Transcriptional enhancers orchestrate cell-type-specific gene expression programs, but how they convey signal-activated transcriptional responses remains poorly understood. Here, the cell-lineage-specific transcription factor FoxA1 is shown to both facilitate and restrict the androgen receptor to act on distinct classes of enhancers. Lowered levels of FoxA1, such as those found in prostate cancer, can reprogram the hormonal response by causing a switch in androgen receptor binding to a set of pre-established, functional enhancers that are marked by enhancer-derived non-coding RNAs (eRNAs). This work points to the existence of a large repository of active enhancers that can be tuned to allow alternative gene expression programs in normal cell development and in disease progression. Mammalian genomes are populated with thousands of transcriptional enhancers that orchestrate cell-type-specific gene expression programs1,2,3,4, but how those enhancers are exploited to institute alternative, signal-dependent transcriptional responses remains poorly understood. Here we present evidence that cell-lineage-specific factors, such as FoxA1, can simultaneously facilitate and restrict key regulated transcription factors, exemplified by the androgen receptor (AR), to act on structurally and functionally distinct classes of enhancer. Consequently, FoxA1 downregulation, an unfavourable prognostic sign in certain advanced prostate tumours, triggers dramatic reprogramming of the hormonal response by causing a massive switch in AR binding to a distinct cohort of pre-established enhancers. These enhancers are functional, as evidenced by the production of enhancer-templated non-coding RNA (eRNA5) based on global nuclear run-on sequencing (GRO-seq) analysis6, with a unique class apparently requiring no nucleosome remodelling to induce specific enhancer–promoter looping and gene activation. GRO-seq data also suggest that liganded AR induces both transcription initiation and elongation. Together, these findings reveal a large repository of active enhancers that can be dynamically tuned to elicit alternative gene expression programs, which may underlie many sequential gene expression events in development, cell differentiation and disease progression.

Genome-wide studies have revealed that mammalian genomes are pervasively transcribed. This has led to the identification and isolation of novel classes of non-coding RNAs (ncRNAs) that influence gene expression by a variety of mechanisms. Here we review the characteristics and functions of regulatory ncRNAs in chromatin remodelling and at multiple levels of transcriptional and post-transcriptional regulation. We also describe the potential roles of ncRNAs in vascular biology and in mediating epigenetic modifications that might play roles in cardiovascular disease susceptibility. The emerging recognition of the diverse functions of ncRNAs in regulation of gene expression suggests that they may represent new targets for therapeutic intervention.

Rationale: Atherosclerotic lesions are known for their cellular heterogeneity, yet the molecular complexity within the cells of human plaques has not been fully assessed. Objective: Using single-cell transcriptomics and chromatin accessibility, we gained a better understanding of the pathophysiology underlying human atherosclerosis. Methods and Results: We performed single-cell RNA and single-cell ATAC sequencing on human carotid atherosclerotic plaques to define the cells at play and determine their transcriptomic and epigenomic characteristics. We identified 14 distinct cell populations including endothelial cells, smooth muscle cells, mast cells, B cells, myeloid cells, and T cells and identified multiple cellular activation states and suggested cellular interconversions. Within the endothelial cell population, we defined subsets with angiogenic capacity plus clear signs of endothelial to mesenchymal transition. CD4 ⁺ and CD8 ⁺ T cells showed activation-based subclasses, each with a gradual decline from a cytotoxic to a more quiescent phenotype. Myeloid cells included 2 populations of proinflammatory macrophages showing IL (interleukin) 1B or TNF (tumor necrosis factor) expression as well as a foam cell-like population expressing TREM2 (triggering receptor expressed on myeloid cells 2) and displaying a fibrosis-promoting phenotype. ATACseq data identified specific transcription factors associated with the myeloid subpopulation and T cell cytokine profiles underlying mutual activation between both cell types. Finally, cardiovascular disease susceptibility genes identified using public genome-wide association studies data were particularly enriched in lesional macrophages, endothelial, and smooth muscle cells. Conclusions: This study provides a transcriptome-based cellular landscape of human atherosclerotic plaques and highlights cellular plasticity and intercellular communication at the site of disease. This detailed definition of cell communities at play in atherosclerosis will facilitate cell-based mapping of novel interventional targets with direct functional relevance for the treatment of human disease.

It is unclear whether bidirectional non-coding RNAs transcribed from enhancer elements (eRNAs) have any functional role; here, the repressive functions of Rev-Erb nuclear receptors in macrophages are shown to be linked to their ability to inhibit the transcription of eRNAs. Bidirectional non-coding RNAs are transcribed from enhancer elements, but it is unclear whether these enhancer-derived RNAs (eRNAs) have a functional role or are merely a reflection of enhancer activity. Two manuscripts in this issue of Nature examine this question in the context of the positive and negative transcriptional functions of different nuclear receptors. Wenbo Li et al. provide evidence for the functional importance of eRNA transcription during the activation of genes by the oestrogen receptor in breast cancer cell lines; and Michael Lam et al. show that the repressive functions of Rev-Erb nuclear receptors in macrophages are linked to their ability to inhibit the transcription of eRNAs. Taken together these studies provide evidence for a role for eRNAs in contributing to enhancer functions. Rev-Erb-α and Rev-Erb-β are nuclear receptors that regulate the expression of genes involved in the control of circadian rhythm1,2, metabolism3,4 and inflammatory responses5. Rev-Erbs function as transcriptional repressors by recruiting nuclear receptor co-repressor (NCoR)–HDAC3 complexes to Rev-Erb response elements in enhancers and promoters of target genes6,7,8, but the molecular basis for cell-specific programs of repression is not known. Here we present evidence that in mouse 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 messenger RNAs, suggesting a direct role of these eRNAs in enhancer function. By precisely defining eRNA start sites using a modified form of global run-on sequencing that quantifies nascent 5′ ends, 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 a direct role of eRNAs in contributing to enhancer functions and suggest that Rev-Erbs act to suppress gene expression at a distance by repressing eRNA transcription.