Bimodal sequence composition underlies functional duality of transcribed enhancers

Background: Recent studies have drawn attention to transcribed enhancers (trEs) as important regulatory elements of gene expression; however, their characteristics and mechanisms of action remain poorly understood. Results: We profiled the characteristics of trEs and obtained insights into their mechanisms of action. We found that trEs harbor functional duality related to bimodal sequence composition. TrEs are composed of nonoverlapping cores and flanking regions (flanks): cores function as regular enhancers, while flanks transcribe enhancer RNAs (eRNAs) that can potentially regulate the expression of their target genes in trans. Cores are evolutionarily conserved and compact, while flanks are significantly longer. We observed that approximately 25% of eRNAs transcribed from the flanks likely contribute to trans DNA:RNA triple helix formation, while another 10% likely employ classical mechanisms of RNA-based regulation. We found that the majority of human enhancers are not transcribed, and trEs are strikingly different from regular enhancers in their functional characteristics. In addition, we found evidence for trEs exhibiting functional duality in regulatory locus encapsulation (RLE), effectively providing localized control over the spread of gene expression upregulation by trE cores and other locus enhancers. Conclusions: In summary, our results advocate for enhancer transcription being an uncommon mechanism of gene regulation, and the duality of transcribed enhancer function being a product of additive, not overlapping, DNA sequence encryption.