Abstract
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.
Highlights
TO ORCHESTRATE the precise gene expression patterns that give rise to the phenotypic and functional diversity of complex biological systems, mammalian genomes utilize millions of regulatory elements known as enhancers
We show that activityinduced expression of enhancer RNAs (eRNAs) from enhancers is significantly correlated with expression from nearby activity-responsive genes across the genome, that Fos eRNAs are localized to distinct loci within the nucleus, and that eRNAs from a distal Fos enhancer are both necessary and sufficient for activityregulated induction of Fos mRNA
Of 12,924 transcribed intergenic regions (TIRs), 3,107 regions overlapped H3K4me1 peaks (a mark of active enhancers (Li et al, 2016)) or overlapped both H3K4me3 and H4K27ac peaks (marks often used to denote poised enhancers (Li et al, 2016)). These transcribed regions, which we designated as transcriptionally active enhancers (TAEs; Fig. 1A-B), exhibited increased levels of non-polyA RNA expression as compared to non-enhancer TIRs, and were enriched for RNA Polymerase II (RNAP2) and the enhancer-linked chromatin looping factor CTCF (CCCTC-binding factor) (Fig. 1B)
Summary
To map neuronal eRNAs across the rat genome, we took advantage of the fact that eRNAs are predominantly non-polyadenylated transcripts. To gain insight into the spatial distribution of gene are bidirectionally modulated by neuronal activity eRNAs and their response to stimulation, we performed states To further explore this relationship, we performed a single molecule fluorescent in situ hybridization (smFISH), KCl stimulation time-course experiment in which cultured a technique that allows visualization of individual eRNA neurons were depolarized with KCl and RNA was isolated and mRNA transcripts on a single cell level. Using this tool, from neurons at multiple time points (15, 30, 45, and 60 min) we investigated whether the number or localization of our after treatment. We designed custom probe sets to selectively target and mark individual Gapdh and Fos mRNA transcripts, as well
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