biostudies-literatureZhou YMinistry of Science and Technology of ChinaScience and Technology Commission of Shanghai MunicipalityNational Natural Science Foundation of China1383-1394https://www.ebi.ac.uk/biostudies/studies/S-EPMC8494205biostudies-literatureUnknown35(19-20)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 <i>PEARL</i> (<i>Pcdh</i> eRNA associated with R-loop formation) that is transcribed from the protocadherin (<i>Pcdh</i>) <i>α HS5-1</i> 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 <i>PEARL</i> regulates <i>Pcdhα</i> gene expression by forming local RNA-DNA duplexes (R-loops) in situ within the <i>HS5-1</i> enhancer region to promote long-distance chromatin interactions between distal enhancers Genes & developmentSystematic functional characterization of antisense eRNA of protocadherin <i>α</i> composite enhancer.PMC8494205919403032017YFA0504203316300392018YFC100450421DZ221020019JC1412500Wu QXu SZhang MZhou YfalseSystematic functional characterization of antisense eRNA of protocadherin <i>α</i> 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 <i>PEARL</i> (<i>Pcdh</i> eRNA associated with R-loop formation) that is transcribed from the protocadherin (<i>Pcdh</i>) <i>α HS5-1</i> 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 <i>PEARL</i> regulates <i>Pcdhα</i> gene expression by forming local RNA-DNA duplexes (R-loops) in situ within the <i>HS5-1</i> enhancer region to promote long-distance chromatin interactions between distal enhancers 2021-01-01T00:00:00Z2021 Oct2025-04-22T09:16:43.865Z2025-04-05T22:57:43.694ZS-EPMC84942053453131710.1101/gad.348621.121