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) 基因的独特之处在于，它们会经历三种不同类型的基因改变，以在骨髓 (BM) 中的早期 B 细胞发育过程中实现功能齐全的体液免疫反应。 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，该过程协同作用。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，染色质环化影响 V(D)J 重组过程中的伴侣选择、CSR，并可能驱动特异性。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，因此我们对整个基因座进行了分析。 Igh 基因座使用基于 3C 的方法与下一代测序技术相结合，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 基因座分为两个独特的亚拓扑结构域。非淋巴细胞 MEF 细胞和原 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，并且都是独特的。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，从而产生空间邻近性。 重排的 DJH 连接和远端 VH 基因对正常条件下和病理疾病状态下的 IgH 库形成具有影响，我们建议充分表征涉及位点 I 的循环相互作用，然后使用这些信息构建具有位点 I 的小鼠。将使用 3C 染色质环化分析、3D FISH 以及 B 细胞发育和 V(D)J 期间 VH 基因使用分析来充分探讨位点 I 被删除或突变的后果。这些研究将为有关体液免疫发展的新见解奠定基础。