Regulation of Ig-kappa recombination during B lymphopoiesis

 描述（由申请方提供）：B淋巴细胞生成的标志是免疫球蛋白重链（IGµ）然后轻链基因座的顺序生产性基因组重排。框内IGµ重组后，前BCR的表达与IL-7依赖性克隆扩增相关。然而，前B细胞必须在启动Igk重组之前退出细胞周期。如果不这样做，就有基因组不稳定和白血病转化的风险。在IL-7 R下游，STAT 5通过诱导细胞周期蛋白D3同时抑制Igk重组来驱动增殖。我们现在已经阐明了STAT 5抑制Igk的三种机制。首先，STAT 5结合Igk内含子增强子作为四聚体，并募集组蛋白甲基转移酶Ezh 2，其用H3 K27 me 3装饰Jk和Ck区域。STAT 5还抑制含有布罗莫结构域的表观遗传阅读器BRWD 1。在没有BRWD 1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增强子处的核小体定位紊乱有关。BRWD 1是从扩展的GAGA基序中清除核小体和将RAG 1/2募集到Jk所必需的。这些发现表明BRWD 1在染色质重塑、基因转录和靶向重组到Igk中具有关键和全面的作用。最后，STAT 5诱导细胞周期蛋白D3的表达，其既驱动增殖又抑制Vk转录。共聚焦和超分辨率显微镜表明，在前B细胞，Vk被嵌入在一个紧密并列的核基质相关的细胞周期蛋白D3和elongating-pol II（转录中心）的网络。在这些细胞中，Vk不进入转录中心。然而，在过渡到小的前B细胞阶段，或当细胞周期蛋白D3不存在时，Vk的一个等位基因环入e-pol II。相比之下，在小的前B细胞中，JkCk更常见地以双等位基因方式与e-pol II相关。这些后来的研究结果表明，Vk的可访问性，而不是JkCk的可访问性，有助于等位基因排斥。总的来说，这些研究结果表明一个全面的模型，其中从根本上不同的镇压机制，控制移动的Vk节段访问e-pol II和表观遗传调控的一个固定的JkCk平台，确定Igk的重组，并确保增殖和重组保持相互排斥。目标1。明确BRWD 1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BRWD 1对GAGA基序的识别如何促进Igk重组。目标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.