Systematic Functional Characterization of Antisense eRNA of Protocadherin alpha HS5-1 Enhancer

Enhancers generate bidirectional noncoding enhancer RNAs (eRNA) that may regulate gene expression. At present, the eRNA function remains enigmatic. Here, we report a 5' capped antisense eRNA PEARL (Pcdh eRNA associated with R-loop formation) that is transcribed from the protocadherin (Pcdh) alpha HS5-1 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 find that PEARL regulates Pcdh alpha gene expression by forming local RNA-DNA duplexes (R-loops) in situ within the HS5-1 enhancer region to promote long-distance chromatin interactions between distal enhancer and target promoter. In particular, increased levels of eRNA PEARL via perturbing transcription elongation factor SPT6 leads to strengthened local three-dimensional chromatin organization within the Pcdh superTAD. These findings have important implications regarding molecular mechanisms by which the HS5-1 enhancer regulates stochastic Pcdh alpha promoter choice in single cells in the brain. Overall design: 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 find that PEARL regulates Pcdh alpha gene expression by forming local RNA-DNA duplexes (R-loops) in situ within the HS5-1 enhancer region to promote long-distance chromatin interactions between distal enhancer and target promoter. In particular, increased levels of eRNA PEARL via perturbing transcription elongation factor SPT6 leads to strengthened local three-dimensional chromatin organization within the Pcdh superTAD.