Control of B cell Development by YY1

 描述（由申请方提供）：B细胞发育需要免疫球蛋白（IG）基因的有序V（D）J重排。IG重排缺陷导致严重的免疫缺陷，而异常的染色体易位导致白血病和淋巴瘤。在发育过程中，IG重排受到严格调控，重链基因座（IgH）可接近性和重排发生在前B细胞阶段，轻链κ基因座（IGκ）可接近性和重排发生在前B细胞阶段。由于重链和轻链基因座巨大（高达3.4 mb），远端V（D）J重排需要额外的发育控制的物理收缩过程，以使远端V区基因接近D和J基因片段。虽然很明显，可及性和轨迹收缩是发育调节的，这些影响的机制基础目前还不清楚。在过去的资助期间，我们已经确定YY 1作为IG位点收缩的关键调节因子。我们证明，YY 1定位在跨越IGκ重排的细胞中的IGκ基因座的20多个位点上，并且YY 1与凝聚素、粘附素和多梳组（PcG）复合物的组分（参与大规模染色体相互作用的蛋白质）共定位在这些位点上。我们最近还发现YY 1与IGκ Cer DNA元件结合，该元件以YY 1依赖的方式调节IGκ位点的收缩并募集凝聚素蛋白。基于我们的累积数据，我们假设YY 1与IG位点结合，并且DNA结合的YY 1与凝聚素、粘附素和PcG复合物的各种组分以及与CTCF（一种已知影响IG位点收缩的YY 1相互作用蛋白）的发育调节的相互作用调节IG位点的时间重排。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PcG、凝聚素、粘附素和CTCF蛋白的发育调节YY 1依赖性募集。使用蛋白质组学、co-IP和修饰特异性抗体， 将（2）确定发育调节YY 1与凝聚素，粘附素，PcG和CTCF蛋白相互作用的机制基础。我们推测（3）YY 1、凝聚素、粘附素和PcG蛋白共定位位点相互作用，并与Cer调控序列相互作用，介导位点收缩和IGκ重排。我们将在pro-B、pre-B和YY 1敲除背景下使用3C检测对此进行测试，并将通过在YY 1缺失细胞中表达具有确定功能的YY 1突变体，然后通过3D-FISH测量基因座收缩来确定YY 1在IG基因座收缩中的功能机制。我们期望我们的研究将提供对YY 1介导的IG位点收缩机制的基础性见解，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的巨大进步。

 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.