Control of B cell Development by YY1

描述(申请人提供)：阴阳-1(YY1)是一种多功能转录因子，因其根据DNA结合位点上下文或细胞类型抑制或激活转录的能力而得名。YY1对B细胞的发育很重要，因为在B细胞谱系中条件性敲除YY1会导致早期的B细胞缺陷，只有极少数成熟的B细胞。最初的阻断是在原B细胞阶段，与免疫球蛋白(Ig)基因位点的缺失有关，染色体的大规模收缩导致远端可变(V)区基因重排减少。然而，其他B细胞缺陷也很明显，因为重排的Ig基因只能部分逆转突变的表型。我们将YY1鉴定为哺乳动物多梳组蛋白(PcG)，为这些效应的机制基础提供了潜在的见解。有趣的是，YY1条件性基因敲除小鼠表现出与PcG蛋白EZH2敲除类似的B细胞缺陷表型。我们开发了一个YY1突变体(YY1 Repo)，它能特异性地去除YY1 PcG功能，并发现与野生型YY1不同，YY1突变体在YY1缺失的骨髓重建系统中有效地挽救了B细胞的发育，而YY1 repo突变体不能支持有效的B细胞发育。我们还发现，YY1 PcG功能所必需的YY1repo结构域能够与参与染色体收缩的凝聚素和粘附素复合体蛋白物理上相互作用。基于这些发现和各种额外的数据，我们假设YY1将PcG和凝集素蛋白招募到免疫球蛋白基因座上，导致远端可变区基因重排所需的染色体收缩。与这一假设一致，我们发现YY1可以与Ig kappa基因座内的多个位置结合，并且值得注意的是，EZH2和凝集素蛋白与YY1在这些位置上共存。凝集素亚单位SMC4的敲除导致免疫球蛋白重排减少，提示这些相互作用与免疫球蛋白基因座的收缩直接相关。我们将利用YY1repo突变体和多个新的YY1缺失的条件敲除系统来评估YY1 PcG功能在体内B细胞发育中的作用(目标1)。我们还将使用我们的YY1-空重组系统来确定YY1 PcG功能在免疫球蛋白基因座收缩和V区远端基因重排中的作用(目标2)。最后，我们将探讨YY1在免疫球蛋白基因座收缩中的作用机制(目标3)。这些研究将提供YY1 PcG功能在B细胞发育和对免疫功能至关重要的过程中所起作用的关键机制细节。了解YY1的这些功能可能为PcG功能障碍或异常干细胞自我更新引起的疾病的可能治疗干预提供多种途径。

DESCRIPTION (provided by applicant): Yin Yang-1 (YY1) is a multifunctional transcription factor that derives it name from its ability to either repress or activate transcription depending upon DNA binding site context or cell type. YY1 is important for B cell development, as conditional knock-out of YY1 in the B cell lineage results in an early B cell defect with very few mature B cells. The initial block is at the pro-B cell stage and is associated with absence of immunoglobulin (Ig) locus large-scale chromosomal contraction resulting in a reduction in rearrangement of distal variable (V) region genes. However, other B cell defects are also evident because a rearranged Ig gene only partially reverts the mutant phenotype. Potential insight into the mechanistic basis for these effects is provided by our identification of YY1 as a mammalian Polycomb Group (PcG) protein. Interestingly, YY1 conditional knockout mice show a B cell defect phenotype similar to that seen by knock-out of the PcG protein, EZH2. We developed a YY1 mutant (YY1 REPO) that specifically ablates YY1 PcG function, and found that, in contrast to wild-type YY1 that efficiently rescued B cell development in a YY1-null bone marrow reconstitution system, the YY1 REPO mutant is incapable of supporting efficient B cell development. We also found that the YY1 REPO domain, necessary for YY1 PcG function, is able to physically interact with condensin and cohesin complex proteins involved in chromosomal contraction. Based upon these findings, and a variety of additional data, we hypothesize that YY1 recruits PcG and condensin proteins to the immunoglobulin loci resulting in the chromosomal contraction needed for rearrangement of distal variable region genes. Consistent with this hypothesis, we found that YY1 can bind to multiple sites within the Ig kappa locus and that, remarkably, EZH2 and condensin proteins colocalize with YY1 at these sites. Knock-down of condensin subunit SMC4 resulted in reduced Ig rearrangement suggesting that these interactions are directly related to Ig locus contraction. We will utilize the YY1 REPO mutant in conjunction with multiple novel YY1-null conditional knock-out systems to assess the role of YY1 PcG function in B cell development in vivo (Aim 1). We will also use our YY1-null reconstitution systems to determine the role of YY1 PcG function in Ig locus contraction and rearrangement of distal V region genes (Aim 2). Finally, we will explore the mechanism of YY1 function in immunoglobulin locus contraction (Aim 3). These studies will provide key mechanistic detail on the role of YY1 PcG function for B cell development and for processes critical for immune function. Understanding these functions of YY1 may provide multiple avenues for possible therapeutic intervention in diseases caused by PcG dysfunction, or aberrant stem cell self renewal.