Transcriptional Mechanisms of Human Insulin Resistance

摘要 胰岛素抵抗(IR)是2型糖尿病的必要条件，也是许多其他疾病的致病因素 各州。IR的分子基础是复杂的，并与许多相互交织的途径有关。我们有 重点研究IR的核机制，包括转录因子和转录因子的联合作用 表观基因组修饰物。在之前的资金周期中，我们已经确定了几个细胞转录调控因子 和使用高级表观基因组学策略的组织IR。对于即将到来的周期，我们建议转向研究 人类胰岛素抵抗；具体地说，识别驱动胰岛素抵抗发展的转录机制 人类脂肪细胞。为此，我们已经生成了原始脂肪细胞的染色质状态图 胰岛素抵抗和敏感的受试者，导致识别出数千种不同的增强剂 IR中的活动。首先，我们将使用一种新的数学模型将这些增强子与它们的目标基因联系起来，并且 然后使用CRISPRi验证其中一些预测。接下来，我们将评估这些增强剂中的哪些 而基因表现出等位基因不平衡的证据，从而暗示了它们差异浓缩的遗传基础。 并预测导致这种影响的SNPs。一项大规模平行的记者分析将进一步牵连 单个SNP和Tf基序作为IR的候选驱动因素，从而使上游预测成为可能 结合并激活增强子的调节器。最后，我们将使用中的基本编辑来验证因果SNP 培养的脂肪细胞检测其对靶基因表达和胰岛素敏感性的影响。我们还将验证 在体外和体内使用增益型和功能损失型相结合的方法的候选上游调节器 活着。这项提案的关键成果将是阐明详细的转录机制。 导致人类IR的潜在非编码变异。

ABSTRACT Insulin resistance (IR) is a sine qua non of Type 2 diabetes and a pathogenic factor in many other disease states. The molecular basis of IR is complex, and associated with many interweaving pathways. We have focused on nuclear mechanisms of IR, comprising the combined actions of transcription factors (TFs) and epigenomic modifiers. In the prior funding cycle we have identified several transcriptional regulators of cellular and tissue IR using advanced epigenomic strategies. For the upcoming cycle, we propose to shift to the study of human IR; specifically, the identification of transcriptional mechanisms that drive the development of IR in human adipocytes. Toward this end, we have generated chromatin state maps of primary adipocytes from insulin resistant and sensitive subjects, leading to the identification of thousands of enhancers with differential activity in IR. First, we will link these enhancers to their target genes using a novel mathematical model, and then validate a number of these predictions using CRISPRi. Next, we will assess which of these enhancers and genes show evidence of allelic imbalance, thus implying a genetic basis for their differential enrichment and predicting SNPs responsible for this effect. A massively parallel reporter assay will further implicate individual SNPs and TF motifs as candidates for drivers of IR, thus enabling the prediction of upstream regulators that bind and activate the enhancers. Finally, we will validate causal SNPs using base editing in cultured adipocytes testing their effects on target gene expression and insulin sensitivity. We will also validate candidate upstream regulators using a combination of gain- and loss-of-function approaches in vitro and in vivo. The key deliverable of this proposal will be the elucidation of the detailed transcriptional mechanisms underlying noncoding variation leading to human IR.