Systematic functional characterization of antisense eRNA of protocadherin composite enhancer.

Enhancers generate bidirectional noncoding enhancer RNAs (eRNAs) that may regulate gene expression. At present, the eRNA function remains enigmatic. Here, we report a 5' capped antisense eRNA ( eRNA associated with R-loop formation) that is transcribed from the protocadherin () enhancer region. Through loss- and gain-of-function experiments with CRISPR/Cas9 DNA fragment editing, CRISPRi, and CRISPRa, as well as locked nucleic acid strategies, in conjunction with ChIRP, MeDIP, DRIP, QHR-4C, and HiChIP experiments, we found that regulates gene expression by forming local RNA-DNA duplexes (R-loops) in situ within the enhancer region to promote long-distance chromatin interactions between distal enhancers and target promoters. In particular, increased levels of eRNA via perturbing transcription elongation factor lead to strengthened local three-dimensional chromatin organization within the superTAD. These findings have important implications regarding molecular mechanisms by which the enhancer regulates stochastic promoter choice in single cells in the brain.