BRWD1 and the molecular regulation of humoral immunity

项目总结 我们的实验室最近证明了血统受限的Bromo和WD40结构域包含 表观遗传阅读器BRWd1打开Igκ，使RAG蛋白能够在Jκ上组装。我现在向大家报告BRWD1 在晚期B细胞发育中有更广泛的作用。在骨髓中，BRWD1重塑了染色质 关闭B细胞发育早期表达的基因增强子并打开基因增强子的景观 在后期表达。在缺乏BRWD1的情况下，超过7000个基因发生了异常表达。虽然很多人 这些基因中的许多通常在早期发育阶段表达，许多也是生发的一部分。 中心(GC)转录程序。BRWD1首先在小的Pre-B细胞中表达。但是，Brwd1表达式 在幼稚滤泡(FO)中较高，在第12天的GC B细胞中最高。这些数据表明，BRWD1可能 也调节GC B细胞程序。我在WT GC B细胞中的数据高度支持这一点。通过使用新的3- 隔离暗区(DZ)、亮区(LZ)和新识别的种群灰色区的种群门控策略 (GZ)，我发现DZ和LZ细胞在转录和基因组可及性方面存在很大差异。广州是 在转录和基因组可获得性方面也是DZ和LZ B细胞的独特之处，表明它可能包含 一个新的GC B细胞亚群(S)。在LZ细胞中，BRWD1在转录因子Myc-cell中表达 被BRWD1压制。最后，对GC、DZ和LZ中差异表达的基因进行了分析 在小的Pre-B细胞中受BRWD1的差异调节，预测BRWD1抑制DZ程序和 引出了LZ程序。这具有免疫学意义，因为免疫的小鼠在GC B中缺乏BRWD1 细胞表现为GC排列紊乱，亲和力成熟严重受损。总体而言，这些数据表明 在发育和免疫球蛋白κ重组中分离增殖和免疫球蛋白DNA重组的遗传程序之间的相似性 在GC中，将增殖与选择分开的那些。基于这些发现，我假设 调节增强子可及性BRWD1对调节新的GC B细胞的转录程序很重要 子集。此外，我假设，通过抑制DZ和诱导LZ遗传程序，BRWD1是至关重要的 依赖GC的体液免疫。这些假设将在以下具体目标中得到检验： 目的1.定义正常生发中心反应的转录调控。 目的2.确定BRWD1在获得性免疫中的作用。

PROJECT SUMMARY Our laboratory recently demonstrated that the lineage-restricted BROMO and WD40 domain containing epigenetic reader BRWD1 opens Igκ and enables assembly of RAG proteins at Jκ. I now report that BRWD1 has a much broader role in late B cell development. In the bone marrow, BRWD1 reshaped chromatin landscape by closing enhancers of genes expressed early in B cell development and opening those of genes expressed in late stages. In the absence of BRWD1, over 7000 genes were aberrantly expressed. While many of these genes are normally expressed in earlier developmental stages, many are also part of the germinal center (GC) transcription program. BRWD1 is first expressed in small pre-B cells. However, Brwd1 expression was much higher in naïve follicular (FO) and highest in day 12 GC B cells. These data suggest BRWD1 could also regulate GC B cell programs. My data in WT GC B cells highly supports this. By using a novel 3- population gating strategy to isolate dark zone (DZ), light zone (LZ), and newly identified population grey zone (GZ), I found large transcriptional and genome accessibility differences between DZ and LZ cells. The GZ is also unique from DZ and LZ B cells based on transcription and genome accessibility, suggesting it may contain a novel GC B cell subpopulation(s). In LZ cells, BRWD1 is expressed in Myc- cells, a transcription factor repressed by BRWD1. Finally, analysis of genes differentially expressed in GC DZ and LZ, compared to those differentially regulated by BRWD1 in small pre-B cells, predicted that BRWD1 represses the DZ program and induces the LZ program. This has immunological implications as immunized mice lacking BRWD1 in GC B cells exhibited disorganized GCs and severely impaired affinity maturation. Overall, these data suggest similarities between genetic programs that separate proliferation and Igκ recombination in development and those that segregate proliferation from selection in GCs. Based on these findings, I hypothesize that by regulating enhancer accessibility BRWD1 is important to regulate transcriptional programs in novel GC B cell subsets. Additionally, I hypothesize that by repressing DZ and inducing LZ genetic programs, BRWD1 is critical for GC-dependent humoral immunity. These hypotheses will be tested in the following Specific Aims: Aim 1. Define transcriptional regulation of normal germinal center responses. Aim 2. Determine the role of BRWD1 in adaptive immunity.