Graphical Abstract Highlights d A conserved antisense promoter is located within each of the Pcdha alternate exons d Antisense lncRNA transcription leads to DNA demethylation of promoters and CBSs d CTCF/cohesin drive the assembly of Pcdha enhancer/ promoter complex via loop extrusion d Coupling lncRNA transcription to DNA demethylation ensures stochastic promoter choice SUMMARYStochastic activation of clustered Protocadherin (Pcdh) a, b, and g genes generates a cell-surface identity code in individual neurons that functions in neural circuit assembly. Here, we show that Pcdha gene choice involves the activation of an antisense promoter located in the first exon of each Pcdha 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 Pcdha gene. Uncoupling DNA demethylation from antisense transcription by Tet3 overexpression in mouse olfactory neurons promotes CTCF binding to all Pcdha 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 Pcdha promoter choice.

Neural type–specific expression of clustered Protocadherin (Pcdh) proteins is essential for the establishment of connectivity patterns during brain development. In mammals, deterministic expression of the same Pcdh isoform promotes minimal overlap of tiled projections of serotonergic neuron axons throughout the brain, while stochastic expression of Pcdh genes allows for convergence of tightly packed, overlapping olfactory sensory neuron axons into targeted structures. How can the same gene locus generate opposite transcriptional programs that orchestrate distinct spatial arrangements of axonal patterns? Here, we reveal that cell type–specific Pcdh expression and axonal behavior depend on the activity of cohesin and its unloader, WAPL (wings apart–like protein homolog). While cohesin erases genomic-distance biases in Pcdh choice, WAPL functions as a rheostat of cohesin processivity that determines Pcdh isoform diversity.

Clustered Protocadherin (Pcdh) proteins act as cell-surface recognition barcodes for neural circuit formation. Neurites expressing the same barcode repel each other, but this mechanism is deployed in two different ways. For instance, convergence of olfactory sensory neuron (OSN) projections requires stochastic expression of distinct Pcdh isoforms in individual cells, while tiling of neural arbors of serotonergic neurons (5-HTs) requires expression of the same isoform, Pcdhαc2. Despite their essential role, however, the molecular mechanisms of cell-type specific Pcdh barcoding remain a mystery. Here, we uncover a new role of cohesin: that of regulating distance-independent enhancer-promoter interactions to enable random Pcdh isoform choice via DNA loop extrusion in OSNs. Remarkably, this step mediates DNA demethylation of Pcdh promoters and their CTCF binding sites, thus directing CTCF to the chosen promoter. In contrast, the uniform pattern of Pcdh expression in 5-HTs is achieved through conventional cohesin-independent, distance-dependent enhancer/promoter interactions, that favor choice of the nearest isoform. Thus, cell-type specific cohesin deployment converts a distance-dependent and deterministic regulatory logic into a distance-independent and stochastic one. We propose that this mechanism provides an elegant strategy to achieve distinct patterns of Pcdh expression that generate wiring instructions to meet the connectivity requirements of different neural classes.

Stochastic and combinatorial activation of clustered Protocadherin (Pcdh) α, β, and γ gene promoters generates a cell-surface identity code in individual neurons that functions in neural circuit assembly. Here we show that Pcdhα promoter choice requires transcription of a long noncoding RNA (lncRNA) initiated from newly identified promoters located in the protein coding sequence of each Pcdhα exon. Antisense transcription of the lncRNA through the sense promoter results in its activation and in DNA demethylation of the binding sites for the CCCTC-binding protein, CTCF, located in close proximity to both sense and antisense promoters. Increased CTCF binding promotes the assembly of long-range DNA contacts between the activated promoter and a neuron-specific enhancer, thus locking in the epigenetic state of the stochastically chosen Pcdhα promoter. Examination of this hierarchical molecular mechanism in differentiating olfactory sensory neurons, suggests that antisense Pcdhα transcription is a key prerequisite for stochastic Pcdhα promoter choice in vivo.