Characterization of chromatin and gene expression changes during fasting and refeeding in mouse liver [RNA-Seq]. Characterization of chromatin and gene expression changes during fasting and refeeding in mouse liver [RNA-Seq]

The discovery of FoxO1 as an effector of insulin action on gene expression filled a gap in our knowledge of insulin signaling. The metabolic impact of hepatic FoxO1 has been demonstrated in genetic mouse models showing that FoxO1 inhibition can reverse diabetes. However, the gamut of FoxO1 targets is unknown, due to the lack of robust genome-wide chromatin occupancy data. To elucidate the genomic architecture of the FoxO1 effect on liver metabolism, we integrated genome-wide ChIP-seq and RNA-seq. During the physiological transition from fasting to refeeding, hepatic FoxO1 translocated from the nucleus to the cytoplasm. At the same time points, cistrome analysis demonstrated that 60% of FoxO1 target sites were cleared by refeeding. RNA-seq from mice following acute liver-specific FoxO1 ablation allowed us to integrate the data in a comprehensive FoxO1 regulome. We identified four distinct classes of FoxO1 targets. Class I targets are enriched in genes regulating cell homeostasis, have FoxO1 sites clustered in promoters and do not show regulation with fasting/refeeding. Class II is comprised of canonical FoxO1 metabolic targets coordinately regulated with other fasting-inducible TFs, such as PPARA, CREB, and GR through promoters. Class III is enriched in glucose metabolic genes regulated through active enhancers, as detected by H3K4me1 and H3K27ac histone marks. Class IV is comprised of triglyceride (TG) and lipoprotein metabolism genes, regulated through intron sites, and characterized by an uneven response to fasting/refeeding regulation. Overall design: mRNA profiles of liver tissues from constitutive or acute liver specific FoxO1 knockout mice during 4h fasted or 4 hr fasted/1 hr refed. For constitutive liver-specific FoxO1 knockouts, we crossed FoxO1lox/lox and Albumin-cre transgenic mice. Acute liver-specific FoxO1 knockout mice were generated by injection of 1×1011 purified viral particles (AAV8.TBG.eGFP or AAV8.TBG.Cre) per mouse via tail vein. We collected samples on day 21 post-injection.

FoxO1作为胰岛素调控基因表达的效应因子的发现，填补了我们在胰岛素信号通路认知中的一处空白。肝脏FoxO1的代谢调控作用已在基因工程小鼠模型中得到验证，该研究显示FoxO1抑制可逆转糖尿病。然而，由于缺乏可靠的全基因组染色质结合数据，FoxO1的全部靶基因范围仍未明确。为阐明FoxO1调控肝脏代谢的基因组调控机制，本研究整合了全基因组染色质免疫共沉淀测序（ChIP-seq）与RNA测序（RNA-seq）数据。在禁食到再喂食的生理转换过程中，肝脏FoxO1会从细胞核转位至细胞质。在相同时间节点，顺式调控组（cistrome）分析显示，60%的FoxO1结合位点会在再喂食过程中消失。对急性肝脏特异性FoxO1敲除小鼠进行RNA测序，使我们能够整合数据构建完整的FoxO1调控组。本研究共鉴定出四类不同的FoxO1靶基因：I类靶基因富集于调控细胞稳态的基因，其FoxO1结合位点聚集在启动子区域，且不受禁食/再喂食过程调控；II类靶基因为经典的FoxO1代谢调控靶点，通过启动子区域与其他禁食诱导型转录因子（Transcription Factor，TF），如过氧化物酶体增殖物激活受体α（PPARA）、环腺苷酸应答元件结合蛋白（CREB）及糖皮质激素受体（GR）协同调控；III类靶基因富集于糖代谢相关基因，其调控依赖于通过组蛋白修饰标记H3K4me1与H3K27ac鉴定出的活性增强子；IV类靶基因包含甘油三酯（TG）与脂蛋白代谢相关基因，通过内含子区域进行调控，其对禁食/再喂食调控的响应并不均一。

实验整体设计：分别采集持续禁食4小时，或禁食4小时/再喂食1小时的组成型肝脏特异性FoxO1敲除小鼠与急性肝脏特异性FoxO1敲除小鼠的肝脏组织，进行mRNA表达谱分析。组成型肝脏特异性FoxO1敲除小鼠通过将FoxO1lox/lox小鼠与Albumin-cre转基因小鼠杂交获得。急性肝脏特异性FoxO1敲除小鼠则通过尾静脉注射每只小鼠1×10^11个纯化病毒颗粒（腺相关病毒AAV8.TBG.eGFP或AAV8.TBG.Cre）构建得到。本研究在病毒注射后第21天采集组织样本。