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.
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.
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