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

描述（由申请人提供）：免疫系统产生高度多样化的抗体库，提供对外来病原体的防御。这一庞大的基因库是在受体位点编码的，这些受体位点以谱系特异性和发育阶段特异性的方式经历高度调控的V(D)J重组过程。IgH位点包含超过100个功能性Vh基因，横跨2.5 Mb的区域。因此，为了创造一个多样化的Ig曲目，V(D)J重组的挑战是将分布在如此大范围内的所有V基因靠近小DJ集群。这是通过多个染色体环化事件来完成Vh位点的收缩。CTCF是一种锌指蛋白，与几个发育调控位点的绝缘子和边界元件有关。重要的是，CTCF已被证明在这些复杂的基因座中通过环形成远程相互作用。由于Vh基因座通过环缩紧，我们假设CTCF可能在形成这些环的过程中起关键作用。通过ChIP-chip，我们证明了在Vh位点有许多CTCF位点，一个在D簇的5‘端，一个在IgH簇的3’端。虽然CTCF不与谱系特异性结合，但最近被证明与CTCF结合的内聚蛋白却可以。我们建议采用ChIP-seq技术来确定CTCF和黏结蛋白在B细胞分化的各个阶段以及在非B细胞中的结合模式。我们将对Vh基因座收缩和远端Vh基因重排所必需的蛋白（YY1, Pax5, Ezh2）以及已知通过基质结合参与染色体环的SATB1进行ChIP-seq。YY1是特别有趣的，因为它已被证明是CTCF的x染色体失活的辅助因子。为了证明CTCF和/或内聚蛋白对于IgH基因座的收缩是否必要，我们将敲除前b细胞中的CTCF或Rad21，并使用3D-FISH评估基因座的收缩是否受到影响。我们将进行染色体构象捕获（3C），以确定在CTCF/内聚蛋白结合的位点，或在与IgH基因座收缩相关的其他蛋白质结合的位点，是否存在远程染色体相互作用。将评估CTCF/内聚点作为控制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重排之前、期间和之后的IgH位点的三维结构，并将深入了解CTCF和内聚蛋白在V(D)J重组中的调控作用。越来越明显的是，癌症可能具有重要的表观遗传成分。解除CTCF的管制可以促进癌症的发展，并且粘接蛋白的突变已被证明是一些严重发育综合征的基础。因此，在V(D)J重组的发育调控过程中提出的研究将拓宽我们对CTCF和黏结蛋白在分化过程中的许多作用的理解。

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.