title: 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.
authors.affiliation: Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, QC H3T 1J4, Canada.
authors.forename: Samir
authors.initials: S
authors.lastname: Rahman
authors.affiliation: Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, QC H3T 1J4, Canada.
authors.forename: Cornelia E
authors.initials: CE
authors.lastname: Zorca
authors.affiliation: Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, QC H3T 1J4, Canada.|Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, QC H3T 1J4, Canada.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Traboulsi
authors.affiliation: Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, QC H3T 1J4, Canada.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Noutahi
authors.affiliation: Montreal Neurological Institute, McGill University, Montréal, QC H3A 2B4, Canada.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Krause
authors.affiliation: Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, QC H3T 1J4, Canada.|Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, QC H3T 1J4, Canada.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Mader
authors.affiliation: Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, QC H3T 1J4, Canada.
authors.forename: Daniel
authors.initials: D
authors.lastname: Zenklusen
mesh_terms: D004742:Enhancer Elements, Genetic; D004958:Estradiol / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000502:physiology; D051858:Forkhead Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D011495:Histone-Lysine N-Methyltransferase / Q000502:physiology; D006801:Humans; D061986:MCF-7 Cells; D008958:Models, Molecular; D051788:Myeloid-Lymphoid Leukemia Protein / Q000502:physiology; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D022661:RNA, Untranslated / Q000096:biosynthesis* / Q000502:physiology; D058473:Receptors, Purinergic P2Y2 / Q000096:biosynthesis / Q000235:genetics; D059010:Single-Cell Analysis; D014158:Transcription, Genetic
chemical_list: D047628:Estrogen Receptor alpha; C112743:FOXC1 protein, human; D051858:Forkhead Transcription Factors; C497422:KMT2A protein, human; C546385:P2RY2 protein, human; D012333:RNA, Messenger; D022661:RNA, Untranslated; D058473:Receptors, Purinergic P2Y2; D051788:Myeloid-Lymphoid Leukemia Protein; D004958:Estradiol; D011495:Histone-Lysine N-Methyltransferase