Molecular Mechanisms of Insulin Resistance Associated Loci

 描述(由申请人提供) 2型糖尿病(T2D)是胰岛素敏感性受损(胰岛素抵抗，IR)和胰岛素分泌不足的不利交集。虽然在识别与T2D相关的遗传变异方面取得了显着的进展，但在定义特定的因果变异和破译因果变异与生物学之间的分子机制方面进展有限。大多数T2D变异体似乎通过影响胰岛β细胞发育或胰岛素的产生、加工或分泌发挥作用。然而，越来越多的这些变体似乎与IR有关。这些IR相关变种尤其重要，因为IR是T2D和心血管疾病的一个强大的危险因素，治疗选择非常有限。现在，了解这些与红外线有关的联系的机制对于在这一领域取得进展至关重要。我们组建了一个具有互补专业知识的独特的研究小组，以加速发现影响IR风险的因果基因和变异，并进行功能研究，以获得对这些因果基因和变异导致疾病的机制的新见解。我们的初步研究基于正交数据对IR相关变异/基因座进行了优先排序，这些数据包括我们独特的GWA2700人的结果，这些个体进行了基于“钳位”的胰岛素敏感性测量，以及IR变异的详细生理特征。在这个提案中，我们将使用方法学来识别因果变异，并全面研究这些变异调控疾病相关细胞类型，即脂肪和骨骼肌组织中因果基因表达的机制。Aim 1的研究将使用组蛋白修饰芯片-SEQ和ATAC-SEQ方法确定胰岛素敏感组织中的活性染色质区域，调查因果变体与具有等位基因特异性表达的因果基因之间的局部相互作用，并利用CHIA-PET在整个基因组结构的背景下确定因果远程相互作用。在目标2中，体外报告基因研究将通过等位基因特异性的转染报告基因研究和染色质免疫沉淀转录因子结合试验来研究原因变异和基因相互作用的特定机制。在目标3中，通过这些研究确定的转录元件的增强子活性，以及这种增强子活性的因果变异修饰的相关性，将通过在相关细胞系中进行基因组编辑，并在体内利用具有以下特性的转基因小鼠进行评估 记者构建了专门对接到小鼠基因组中的结构。综上所述，建议的研究将建立在GWAS发现的基础上，以确定IR相关基因中的因果变异和基因，这些变异和基因从根本上与胰岛素抵抗有关，这将促进开发工具 风险预测和疾病的早期治疗，并可能确定新的治疗目标。

 DESCRIPTION (provided by applicant) Type 2 diabetes (T2D) results from the unfavorable intersection of impaired insulin sensitivity (insulin resistance, IR) and inadequate insulin secretion. While there has been phenomenal progress in identifying T2D associated genetic variants, there has been limited progress towards defining the specific causal variants and deciphering the molecular mechanisms between causal variation and biology. Most T2D variants appear to be acting through effects on pancreatic beta cell development or insulin production, processing or secretion. However, a growing number of these variants appear to relate to IR. These IR-associated variants are particularly important as IR is a powerful risk factor for T2D and cardiovascular disease with very limited therapeutic options. Understanding the mechanisms for these IR-related associations is now critical for progress in the field. We have assembled a unique group of investigators with complementary expertise both to accelerate the discovery of causal genes and variants that influence the risk for IR and to conduct functional studies to gain novel insight into the mechanisms by which these causal genes and variants contribute to disease. Our preliminary studies have prioritized IR related variants/loci based on orthogonal data including results from our unique GWAS of over 2,700 individuals with "clamp" based measures of insulin sensitivity as well as detailed physiologic characterization of IR variants. In this proposal, we wll employ methodology to identify causal variation and comprehensively investigate the mechanisms by which these variants modulate expression of causal genes in disease relevant cell types, i.e. adipose and skeletal muscle tissue. Studies in Aim 1 will determine regions of active chromatin in insulin sensitive tissues with histone modification ChIP-Seq and ATAC-Seq methods, investigate local interactions between the causal variant and causal gene with allele- specific expression, and identify causal long-range interactions in the context of whole genome architecture with ChIA-PET. In Aim 2, in vitro reporter gene studies will investigate the specific mechanisms for the causal variant and gene interactions as assayed by allele-specific transfection reporter gene studies and chromatin immunoprecipitation transcription factor binding assays. In Aim 3, the enhancer activity of transcriptional elements identified through these studies, and the relevance of causal variant modification of this enhancer activity, will be evaluated through genome editing in relevant cell lines, and in vivo utilizing transgenic mice with reporter constructs specifically docked into the mouse genome. Taken together, studies proposed will build on GWAS findings to identify causal variants and genes in IR associated loci that are fundamentally linked to insulin resistance, which will facilitate development of tools for risk prediction and early treatment of disease and potentially identify new therapeutic targets.