B cell development requires the ordered V(D)J rearrangement of immunoglobulin (Ig) genes. Defects in Ig rearrangement lead to severe immunodeficiencies while aberrant chromosomal translocations result in leukemia and lymphoma. During development, Ig rearrangement is tightly regulated with heavy chain locus (IgH) accessibility and rearrangement occurring at the pro-B cell stage and light chain kappa locus (Ig?) accessibility and rearrangement at the pre-B cell stage. As the heavy and light chain loci are huge (up to 3.4 mb), distal V(D)J rearrangement requires an additional developmentally controlled physical contraction process to bring distal V region genes into the proximity of D and J gene segments. While it is clear that accessibility and locus contraction are developmentally regulated, the mechanistic basis for these effects is currently unclear. During the past funding period, we have identified YY1 as a critical regulator of Ig locus contraction. We demonstrated that YY1 localizes at over 20 sites spanning the Ig? locus in cells poised to undergo Ig? rearrangement, and that YY1 co-localizes at these sites with components of the condensin, cohesin, and Polycomb Group (PcG) complexes, proteins involved in large-scale chromosomal interactions. We recently also found that YY1 binds to the Ig? Cer DNA element that regulates Ig? locus contraction and recruits condensin proteins in a YY1-dependent manner. Based on our cumulative data, we hypothesize that YY1 binds to Ig loci, and that developmentally regulated interactions of DNA-bound YY1 with various components of the condensin, cohesin, and PcG complexes, as well as with CTCF, a YY1-interacting protein known to impact Ig locus contraction, regulates the temporal rearrangement of the Ig loci. By ChIP-seq approaches we will determine (1) whether developmentally regulated YY1-dependent recruitment of PcG, condensin, cohesin, and CTCF proteins is required for differential recombination of the IgH and Ig? loci during B cell maturation. Using proteomic, co-IP, and modification-specific antibodies we will (2) determine the mechanistic basis for developmentally regulated YY1 interactions with condensin, cohesin, PcG, and CTCF proteins. We hypothesize that (3) the YY1, condensin, cohesin, and PcG protein co-localization sites physically interact with each other, and with the Cer regulatory sequence to mediate locus contraction and Ig? rearrangement. We will test this using 3C assays in pro-B, pre-B, and YY1 knock-out backgrounds, and will define the mechanism of YY1 function in Ig locus contraction by expressing YY1 mutants with defined functions in YY1-null cells followed by 3D-FISH to measure locus contraction. We anticipate that our studies will provide foundational insight into the mechanisms of YY1-mediated Ig locus contraction and will result in a tremendous advances in our understanding of B cell development and immune function, as well as mechanisms resulting in leukemia and lymphoma.
Our studies will determine how YY1 controls immunoglobulin (Ig) locus contraction, a process required for the generation of antibodies that protect the body from infection. As defects in Ig locus contraction can cause immune deficiencies and translocations involved in leukemia and lymphoma, these studies may identify new mechanisms to regulate these DNA interactions, thereby preventing disease development in the immune system, as well as other systems in which long-range DNA interactions are required.
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