Role of CTCF and Cohesin in V(D)J Rearrangement

CTCF 和粘连蛋白在 V(D)J 重排中的作用

• 批准号: 8636390
• 负责人: ANN J FEENEY
• 金额: $ 47万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8636390
• 关键词: 3-Dimensional; Affect; Antibody Repertoire; Architecture; Area; B cell differentiation; B-Lymphocytes; Binding; Binding Sites; Boundary Elements; Bruck-de Lange syndrome; Cells; ChIP-on-chip; ChIP-seq; Chromatin; Chromosomes; Complex; Contracts; DNA Sequence Rearrangement; Development; Distal; Epigenetic Process; Event; Gene Expression; Gene Expression Regulation; Gene Rearrangement; Gene Targeting; Genes; Genetic Recombination; Globin; Heavy-Chain Immunoglobulins; IGH@ gene cluster; Immune system; Immunologic Receptors; Location; Malignant Neoplasms; Modification; Molecular Conformation; Movement; Mutation; Pattern; Phase; Play; Process; Protein Binding; Proteins; Regulation; Role; Site; Specificity; Staging; Syndrome; Techniques; Technology; Time; V(D)J Recombination; Work; X Inactivation; YY1 Transcription Factor; Zinc Fingers; base; cell type; cohesin; histone modification; insight; interest; knock-down; pathogen; prevent; public health relevance; receptor; three dimensional structure; thymocyte

DESCRIPTION (provided by applicant): The immune system creates a highly diverse repertoire of antibodies to provide defense against foreign pathogens. This vast repertoire is encoded at receptor loci which undergo the highly regulated process of V(D)J recombination in a lineage-specific and developmental stage-specific manner. The IgH locus contains over 100 functional Vh genes spanning a 2.5 Mb region. Hence, to create a diverse repertoire of Ig, the challenge of V(D)J recombination is to bring all the V genes, spread over such a large area, close to the small DJ cluster. This is accomplished by contraction of the Vh locus via multiple chromosomal looping events. CTCF is a zinc finger protein that is associated with insulators and boundary elements at several well-studied developmentally regulated loci. Importantly, CTCF has been demonstrated to form long-range interactions in these complex loci via looping. Since the Vh gene locus compacts by looping, we hypothesized that CTCF may be playing a critical role in forming these loops. By ChIP-chip, we demonstrated that there are many CTCF sites in the Vh locus, one at the 5' end of the D cluster, and one at the 3' end of the IgH locus. Although CTCF did not bind with lineage specificity, cohesin, which has recently been demonstrated to bind to CTCF did. We propose to perform ChIP-seq to determine the binding patterns of CTCF and cohesin at various stages of B cell differentiation and in non-B cells. We will perform ChIP-seq for proteins (YY1, Pax5, Ezh2) which have been found to be essential for Vh locus contraction and for distal Vh gene rearrangement, as well as for SATB1, which is known to be involved in chromosomal looping via matrix binding. YY1 is of particular interest since it has been shown to be a co-factor with CTCF for X-chromosome inactivation. To demonstrate whether CTCF and/or cohesin are necessary for IgH locus contraction, we will knock down CTCF or Rad21 in pro-B cells and assess whether locus contraction is affected using 3D-FISH. We will perform chromosome conformation capture (3C) to determine if there are long-range chromosomal interactions at the sites of the CTCF/cohesin binding, or at the sites of binding of other proteins implicated in IgH locus contraction which bind at the Vh locus. The role of CTCF/cohesin sites as boundary elements controlling accessibility to V or DJ sub regions will be assessed. Together, these studies will give us a deeper understanding of the three dimensional structure of the IgH locus before, during and after V(D)J rearrangement and will give insights into the role of CTCF and cohesin in the regulation of V(D)J recombination. It is becoming increasingly apparent that cancer can have a significant epigenetic component. Deregulation of CTCF can contribute to cancer development, and mutations in cohesin have been demonstrated to be the basis of some severe developmental syndromes. Thus, studies such as the ones proposed in this application in the developmentally regulated process of V(D)J recombination will broaden our understanding of the many roles of CTCF and cohesin during differentiation.

描述（由申请人提供）：免疫系统产生高度多样化的抗体库，以防御外来病原体。 这一庞大的库是在受体基因座编码的，这些基因座以谱系特异性和发育阶段特异性的方式经历高度调节的V（D）J重组过程。 IgH基因座包含超过100个功能性Vh基因，跨越2.5 Mb区域。 因此，为了创建多样化的IG库，V（D）J重组的挑战是使分布在如此大的区域上的所有V基因靠近小的DJ簇。 这是通过多个染色体成环事件使Vh基因座收缩来实现的。CTCF是一种锌指蛋白，在几个发育调控位点与绝缘子和边界元件相关。 重要的是，CTCF已被证明通过成环在这些复杂基因座中形成长程相互作用。 由于Vh基因位点通过成环而紧凑，我们假设CTCF可能在形成这些环中起关键作用。 通过ChIP芯片检测，我们发现在Vh基因座上存在多个CTCF位点，一个位于D簇的5'端，一个位于IgH基因座的3'端。 尽管CTCF不以谱系特异性结合，但最近被证明与CTCF结合的粘附素却以谱系特异性结合。 我们建议进行ChIP-seq以确定CTCF和粘附素在B细胞分化的各个阶段和非B细胞中的结合模式。 我们将对蛋白质（YY 1，Pax 5，Ezh 2）进行ChIP-seq，这些蛋白质已被发现对Vh基因座收缩和远端Vh基因重排以及SATB 1至关重要，SATB 1已知通过基质结合参与染色体成环。 YY 1是特别感兴趣的，因为它已被证明是一个与CTCF的X染色体失活的辅因子。 为了证明CTCF和/或粘着蛋白是否是IgH基因座收缩所必需的，我们将敲低pro-B细胞中的CTCF或Rad 21，并使用3D-FISH评估基因座收缩是否受到影响。 我们将进行染色体构象捕获（3C），以确定在CTCF/粘附素结合位点，或在与IgH基因座收缩相关的其他蛋白质（结合在Vh基因座）的结合位点，是否存在长程染色体相互作用。 将评估CTCF/cohesin位点作为控制V或DJ次区域可达性的边界元素的作用。 总之，这些研究将使我们更深入地了解V（D）J重组之前，期间和之后的IgH位点的三维结构，并将深入了解CTCF和cohesin在V（D）J重组调控中的作用。 越来越明显的是，癌症可能具有重要的表观遗传成分。 CTCF的失调可能有助于癌症的发展，并且已经证明粘附素的突变是一些严重发育综合征的基础。 因此，在V（D）J重组的发育调节过程中，诸如本申请中提出的研究将拓宽我们对CTCF和粘附素在分化期间的许多作用的理解。

项目成果

New insights emerge as antibody repertoire diversification meets chromosome conformation.

• DOI: 10.12688/f1000research.17358.1
• 发表时间: 2019-01-01
• 期刊: F1000Research
• 作者: Kenter, Amy L;Feeney, Ann J
• 通讯作者: Feeney, Ann J

Editorial overview. Lymphocyte development.

编辑概述。

• DOI: 10.1016/j.coi.2012.03.003
• 发表时间: 2012
• 期刊: Current opinion in immunology
• 影响因子: 7
• 作者: Corcoran,AnneE;Feeney,AnnJ
• 通讯作者: Feeney,AnnJ

Germline deletion of Igh 3' regulatory region elements hs 5, 6, 7 (hs5-7) affects B cell-specific regulation, rearrangement, and insulation of the Igh locus.

• DOI: 10.4049/jimmunol.1102763
• 发表时间: 2012-03-15
• 期刊: Journal of immunology (Baltimore, Md. : 1950)
• 作者: Volpi SA;Verma-Gaur J;Hassan R;Ju Z;Roa S;Chatterjee S;Werling U;Hou H Jr;Will B;Steidl U;Scharff M;Edelman W;Feeney AJ;Birshtein BK
• 通讯作者: Birshtein BK

CTCF and ncRNA Regulate the Three-Dimensional Structure of Antigen Receptor Loci to Facilitate V(D)J Recombination.

• DOI: 10.3389/fimmu.2014.00049
• 发表时间: 2014
• 期刊: Frontiers in immunology
• 影响因子: 7.3
• 作者: Choi NM;Feeney AJ
• 通讯作者: Feeney AJ