Mechanisms Underlying the Control of Recombination and Gene Regulation

摘要 我的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起着至关重要的作用。在此应用程序中，我们有 合并了两个不同的项目，扩展了这项工作。第一个项目的重点是了解 单个淋巴细胞RAG活性的反馈控制机制及其机制 去调控卵裂的后果。V(D)J重组必须受到严格监管，以确保 在重组的顺式或反式可及靶基因上，切割不会继续。 重叠的发育阶段以及通过隐蔽重组活跃转录的脱靶基因座 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末端在调控单个细胞内的裂解过程中起着重要作用 核组织的成员。这限制了用于转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末端的缺失和 抑制ATM激酶活性导致类似的表型，我们假设它们可能在相同的作用中发挥作用 路径。在我们最新未发表的工作中，我们发现了一个保守的SQ靶标磷酸化位点。 RAG2(残基365-366)，概括了RAG2 C末端和ATM在调节中的功能 乳沟。然而，与这两个突变体相比，RAG2-S356A具有稳定的RAG后切割复合体。 因此，我们第一次有了一个工具，可以在没有任何混乱修复的情况下研究反馈调节 叛逃。在这里，我们的目标是确定！(I)个体RAG活动反馈控制的潜在机制 细胞，(二)去卵裂化对等位基因排除、基因组不稳定和基因的影响 调节和(Ii)解除调节的RAG活性促进肿瘤发生的机制。第二 项目的重点是了解增强子在控制基因调控中的作用模式。 3D染色质结构的上下文。增强剂在确保精确控制 发育和分化中的转录模式。基因和这些基因之间的物理接触 调控元件对于适当的转录控制和维持这些相互作用是必不可少的。 对于防止生理过程中可能表现为疾病状态的异常至关重要。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捉(4C)的相互作用。 具体地说，我们的目标是研究促进剂在下列情况下的作用模式：(I) 控制一个以上靶基因的调控；(Ii)具有以下作用的增强子簇的功能相关性 构成调控靶基因座的超级增强子和促进其进化的因素，最终 (3)转座元件在驱动健康和疾病的基因调控网络方面的潜在作用。

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 In our most recent unpublished work we identified a conserved SQ target phosphorylation site on RAG2 (residues 365-366) that recapitulates the function of the RAG2 C-terminus and ATM in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.