Transcriptional Regulation of the Insulin Gene in Health and Disease

描述(由申请人提供)： 胰岛素在细胞中的产生主要受转录水平的控制，这一过程是通过细胞中特异表达的转录因子的作用和胰岛素基因染色质结构的组蛋白修饰来调节的。该项目的总体目标将是进一步研究胰岛素基因激活的过程，并确定染色质结构对葡萄糖等营养物质的响应发生的急性和慢性变化。我假设胰岛素基因的转录在一定程度上是通过染色质的变化来调节的，染色质的变化是由PDX-1介导的，PDX-1是同源盒转录因子类的成员。我进一步假设，在疾病状态下，如糖尿病，染色质水平存在缺陷，从而改变基因表达模式。在这项建议的目标1中，我将通过对整个动物和分离的人胰岛进行PDX-1缺失研究，来表征PDX-1在葡萄糖刺激的胰岛素基因反式激活中的作用。在这个目标中，我将确定PDX-1如何在功能上将细胞外葡萄糖水平与胰岛素转录、组蛋白修饰和代谢控制联系起来。目的2将描述PDX-1与细胞内基本转录机制的新成分的相互作用，首先集中在PDX-1与与RNA聚合酶II相关的蛋白质Paf1的相互作用。其他新的相互作用蛋白将通过MALDI-TOF质谱仪鉴定，并将通过哺乳动物双杂交实验验证相互作用。这些相互作用在细胞中的生物学相关性将使用siRNA和染色质免疫沉淀分析来测试。在目标3中，我将使用染色质分析技术，如微球菌消化试验和染色质免疫沉淀，表征高胰岛素和低胰岛素2型糖尿病啮齿动物模型和长期暴露在高糖环境中的人胰岛的组蛋白修饰和更新缺陷。相关：糖尿病(DM)是一种代谢性疾病，由激素胰岛素的完全或相对缺乏引起。糖尿病目前在美国影响着2080万人，而且发病率还在增加。治疗这种疾病的费用是巨大的，每年超过1000亿美元。在这项建议中，将在编码胰岛素的基因水平上探索胰腺产生胰岛素激素的确切机制。这种类型的研究有可能指导糖尿病新疗法的开发。

DESCRIPTION (provided by applicant): Insulin production in the ¿ cell is controlled primarily at the level of transcription, a process which is regulated through the action of transcription factors specifically expressed in the ¿ cell and via histone modification of insulin gene chromatin structure. The overall goal of this project will be to further investigate the process whereby insulin gene activation occurs and to define acute and chronic changes that occur in chromatin structure in response to nutrients such as glucose. I hypothesize that transcription of the insulin gene is regulated, in part, through changes in chromatin, which are mediated by Pdx-1, a member of the homeobox class of transcription factors. I further hypothesize that in states of disease, like diabetes mellitus, there are defects at the chromatin level that alter gene expression patterns. In Aim 1 of this proposal, I will characterize the role of Pdx-1 in glucose-stimulated insulin gene transactivation, using Pdx-1 deletion studies in whole animals and isolated human islets. In this aim, I will establish how Pdx-1 functionally links extracellular glucose levels to insulin transcription, histone modifications, and metabolic control. Aim 2 will characterize the interactions of Pdx-1 with novel components of the basal transcriptional machinery in the ¿ cell, concentrating initially on the interaction of Pdx-1 with Paf1, a protein that associates with RNA polymerase II. Other novel interacting proteins will be identified using MALDI-TOF mass spectrometry, and interactions will be verified by mammalian two-hybrid assay. The biological relevance of these interactions in the ¿ cell will be tested using siRNA and chromatin immunoprecipitation assays. In Aim 3, I will characterize defects in histone modification and turnover in hyperinsulinemic and hypoinsulinemic rodent models of Type 2 diabetes and human islets chronically exposed to high glucose, using chromatin analysis techniques, such as the micrococcal digest assay and chromatin immunoprecipitation. Relevance: Diabetes mellitus (DM) is a metabolic disease that results from either a complete or relative deficiency of the hormone insulin. DM currently affects 20.8 million people in the U.S. and is increasing in incidence. The cost of caring for this disease is enormous and exceeds over 100 billion dollars annually. In this proposal, the precise mechanisms by which the pancreas produces the hormone insulin will be explored at the level of the gene encoding insulin. Research of this type has the potential to guide the development of new therapies for DM.