Control of B cell Development by YY1

 描述(由申请人提供)：B细胞的发育需要免疫球蛋白(Ig)基因的有序V(D)J重排。免疫球蛋白重排缺陷会导致严重的免疫缺陷，而染色体异常易位则会导致白血病和淋巴瘤。在发育过程中，Ig重排受到严格调控，重链基因(IGH)可及性和重排发生在前B细胞阶段，轻链kappa基因(Igκ)可及和重排发生在前B细胞阶段。由于重链和轻链基因都很大(长达3.4mb)，V(D)J远端重排需要一个额外的发育控制的物理收缩过程，以使远端V区基因进入D和J基因片段的附近。虽然可及性和局部收缩显然是发育调节的，但这些影响的机制基础目前尚不清楚。在过去的资助期间，我们已经确定YY1是免疫球蛋白基因座收缩的关键调节因子。我们证明了YY1定位于跨越Igκ基因座的20多个位置，在准备进行Igκ重排的细胞中，YY1与凝集素、粘附素和聚梳基(PcG)复合体的成分共同定位在这些位置，这些蛋白质参与大规模的染色体相互作用。我们最近还发现，YY1与调节Igκ位点收缩的IgκCerDNA元件结合，并以YY1依赖的方式招募凝集素蛋白。根据我们的累积数据，我们假设YY1与Ig基因座结合，并且DNA结合的YY1与凝集素、粘附素和PcG复合体的各种成分以及与CTCF的发育调控相互作用，CTCF是一种已知影响Ig基因座收缩的YY1相互作用蛋白，调节Ig基因座的时间重排。通过CHIP-SEQ方法，我们将确定：(1)在B细胞成熟过程中，IgH和Igκ基因座的差异重组是否需要依赖于YY1的发育调节的PcG、凝聚素、粘附素和CTCF四种蛋白的招募。使用蛋白质组、共IP和修饰特异性抗体 将(2)确定发育调节的YY1与凝聚素、粘附素、PcG和CTCF蛋白相互作用的机制基础。我们假设(3)YY1、凝聚素、粘附素和PcG蛋白共定位位点在物理上相互作用，并与Cer调控序列相互作用，介导基因座收缩和Igκ重排。我们将在Pro-B、Pre-B和YY1基因敲除背景下使用3C分析来测试这一点，并将通过在YY1-空细胞中表达具有特定功能的YY1突变体，然后使用3D-FISH来测量基因座收缩，以确定YY1在Ig基因座收缩中的作用机制。我们预计，我们的研究将为YY1介导的免疫球蛋白基因座收缩机制提供基础性的见解，并将导致我们对B细胞发育和免疫功能以及导致白血病和淋巴瘤的机制的理解取得巨大进展。

 DESCRIPTION (provided by applicant): 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 (Igκ) 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 Igκ locus in cells poised to undergo Igκ 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 Igκ Cer DNA element that regulates Igκ 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 Igκ 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 Igκ 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.