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

项目概要 我们希望揭示维持 B 细胞稳态的新型基本 B 细胞内在表观遗传机制 并告知对外源抗原（例如病毒、细菌）的抗体反应至关重要的 B 细胞过程 和自身抗原（例如系统性狼疮中的 dsDNA、RNP）。正如我们在本次拨款的第一个周期中所示， 表观遗传机制与遗传程序相互作用来调节 AID（由 AICDA/Aicda 编码）表达， 对于 SHM/CSR 至关重要。事实上，Zn2 依赖性 I、II 和 IV 类 HDAC 促进 AID 诱导和 SHM/CSR 以 B 细胞固有的方式。 AID 诱导受到 I、II 和 IV 类 HDAC 抑制剂的抑制，例如短效 肠道微生物群通过膳食纤维加工产生的链脂肪酸，通过上调选择 靶向 AICDA/Aicda 3’UTR 的 microRNA，导致抗体/自身抗体反应失效。 受我们对 Sirt1（一种依赖于 NAD 的 III 类 HDAC）的最新且令人信服的发现的推动， 代谢传感器与衰老、癌症和糖尿病有关，我们假设这种 Sirtuin 会抑制 AID（一种 以 B 细胞固有方式发挥与 I、II 和 IV 类 HDAC 相反的作用，以及 Sirt1 表达的调节 或活性有效调节 SHM/CSR 和抗体/自身抗体反应。正如我们所说，高 Sirt1 水平/活性会影响静息 B 细胞中的稳态 Aicda 沉默，这是三相的第一阶段 Sirt1 和 AID 表达相互波动，随后激活的 B 细胞中 Sirt1 较低且 AID 较高 浆细胞和记忆 B 细胞中从高 Sirt1 到低 AID。 Sirt1 会调节 AID 表达 通过三管齐下的组蛋白和非组蛋白去乙酰化。它还会强制执行 B 细胞固有的 自 Sirt1 辅因子 NAD 整合代谢线索以来，AID 上调的代谢-表观遗传检查点。 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-tdTomato 和 Cd19 /Cre-Ert2Rosa26fl-STOP-fl-Sirt1-IRES-Gfp 小鼠）、 成像（AicdaCreRosa26fl-STOP-fl-荧光素酶报告小鼠）和人类抗体/自身抗体的动物模型 反应（人源化 NSG/cKitW-41J 小鼠）使我们有能力检验我们的假设。目标 1 地址 Sirt1 表达和 NAD 水平的人类和小鼠 B 细胞分化阶段特异性调节，以及 潜在机制，重点关注转录激活子 USF1、转录抑制子 c-Myb 和 靶向 Sirt1 3’UTR 的 microRNA。目标 2 解决 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，并定义了潜在的 H3K4Ac、H3K36Ac、Dnmt1 和 NF- kB 脱乙酰机制；目标 3 解决 B 抑制抗体和自身抗体反应的问题 细胞 Sirt1 和 Sirt1 激活剂，并探索 Sirt1 激活剂作为系统性狼疮的治疗方法。我们的高度 重要且创新的实验将为 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.!