The role of LSD1 in the epigenetic regulation of B cell terminal differentiation

项目摘要 B细胞终末分化产生抗体分泌细胞(ASCs)，提供对 病原体。疫苗诱导B细胞终末分化以提供靶向免疫。当B细胞终末 分化失调，导致自身免疫和癌症等疾病。改进疫苗设计 对于B细胞疾病的治疗，我们必须了解调节B细胞的分子机制 末端分化。最近的证据表明，在关键转录的引导下，表观遗传重编程 影响B细胞终末分化的因素。值得注意的是，组蛋白去甲基酶LSD1已被证明直接 与BLIMP1相互作用，介导抑制ASCs中B细胞的转录程序。LSD1脱甲基化物 组蛋白3赖氨酸4单甲基化和双甲基化(H3K4me1/2)以使活性增强剂和 推动者。深入分析特定表观遗传修饰蛋白对B细胞终末的影响 分化以及它们施加的染色质调节将有助于阐明分子机制。 因此，我们建议进一步研究LSD1在这一过程中的作用。初步 实验表明，在体外和体内分化产生的ASCs分别减少了65%和58% 提示LSD1在B细胞终末分化过程中起重要作用。 此外，RNA-seq和atac-seq表明，没有LSD1，幼稚B细胞和ASCs都表现出靶向基因 染色质可及性上调和获得，提示LSD1通过 促进染色质关闭的机制。这些数据让我们假设LSD1有助于 ASCs中B细胞程序基因去甲基化抑制B细胞终末分化 H3K4me1/2位于启动子和增强子区域。在目标1中，我们将进一步定义LSD1的功能角色 B细胞终末分化贯穿始终。LSD1和B细胞的终末分化在 因此，我们将利用一个小鼠模型，在B细胞中有条件地删除LSD1。诱使 B细胞终末分化时，我们将用T细胞依赖和非依赖模型抗原免疫小鼠。 我们将把这些系统与基于流式细胞术的检测、血清学检测和 免疫组织化学方法研究LSD1在B细胞终末分化阶段中的作用 激活、增殖、生发中心反应，以及激活的B细胞向ASCs的转变。在目标2中， 我们将通过以下方式确定LSD1调节B细胞终末分化的分子机制 对LSD1充足的和LSD1缺乏的B细胞群体进行前后的芯片序列分析 免疫接种。初步测序数据和芯片序列数据分析将用于指导开发 通过遗传学和分子检测来探索与LSD1调控B细胞末端相关的基因 差异化。这项工作将提高我们对人类B细胞终末分化是如何调节的了解 并可用于进一步改进B细胞疾病的疫苗和治疗方法的开发。

Project Summary B cell terminal differentiation gives rise to antibody-secreting cells (ASCs) that provide immunity against pathogens. Vaccines induce B cell terminal differentiation to provide targeted immunity. When B cell terminal differentiation is dysregulated, diseases such as autoimmunity and cancer result. To improve vaccine design and treatments for B cell-based diseases, we must understand the molecular mechanisms that regulate B cell terminal differentiation. Recent evidence suggests that epigenetic reprogramming guided by key transcription factors drives B cell terminal differentiation. Of note, the histone demethylase LSD1 has been shown to directly interact with BLIMP1 to mediate suppression of the B cell transcriptional program in ASCs. LSD1 demethylates histone 3 lysine 4 mono- and di-methylation (H3K4me1/2) in order to decommission active enhancers and promoters. In depth analysis of the impact that specific epigenetic modifying proteins have on B cell terminal differentiation as well as the chromatin modulations they impose will shed light on the molecular mechanisms that regulate this process, thus we propose to further study the role of LSD1 in this process. Preliminary experiments show a 65% and 58% reduction in ASCs generated from ex vivo and in vivo differentiation of LSD1-deficient B cells, respectively, indicating that LSD1 is highly important for B cell terminal differentiation. Also, RNA-seq and ATAC-seq show that without LSD1, both naive B cells and ASCs exhibit targeted gene upregulation and gain of chromatin accessibility, suggesting that LSD1 mediates gene silencing through mechanisms that promote the closing of chromatin. These data lead us to hypothesize that LSD1 facilitates proper B cell terminal differentiation by repressing B cell program genes in ASCs via demethylation of H3K4me1/2 at promoter and enhancer regions. In aim 1, we will further define the functional role of LSD1 throughout B cell terminal differentiation. LSD1 and B cell terminal differentiation are conserved between humans and mice, so we will utilize a mouse model in which Lsd1 is conditionally deleted in B cells. To induce B cell terminal differentiation, we will immunize mice with T cell-dependent and -independent model antigens. We will use these systems in conjunction with flow cytometry-based assays, serological assays, and immunohistochemistry to assess the contribution of LSD1 to the B cell terminal differentiation stages of activation, proliferation, the germinal center reaction, and the transition of activated B cells into ASCs. In aim 2, we will determine the molecular mechanism by which LSD1 regulates B cell terminal differentiation by performing ChIP-seq on LSD1-sufficient and LSD1-deficient B cell populations prior to and following immunization. Preliminary sequencing data and ChIP-seq data analyses will be used to guide the development of genetic and molecular assays to probe gene relevance to LSD1-based regulation of B cell terminal differentiation. This work will improve our knowledge of how human B cell terminal differentiation is regulated and can be used to further improve the development of vaccines and treatments for B cell-based diseases.