Mechanism of HS5-1 eRNA Regulation of Protocadherin α Cluster via R-loop Formation

Enhancers generate bidirectional noncoding enhancer RNAs that may regulate gene expression. At present, mechanisms of eRNA functions are not fully understood. Here, we report an antisense eRNA PEARL that is transcribed from the protocadherin HS5-1 enhancer region. Through loss- and gain-of-function experiments with CRISPR/Cas9 DNA-fragment editing, CRISPRi, and CRISPRa strategies, in conjunction with ChIRP, MeDIP, and DRIP experiments, we find that PEARL regulates Pcdh expression by forming local R-loop in situ within the HS5-1 enhancer region to promote long-distance chromatin interactions between distal enhancer and target promoters. These findings have important implications regarding mechanisms by which the HS5-1 enhancer regulates stochastic Pcdh promoter choice in single cells in the brain.