Cell-specific exon methylation and CTCF binding in neurons regulates calcium ion channel splicing and function

SummaryCell-specific alternative splicing modulates myriad cell functions and this process is disrupted in disease. The mechanisms governing alternative splicing are known for relatively few genes and typically focus on RNA splicing factors. In sensory neurons, cell-specific alternative splicing of the presynaptic voltage-gated calcium channel Cacna1b gene modulates opioid sensitivity. How this splicing is regulated has remained unknown. We find that cell-specific exon DNA hypomethylation permits binding of CTCF, the master regulator of chromatin structure in mammals, which, in turn, controls splicing in noxious heat-sensing nociceptors.Hypomethylation of an alternative exon specifically in nociceptors allows for CTCF binding, and expression of CaV2.2 channels with increased opioid sensitivity. Following nerve injury, exon methylation is increased, and splicing is disrupted. Our studies define the molecular mechanisms of cell-specific alternative splicing of a functionally validated exon in normal and disease states – and reveal a potential target for the treatment of chronic pain.HighlightsThe molecular basis of cell-specific splicing of a synaptic calcium channel gene.Splicing controlled by cell-specific exon hypomethylation and CTCF binding.Peripheral nerve injury disrupts exon hypomethylation and splicing.Targeted demethylation of exon by dCAS9-TET modifies alternative splicing.GRAPHICAL ABSTRACTCell-specific epigenetic modifications in a synaptic calcium ion channel gene controls cell-specific splicing in normal and neuropathic pain.In naïve animals, in most neurons, Cacna1b e37a locus is hipermethylated (5-mC) and CTCF does not bind this locus. During splicing, e37a is skipped and Cacna1b mRNAs include e37b. In contrast, in Trpv1-lineage neurons, Cacna1b e37a locus is hypomethylated and is permissive for CTCF binding. CTCF promotes e37a inclusion and both Cacna1b e37a and e37b mRNAs are expressed. E37a confers strong sensitivity to the Cav2.2 channel to inhibition by μ-opioid receptors (μOR). Morphine is more effective at inhibiting e37a-containing Cav2.2 channels. After peripheral nerve injury that results in pathological pain, methylation level of Cacna1b e37a locus is increased, CTCF binding is impaired, and Cacna1b e37a mRNA levels are decreased. This disrupted splicing pattern is associated with reduced efficacy of morphine in vivo.