Genome-wide binding of the orphan nuclear receptor TR4 suggests its general role in fundamental biological processes. Homo sapiens

Background: The orphan nuclear receptor TR4 (human testicular receptor 4 or NR2C2) plays a pivotal role in a variety of biological and metabolic processes. With no known ligand and few known target genes, the mode of TR4 function was unclear. Results: We report the first genome-wide identification and characterization of TR4 in vivo binding. Using chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq), we identified TR4 binding sites in 4 different human cell types and found that the majority of target genes were shared among different cells. TR4 target genes are involved in fundamental biological processes such as RNA metabolism and protein translation. In addition, we found that a subset of TR4 target genes exerts cell-type specific functions. Analysis of the TR4 binding sites revealed that less than 30% of the peaks from any of the cell types contained the DR1 motif previously derived from in vitro studies, suggesting that TR4 may be recruited to the genome via interaction with other proteins. A bioinformatics analysis of the TR4 binding sites predicted a cis regulatory module involving TR4 and ETS transcription factors. To test this prediction, we performed ChIP-seq for the ETS factor ELK4 and found that 30% of TR4 binding sites were also bound by ELK4. Motif analysis of the sites bound by both factors revealed a lack of the DR1 element, suggesting that TR4 binding at a subset of sites is facilitated through the ETS transcription factor ELK4. Further studies will be required to investigate the functional interdependence of these two factors. Conclusions: Our data suggest that TR4 plays a pivotal role in fundamental biological processes across different cell types. In addition, the identification of cell type specific TR4 binding sites enables future studies of the pathways underlying TR4 action and its possible role in metabolic diseases. Overall design: This is one part of the study that involved the examination of HepG2 gene expression. Biological triplicate Illumina Sentrex bead arrays.

背景：孤儿核受体TR4（人睾丸受体4，亦称NR2C2）在多种生物学与代谢过程中发挥关键调控作用。由于其尚未发现天然配体且已知靶基因数量极少，此前TR4的作用机制始终不明。结果：本研究首次完成了TR4体内结合位点的全基因组鉴定与特征解析。采用染色质免疫共沉淀结合高通量测序（chromatin immunoprecipitation followed by high throughput sequencing, ChIP-seq）技术，我们在4种不同的人类细胞系中鉴定出TR4的结合位点，并发现绝大多数靶基因在不同细胞间共享。TR4靶基因参与RNA代谢、蛋白质翻译等核心生物学过程。此外，本研究还发现部分TR4靶基因具备细胞类型特异性的功能。对TR4结合位点的分析显示，所有细胞系中仅不到30%的测序峰包含此前体外研究鉴定得到的DR1基序，这提示TR4可能通过与其他蛋白质相互作用被招募至基因组。对TR4结合位点的生物信息学分析预测，存在一个包含TR4与ETS转录因子（ETS transcription factors）的顺式调控模块。为验证该预测，我们针对ETS家族转录因子ELK4开展了ChIP-seq实验，结果显示30%的TR4结合位点同时被ELK4结合。对两种因子共同结合的位点进行基序分析后发现，该类位点缺乏DR1元件，这表明部分位点的TR4结合是通过ETS转录因子ELK4介导实现的。后续仍需开展深入研究以阐明这两种因子的功能互作关系。结论：本研究数据表明，TR4在不同细胞类型的核心生物学过程中均发挥关键调控作用。此外，细胞类型特异性TR4结合位点的鉴定，为后续探究TR4的作用通路及其在代谢疾病中的潜在功能提供了重要基础。整体实验设计：本研究的该部分内容旨在检测HepG2细胞的基因表达，实验采用3次生物学重复的Illumina Sentrex微球芯片技术。