A highly diverse antibody repertoire is achieved through V(D)J rearrangement, which involves the contraction and looping of the large ~3 Mb Ig heavy chain (Igh) and kappa (IgK) loci to bring all V genes in proximity with D or J genes to recombine. The nuclear factors controlling Ig loci contraction are not known. CTCF (CCCTC-binding factor) is a nuclear protein that participates in long-range chromosomal interactions. We hypothesize that the CTCF complex may be critical for locus contraction and formation of loops of the large multi-gene immunoglobulin receptor loci during appropriate stages of B cell differentiation.
In Specific Aim 1, we will investigate whether or not CTCF facilitates long-range interactions, looping, and contraction of immunoglobulin receptor loci. To test this, CTCF will be knocked-down with shRNA lentiviruses in pro-B cells. Following CTCF knock-down, 3D-fluorescent in situ hybridization will be used to measure locus contraction. The chromosomal conformation capture (3C) assay will be performed to evaluate interactions, reflecting chromosomal looping during different stages of B cell differentiation and thymocytes. We expect to observe maximum looping interactions during the stage when loci are undergoing V(D)J rearrangement. We predict that knock-down of CTCF will reduce contraction and long- range interactions of the loci.
In Specific Aim 2, we will determine the binding sites of other potential factors involved in Ig loci contraction using chromatin immunoprecipitation on chip (ChlP-Chip) genomic tiling arrays. We will perform ChlP-Chip analyses through stages of B cell differentiation with factors that could inhibit or enhance CTCF function, including cohesin, Kaiso, chromodomain helicase 8 (CHD8), and YY1. Our preliminary data indicate that cohesin binding is present primarily at the stage when the locus is undergoing rearrangement, whereas some of the CTCF sites display similar levels of occupancy at different stages of differentiation. In addition, other factors involved in stage-specific looping of the Igh locus include Pax5, Ezh2, and Ikaros. Binding of these factors throughout immunoglobin loci will be examined by ChlP- Chip. Together, these experiments will provide insight into the role of the CTCF complex in contraction and looping event which enable V(D)J rearrangement at the appropriate stage of B cell differentiation.
e production of antibodies is a critical component of the immune system. Antibodies are produced from a highly regulated complex series of events, including rearrangement of V, D, and J genes. We are investigating if CTCF brings these genes in proximity with each other to rearrange by facilitating the formation of loops.
|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|
|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|