Single-cell profiling reveals that eRNA accumulation at enhancer–promoter loops is not required to sustain transcription

Abstract

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 ERα, 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.

Transcriptional control by enhancers and enhancer RNAs

“…Transcription of eRNAs occurs via RNA Pol II and temporally precedes the activation of target gene promoters [8,78,82,[88][89][90]. These transcripts exist in low abundance and are primarily found in nuclear and chromatin-bound fractions [78,[91][92][93]. Quantification of eRNAs that are thought to exist in higher abundance suggests a range of 0.5-20 copies per cell [94].…”

Diversity and Emerging Roles of Enhancer RNA in Regulation of Gene Expression and Cell Fate

“…There are several potential explanations for these conflicting studies. Some basal level eRNA transcription can occur and ongoing eRNA transcription may not be necessary during mRNA transcription (Rahman et al, 2017). Thus, it may be that enhancer-promoter interactions were established and maintained at these loci before eRNA knockdown.…”

Independent transposon exaptation is a widespread mechanism of shadow enhancer evolution in the mammalian genome

“…Whether this is a property that every active enhancer shows, is still a matter of debate. However, it is likely that not all active enhancers have this property [48]. In any case, the features distinguishing these alleged two classes of enhancers remain unclear, and therefore, it is difficult to assess to what degree our findings could be extended to enhancers that are not transcribed.…”