Functional follow-up of the association between TCF7L2 and T2D

描述（由申请人提供）：本提案旨在从功能上确定Tcf 7 l2对葡萄糖代谢的作用。全基因组关联研究（GWAS）一直认为TCF 7 L2基因座内的非编码变异与2型糖尿病（T2 D）的风险有关。虽然这代表了人类T2 D风险的最强遗传决定因素，但目前尚不清楚这些非编码变体如何影响疾病病因。我们推测，这些相关的非编码区含有改变控制TCF 7 L2表达的长程顺式调控元件的活性的变体，将介导疾病风险的增加。在过去的3年里，该应用程序的PI已经产生了大量的资源和数据来支持这一想法。我们发现，与T2 D相关的基因组间隔含有调节TCF 7 L2时空表达模式的长距离增强子，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Tcf 7 l2表达的变化是否导致葡萄糖稳态改变，我们在小鼠中开发了Tcf 7 l2拷贝数等位基因系列。我们表明，无效Tcf 7 l2等位基因（敲除小鼠）导致，以剂量依赖性的方式，更好的葡萄糖耐量。相反，携带额外Tcf 7 l2拷贝（基因表达增加）的转基因小鼠显示相反的表型，包括葡萄糖耐受不良。我们还显示了这些小鼠的代谢表型主要是由胰腺外组织介导的。这些结果直接证明Tcf 712是葡萄糖耐量的关键调节剂，并突出了其在外周组织中作为人类T2 D风险的介导物的作用。在本申请中，我们提出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、基于体外细胞和基于体内小鼠的方法来解决突出的问题：（i）Tcf 7 l2调节体内葡萄糖耐量的机制是什么？(ii)哪些组织参与Tcf 7 l2介导的葡萄糖耐量调节？(iii)Tcf 7 l2在这些组织中调控的分子下游靶点和途径是什么？为了解决这些问题，我们提出了两个目标。在目标1中，我们将在我们的Tcf 7 l2拷贝数小鼠组中进行详细的生理、分子和形态学评价。我们还将测试Tcf 7 l2的不同剪接异构体可能导致葡萄糖代谢表型的假设。在目标2中，我们将建立在Tcf 7 l2介导的葡萄糖代谢控制中发挥作用的组织。我们将研究Tcf 7 L2在脑/下丘脑，脂肪组织和肝脏的组织特异性消融这个基因的假定的生理作用。这些研究的结果将极大地影响我们对 T2 D的发病机制，提供了一个新的范例，GWAS与非编码变异的实验后续，并确定新的治疗途径，在组织与尚未未知的Tcf 7 l2介导的功能。

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.