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

描述(申请人提供)：免疫系统产生高度多样化的抗体，以提供对外来病原体的防御。这一庞大的谱系是在受体基因座上编码的，这些受体基因以特定的谱系和发育阶段特定的方式经历了V(D)J重组的高度调控过程。IgH基因座包含100多个VH功能基因，跨度为2.5Mb。因此，为了创造一个多样化的免疫球蛋白谱系，V(D)J重组的挑战是使分布在如此大的区域内的所有V基因接近小的DJ簇。这是通过多个染色体环事件收缩VH基因座来实现的。CTCF是一种锌指蛋白，它与绝缘体和边界元件相关，位于几个研究得很好的发育调节基因座上。重要的是，CTCF已被证明通过环在这些复杂的基因座上形成远程相互作用。由于VH基因位点通过环的方式紧凑，我们推测CTCF可能在形成这些环的过程中起着关键作用。通过芯片分析，我们发现在VH基因座上存在多个CTCF位点，其中一个位于D簇的5‘端，一个位于IGH基因的3’端。虽然CTCF不与谱系特异性结合，但最近被证明与CTCF结合的粘附素具有。我们建议进行CHIP-SEQ来确定CTCF和粘附素在B细胞分化的不同阶段和非B细胞中的结合模式。我们将对已被发现对VH位点收缩和远端VH基因重排至关重要的蛋白质(YY1、Pax5、Ezh2)以及SATB1进行CHIP-SEQ，SATB1被认为通过基质结合参与染色体环。YY1特别令人感兴趣，因为它已经被证明是X染色体失活的一个与CTCF有关的辅助因子。为了证明CTCF和/或粘附素是否是IgH基因座收缩所必需的，我们将敲除Pro-B细胞中的CTCF或Rad21，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捉(3C)，以确定在CTCF/粘附素结合部位是否存在染色体长程相互作用，或者在VH基因座上与IGH位点收缩有关的其他蛋白质的结合部位是否存在染色体长程相互作用。将评估CTCF/粘附素位点作为控制V或DJ亚区可及性的边界因素的作用。综上所述，这些研究将使我们更深入地了解V(D)J重排前、中和后IgH基因的三维结构，并将深入了解CTCF和粘附素在V(D)J重排调控中的作用。越来越明显的是，癌症可以有一个重要的表观遗传成分。CTCF的失控可能导致癌症的发展，而粘附素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，这类应用中提出的研究将拓宽我们对CTCF和粘附素在分化过程中的作用的理解。 与公共卫生相关：癌症可能具有重要的表观遗传学成分，这一点越来越明显。CTCF的失控会导致癌症的发生，V(D)J重组机制的失控也会导致癌症的发生，而粘附素的突变已被证明是一些严重的发育综合征的基础，如Cornelia de Lange综合征。因此，像本申请中提出的在V(D)J重组的高度复杂和发育调控过程中的研究将拓宽我们对CTCF和粘附素的多种作用的理解。

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. PUBLIC HEALTH RELEVANCE: It is becoming increasingly apparent that cancer can have a significant epigenetic component. Deregulation of CTCF can contribute to cancer development, as can misregulation of the V(D)J recombination mechanism, and mutations in cohesin have been demonstrated to be the basis of some severe developmental syndromes such as Cornelia de Lange syndrome. Thus, studies such as the ones proposed in this application in the highly complex and developmentally regulated process of V(D)J recombination will broaden our understanding of the many roles of CTCF and cohesin.