。虽然扫描突触可能通过在所有座位上的缺失来介导V-to-RC重排， 它不能指导通过Tcrb和Igk基因座倒置而发生的长程V-to-RC重排。基于 关于申请人已发表的IGH研究和未发表的TCRB数据之间的差异，他假设 粘附素介导的环穿过某个轨迹的方向决定了是扫描突触还是结构突触 调节远距离的重排。为了检验这一假设，申请人建议：目标1)确定 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 每种机制对长程突触的修饰。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 长程V-to-RC重组。这将作为确定功能的实验的基础 在调节基因组的基因表达和修复、复制和分离中的环挤出方向。