Rev-Erbs repress macrophage gene expression by inhibiting enhancer-directed transcription. Mus musculus

Rev-Erba and Rev-Erbb are nuclear receptors that regulate the expression of genes involved in the control of circadian rhythm, metabolism, and inflammatory responses. Rev-Erbs function as transcriptional repressors by recruiting NCoR/HDAC3 co-repressor complexes to Rev-Erb response elements in enhancers and promoters of target genes, but the molecular basis for cell-specific programs of repression is not known. Here, we present evidence that in macrophages, Rev-Erbs regulate target gene expression by inhibiting the functions of distal enhancers that are selected by macrophage lineage-determining factors, thereby establishing a macrophage-specific program of repression. Remarkably, the repressive functions of Rev-Erbs are associated with their ability to inhibit the transcription of enhancer-derived RNAs (eRNAs). Furthermore, targeted degradation of eRNAs at two enhancers subject to negative regulation by Rev-Erbs resulted in reduced expression of nearby mRNAs, implying a direct role of these eRNAs in enhancer function. By precisely defining eRNA start sites using a method that quantifies nascent 5' ends (5'-GRO-Seq), we show that transfer of full enhancer activity to a target promoter requires both the sequences mediating transcription factor binding and the specific sequences encoding the eRNA transcript. These studies provide evidence for direct roles of eRNAs in contributing to enhancer functions and suggest that Rev-Erbs act to suppress gene expression at a distance by repressing eRNA transcription. Overall design: Using ChIPseq, GRO-seq, and 5'GRO-seq to determine mechanism of RevErb in transcriptional regulation in macrophages

Rev-Erbα（Rev-Erba）与Rev-Erbβ（Rev-Erbb）是核受体（nuclear receptors），可调控参与昼夜节律、代谢及炎症应答调控的基因的表达。Rev-Erb家族受体可通过招募NCoR/HDAC3共阻遏复合物（co-repressor complexes），结合靶基因增强子（enhancers）与启动子（promoters）中的Rev-Erb应答元件（Rev-Erb response elements），充当转录阻遏物（transcriptional repressors），但目前尚不明确细胞特异性阻遏程序的分子基础。本研究提供证据表明，在巨噬细胞（macrophages）中，Rev-Erb受体通过抑制由巨噬细胞谱系决定因子（macrophage lineage-determining factors）选定的远端增强子的功能，调控靶基因的表达，进而建立巨噬细胞特异性的阻遏程序。值得注意的是，Rev-Erb受体的阻遏功能与其抑制增强子衍生RNA（enhancer-derived RNAs, eRNAs）转录的能力密切相关。此外，在两个受Rev-Erb负调控的增强子处进行eRNA的靶向降解，会导致邻近mRNA的表达水平降低，这暗示这些eRNAs在增强子功能中发挥直接作用。通过使用一种可量化新生5'端（nascent 5' ends）的方法（5'-GRO-Seq）精准确定eRNA的起始位点，本研究证实：将完整的增强子活性转移至靶启动子，同时需要介导转录因子结合的序列，以及编码eRNA转录本的特定序列。本研究为eRNAs在增强子功能中的直接作用提供了证据，并表明Rev-Erb受体可通过抑制eRNA转录，在远端发挥抑制基因表达的作用。实验整体设计：采用染色质免疫沉淀测序（ChIP-seq）、全局运行-on测序（GRO-seq）以及5'端全局运行-on测序（5'-GRO-seq），解析Rev-Erb在巨噬细胞中转录调控的分子机制。