Mechanisms Underlying the Control of Recombination and Gene Regulation

总结 我的实验室的工作一直处于研究的前沿，表明核组织和长距离染色质 相互作用在重组和基因调控中起重要作用。在这个应用程序中，我们有 结合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 反馈控制单个淋巴细胞中RAG活性的潜在机制， 解调控的结果。必须严格控制V（D）J重组，以确保 切割在可接近的靶基因座上不以顺式或反式继续，所述靶基因座在 重叠的发育阶段以及在具有隐蔽重组的活跃转录的脱靶基因座上 信号序列（RSS）位点结合RAG蛋白。我们最近的研究表明，ATM和C RAG 2末端通过调节在单个细胞中对切割的反馈控制中具有重要作用 核组织。这限制了用于易位的潜在底物的数量，并提供了一种有效的方法。 保护基因组稳定性的重要机制。考虑到RAG 2的C末端的缺失， ATM激酶活性的抑制导致相似的表型，我们假设它们可以在相同的细胞中起作用。 通路在我们最近未发表的工作中，我们鉴定了一个保守的SQ靶磷酸化位点， RAG 2（残基365-366），其概括RAG 2 C-末端和ATM在调节 乳沟然而，与这两个突变体相反，RAG 2-S356 A具有稳定的RAG切割后复合物。 因此，我们第一次有一个工具来研究反馈调节，在没有任何混杂修复的情况下， 缺损在这里，我们的目标是确定！(i)个体RAG活性反馈控制机制 细胞，（ii）切割失调对等位基因排斥、基因组不稳定性和基因 调节和（ii）RAG活性失调有助于肿瘤发生的机制。第二 该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染色质结构的背景。增强子在确保精确控制 转录模式的发展和分化。基因和这些细胞之间的物理接触 调节元件对于适当的转录控制是必不可少的，并且这些相互作用的维持是 这对于防止可能表现为疾病状态的生理过程中的畸变至关重要。使用新 在GM 086852和GM 112192的支持下开发的工具，我们现在能够调查这些 使用实时成像系统和高分辨率染色体构象捕获（4C）的相互作用。 具体地，我们的目的是研究增强子在以下情况下的作用模式：（i）增强子， 控制一个以上靶基因的调节，（ii）增强子簇的功能相关性， 在调节靶基因座和促进其进化的因子中构成超级增强子， (iii)转座因子在健康和疾病中驱动基因调控网络的潜在作用。

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 In our most recent unpublished work we identified a conserved SQ target phosphorylation site on RAG2 (residues 365-366) that recapitulates the function of the RAG2 C-terminus and ATM in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