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（由申请方提供）：免疫球蛋白（IG）基因的独特之处在于，它们经历三种不同类型的基因改变以实现全功能的体液免疫应答。在骨髓（BM）中的早期B细胞发育期间，V（D）J或VJ连接分别发生在IgH和L链基因上，并且由RAG重组酶介导。我们的新研究描述了一个逐步的染色体构象改变的过程，合作创造条件，适合于组装成连续的V（D）J外显子编码IgH分子的抗原结合部分的V-D-J基因片段。最近的研究表明，染色质成环影响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Igh基因座识别出一小部分环，但无法对基因座环进行无偏检查。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。这使我们能够系统地表征三维（3D）染色质组织的几个基因组规模。我们已经发现，Igh轨迹被划分成两个独特的子拓扑结构域分开的一个相对非结构化的区域。非淋巴MEF细胞和pro-B淋巴细胞的比较揭示了一组非常长范围的成环相互作用，其用于桥接亚拓扑结构域，并且对于pro-B细胞和Pax 5依赖性都是独特的。因此，我们已经鉴定了Pax 5依赖性成环相互作用，称为位点I、II、II.5和III，其负责Igh基因座收缩，其用于在Igh基因座之间产生空间接近。 重排的DJH连接和远端VH基因。这些发现对正常条件下和病理疾病状态下IgH库的形成具有影响。我们建议充分表征循环的相互作用，涉及网站I，然后使用这些信息来构建网站I已被删除或突变的小鼠。位点I的靶向缺失的后果将使用3C染色质成环测定、3D FISH以及B细胞发育和V（D）J连接期间VH基因使用的分析来充分探索。这些研究将为关于体液免疫发展的新见解奠定基础。