Functional follow-up of the association between TCF7L2 and T2D

描述（由申请人提供）：本提案旨在功能性地确定Tcf7l2在葡萄糖代谢中的作用。全基因组关联研究（GWAS）一致表明，TCF7L2位点的非编码变异与2型糖尿病（T2D）的风险有关。虽然这代表了人类T2D风险的最强遗传决定因素，但这些非编码变异如何影响疾病病因尚不清楚。我们假设这些相关的非编码区含有改变控制TCF7L2表达的远程顺式调控元件活性的变异，从而介导疾病风险的增加。在过去的3年里，这个应用程序的pi已经产生了大量的资源和数据来支持这个想法。我们发现，与T2D相关的基因组间隔中含有远程增强子，可以调节TCF7L2的时空表达模式，包括在葡萄糖稳态中起作用的组织中的表达。为了进一步确定Tcf7l2表达的变化是否会导致葡萄糖稳态的改变，我们在小鼠中开发了Tcf7l2拷贝数等位基因系列。我们发现，一个无效的Tcf7l2等位基因（敲除小鼠）以剂量依赖的方式导致更好的葡萄糖耐量。相反，携带额外Tcf7l2拷贝（基因表达增加）的转基因小鼠表现出相反的表型，包括葡萄糖耐受不良。我们还展示了这些小鼠的代谢表型是如何主要由胰腺外组织介导的。这些结果直接证明了Tcf7l2是葡萄糖耐量的关键调节因子，并突出了其作为人类T2D风险介质在外周组织中的作用。在这个应用中，我们建议利用我们已经开发的独特的分子和活性试剂，并使用基因组学，体外细胞和体内小鼠为基础的方法来解决悬而未决的问题：(i) Tcf7l2调节体内葡萄糖耐量的机制是什么？（ii）哪些组织参与了tcf712介导的糖耐量调节？（iii） Tcf7l2在这些组织中调控的分子下游靶点和通路是什么？为了解决这些问题，我们提出了两个目标。在Aim 1中，我们将在Tcf7l2拷贝数小鼠面板中进行详细的生理、分子和形态计量学评估。我们还将验证Tcf7l2的不同剪接异构体可能导致葡萄糖代谢表型的假设。在Aim 2中，我们将建立在Tcf7l2介导的葡萄糖代谢控制中发挥作用的组织。我们将通过组织特异性切除Tcf7l2基因来研究Tcf7l2在脑/下丘脑、脂肪组织和肝脏中可能的生理作用。这些研究的结果将显著影响我们对

DESCRIPTION (provided by applicant): This proposal aims to functionally determine the roles that Tcf7l2 play on glucose metabolism. Genome-wide association studies (GWAS) have consistently implicated non-coding variation within the TCF7L2 locus with risk of type 2 diabetes (T2D). While this represents the strongest genetic determinant for T2D risk in humans, it remains unclear how these non-coding variants affect disease etiology. We hypothesize that these associated non-coding regions harbor variants that alter the activity of long-range cis-regulatory elements controlling TCF7L2 expression would mediate the increased risk to disease. Over the past 3 years the PIs on this application have generated extensive resources and data that support this idea. We show that genomic interval associated with T2D harbors long-range enhancers regulating the temporal- spatial expression patterns of TCF7L2, including expression in tissues with a role in glucose homeostasis. To further determine whether variation in Tcf7l2 expression results in altered glucose homeostasis, we developed a Tcf7l2 copy number allelic series in mice. We show that a null Tcf7l2 allele (knockout mice) leads, in a dose- dependent manner, to better glucose tolerance. Conversely, transgenic mice harboring extra Tcf7l2 copies (with increased expression of the gene) display opposite phenotypes, including glucose intolerance. We also show how the metabolic phenotypes in these mice are mediated primarily from extra-pancreatic tissues. These results directly demonstrate that Tcf7l2 is a key regulator of glucose tolerance, and highlight its roles in peripheral tissues as mediators of T2D risk in humans. In this application, we propose to capitalize on the unique molecular and live reagents that we have developed and address outstanding questions using genomics, in vitro cellular-based and in vivo mouse-based approaches: (i) What is the mechanism by which Tcf7l2 regulates glucose tolerance in vivo?, (ii) Which tissues participate in the Tcf7l2-mediate regulation of glucose tolerance?, (iii) What are the molecular downstream targets and pathways regulated by Tcf7l2 in these tissues? To address these questions we propose 2 aims. In Aim 1 we will carry out a detailed physiological, molecular and morphometric evaluation in our Tcf7l2 copy number mouse panel. We will also test the hypothesis that different splice isoforms of Tcf7l2 may result in glucose metabolism phenotypes. In Aim 2, we will establish the tissues that play a role in Tcf7l2- mediated control of glucose metabolism. We will investigate the putative physiological roles of Tcf7l2 in brain/hypothalamus, adipose tissue, and liver by tissue-specific ablations of this gene. The results of these studies will significantly impact our understanding of T2D pathogenesis, provide a new paradigm for the experimental follow-up of GWAS with noncoding variation, and identify new therapeutic pathways in tissues with yet unknown Tcf7l2-mediated functions.