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 states. IR的分子基础是复杂的，并且与许多交织的途径相关。我们有 着重于IR的核机制，包括转录因子（TF）和 表观基因组修饰物。在之前的资助周期中，我们已经确定了几种细胞转录调控因子， 和组织IR使用先进的表观基因组策略。在下一个周期，我们建议转向研究 特别是，识别驱动IR发展的转录机制， 人类脂肪细胞为此，我们已经生成了来自以下细胞的原代脂肪细胞的染色质状态图： 胰岛素抵抗和敏感的受试者，导致鉴定了数千种具有差异的增强子。 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靶基因联系起来， 然后使用CRISPRi验证这些预测中的一些。接下来，我们将评估这些增强剂中的哪一种 和基因显示等位基因不平衡的证据，从而暗示了它们差异富集的遗传基础 并预测导致这种效应的SNP。一个大规模平行的报告分析将进一步暗示 单个SNP和TF基序作为IR驱动因子的候选者，从而能够预测上游 结合并激活增强子的调节子。最后，我们将使用碱基编辑验证因果SNP， 培养的脂肪细胞测试它们对靶基因表达和胰岛素敏感性的影响。我们还将验证 使用功能获得和功能丧失方法的组合在体外和体内筛选候选上游调节子。 vivo.该提案的关键交付将是详细的转录机制的阐明 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.