Cell type-dependent directional transcription at enhancers

Enhancers are noncoding regulatory regions in the genome that play essential roles in modulating gene expression. Previous work showed that enhancers are not transcriptionally silent but are characterized by bidirectional expression of short capped noncoding RNAs. Balanced bidirectional expression has therefore been used as a key feature for the detection of enhancers from transcriptome data. Instead, by analyzing FANTOM5 and other deep cap analysis gene expression transcriptome datasets, we find enhancer transcription preferentially in one direction in individual cell types. As the preferred direction of transcription of an enhancer can switch between cell types, balanced bidirectional enhancer expression may appear if transcriptome data are aggregated over cell types. 5' single-cell RNA sequencing data showed that enhancers were almost exclusively expressed unidirectionally in a single cell. Reporter assay data demonstrated that the regulatory function of an enhancer does not depend on its preference for unidirectional or bidirectional expression. We conclude that requiring balanced bidirectional transcription for enhancer detection may discard most valid enhancers when applied to transcriptome data of a single cell type.