Characterization of chromatin loops responsible for Igh locus contraction

 说明(申请人提供)：免疫球蛋白(Ig)基因是独一无二的，因为它们受到三种不同类型的基因改变的影响，以实现完全功能的体液免疫反应。在骨髓中B细胞发育的早期，V(D)J或VJ连接分别发生在IgH和L链基因上，并由RAG重组酶介导。我们的新研究描述了染色体构象改变的逐步过程，这些改变协作创造了条件，使V-D-J基因片段组装成连续的V(D)J外显子，编码IgH分子的抗原结合部分。最近的研究表明，染色质环影响V(D)J重组、CSR过程中的伴侣选择，并可能驱动特定的染色体易位事件。尽管已经为IgH基因识别出一小部分环路，但对该基因座环路的无偏检查是不可用的。因此，我们使用基于3C的方法结合下一代测序技术对整个IgH基因座进行了分析。这使得我们能够在几个基因组尺度上系统地描述三维(3D)染色质组织。我们发现，IGH轨迹被划分成两个独特的子拓扑域，由一个相对非结构化的区域隔开。对非淋巴样MEF细胞和前B淋巴细胞的比较揭示了一组非常长的环状相互作用，它们起到了桥接亚拓扑域的作用，并且都是前B细胞和Pax5依赖细胞所独有的。因此，我们已经确定了Pax5依赖的环状相互作用，称为站点I、II、II、5和III，负责IGH基因座的收缩，从而在 重排的DJH连接了VH基因和远端VH基因。这些发现对正常情况下和病理疾病状态下的IgH谱系的形成有一定的意义。我们建议完全表征涉及I位点的循环相互作用，然后使用这些信息来构建I位点缺失或突变的小鼠。通过3C染色质环分析、3D FISH以及对V(D)J连接过程中B细胞发育和VH基因使用的分析，将充分探索定点I定向缺失的后果。这些研究将形成对体液免疫发展的新见解的基础。

 DESCRIPTION (provided by applicant): Immunoglobulin (Ig) genes are unique in that they are subject to three different types of gene alterations to achieve a fully functional humoral immune response. During early B cell development in the bone marrow (BM), V(D)J or VJ joining occurs on the IgH and L chain genes, respectively and is mediated by the RAG recombinase. Our new studies describe a stepwise process of chromosomal conformational alterations which collaborate to create conditions amenable for the assembly of V-D-J gene segments into contiguous V(D)J exons that encode the antigen binding portion of IgH molecules. Recent studies indicate that chromatin looping influences partner selection during V(D)J recombination, CSR and may drive specific chromosomal translocation events. Although a small subset of loops have been discerned for the Igh locus an unbiased examination of locus looping was unavailable. We therefore undertook an analysis of the entire Igh locus using 3C based methodology in combination with next generation sequencing technologies. This has permitted us to systematically characterize three dimensional (3D) chromatin organization on several genomic scales. We have found that the Igh locus is compartmentalized into two unique sub-topological domains separated by a relatively unstructured region. Comparison of non-lymphoid MEF cells and pro-B lymphocytes has revealed a set of very-long range looping interactions that serve to bridge the sub- topological domains and are both unique to pro-B cells and Pax5 dependent. Thus, we have identified the Pax5 dependent looping interactions, termed sites I, II, II.5 and III, responsible for Igh locus contraction that serves to create spatial proximity between the rearranged DJH joins and distal VH genes. These findings have implications for IgH repertoire formation under normal conditions and in pathological disease states. We propose to fully characterize looping interactions involving Site I and then to use this information to construct mice in which Site I has been deleted or mutated. The consequences of targeted deletion of Site I will be fully explored using 3C chromatin looping assays, 3D FISH, and analysis of B cell development and VH gene usage during V(D)J joining. These studies will form the basis for new insights regarding development of humoral immunity.