Antidiabetic Rosiglitazone Remodels the Adipocyte Transcriptome by Redistributing Transcription to PPARg-Driven Enhancers [HTS]

Rosiglitazone (rosi) is a powerful insulin sensitizer, but serious toxicities have curtailed its widespread clinical use. Rosi functions as a high-affinity ligand for PPARg, the adipocyte-predominant nuclear receptor (NR). The classic model, involving binding of ligand to the NR on DNA, explains positive regulation of gene expression, but ligand-dependent repression is not well understood. We have now addressed this issue by studying the direct effects of rosiglitazone on gene transcription, using global run-on sequencing (GRO-seq). Rosi-induced changes in gene body transcription were pronounced after 10 minutes and correlated with steady-state mRNA levels as well as with transcription at nearby enhancers (eRNAs). Upregulated eRNAs occurred almost exclusively at PPARg binding sites, to which rosi treatment recruited the coactivator MED1. By contrast, transcriptional repression by rosi involved a loss of MED1 from eRNA sites devoid of PPARg and enriched for other TFs including AP-1 factors and C/EBPs. Thus, rosi activates and represses transcription by fundamentally different mechanisms that could inform the future development of antidiabetic drugs. 3T3-L1 matuer adipocyte were treated with rosi, and nascent transcripts were measured at various time points using GRO-seq. ChIP-seq experiments for various coactivators, corepressor, and transcription factors also have been done to monitor initial occupancy or change before and after treatment.

罗格列酮（Rosiglitazone，简称rosi）是一种强效胰岛素增敏剂，但严重的毒副作用限制了其临床广泛应用。罗格列酮作为脂肪细胞优势表达的核受体（NR）——过氧化物酶体增殖物激活受体γ（PPARg）的高亲和力配体发挥功能。经典的配体结合核受体于DNA的模型可解释基因表达的正向调控，但配体依赖的转录抑制机制仍未被充分阐明。本研究通过全局延伸测序（GRO-seq）探究罗格列酮对基因转录的直接作用，以此解决这一科学问题。罗格列酮诱导的基因本体转录变化在处理10分钟后即十分显著，且与稳态mRNA水平以及邻近增强子区域的增强子RNA（eRNAs）转录水平相关。上调的增强子RNA几乎全部出现在PPARg结合位点，罗格列酮处理可在此类位点招募共激活因子MED1。与之相反，罗格列酮介导的转录抑制则涉及从不含PPARg、但富集包括激活蛋白1（AP-1）因子和CCAAT增强子结合蛋白（C/EBPs）在内的其他转录因子的增强子RNA位点中丢失共激活因子MED1。综上，罗格列酮通过两种截然不同的机制分别激活和抑制基因转录，这一发现可为未来抗糖尿病药物的开发提供理论参考。本研究以3T3-L1成熟脂肪细胞为模型，经罗格列酮处理后，利用GRO-seq在多个时间点检测新生转录本的表达水平；同时还开展了针对多种共激活因子、共抑制因子及转录因子的染色质免疫共沉淀测序（ChIP-seq）实验，以监测处理前后转录因子的初始结合状态及其丰度变化。