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，防止导致白血病和淋巴瘤的损伤和易位的发生。我们最近的工作集中在确定单个细胞中RAG切割调节的机制。这些研究表明，抗原受体位点的高阶环和核组织促进了重组中心的调节和协调重排。简而言之，我们发现大的高阶单等位基因RAG依赖环的形成，将Tcra的3'端与其染色体区域分开，与RAG结合的靶向性和该区域的单等位基因切割有关。RAG依赖的高阶环在重组时促进了同源（Tcra）和异源（Tcra/Igh）抗原受体等位基因的RAG依赖结合。此外，单等位基因、单位点切割和基因组稳定性的维持与atm介导的高阶单等位基因、单位点环的形成和Tcra和Igh的核组织的调节有关。这些数据支持在局部重组中心发生的RAG活性的反馈控制模型。在这次更新中，我们的目标是扩展我们之前的发现，进一步测试我们的模型，并探索ATM和rag介导的卵裂控制的机制。高阶环出Tcra从染色体领域定义了一种环重组形式，不同于我们和其他人之前发现的众所周知的位点内环/收缩。因此，我们的发现为重排过程增加了一个全新的监管层。我们将通过在存在和不存在RAG的情况下对不同发育阶段的Tcra/d基因座进行详细分析，来检验结构和功能之间的关系。我们的方法将结合新的实验和理论模拟，使用高通量自动化分析和最高分辨率FISH (hthrr -FISH)，以及通过染色体构象捕获（4C-seq）高通量监测染色体内/染色体间接触。为了确定高阶环如何与(i)焦点RAG结合，（ii）染色质修饰，（iii） RNA水平和（iv）核可及性相结合以控制RAG切割以保持基因组稳定性，我们将研究不同的修复蛋白（ATM， 53BP1和Artemis）在调节Tcra/d， Igh， Tcrb和Tcrg配对的染色体动力学中的作用。高阶环化和重定位抑制中心周围异染色质，以控制这些位点的断裂和损伤。这将深入了解在缺乏个体修复因子的情况下，改变的调控如何影响突变细胞的易位特征。最后，我们的目的是确定atm介导的RAG切割负反馈调节是否涉及抑制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. !