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.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01AI082918-05
Application #
8636390
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Nasseri, M Faraz
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Choi, Nancy M; Feeney, Ann J (2014) CTCF and ncRNA Regulate the Three-Dimensional Structure of Antigen Receptor Loci to Facilitate V(D)J Recombination. Front Immunol 5:49
Predeus, Alexander V; Gopalakrishnan, Suhasni; Huang, Yue et al. (2014) Targeted chromatin profiling reveals novel enhancers in Ig H and Ig L chain Loci. J Immunol 192:1064-70
Gopalakrishnan, Suhasni; Majumder, Kinjal; Predeus, Alexander et al. (2013) Unifying model for molecular determinants of the preselection V* repertoire. Proc Natl Acad Sci U S A 110:E3206-15
Kumar, Satyendra; Wuerffel, Robert; Achour, Ikbel et al. (2013) Flexible ordering of antibody class switch and V(D)J joining during B-cell ontogeny. Genes Dev 27:2439-44
Choi, Nancy M; Loguercio, Salvatore; Verma-Gaur, Jiyoti et al. (2013) Deep sequencing of the murine IgH repertoire reveals complex regulation of nonrandom V gene rearrangement frequencies. J Immunol 191:2393-402
Volpi, Sabrina A; Verma-Gaur, Jiyoti; Hassan, Rabih et al. (2012) 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. J Immunol 188:2556-66
Feeney, Ann J; Verma-Gaur, Jiyoti (2012) CTCF-cohesin complex: architect of chromatin structure regulates V(D)J rearrangement. Cell Res 22:280-2
Degner, Stephanie C; Verma-Gaur, Jiyoti; Wong, Timothy P et al. (2011) CCCTC-binding factor (CTCF) and cohesin influence the genomic architecture of the Igh locus and antisense transcription in pro-B cells. Proc Natl Acad Sci U S A 108:9566-71
Feeney, Ann J (2011) Epigenetic regulation of antigen receptor gene rearrangement. Curr Opin Immunol 23:171-7
Degner-Leisso, Stephanie C; Feeney, Ann J (2010) Epigenetic and 3-dimensional regulation of V(D)J rearrangement of immunoglobulin genes. Semin Immunol 22:346-52