Characterization of chromatin loops responsible for Igh locus contraction

负责 Igh 基因座收缩的染色质环的表征

• 批准号: 8873312
• 负责人: Amy L Kenter
• 金额: $ 23.97万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-15 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8873312
• 关键词: Address; Affect; Affinity; Antibodies; B-Cell Development; B-Lymphocytes; Biological Assay; Bone Marrow; Cell Line; Cell Nucleus; Cells; Chromatin; Chromatin Loop; Chromatin Structure; Chromosomal translocation; Chromosome Structures; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; DNA; Data; Development; Disease; Distal; Embryo; Evaluation; Event; Exons; Fibroblasts; Fluorescent in Situ Hybridization; Gene Mutation; Gene Rearrangement; Genes; Genome; Genomics; Humoral Immunities; Immune response; Immunoglobulin Class Switching; Immunoglobulin Genes; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Immunoglobulins; J segment gene; Link; Lymphoid; Mature B-Lymphocyte; Mediating; Methodology; Molecular Conformation; Mus; Mutagenesis; Mutate; Nuclear; Organ; Phase; Process; Property; Publishing; Regulation; Regulatory Element; Site; Staging; Structure of germinal center of lymph node; Structure-Activity Relationship; Technology; Time; V(D)J Recombination; antigen binding; base; conformational alteration; design; genetic element; homologous recombination; insight; next generation sequencing; programs; public health relevance; recombinase; spatial relationship

 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.

 描述（由申请人提供）：免疫球蛋白（Ig）基因是独特的，因为它们经历三种不同类型的基因改变以实现功能齐全的体液免疫反应。在骨髓 (BM) 的早期 B 细胞发育过程中，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，这些过程共同创造了适合将 V-D-J 基因片段组装成编码 IgH 分子抗原结合部分的连续 V(D)J 外显子的条件。最近的研究表明，染色质环化会影响 V(D)J 重组、CSR 过程中伴侣的选择，并可能驱动特定的染色体易位事件。尽管已经识别出 Igh 基因座的一小部分环，但无法对基因座环进行公正的检查。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。这使我们能够在多个基因组尺度上系统地表征三维 (3D) 染色质组织。我们发现 Igh 基因座被划分为两个独特的子拓扑域，由相对非结构化的区域隔开。非淋巴细胞 MEF 细胞和 pro-B 淋巴细胞的比较揭示了一组非常长范围的循环相互作用，这些相互作用用于桥接亚拓扑域，并且都是 pro-B 细胞所独有的并且依赖于 Pax5。因此，我们已经确定了 Pax5 依赖的循环相互作用，称为位点 I、II、II.5 和 III，负责 Igh 基因座收缩，从而在 重新排列的 DJH 连接和远端 VH 基因。这些发现对于正常条件下和病理疾病状态下 IgH 库的形成具有重要意义。我们建议充分表征涉及位点 I 的循环相互作用，然后使用此信息构建位点 I 已被删除或突变的小鼠。将使用 3C 染色质环化分析、3D FISH 以及 B 细胞发育分析和 V(D)J 连接过程中 VH 基因使用分析来充分探讨位点 I 定向删除的后果。这些研究将为有关体液免疫发展的新见解奠定基础。