Mechanisms Underlying the Control of Recombination and Gene Regulation

重组控制和基因调控的机制

• 批准号: 9276345
• 负责人: Jane Amanda Skok
• 金额: $ 49.6万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-03 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9276345
• 关键词: Alleles; Architecture; Automobile Driving; Binding Sites; Cells; Chromatin; Chromatin Structure; Chromosomes; Complex; DNA Transposable Elements; Defect; Development; Disease; Enhancers; Ensure; Ephrin-A5; Event; Evolution; Exclusion; Feedback; Gene Expression Regulation; Gene Targeting; Genes; Genetic Recombination; Genome Stability; Genomic Instability; Health; Individual; Lead; Lymphocyte; Maintenance; Mediating; Nuclear; Pathway interactions; Pattern; Peptide Signal Sequences; Phenotype; Phosphorylation Site; Physiological Processes; Play; Positioning Attribute; Regulation; Regulator Genes; Regulatory Element; Resolution; Role; Time; Transcriptional Regulation; V(D)J Recombination; Work; ataxia telangiectasia mutated protein; chromosome conformation capture; genome integrity; imaging system; mutant; prevent; repaired; tool; tumorigenesis; unpublished works

Summary My lab's work has been at the forefront of studies showing that nuclear organization and long-range chromatin interactions play an essential role in recombination and gene regulation. In this application we have incorporated two distinct projects that extend this work. The first project focuses on understanding the mechanisms underlying feedback control of RAG activity in individual lymphocytes and the consequence of de-regulated cleavage. V(D)J recombination has to be tightly regulated to ensure that cleavage does not continue in cis, or in trans on accessible target loci that undergo recombination at overlapping stages of development as well as on actively transcribed off-target loci with cryptic recombination signal sequence (RSS) sites that bind the RAG proteins. Our recent studies reveal that ATM and the C terminus of RAG2 have an important role in feedback control of cleavage in individual cells through modulation of nuclear organization. This limits the number of potential substrates for translocation and provides an important mechanism for protecting genome stability. Given that an absence of the C terminus of RAG2 and inhibition of ATM kinase activity lead to similar phenotypes we hypothesized they could act in the same pathway. In our most recent unpublished work we identified a conserved SQ target phosphorylation site on RAG2 (residues 365-366) that recapitulates the function of the RAG2 C-terminus and ATM in regulating cleavage. However, in contrast to these two mutants, RAG2-S356A has a stable RAG post cleavage complex. Thus, for the first time we have a tool to study feedback regulation in the absence of any confounding repair defect. Here we aim to determine!(i) the mechanism underlying feedback control of RAG activity in individual cells, (ii) the consequences of cleavage deregulation on allelic exclusion, genome instability and gene regulation and (ii) the mechanism by which deregulated RAG activity contributes to oncogenesis. The second project focuses on understanding the mode of action of enhancers in controlling gene regulation in the context of 3D chromatin structure. Enhancers play a fundamental role in ensuring precise control of transcriptional patterns in development and differentiation. Physical contacts between genes and these regulatory elements are essential for proper transcriptional control and maintenance of these interactions is critical for preventing aberrations in physiological processes that could manifest as disease states. Using new tools developed under the support of GM086852 and GM112192 we are now able to investigate these interactions using live imaging systems and high-resolution chromosome conformation capture (4C). Specifically, our aim is to investigate the mode of action of enhancers in the context of: (i) enhancers that control the regulation of more than one target gene, (ii) the functional relevance of clusters of enhancers that constitute super-enhancers in regulating target loci and the factors that contribute to their evolution, and finally (iii) the potential role of transposable elements in driving gene regulatory networks in health and disease.

概括 我实验室的工作一直处于研究的前沿，表明核组织和远程染色质 相互作用在重组和基因调控中发挥着重要作用。在这个应用程序中我们有 合并了两个不同的项目来扩展这项工作。第一个项目的重点是了解 个体淋巴细胞 RAG 活性反馈控制的机制 解除管制的裂解的结果。 V(D)J 重组必须受到严格监管，以确保 在可及的目标基因座上，顺式或反式切割不会继续，这些基因座在 重叠的发育阶段以及主动转录的脱靶位点与神秘重组 结合 RAG 蛋白的信号序列 (RSS) 位点。我们最近的研究表明，ATM 和 C RAG2 末端通过调节在单个细胞裂解的反馈控制中发挥重要作用 核组织。这限制了易位的潜在底物的数量，并提供了 保护基因组稳定性的重要机制。鉴于 RAG2 的 C 末端缺失且 ATM 激酶活性的抑制导致相似的表型，我们假设它们可以以相同的方式发挥作用 途径。在我们最近未发表的工作中，我们发现了一个保守的 SQ 目标磷酸化位点 RAG2（残基 365-366）概括了 RAG2 C 末端和 ATM 在调节中的功能 分裂。然而，与这两个突变体相比，RAG2-S356A 具有稳定的 RAG 裂解后复合物。 因此，我们第一次拥有了在没有任何混杂修复的情况下研究反馈调节的工具 缺点。在这里，我们的目标是确定！（i）个体 RAG 活动反馈控制的机制 细胞，(ii) 裂解失调对等位基因排除、基因组不稳定性和基因的影响 (ii) RAG 活性失调导致肿瘤发生的机制。第二个 项目重点是了解增强子在控制基因调控中的作用模式 3D 染色质结构的背景。增强剂在确保精确控制方面发挥着基础作用 发育和分化的转录模式。基因与这些物质之间的物理接触 调控元件对于适当的转录控制和维持这些相互作用至关重要 对于防止可能表现为疾病状态的生理过程异常至关重要。使用新的 在 GM086852 和 GM112192 的支持下开发的工具，我们现在能够研究这些 使用实时成像系统和高分辨率染色体构象捕获 (4C) 进行相互作用。 具体来说，我们的目标是研究增强剂在以下情况下的作用模式：（i）增强剂 控制多个靶基因的调节，（ii）增强子簇的功能相关性 构成调节目标位点及其进化因素的超级增强子，最后 (iii) 转座因子在驱动健康和疾病基因调控网络中的潜在作用。