Antisense lncRNA Transcription Mediates DNA Demethylation to Drive Stochastic Protocadherin α Promoter Choice

Stochastic activation of clustered Protocadherin (Pcdh) alpha, beta, and gamma genes generates a cell-surface identity code in individual neurons that functions in neural circuit assembly. Here, we show that Pcdh alpha gene choice involves the activation of an antisense promoter located in the first exon of each Pcdh alpha alternate gene. Transcription of an antisense long noncoding RNA (lncRNA) from this antisense promoter extends through the sense promoter, leading to DNA demethylation of the CTCF binding sites proximal to each promoter. Demethylation-dependent CTCF binding to both promoters facilitates cohesin-mediated DNA looping with a distal enhancer (HS5-1), locking in the transcriptional state of the chosen Pcdh alpha gene. Uncoupling DNA demethylation from antisense transcription by Tet3 overexpression in mouse olfactory neurons promotes CTCF binding to all Pcdh alpha promoters, resulting in proximity-biased DNA looping of the HS5-1 enhancer. Thus, antisense transcription-mediated promoter demethylation functions as a mechanism for distance-independent enhancer/promoter DNA looping to ensure stochastic Pcdh alpha promoter choice.