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 由于约 10% 的人类疾病都会出现，因此阐明控制染色体拓扑的机制非常有意义 来自基因组结构的变化。 Cohesin 和 CTCF 蛋白建立和修改染色体环 指导启动子/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连蛋白介导的环挤出形成 由 CTCF 结合的结合元件 (CBE)。然而，环形成的方向性的任何潜在作用尚未得到证实。 已被考虑。抗原受体 (AgR) 位点是研究染色体拓扑功能的绝佳模型 因为淋巴细胞发育过程中其结构的变化有助于建立对 AgR 至关重要的库 免疫。 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，研究 AgR 位点还可以深入了解染色体拓扑如何控制 V(D)J 重组的启动以及抑制转化的细胞对 DNA 损伤的反应 所有细胞。 AgR 基因座包含许多 V 片段和 CBE，跨越位于上游的巨大基因组距离 (D)J 簇的侧面是与 V CBE 收敛方向的 CBE。当 AgR 基因座激活时，RAG 核酸内切酶结合 (D)J 片段建立重组中心 (RC)，CTCF 和粘连蛋白形成环 将所有 V 段重新定位在与 RC 相似的空间邻近范围内。早期研究制定了一个模型，其中 V 和 RC CBE 之间的 Cohesin/CTCF 介导的环生成压缩的基因座结构，驱动长链 通过增加 V 段之间基于扩散的碰撞的机会来距离 V 到 RC 重组 和 RC（结构联会）。该机制将支持通过删除或倒置进行重组。更多的 最近对 Igh 和 Tcra/d 的下一代测序 (NGS) 研究表明，粘连蛋白引导的环挤出 通过允许 RAG 单向扫描轨迹并捕获 V，从 RC 引导 V 到 RC 重组 RSS（扫描突触）。该机制规定重组仅通过删除发生并使用 V CBE 阻止环挤出。虽然扫描突触可能通过删除所有位点来介导 V-to-RC 重排， 它不能指导通过 Tcrb 和 Igk 位点倒位发生的长程 V-to-RC 重排。基于 申请人已发表的 Igh 研究与未发表的 Tcrb 数据之间存在差异，他假设 粘连蛋白介导的环挤出穿过基因座的方向决定了扫描还是结构突触 介导长程重排。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 和目标 2 中运行的程度阐明了 Vb CBE 的影响 每种机制对远程突触的修改。预期成果将取得重大进展 通过提供强有力的证据证明染色体环挤出的方向决定了潜在的机制 长程 V 到 RC 重组。这将作为确定函数的实验的基础 环挤出方向在调节基因表达以及基因组修复、复制和分离中的作用。