Intrinsic B cell epigenetic regulation of antibody and autoantibody responses by Sirt1

项目总结 我们想要揭示维持B细胞动态平衡的新的基本B细胞内在表观遗传机制 并告知对外源性抗原(如病毒、细菌)的抗体反应至关重要的B细胞过程 和自身抗原(如系统性红斑狼疮中的dsDNA、RNPs)。正如我们在这笔赠款的第一个周期中所表明的， 表观遗传机制与遗传程序相互作用以调节AID(由AICDA/Aicda编码)表达， 对SHM/CSR至关重要。事实上，依赖锌的I、II和IV类HDAC促进AID诱导和SHM/CSR 以B细胞固有的方式。辅助诱导受到I、II和IV类HDAC抑制剂的抑制，如Short- 肠道微生物通过加工膳食纤维，通过上调SELECT基因表达而产生的链脂肪酸 靶向AICDA/Aicda 3‘非编码区的microRNAs，导致抗体/自身抗体反应的消除。 根据我们关于Sirt1的最新和令人信服的发现，依赖NAD的III类HDAC和 代谢感受器与衰老、癌症和糖尿病有关，我们假设这种Sirtuin抑制AID(a 与I、II和IV类HDAC相反的作用)在B细胞固有的方式中，以及Sirt1表达的调节 OR活性有效地调节SHM/CSR和抗体/自身抗体反应。正如我们所争辩的，高Sirt1 水平/活动将影响静息B细胞的动态平衡Aicda沉默，这是三相B细胞的第一阶段 激活的B细胞中Sirt1和AID的相互表达以及随后的低Sirt1和高AID的波动 浆细胞和记忆B细胞由高Sirt1到低AID。SIRT1将调控艾滋病病毒的表达 通过三管齐下的组蛋白和非组蛋白去乙酰化。它还将强制B细胞固有的 自Sirt1辅因子NAD整合代谢信号以来，艾滋病上调的代谢-表观遗传学检查点。 我们在B细胞生物学、分子SHM/CSR机制和自身免疫方面的优势，以及我们的切割- 边缘表观遗传学方法(CHIP-SEQ、甲基DNA-SEQ和ATAC-SEQ)、生物化学新工具(NAD 生物传感器)、遗传学(CD19/Cre-Ert2Sirt16fl/flRosa26fl-Stop-fl-td番茄和CD19/Cre-Ert2Rosa26fl-Stop-fl-Sirt1-IRES-GFP小鼠)， 成像(AicdaCreRosa26fl-Stop-fl-荧光素酶报告鼠)和人类抗体/自身抗体的动物模型 反应(人源化的NSG/cKitW-41J小鼠)使我们独一无二地准备检验我们的假设。AIM1地址 人和小鼠B细胞分化阶段特异性调节Sirt1表达和NAD水平，以及 潜在的机制，重点是转录激活因子USF1，转录抑制因子c-Myb和 靶向Sirt1 3‘非编码区的microRNA。目的2探讨B细胞Sirt1在抑制AICDA/Aicda表达中的作用 通过遗传学和/或化合物/代谢物，并定义潜在的H3K4Ac，H3K36Ac，Dnmt1和NF- KB去乙酰化机制；目标3解决B对抗体和自身抗体反应的抑制 细胞Sirt1和Sirt1激活剂，并探索Sirt1激活剂在系统性红斑狼疮治疗中的作用。我们的高度重视 重大和创新的实验将提供对B细胞表观遗传学和 免疫调节，并产生代谢-表观遗传检查点调节剂作为自身免疫的新疗法。

PROJECT SUMMARY We want to unveil novel fundamental B cell-intrinsic epigenetic mechanisms that maintain B cell homeostasis and inform B cell processes critical to the antibody response to exogenous antigens (e.g., viruses, bacteria) and self-antigens (e.g., dsDNA, RNPs in systemic lupus). As we have shown in the first cycle of this grant, epigenetic mechanisms interact with genetic programs to modulate AID (encoded by AICDA/Aicda) expression, critical for SHM/CSR. Indeed, Zn2+-dependent Class I, II and IV HDACs promote AID induction and SHM/CSR in a B cell-intrinsic fashion. AID induction is dampened by Class I, II and IV HDAC inhibitors, such as short- chain fatty acids produced by gut microbiota via processing of dietary fibers, through upregulation of select microRNAs that target AICDA/Aicda 3’UTR, leading to abrogation of antibody/autoantibody responses. Prompted by our most recent and compelling findings on Sirt1, a NAD+-dependent Class III HDAC and metabolic sensor implicated in aging, cancer and diabetes, we hypothesize that this Sirtuin dampens AID (a role opposite to that of Class I, II and IV HDACs) in a B cell-intrinsic fashion, and regulation of Sirt1 expression or activity effectively modulates SHM/CSR and antibody/autoantibody responses. As we contend, high Sirt1 levels/activity would effect homeostatic Aicda silencing in resting B cells, the first phase of a tri-phasic fluctuation of reciprocal Sirt1 and AID expression, as followed by low Sirt1 and high AID in activated B cells and back to high Sirt1 to low AID in plasma cells and memory B cells. Sirt1 would modulate AID expression through a three-prong histone and non-histone protein deacetylation. It would also enforce a B cell-intrinsic metabolic–epigenetic checkpoint of AID upregulation since Sirt1 cofactor NAD+ integrates metabolic cues. Our strengths in B cell biology, molecular SHM/CSR mechanisms and autoimmunity, as well as our cutting- edge epigenetic approaches (ChIP-Seq, methylDNA-Seq and ATAC-Seq), new tools in biochemistry (NAD+ biosensor), genetics (Cd19+/Cre-Ert2Sirt16fl/flRosa26fl-STOP-fl-tdTomato and Cd19+/Cre-Ert2Rosa26fl-STOP-fl-Sirt1-IRES-Gfp mice), imaging (AicdaCreRosa26fl-STOP-fl-luciferase reporter mice), and animal models of human antibody/autoantibody responses (humanized NSG/cKitW-41J mice) make us uniquely poised to test our hypotheses. Aim1 addresses human and mouse B cell differentiation stage-specific regulation of Sirt1 expression and NAD+ levels, and underlying mechanisms, with focus on transcription activator USF1, transcription repressor c-Myb and microRNAs targeting Sirt1 3’UTR. Aim 2 addresses B cell Sirt1 role in dampening AICDA/Aicda expression through genetics and/or compounds/metabolites, and defines underlying H3K4Ac, H3K36Ac, Dnmt1 and NF- kB deacetylation mechanisms; Aim 3 addresses the inhibition of antibody and autoantibody responses by B cell Sirt1 and Sirt1 activators, and explores Sirt1 activators as therapeutics in systemic lupus. Our highly significant and innovative experiments will provide novel mechanistic insights into B cell epigenetics and immunoregulation, and yield metabolic-epigenetic checkpoint modulators as new therapeutics in autoimmunity.!