Co-ordination of recombination and allelic exclusion at IgH and Igk loci

描述（由申请人提供）：需要严格控制抗原受体基因重排，以保持基因组完整性并防止导致白血病和淋巴瘤的损伤和易位的发生。我们最近的努力集中在确定RAG切割在单个细胞中的调节机制。这些研究表明，抗原受体基因座的高阶成环和核组织有利于重组中心的调节、协调重排。简而言之，我们发现将Tcra的3'末端与其染色体区域分开的大的高阶单等位基因RAG依赖性环的形成与RAG结合的靶向和该区域中的单等位基因切割相关。RAG依赖性高阶成环促进重组时同源（Tcra）和异源（Tcra/Igh）抗原受体等位基因的RAG依赖性缔合。此外，单等位基因、单基因座切割和基因组稳定性的维持与Tcra和Igh的高级单等位基因、单基因座环形成和核组织的ATM介导的调节有关。这些数据支持的RAG活动发生在本地化重组中心的反馈控制模型。在这次更新中，我们的目标是扩大我们以前的研究结果，以进一步测试我们的模型，并探讨ATM和RAG介导的切割控制的机制。Tcra从染色体区域中的高阶成环定义了重组中的成环形式，其不同于我们和其他人先前发现的众所周知的基因座内成环/收缩。因此，我们的发现为重排过程增加了一个全新的调节层。我们将通过在存在和不存在RAG的不同发育阶段对Tcra/d基因座进行详细分析来研究结构与功能之间的关系。我们的方法将是联合收割机新的实验和理论模拟使用高通量自动化分析与最高分辨率FISH（HTHR-FISH），和高通量监测内/间染色体接触的染色体构象捕获（4C-seq）。为了确定高阶成环如何与（i）局部RAG结合、（ii）染色质修饰、（iii）RNA水平和（iv）核可及性整合以控制RAG切割以保持基因组稳定性，我们将检查不同修复蛋白的作用（ATM，53 BP 1和Artemis）在调节Tcra/d，Igh，Tcrb和Tcrg的染色体动力学中的作用（配对、高阶成环和重新定位至抑制性着丝粒周围异染色质）以控制这些基因座上的断裂和损伤。这将提供洞察如何改变调节，在没有个人的排斥因子，影响突变细胞的易位签名。最后，我们的目的是确定是否ATM介导的负反馈调节RAG切割涉及抑制RAG酶活性。!

DESCRIPTION (provided by applicant): Tight control of antigen-receptor gene rearrangement is required to preserve genome integrity and prevent the occurrence of damage and translocations that lead to leukemia and lymphoma. Our most recent efforts have focused on identifying the mechanism underlying regulation of RAG cleavage in individual cells. These studies have revealed that higher-order looping and nuclear organization of antigen receptor loci facilitates regulated, coordinated rearrangement in recombination centers. In brief, we discovered that the formation of large higher-order mono-allelic RAG-dependent loops, which separate the 3' end of Tcra from its chromosome territory, correlates with targeting of RAG binding and mono-allelic cleavage in this region. RAG- dependent higher-order looping facilitates RAG-dependent association of homologous (Tcra) and heterologous (Tcra/Igh) antigen receptor alleles at the time of recombination. Moreover, mono-allelic, mono- locus cleavage and the maintenance of genome stability are linked to ATM-mediated regulation of higher- order mono-allelic, mono-locus loop formation and nuclear organization of Tcra and Igh. These data support a model for feedback control of RAG activity occurring in localized recombination centers. In this renewal we aim to expand on our previous findings to further test our model and explore the mechanisms underlying ATM and RAG-mediated control of cleavage. Higher-order looping out of Tcra from the chromosome territory defines a form of looping in recombination that is distinct from the well-known intra-locus looping / contraction tha we and others previously discovered. Thus, our finding adds an entirely new layer of regulation to the rearrangement process. We will examine the relationship between structure and function by performing a detailed analysis of the Tcra/d locus at different stages of development in the presence and absence of RAG. Our approach will be to combine novel experimental and theoretic simulation using high-throughput automated analyses with highest resolution FISH (HTHR-FISH), and high-throughput monitoring of intra/inter chromosomal contacts by chromosome conformation capture (4C-seq). To determine how higher-order looping is integrated with (i) focal RAG binding, (ii) chromatin modifications, (iii) RNA levels and (iv) nuclear accessibility to control RAG cleavage in the interest of preserving genomic stability we will examine the effects of different repair proteins (ATM, 53BP1 and Artemis) in regulating chromosome dynamics of Tcra/d, Igh, Tcrb, and Tcrg (pairing, higher order looping and repositioning to repressive pericentromeric heterochromatin) to control breaks and damage on these loci. This will provide insight into how altered regulation, in the absence of individual repir factors, impacts on the translocation signature of mutant cells. Finally, we aim to determine whether ATM-mediated negative feedback regulation of RAG cleavage involves inhibition of RAG enzymatic activity. !