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. 2017 Apr 7;45(6):3017-3030.
doi: 10.1093/nar/gkw1220.

Single-cell Profiling Reveals That eRNA Accumulation at Enhancer-Promoter Loops Is Not Required to Sustain Transcription

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Single-cell Profiling Reveals That eRNA Accumulation at Enhancer-Promoter Loops Is Not Required to Sustain Transcription

Samir Rahman et al. Nucleic Acids Res. .
Free PMC article

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.

Figures

Figure 1.
Figure 1.
eRNAs are low-abundance nuclear transcripts that are induced with similar kinetics as their target genes. (A) Diagram of the genomic organization of the FOXC1 and P2RY2 loci. (B) smFISH showing the expression and localization of FOXC1 (left) and P2RY2 (right) eRNAs and mRNAs before and after 40 min of E2 induction in MCF-7 cells. Arrows indicate different eRNA and transcription site configurations. (C) Frequency distributions of FOXC1 (left panels) and P2RY2 (right panels) eRNA and active mRNA transcription sites during E2 induction. Representative data of three independent experiments; n = 100 cells per time point.
Figure 2.
Figure 2.
Induction of FOXC1 eRNA and mRNA transcription requires ERα. (A) FOXC1 eRNA and mRNA expression in the presence or absence of a 3 h pre-treatment with Tamoxifen (100 nM) or ICI (100 nM) followed by E2 induction (5 nM) for 40 min. (B) Quantification of data from (A). Frequency distributions of FOXC1 antisense eRNAs and nascent FOXC1 transcripts, representative of two independent experiments; n = 70–200 cells for each condition.
Figure 3.
Figure 3.
MLL1 is required for E2-induced eRNA transcription. (A) smFISH analysis for P2RY2 sense eRNA and mRNA combined with MLL1 immunofluorescence in MLL1-depleted or non-specific siRNA-treated cells before and after E2 induction. (B) Frequency distribution of P2RY2 sense eRNAs and mRNA in MLL1-depleted or non-specific siRNA-treated cells before and after E2 induction. Data are representative of four independent experiments; n = 160–200 cells for each condition. (C) ChIP analysis for H3K4me1 (left) and ERα (right) presence at the P2RY2 enhancer in MLL1-depleted or non-specific siRNA-treated cells in the presence or absence of E2 treatment; n = 2 independent experiments; error bars indicate SD.
Figure 4.
Figure 4.
Simultaneous expression of eRNAs and mRNAs is infrequent in single cells and is not required to maintain transcription. (A) Frequency of co-localization between FOXC1 (top panel) and P2RY2 (bottom panel) mRNA transcription sites and their cognate eRNAs using the transcription site as a reference. eRNA-mRNA transcription site co-localization was scored within a 400 nm radius. (B) Frequency of co-localization of eRNAs with mRNA transcription sites using the eRNA signal as a reference. Panels A and B indicate the mean and SD from three independent experiments. (C) Quantification of the relationship between eRNA-transcription site co-localization and the RNAPII density on individual alleles. Outlines represent the relative density of data points. Data was pooled from three independent experiments; active transcription sites from n = 300 cells per time point were analyzed.
Figure 5.
Figure 5.
eRNA-mRNA co-expressing alleles are infrequently found in a closed enhancer–promoter loop configuration. (A) Determination of the upper limit of co-localization detection (63 nm) using smFISH probes spanning the P2RY2 5΄ intron labeled in two different colors and 2D Gaussian fitting. (B) Frequency distribution plots displaying the distances between mRNA transcription sites and nascent eRNAs within a 400 nm radius (red) overlaid with the localization precision plot (green) shown in (A). The data represent the 40 min E2 induction point combined from three independent experiments. (C) Cartoon illustrating the spatial organization of nascent eRNAs relative to the transcription site. Three different scenarios of eRNA and target mRNA co-expression are observed at single alleles. (i) Simultaneous eRNA and mRNA transcription consistent with a closed enhancer–promoter loop conformation; least frequent. (ii) Simultaneous eRNA and mRNA transcription in an open enhancer-promoter loop conformation, and (iii) mRNA transcription from alleles that are not co-expressed with an eRNA; most prevalent. Factors involved in enhancer-promoter communication are shown schematically.

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