Transcriptional Regulation of the Insulin Gene in Health and Disease

描述（由申请人提供）： 胰岛素的生产细胞主要在转录水平上受到控制，这是一个通过细胞中特异性表达的转录因子的作用和通过胰岛素基因染色质结构的组蛋白修饰来调节的过程。该项目的总体目标是进一步研究胰岛素基因激活发生的过程，并确定响应营养物质（如葡萄糖）的染色质结构发生的急性和慢性变化。我假设胰岛素基因的转录是受调控的，部分是通过染色质的变化，这是由Pdx-1，转录因子的同源异型盒类的成员介导的。我进一步假设，在疾病状态下，如糖尿病，在染色质水平上存在改变基因表达模式的缺陷。在本提案的目标1中，我将使用完整动物和分离的人胰岛中的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，我将建立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与细胞中基础转录机制的新组分的相互作用，最初集中在Pdx-1与Paf 1的相互作用上，Paf 1是一种与RNA聚合酶II相关的蛋白质。其他新的相互作用蛋白将使用MALDI-TOF质谱法进行鉴定，并通过哺乳动物双杂交试验验证相互作用。将使用siRNA和染色质免疫沉淀测定来测试这些相互作用在细胞中的生物学相关性。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2型糖尿病和人类胰岛长期暴露于高糖的高胰岛素血症和低胰岛素血症啮齿动物模型中组蛋白修饰和周转的缺陷。相关性：糖尿病（DM）是一种代谢性疾病，由胰岛素激素的完全或相对缺乏引起。糖尿病目前影响着美国2080万人，发病率正在上升。治疗这种疾病的费用是巨大的，每年超过1000亿美元。在这个提议中，胰腺产生胰岛素的精确机制将在编码胰岛素的基因水平上进行探索。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.