A novel link between gene regulation and histone modifications governing islet beta-cell development and function

基因调控与控制胰岛β细胞发育和功能的组蛋白修饰之间的新联系

• 批准号: 10365325
• 负责人: Chad S Hunter
• 金额: $ 45.83万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-01 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10365325
• 关键词: Adult; Affect; American; Architecture; Autoimmune; B-Cell Development; B-Lymphocytes; Biological Assay; Cell Line; Cell Lineage; Cell Maturation; Cell Survival; Cell physiology; Cells; Chromatin; Code; Complex; Data; Data Set; Deposition; Development; Diabetes Mellitus; Disease; Economic Burden; Economics; Embryo; Enhancers; Epidemic; Epigenetic Process; Essential Genes; Functional disorder; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic Transcription; Glucose; Goals; Healthcare; Histone H2B; Histone H3; Histones; Homo; Hormones; Human; Hyperglycemia; Immunoprecipitation; In Vitro; Individual; Insulin; Insulin-Dependent Diabetes Mellitus; Islet Cell; Islets of Langerhans; Knockout Mice; Knowledge; LIM Domain; Link; Lysine; Mass Spectrum Analysis; Mediating; Messenger RNA; Modeling; Modification; Monoubiquitination; Mus; Mutation; Non-Insulin-Dependent Diabetes Mellitus; Nuclear; Pathway interactions; Patients; Pharmaceutical Preparations; Physiological; Production; Property; Protein Binding Domain; Proteins; Publishing; Quality of life; Regulation; Regulator Genes; Reporting; Repression; Ring Finger Domain; Role; SS DNA BP; Structure of beta Cell of islet; Therapeutic; Transactivation; Transcriptional Activation; Ubiquitin; Work; blood glucose regulation; care burden; chromatin remodeling; combat; crosslink; design; diabetic; dimer; endocrine pancreas development; expectation; experimental study; functional loss; glucose tolerance; histone modification; homeodomain; improved; in vitro Assay; insight; insulin regulation; insulin secretion; islet; knock-down; lentiviral-mediated; mouse model; novel; postnatal; preservation; promoter; recruit; small hairpin RNA; therapy development; transcription factor; transcriptome; ubiquitin ligase; ubiquitin-protein ligase

ABSTRACT Insulin-secreting b-cells within the pancreatic islets of Langerhans are absolutely required for glucose homeostasis. Loss of b-cell survival or function are hallmarks of type 1 or type 2 diabetes mellitus, respectively, which affects millions of Americans, with numbers expected to greatly increase. This has created enormous economic and health care burdens. Improvements in diabetic therapies will require the deeper understanding of novel mouse and human b-cell regulators and/or pathways directly implicated in b-cell function, including glucose-stimulated insulin secretion (GSIS). The LIM-Homeodomain class transcription factor Islet-1 (Isl1) is an islet-enriched regulator of pancreatic islet cell development, maturation, and function. Despite this, little is known of the transcriptional mechanisms or protein interactors employed by Isl1 to elicit these functions in mouse or human b-cells. To identify components of Isl1 transcriptional complexes, we performed reverse-crosslinked immunoprecipitation and mass spectroscopy experiments using mouse b-cells. We found the novel Isl1 interactors Ring Finger (Rnf)20 and Rnf40, which are E3 ubiquitin ligases that act as a homo- or hetero-dimeric complex to promote transcription via specifically mono-ubiquitinating histone H2B (H2Bub1), a precursor to active histone 3 lysine 4 trimethylation (H34me3). In vitro assays with b-cell lines revealed that (at least) Rnf20 is required for the expression of many Isl1 target genes (e.g., Glut2, MafA, Ins1), as well as GSIS. Strikingly, we also found that reduction of Rnf20 or Isl1 in b-cell lines reduced H2Bub1 and H3K4me3 marks in vitro, thus linking Isl1:Rnf20 to b-cell epigenetics. Our preliminary mouse knockout data support that Rnf20 may act as a homodimer (i.e., without Rnf40) to drive Isl1-mediated target gene regulation in mouse b-cells. Therefore, in this proposal we will compare the functional and transcriptional impacts in mouse models of Isl1 or Rnf20 deficiency, as well as in human b-cells lacking ISL1, RNF20, or RNF40. Our central hypothesis is that b-cell formation and function requires deposition of the H2Bub1 modification by recruited Isl1:Rnf20. We designed three specific aims that will examine the relative functional and gene expression importance of Isl1 and Rnf20 (and thus H2Bub1) in embryonic and adult mouse b-cells, and also in adult human b-cells. Results reported from these studies will establish Rnf20 and the H2Bub1 epigenetic mark as fundamental gene regulatory effectors of Isl1, thus impacting mouse and human b-cell function. Concepts learned will yield new insight into development of novel diabetes drugs or enhance strategies to produce therapeutic b-like cells in vitro.

抽象的 Langerhans胰岛内的胰岛素分泌B细胞绝对需要葡萄糖 稳态。 B细胞存活或功能的丧失分别是1型或2型糖尿病的标志 这会影响数百万美国人，预计数字将大大增加。这创造了巨大的 经济和医疗保健负担。糖尿病疗法的改善将需要更深入的了解 新型小鼠和人类B细胞调节剂和/或直接与B细胞功能有关的途径，包括 葡萄糖刺激的胰岛素分泌（GSIS）。 Lim-Homeodomain类转录因子Islet-1（ISL1）是胰岛增强的调节剂 胰岛细胞的发育，成熟和功能。尽管如此，对转录的知之甚少 ISL1使用的机制或蛋白质相互作用者在小鼠或人B细胞中引起这些功能。到 识别ISL1转录复合物的组成部分，我们进行了反向交叉的免疫沉淀 使用小鼠B细胞进行质谱实验。我们发现小说ISL1相互作用者无名指 （RNF）20和RNF40，是E3泛素连接酶，充当同型或异二二聚体复合物来促进 通过特异性单泛素化组蛋白H2B（H2BUB1）的转录，这是活性组蛋白3赖氨酸4的前体 三甲基化（H34me3）。用B细胞系的体外测定表明（至少）RNF20是必需的 许多ISL1靶基因（例如GLUT2，MAFA，INS1）以及GSI的表达。令人惊讶的是，我们还发现 在B细胞系中降低RNF20或ISL1的降低H2BUB1和H3K4ME3体外标记了ISL1：RNF20 到B细胞表观遗传学。我们的初步鼠标敲除数据支持RNF20可能充当同型二聚体（即 没有RNF40）以驱动小鼠B细胞中ISL1介导的靶基因调节。因此，在这个建议中我们 将比较ISL1或RNF20缺陷的鼠标模型中的功能和转录影响，以及 在缺少ISL1，RNF20或RNF40的人类B细胞中。我们的中心假设是B细胞的形成和功能 需要通过招募的ISL1：RNF20对H2BUB1修饰进行沉积。我们设计了三个特定目标 将检查ISL1和RNF20（因此H2BUB1）在 胚胎和成年小鼠B细胞，以及成人人类B细胞。这些研究报告的结果将 建立RNF20和H2BUB1表观遗传标记作为ISL1的基因调节效应子，因此 影响小鼠和人类B细胞功能。学到的概念将产生新的见解，以了解新颖的发展 糖尿病药物或增强体外产生治疗性B样细胞的策略。