Regulation of Ig-kappa recombination during B lymphopoiesis

 描述(由申请人提供)：B淋巴细胞生成的标志是免疫球蛋白重链(Ig？)和轻链基因座的连续生产性基因组重排。在框内Igµ重组后，Pre-BCR的表达与IL-7依赖的克隆性扩张有关。然而，前B细胞在启动Igk重组之前必须退出细胞周期。如果做不到这一点，就会面临基因组不稳定和白血病转化的风险。在IL-7R下游，STAT5通过诱导细胞周期蛋白D3同时抑制Igk重组来驱动细胞增殖。我们现在已经阐明了STAT5抑制IgK的三种机制。首先，STAT5以四聚体的形式结合IgK内含子增强子，并招募组蛋白甲基转移酶Ezh2，该酶用H3K27me3装饰JK和CK区域。Stat5还抑制了包含表观遗传阅读器BRWD1的溴域。在没有BRWD1的情况下，IgK不能有效地重组，这与JK可及性降低以及JK外显子和IgK增强子上核小体定位紊乱有关。BRWD1是从扩展的GagA基序中清除核小体和将RAG1/2招募到JK所必需的。这些发现表明BRWD1在染色质重塑、基因转录和针对Igk的靶向重组中发挥着关键和全面的作用。最后，STAT5诱导细胞周期蛋白D3的表达，而细胞周期蛋白D3既能促进细胞增殖，又能抑制VK的转录。共聚焦显微镜和超分辨显微镜显示，在Pro-B细胞中，VK嵌入在紧密并列的与核基质相关的细胞周期蛋白D3和延长的PolII(转录中心)的网络中。在这些细胞中，VK没有进入转录中心。然而，当过渡到小的前B细胞阶段时，或者当周期蛋白D3缺失时，VK的一个等位基因循环进入e-polII。相反，在小的前B细胞中，JkCk更多地以双等位方式与e-polII相关。这些后来的发现表明，Vk可及性，而不是JkCk可及性，有助于等位基因排斥。总之，这些发现提出了一个全面的Igk调控模型，在该模型中，完全不同的抑制机制、对移动Vk片段访问e-polII的控制以及对固定JkCk平台的表观遗传调控，决定了Igk对重组的可及性，并确保增殖和重组保持互斥。目的1.明确BRWD1介导的染色质重塑在B淋巴细胞生成中的功能重要性。目的2：确定BRWD1对GagA基序的识别如何促进Igk重组。目的3.确定调节VK可及性的机制。

 DESCRIPTION (provided by applicant): The hallmark of B lymphopoiesis is the sequential productive genomic rearrangement of immunoglobulin heavy (Igµ) then light chain loci. Following in-frame Igµ recombination, expression of the pre-BCR is associated with IL-7 dependent clonal expansion. However, pre-B cells must exit cell cycle before initiating Igk recombination. Failure to do so risks genomic instability and leukemic transformation. Downstream of the IL-7R, STAT5 drives proliferation by inducing cyclin D3 while simultaneously repressing Igk recombination. We have now elucidated three mechanisms by which STAT5 represses Igk. First, STAT5 binds the Igk intronic enhancer as a tetramer and recruits the histone methyltransferase Ezh2 that decorates the Jk and Ck regions with H3K27me3. STAT5 also represses the bromodomain containing epigenetic reader BRWD1. In the absence of BRWD1, Igk did not recombine efficiently and this was associated with diminished Jk accessibility and disordered nucleosome positioning at both Jk exons and the Igk enhancers. BRWD1 was required for clearing nucleosomes from extended GAGA motifs and for recruiting RAG1/2 to Jk. These findings suggest critical and comprehensive roles for BRWD1 in chromatin remodeling, gene transcription and in targeting recombination to Igk. Finally, STAT5 induces expression of cyclin D3, which both drives proliferation and represses Vk transcription. Confocal and super-resolution microscopy demonstrated that in pro-B cells, Vk was imbedded within a web of closely juxtaposed nuclear matrix-associated cyclin D3 and elongating-pol II (transcription centers). In these cells, Vk did not access the transcription centers. However, upon transition to the small pre-B cell stage, or when cyclin D3 was absent, one allele of Vk looped into e-pol II. In contrast, in small pre-B cells JkCk more commonly associated with e-pol II in a biallelic manner. These later findings suggest that Vk accessibility, and not JkCk accessibility, contributes to allelic exclusion. In toto, these findings suggest a comprehensive model of Igk regulation in which radically different mechanisms of repression, control of mobile Vk segments access to e-pol II and epigenetic regulation of a fixed JkCk platform, determine Igk accessibility to recombination, and ensure that proliferation and recombination remain mutually exclusive. Aim 1. Define the functional importance of BRWD1-mediated chromatin remodeling in B lymphopoiesis. Aim 2: Determine how GAGA motif recognition by BRWD1 contributes to Igk recombination. Aim 3. Determine the mechanisms regulating Vk accessibility.