Abstract
Enhancer RNAs (eRNA) are unstable non-coding RNAs, transcribed bidirectionally from active regulatory sequences, whose expression levels correlate with enhancer activity. We use capped-nascent-RNA sequencing to efficiently capture bidirectional transcription initiation across several human lymphoblastoid cell lines (Yoruba population) and detect ~75,000 eRNA transcription sites with high sensitivity and specificity. The use of nascent-RNA sequencing sidesteps the confounding effect of eRNA instability. We identify quantitative trait loci (QTLs) associated with the level and directionality of eRNA expression. High-resolution analyses of these two types of QTLs reveal distinct positions of enrichment at the central transcription factor (TF) binding regions and at the flanking eRNA initiation regions, both of which are associated with mRNA expression QTLs. These two regions—the central TF-binding footprint and the eRNA initiation cores—define a bipartite architecture of enhancers, inform enhancer function, and can be used as an indicator of the significance of non-coding regulatory variants.
Highlights
Outside transcription regulatory elements (TREs) SNP frequencyRealtive position from transcriptional regulatory elements (tTREs) midpoint c promoter tTRE
The central transcription initiation QTLs (tiQTLs) are significantly enriched for transcription factor binding QTLs, while the transcription start sites (TSS)-proximal Directional initiation QTL (diQTL) show no enrichment in most of the binding QTLs26 (bQTLs). These results indicate that both tiQTLs and diQTLs are associated with gene expression and, given the different positioning of tiQTLs and diQTLs within enhancers and overlap with bQTLs, that sequences at both the central transcription-factor-binding site (TFBS) and the regions surrounding the TSSs may affect enhancer function in gene regulation through distinct mechanisms
We explored the activity and architecture of transcribed transcriptional regulatory elements by studying the variation in transcription initiation across human lymphoblastoid cell lines (LCLs)
Summary
We combined all PRO-cap datasets and identified transcribed transcriptional regulatory elements (tTREs), including both enhancers and promoters, with bidirectional divergent nascent transcription within 300 base pairs (bp) of one another (Fig. 1a, Supplementary Fig. 1a). We compared our approach to LCL data from another nascent-sequencing method, NET-CAGE28, and found that PRO-cap identifies EP300-bound enhancers marginally better than NET-CAGE (Fig. 1c–g). Regulatory variants are more enriched in enhancers than promoters This result was reproduced using DNase hypersensitive sites (DHSs) to identify enhancers, illustrating that it is not a function of our enhancer identification approach (Supplementary Fig. 2j, k). The peak of tiQTL enrichment is at the midpoint (Supplementary Fig. 5a), where we would expect transcription factors to frequently bind Based on these results, we hypothesize that the overall transcriptional activity of enhancers is generally regulated from the central TF
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