Abstract
BackgroundEnhancers are distal cis-regulatory elements that control gene expression. Despite an increasing appreciation of the importance of enhancers in cellular function and disease, our knowledge of enhancer-regulated transcription is very limited. Nascent RNA sequencing technologies, such as global nuclear run-on sequencing (GRO-seq) and precision run-on sequencing (PRO-seq), not only provide a direct and reliable measurement of enhancer activity, but also allow for quantifying transcription of enhancers and target genes simultaneously, making these technologies extremely useful for exploring enhancer-mediated regulation.ResultsNascent RNA sequencing analysis (NRSA) provides a comprehensive view of enhancer-mediated gene regulation. NRSA not only outperforms existing methods for enhancer identification, but also enables annotation and quantification of active enhancers, and prediction of their target genes. Furthermore, NRSA identifies functionally important enhancers by integrating 1) nascent transcriptional changes in enhancers and their target genes and 2) binding profiles from regulator(s) of interest. Applied to wildtype and histone deacetylase 3 (Hdac3) knockout mouse livers, NRSA showed that HDAC3 regulates RNA polymerase recruitment through both proximal (promoter) and distal (enhancer) regulatory elements. Integrating ChIP-seq with PRO-seq data, NRSA prioritized enhancers based on their potential contribution to mediating HDAC3 regulation.ConclusionsNRSA will greatly facilitate the usage of nascent RNA sequencing techniques and accelerate the study of enhancer-mediated regulation.
Highlights
Enhancers are distal cis-regulatory elements that control gene expression
nascent RNA sequencing analysis (NRSA) identifies enhancers accurately and reproducibly NRSA was first applied to public K562 and GM12878 global nuclear run-on sequencing (GRO-seq) data to assess its performance in enhancer identification. 358 enhancers were detected in K562 data (Additional file 1: Table S1a), and 2654 in GM12878 (Additional file 1: Table S1b)
Enhancers contribute to HDAC3-regulated RNA polymerase recruitment to the promoter-proximal region To evaluate whether enhancers mediate HDAC3 regulation, we focused on the 582 enhancers upregulated upon Hdac3 deletion
Summary
Enhancers are distal cis-regulatory elements that control gene expression. Despite an increasing appreciation of the importance of enhancers in cellular function and disease, our knowledge of enhancer-regulated transcription is very limited. Nascent RNA sequencing technologies, such as global nuclear run-on sequencing (GRO-seq) and precision run-on sequencing (PRO-seq), provide a direct and reliable measurement of enhancer activity, and allow for quantifying transcription of enhancers and target genes simultaneously, making these technologies extremely useful for exploring enhancer-mediated regulation. RNA transcription in eukaryotic cells is actively regulated in multiple stages, including RNA polymerase recruitment, transcription initiation, elongation, and termination. Nascent RNA sequencing technologies, such as precision nuclear run-on sequencing (PRO-seq) [6] and global run-on sequencing (GRO-seq) [5], enable the measurement of transient RNA transcription at multiple stages, on a genome-wide scale, for multiple RNA species, including protein-coding genes, Like promoters, enhancers are key regulatory components bound by transcriptional regulators to enable temporal and spatial control of gene expression. 43,011 active, in vivo-transcribed enhancers have been identified by CAGE (cap analysis gene expression)
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