Chromatin occupancy of TCF7L2 in hepatocytes

TCF7L2 is one of the strongest type 2 diabetes (T2DM) candidate genes to emerge from GWAS studies, but the mechanisms by which it regulates the pathways which are important in the pathogenesis of type 2 diabetes are unknown. Previous in vitro and in vivo studies have focused on the link between TCF7L2 and insulin secretion as an explanation for the association between TCF7L2 and T2DM. However, TCF7L2 and the Wnt/ß-catenin pathway are important for metabolic zonation in the liver. This raises the interesting possibility that TCF7L2 may influence glucose homeostasis by regulating hepatic glucose production (HGP). To examine this question, we utilized the H4IIE cell as a model of HGP. Inhibition of HGP in H4IIE cells from lactate and pyruvate was highly sensitive to physiological concentrations of insulin and metformin. Silencing of TCF7L2 protein expression induced a 5-fold increase in basal HGP (P<0.0001), and this was accompanied by marked increase in the expression of several key gluconeogenic genes. FBPase, PEPCK and G6Pase mRNA were up-regulated 2.5-fold (P<0.0001), 1.4-fold (P<0.01) and 2.3-fold (P<0.0001), respectively, compared to scramble siRNA. Compared to their respective baseline values, insulin and metformin suppressed HGP equally in the scramble and TCF7L2 siRNA cells, but HGP remained elevated in TCF7L2 silenced cells due to the increased baseline HGP. Using chromatin immunoprecipitation sequencing (ChIP-Seq), we investigated the direct transcriptional targets of TCF7L2 in hepatocytes. A total of 2119 ChIP peaks were detected, of which 36% were located inside gene boundaries and, overall, a total of 65% of all binding events were within 50 Kb of a gene. De novo motif analysis revealed remarkable conservation of the long and short TCF7L2 consensus binding sites in the rat hepatocytes. Pathway analysis showed that the top two disease categories over-represented in our dataset were “non-insulin dependent diabetes” (155 genes; P = 1.63 x 10-10) and “diabetes mellitus” (245 genes; P = 7.4 x 10-12). Inspection of genes in these categories revealed that TCF7L2 directly binds to multiple genes important in the regulation of glucose metabolism in the liver, including PEPCK, FBP1, IRS1, IRS2, AKT2 ADIPOR1, PDK4 and CPT1A. Our findings suggest a novel mechanism for the regulation of HGP by TCF7L2, and provide a possible explanation for the association of TCF7L2 polymorphisms with the incidence of T2DM.TCF7L2是全基因组关联研究（Genome-Wide Association Study, GWAS）中发现的最强效2型糖尿病（type 2 diabetes, T2DM）候选基因之一，但其调控2型糖尿病发病机制中关键通路的分子机制仍未明确。既往体外及体内研究多聚焦于TCF7L2与胰岛素分泌的关联，以此解释TCF7L2与T2DM之间的相关性。然而，TCF7L2及Wnt/β-连环蛋白通路（Wnt/ß-catenin pathway）对肝脏代谢分区具有重要调控作用，这引出了一个有趣的可能性：TCF7L2或可通过调控肝葡萄糖生成（hepatic glucose production, HGP）影响机体葡萄糖稳态。为验证这一假说，本研究选用H4IIE细胞作为HGP研究模型。由乳酸及丙酮酸介导的H4IIE细胞中HGP抑制效应，对生理浓度的胰岛素及二甲双胍具有高度敏感性。沉默TCF7L2的蛋白表达可使基础HGP水平升高5倍（P<0.0001），同时伴随多个关键糖异生基因表达的显著上调。与乱序阴性对照小干扰RNA（scramble siRNA）组相比，果糖-1,6-二磷酸酶（FBPase）、磷酸烯醇式丙酮酸羧激酶（PEPCK）及葡萄糖-6-磷酸酶（G6Pase）的mRNA水平分别上调2.5倍（P<0.0001）、1.4倍（P<0.01）及2.3倍（P<0.0001）。与各自的基线值相比，胰岛素与二甲双胍可同等抑制阴性对照组及TCF7L2沉默组细胞的HGP，但由于TCF7L2沉默细胞的基础HGP水平升高，其HGP仍维持在较高水平。本研究通过染色质免疫沉淀测序（chromatin immunoprecipitation sequencing, ChIP-Seq）探究了TCF7L2在肝细胞中的直接转录靶标。共检测到2119个ChIP峰，其中36%位于基因区域内，整体而言65%的结合事件发生在距离基因50kb范围内。从头基序分析显示，大鼠肝细胞中TCF7L2的长、短共识结合位点具有显著保守性。通路分析显示，本数据集富集程度最高的两大疾病类别分别为“非胰岛素依赖型糖尿病”（155个基因；P=1.63×10^-10）与“糖尿病”（245个基因；P=7.4×10^-12）。对上述类别中的基因进行分析后发现，TCF7L2可直接结合多个调控肝脏葡萄糖代谢的关键基因，包括PEPCK、FBP1、IRS1、IRS2、AKT2、ADIPOR1、PDK4及CPT1A。本研究结果揭示了TCF7L2调控HGP的全新机制，可为解释TCF7L2基因多态性与T2DM发病风险的关联提供新的理论依据。