Mechanisms of Nkx6.1 Governing Beta-cell Differentiation

描述：（申请人提供）1型和2型的发病率 糖尿病在美国正上升到令人担忧的程度。与 近年来分子生物学和基因治疗的重大进展， 治疗糖尿病的方法来自于对胰腺B细胞的研究， 发展一种非常有前途的方法是迫使前体细胞类型 通过使用“基因调节剂”发育成胰岛素产生细胞，或 转录因子我们实验室的长期目标是 理解转录因子指导转录的机制。 胰岛内产生胰岛素的β细胞的分化 朗格汉斯的 我们的策略是专注于Nkx 6。1，转录因子 控制着B细胞分化的最后一步靶向破坏 NKX6。1导致胚胎缺乏B细胞，在胚胎发育过程中没有变化。 其他细胞类型，包括胰岛。 基于我们的初步数据，我们假设转录功能 关于NKX 6 1是特异性调节的，并控制13-细胞分化 该因子经历一系列分子内结构调整， 分子间蛋白质相互作用，用于调节其DNA结合， 反式激活功能因此，我们的具体目标是 Nkx 6的DNA结合和反式激活特性的系统分析。 1，为了形成这种因素如何在分子水平上发挥作用的模型 导致B细胞的最终分化 1.确定Nkx 6的羧基末端。I调节DNA结合， 序列识别 2.确定转录抑制和DNA结合活性是如何 Nkx6.1通过蛋白质-蛋白质相互作用进行修饰。 3.测定结合的靶基因的体内和体外分布 NKX6。1. 为了实现这些目标，我们将利用体外和体内测定蛋白质和 DNA相互作用，13细胞的细胞培养模型和染色质 免疫沉淀测定。这里提出的研究将提供 框架，以了解分化的分子事件， B细胞谱系，并最终可以应用于工程新的B细胞 对于糖尿病患者。.

DESCRIPTION: (Provided By Applicant) The incidence of both Type 1 and Type 2 diabetes mellitus is rising to alarming rates in the United States. With significant advances in molecular biology and gene therapy in recent years, new approaches for therapy of diabetes are coming from studies of pancreatic B-cell development. One very promising approach is to force precursor cell types to develop into insulin-producing cells by use of "gene regulators," or transcription factors. The long range objective of our laboratory is to understand the mechanisms by which transcription factors direct the differentiation of the insulin-producing 13-cells within the pancreatic islets of Langerhans. Our strategy for this proposal is to focus on Nkx6. 1, a transcription factor that controls the final step of B-cell differentiation. Targeted disruption of Nkx6. 1 in mice leads to embryos that lack B-cells, with no changes in the other cell types that comprise the islets of Langerhans. Based on our preliminary data, we hypothesize that the transcriptional function of Nkx6. 1 is specifically regulated, and to control 13-cell differentiation this factor undergoes a series of intramolecular structural adjustments and intermolecular protein interactions that serves to modulate its DNA binding and transactivation functions. Our specific aims are therefore directed toward a systematic analysis of the DNA binding and transactivation properties of Nkx6. 1, in order to form a model of how this factor functions at the molecular level to cause the final differentiation of B-cells. 1. Determine how the carboxyl terminus of Nkx6. I modulates DNA binding and sequence recognition. 2. Determine how the transcriptional repression and DNA binding activities of Nkx6.1 are modified by protein-protein interactions. 3. Determine the in vivo and in vitro distribution of target genes bound by Nkx6. 1. To achieve these aims, we will utilize in vitro and in vivo assays protein and DNA interactions, cell culture models of 13-cells, and chromatin immunoprecipitation assays. The studies proposed here will provide the framework for understanding the molecular events governing differentiation in the B-cell lineage, and can eventually be applied to engineering new B-cells for patients with diabetes. .