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

摘要 胰岛内分泌胰岛素的b细胞是葡萄糖绝对必需的 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，预计人数将大幅增加。这创造了巨大的 经济和医疗负担。糖尿病治疗的改进需要更深入的了解 新的小鼠和人类B细胞调节剂和/或直接参与B细胞功能的途径，包括 葡萄糖刺激的胰岛素分泌（GSIS）。 LIM-同源结构域类转录因子Islet-1（Isl 1）是胰岛富集的调节因子， 胰岛细胞发育、成熟和功能。尽管如此，人们对转录的 Isl 1在小鼠或人B细胞中引发这些功能的机制或蛋白质相互作用物。到 为了鉴定Isl 1转录复合物的成分，我们进行了反向交联免疫沉淀 和使用小鼠B细胞的质谱实验。我们发现了小说Isl 1互动演员无名指 （Rnf）20和Rnf 40，它们是E3泛素连接酶，其作为同源或异源二聚体复合物起作用，以促进 通过特异性单泛素化组蛋白H2 B（H2 Bub 1）转录，活性组蛋白3赖氨酸4的前体 三甲基化（H34 me 3）。用b细胞系进行的体外试验表明，（至少）Rnf 20是细胞增殖所必需的。 许多Isl 1靶基因的表达（例如，Glut 2、MafA、Ins 1）以及GSIS。引人注目的是，我们还发现， b细胞系中Rnf 20或Isl 1的减少在体外减少了H2 Bub 1和H3 K4 me 3标记，从而将Isl 1：Rnf 20连接起来 B细胞表观遗传学我们初步的小鼠敲除数据支持Rnf 20可以作为同源二聚体（即， 不含Rnf 40）来驱动小鼠b细胞中Isl 1介导的靶基因调控。因此，在本提案中， 将比较Isl 1或Rnf 20缺陷小鼠模型的功能和转录影响，以及 在缺乏ISL 1、RNF 20或RNF 40的人B细胞中。我们的中心假设是b细胞的形成和功能 需要通过募集的Isl 1：Rnf 20沉积H2 Bub 1修饰。我们设计了三个具体目标， 将检查Isl 1和Rnf 20（以及H2 Bub 1）在 胚胎和成年小鼠B细胞，以及成年人B细胞。这些研究报告的结果将 确立Rnf 20和H2 Bub 1表观遗传标记为Isl 1的基本基因调控效应物， 影响小鼠和人类B细胞功能。所学到的概念将对小说的发展产生新的见解 糖尿病药物或增强策略以在体外产生治疗性B样细胞。

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.