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

基本信息

批准号：
10532763
负责人：
Chad S Hunter
金额：
$ 45.72万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
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/10532763
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 Islet Cell Islets of Langerhans Knockout Mice Knowledge LIM Domain Learning Link Lysine Mass Spectrum Analysis Mediating Messenger RNA Methylation 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 Work blood glucose regulation care burden chromatin remodeling combat crosslink design diabetic endocrine pancreas development expectation experimental study functional loss glucose tolerance histone modification homeodomain humoral immunity deficiency 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

项目摘要

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.

抽象的朗格汉斯胰岛内分泌胰岛素的 B 细胞对于葡萄糖是绝对必需的体内平衡。 B 细胞存活或功能丧失分别是 1 型或 2 型糖尿病的标志，这影响了数百万美国人，预计人数还会大幅增加。这创造了巨大的经济和医疗保健负担。糖尿病治疗的改进需要更深入的了解直接涉及 b 细胞功能的新型小鼠和人类 B 细胞调节因子和/或通路，包括葡萄糖刺激的胰岛素分泌（GSIS）。 LIM-同源域类转录因子 Islet-1 (Isl1) 是一种富含胰岛的调节因子胰岛细胞的发育、成熟和功能。尽管如此，人们对转录过程知之甚少。 Isl1 在小鼠或人类 b 细胞中引发这些功能的机制或蛋白质相互作用因子。到为了鉴定 Isl1 转录复合物的成分，我们进行了反向交联免疫沉淀以及使用小鼠 b 细胞进行的质谱实验。我们发现了小说 Isl1 交互者无名指 (Rnf)20 和 Rnf40，它们是 E3 泛素连接酶，充当同源或异源二聚体复合物以促进通过特异性单泛素化组蛋白 H2B (H2Bub1)（活性组蛋白 3 赖氨酸 4 的前体）进行转录三甲基化（H34me3）。 b 细胞系的体外测定表明（至少）Rnf20 是许多 Isl1 靶基因（例如 Glut2、MafA、Ins1）以及 GSIS 的表达。令人惊讶的是，我们还发现 b 细胞系中 Rnf20 或 Isl1 的减少可减少体外 H2Bub1 和 H3K4me3 标记，从而将 Isl1:Rnf20 连接起来 b细胞表观遗传学。我们的初步小鼠敲除数据支持 Rnf20 可能充当同二聚体（即，没有Rnf40）驱动小鼠b细胞中Isl1介导的靶基因调节。因此，在本提案中我们将比较 Isl1 或 Rnf20 缺陷小鼠模型的功能和转录影响，以及存在于缺乏 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 沉积 H2Bub1 修饰。我们设计了三个具体目标将检查 Isl1 和 Rnf20（以及 H2Bub1）在胚胎和成年小鼠 B 细胞，以及成人 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 的基本基因调控效应子，从而影响小鼠和人类 B 细胞功能。学到的概念将为小说的发展带来新的见解糖尿病药物或增强体外生产治疗性 B 样细胞的策略。