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

Abstract

Cell-specific alternative splicing modulates myriad cell functions and 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 CaV channel Cacna1b gene modulates opioid sensitivity. How this splicing is regulated is unknown. We find that cell and exon-specific DNA hypomethylation permits CTCF binding, the master regulator of mammalian chromatin structure, which, in turn, controls splicing in a DRG-derived cell line. In vivo, hypomethylation of an alternative exon specifically in nociceptors, likely permits CTCF binding and expression of CaV2.2 channel isoforms with increased opioid sensitivity in mice. 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.

Emerging Roles of Activity-Dependent Alternative Splicing in Homeostatic Plasticity

“…Although it is not known how network activity regulates this splicing event, it has recently been proposed that inclusion of exon 37a or 37b in Ca V 2.2 is consequent to differences in chromatin structure and transcription rates, rather than being directly regulated at the mRNA level (Javier et al, 2019;Lopez Soto and Lipscombe, 2020). Because splicing occurs mostly co-transcriptionally (Luco et al, 2011), rapid transcription of Cacna1b (the gene for the α 1 subunit of Ca V 2.2) would lead to simultaneous availability to the splicing machinery of the two mutually exclusive exons.…”

“…Conversely, a slow transcription rate would favor recruitment of the splicing machinery to the first upstream exon 37a, thus leading to inclusion of this weaker exon into the final transcript. Indeed, the zinc finger DNA-binding protein CCCTC-binding factor (CTCF), a well-known organizer of chromatin architecture, binds to the exon 37a locus of Cacna1b to promote inclusion of this exon (Javier et al, 2019;Lopez Soto and Lipscombe, 2020). This is likely because CTCF favors the formation of intragenic chromatin loops and slows down the elongation rate of the RNA polymerase II (Pol II; Shukla et al, 2011;Ruiz-Velasco et al, 2017).…”

“…This is likely because CTCF favors the formation of intragenic chromatin loops and slows down the elongation rate of the RNA polymerase II (Pol II; Shukla et al, 2011;Ruiz-Velasco et al, 2017). Importantly, CTCF binding is not constitutive but prevented by methylation of the Cacna1b exon 37a locus, which consequently leads to exon 37a exclusion (Javier et al, 2019;Lopez Soto and Lipscombe, 2020).…”

Epigenetic control of ion channel expression and cell-specific splicing in nociceptors: Chronic pain mechanisms and potential therapeutic targets

“…In neuropathic pain, injured neurons exhibit altered histone modification patterns that impact protein access to gene loci [ 16 , 21 , 23 ] including transcription factor occupancy at gene promotor regions [ 21 , 24 ], and altered DNA methylation of gene promoters and alternatively spliced exons [ 19 , 25–27 ].…”

“…As summarized in Figures 1 and 2 , in heat-sensitive Trpv1 nociceptors, the e37a splice form of Ca V 2.2 is expressed and it is more strongly inhibited by μ-opioid receptor activation especially under conditions of prolonged excitation. By comparison, the more commonly expressed splice form of Ca V 2.2, e37b, dominates in most other neurons and it is less sensitive to μ-opioid receptor inhibition [ 25 , 35 , 42–46 ]. This difference in morphine efficacy, between Ca V 2.2 splice isoforms impacts behavior.…”