Global transcriptional activity dynamics reveal functional enhancer RNAs.
ABSTRACT: Active enhancers of the human genome generate long noncoding transcripts known as enhancer RNAs (eRNAs). How dynamic transcriptional changes of eRNAs are physically and functionally linked with target gene transcription remains unclear. To investigate the dynamic functional relationships among eRNAs and target promoters, we obtained a dense time series of GRO-seq and ChIP-seq data to generate a time-resolved enhancer activity map of a cell undergoing an innate antiviral immune response. Dynamic changes in eRNA and pre-mRNA transcription activities suggest distinct regulatory roles of enhancers. Using a criterion based on proximity and transcriptional inducibility, we identified 123 highly confident pairs of virus-inducible enhancers and their target genes. These enhancers interact with their target promoters transiently and concurrently at the peak of gene activation. Accordingly, their physical disassociation from the promoters is likely involved in post-induction repression. Functional assessments further establish that these eRNAs are necessary for full induction of the target genes and that a complement of inducible eRNAs functions together to achieve full activation. Lastly, we demonstrate the potential for eRNA-targeted transcriptional reprogramming through targeted reduction of eRNAs for a clinically relevant gene, TNFSF10, resulting in a selective control of interferon-induced apoptosis.
Project description:Enhancers are intergenic DNA elements that regulate the transcription of target genes in response to signaling pathways by interacting with promoters over large genomic distances. Recent studies have revealed that enhancers are bi-directionally transcribed into enhancer RNAs (eRNAs). Using single-molecule fluorescence in situ hybridization (smFISH), we investigated the eRNA-mediated regulation of transcription during estrogen induction in MCF-7 cells. We demonstrate that eRNAs are localized exclusively in the nucleus and are induced with similar kinetics as target mRNAs. However, eRNAs are mostly nascent at enhancers and their steady-state levels remain lower than those of their cognate mRNAs. Surprisingly, at the single-allele level, eRNAs are rarely co-expressed with their target loci, demonstrating that active gene transcription does not require the continuous transcription of eRNAs or their accumulation at enhancers. When co-expressed, sub-diffraction distance measurements between nascent mRNA and eRNA signals reveal that co-transcription of eRNAs and mRNAs rarely occurs within closed enhancer-promoter loops. Lastly, basal eRNA transcription at enhancers, but not E2-induced transcription, is maintained upon depletion of MLL1 and ER?, suggesting some degree of chromatin accessibility prior to signal-dependent activation of transcription. Together, our findings suggest that eRNA accumulation at enhancer-promoter loops is not required to sustain target gene transcription.
Project description: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. Using ChIPseq, GRO-seq, and 5'GRO-seq to determine mechanism of RevErb in transcriptional regulation in macrophages
Project description:Enhancer RNAs (eRNAs) are a class of long noncoding RNAs (lncRNA) expressed from active enhancers, whose function and action mechanism are yet to be firmly established. Here we show that eRNAs facilitate the transition of paused RNA polymerase II (RNAPII) into productive elongation by acting as a decoy for the negative elongation factor (NELF) complex upon induction of immediate early genes (IEGs) in neurons. eRNAs are synthesized prior to the culmination of target gene transcription and interact with the NELF complex. Knockdown of eRNAs expressed at neuronal enhancers impairs transient release of NELF from the specific target promoters during transcriptional activation, coinciding with a decrease in target mRNA induction. The enhancer-promoter interaction was unaffected by eRNA knockdown. Instead, chromatin looping might enable eRNAs to act locally at a specific promoter. Our findings highlight the spatiotemporally regulated action mechanism of eRNAs during early transcriptional elongation.
Project description:Rev-Erb-? and Rev-Erb-? 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 nuclear receptor co-repressor (NCoR)-HDAC3 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 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.
Project description:The bromodomain and extra-terminal motif (BET) protein BRD4 binds to acetylated histones at enhancers and promoters via its bromodomains (BDs) to regulate transcriptional elongation. In human colorectal cancer cells, we found that BRD4 was recruited to enhancers that were co-occupied by mutant p53 and supported the synthesis of enhancer-directed transcripts (eRNAs) in response to chronic immune signaling. BRD4 selectively associated with eRNAs that were produced from BRD4-bound enhancers. Using biochemical and biophysical methods, we found that BRD4 BDs function cooperatively as docking sites for eRNAs and that the BDs of BRD2, BRD3, BRDT, BRG1, and BRD7 directly interact with eRNAs. BRD4-eRNA interactions increased BRD4 binding to acetylated histones in vitro and augmented BRD4 enhancer recruitment and transcriptional cofactor activities. Our results suggest a mechanism by which eRNAs are directly involved in gene regulation by modulating enhancer interactions and transcriptional functions of BRD4.
Project description:Genomic enhancer elements regulate gene expression programs important for neuronal fate and function and are implicated in brain disease states. Enhancers undergo bidirectional transcription to generate non-coding enhancer RNAs (eRNAs). However, eRNA function remains controversial. Here, we combined Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq) and RNA-Seq datasets from three distinct neuronal culture systems in two activity states, enabling genome-wide enhancer identification and prediction of putative enhancer-gene pairs based on correlation of transcriptional output. Notably, stimulus-dependent enhancer transcription preceded mRNA induction, and CRISPR-based activation of eRNA synthesis increased mRNA at paired genes, functionally validating enhancer-gene predictions. Focusing on enhancers surrounding the Fos gene, we report that targeted eRNA manipulation bidirectionally modulates Fos mRNA, and that Fos eRNAs directly interact with the histone acetyltransferase domain of the enhancer-linked transcriptional co-activator CREB-binding protein (CBP). Together, these results highlight the unique role of eRNAs in neuronal gene regulation and demonstrate that eRNAs can be used to identify putative target genes.
Project description:Recent studies have disclosed the function of enhancer RNAs (eRNAs), which are long non-coding RNAs transcribed from gene enhancer regions, in transcriptional regulation. However, it remains unclear whether eRNAs are involved in the regulation of human heme oxygenase-1 gene (HO-1) induction. Here, we report that multiple nuclear-enriched eRNAs are transcribed from the regions adjacent to two human HO-1 enhancers (i.e. the distal E2 and proximal E1 enhancers), and some of these eRNAs are induced by the oxidative stress-causing reagent diethyl maleate (DEM). We demonstrated that the expression of one forward direction (5' to 3') eRNA transcribed from the human HO-1 E2 enhancer region (named human HO-1enhancer RNA E2-3; hereafter called eRNA E2-3) was induced by DEM in an NRF2-dependent manner in HeLa cells. Conversely, knockdown of BACH1, a repressor of HO-1 transcription, further increased DEM-inducible eRNA E2-3 transcription as well as HO-1 expression. In addition, we showed that knockdown of eRNA E2-3 selectively down-regulated DEM-induced HO-1 expression. Furthermore, eRNA E2-3 knockdown attenuated DEM-induced Pol II binding to the promoter and E2 enhancer regions of HO-1 without affecting NRF2 recruitment to the E2 enhancer. These findings indicate that eRNAE2-3 is functional and is required for HO-1 induction.
Project description:Enhancer-derived RNAs (eRNAs) are a group of RNAs transcribed by RNA polymerase II from the domain of transcription enhancers, a major type of cis-regulatory elements in the genome. The correlation between eRNA production and enhancer activity has stimulated studies on the potential role of eRNAs in transcriptional regulation. Additionally, eRNA has also served as a marker for global identification of enhancers. Here I review the brief history and fascinating properties of eRNAs.
Project description:Epigenetic and transcriptional alterations are both implicated in Huntington's disease (HD), a progressive neurodegenerative disease resulting in degeneration of striatal neurons in the brain. However, how impaired epigenetic regulation leads to transcriptional dysregulation in HD is unclear. Here, we investigated enhancer RNAs (eRNAs), a class of long non-coding RNAs transcribed from active enhancers. We found that eRNAs are expressed from many enhancers of mouse striatum and showed that a subset of those eRNAs are deregulated in HD vs control mouse striatum. Enhancer regions producing eRNAs decreased in HD mouse striatum were associated with genes involved in striatal neuron identity. Consistently, they were enriched in striatal super-enhancers. Moreover, decreased eRNA expression in HD mouse striatum correlated with down-regulation of associated genes. Additionally, a significant number of RNA Polymerase II (RNAPII) binding sites were lost within enhancers associated with decreased eRNAs in HD vs control mouse striatum. Together, this indicates that loss of RNAPII at HD mouse enhancers contributes to reduced transcription of eRNAs, resulting in down-regulation of target genes. Thus, our data support the view that eRNA dysregulation in HD striatum is a key mechanism leading to altered transcription of striatal neuron identity genes, through reduced recruitment of RNAPII at super-enhancers.
Project description:Enhancers are closely positioned with actively transcribed target genes by chromatin looping. Non-coding RNAs are often transcribed on active enhancers, referred to as eRNAs (enhancer RNAs). To explore the kinetics of enhancer-promoter looping and eRNA transcription during transcriptional activation, we induced the ?-globin locus by chemical treatment and analysed cross-linking frequency between the ?-globin gene and locus control region (LCR) and the amount of eRNAs transcribed on the LCR in a time course manner. The cross-linking frequency was increased after chemical induction but before the transcriptional activation of gene in the ?-globin locus. Transcription of eRNAs was increased in concomitant with the increase in cross-linking frequency. These results show that chromatin looping and eRNA transcription precedes the transcriptional activation of gene. Concomitant occurrence of the two events suggests functional relationship between them.