MBD1 Contributes to the Genesis of Acute Pain and Neuropathic Pain by Epigenetic Silencing ofOprm1andKcna2Genes in Primary Sensory Neurons

Abstract

The transmission of normal sensory and/or acute noxious information requires intact expression of pain-associated genes within the pain pathways of nervous system. Expressional changes of these genes after peripheral nerve injury are also critical for neuropathic pain induction and maintenance. Methyl-CpG-binding domain protein 1 (MBD1), an epigenetic repressor, regulates gene transcriptional activity. We report here that MBD1 in the primary sensory neurons of DRG is critical for the genesis of acute pain and neuropathic pain as DRG MBD1-deficient mice exhibit the reduced responses to acute mechanical, heat, cold, and capsaicin stimuli and the blunted nerve injury-induced pain hypersensitivities. Furthermore, DRG overexpression of MBD1 leads to spontaneous pain and evoked pain hypersensitivities in the WT mice and restores acute pain sensitivities in the MBD1-deficient mice. Mechanistically, MDB1 represses Oprm1 and Kcna2 gene expression by recruiting DNA methyltransferase DNMT3a into these two gene promoters in the DRG neurons. DRG MBD1 is likely a key player under the conditions of acute pain and neuropathic pain.

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

“…DNMT3a is a strong candidate for mediating increased levels of methylation of Cacna1b e37a locus in Trpv1 -lineage neurons. DNMT3a is upregulated in models of neuropathic pain and is associated with hypermethylation at promotor regions of neuronal genes including Oprm1 and Kcna2 (Mo et al, 2018; Zhao et al, 2017); we show that DNMT3a, but not DNMT1 or DNMT3b, shifts the pattern of Cacna1b e37a splicing in F11 cells. Global and highly localized changes in TET1 can also modify the methylation levels of exons (Liu et al, 2016; Liu et al, 2018; Marina et al, 2016) and, as we show for dCAS9-TET1 targeted to Cacna1b e37a, can promote the action of CTCF to shift the pattern of alternative splicing.…”

Contribution of DNMT1 to Neuropathic Pain Genesis Partially through Epigenetically Repressing Kcna2 in Primary Afferent Neurons

“…Functional locomotor activities in mice were assessed through three reflex tests as previously described Mo et al, 2018). (1) Placing reflex: The mice were held with the hind limbs slightly lower than the forelimbs, and the dorsal surfaces of the hindpaws were brought into contact with the edge of a table.…”

“…Control experiments for rabbit anti-DNMT1 included substitution of normal rabbit serum for the primary antiserum, omission of the primary antiserum, and preabsorption of the primary antibody with overdose of the antigen. The specificity of remaining primary antisera has been identified previously Shao et al, 2017;Sun et al, 2017;Xu et al, 2017;Zhao et al, 2017;Mo et al, 2018;Mao et al, 2019;Yuan et al, 2019). The sections finally mounted using VectaMount permanent mounting medium (Vector Laboratories) or Vectashield plus DAPI mounting medium (Vector Laboratories).…”

“…The specificity of rabbit anti-DNMT1 was examined in the following control experiments, including substitution of normal rabbit serum for the primary antiserum, omission of the primary antiserum, and preabsorption of the primary antibody with overdose of the antigen. The specificity of remaining primary antisera was reported previously (Lee et al, 2011;Zhao et al, 2013Zhao et al, , 2017Fan et al, 2014;Li et al, 2015Sun et al, 2017;Mo et al, 2018;Du et al, 2019;Mao et al, 2019) or from vendors' data sheets. Membranes were further incubated with anti-mouse or anti-rabbit HRP-conjugated secondary antibody (1: 3000, Jackson ImmunoResearch Laboratories) for 2 h at room temperature and visualized by Western peroxide reagent and luminol/enhancer reagent (Clarity Western ECL Substrate, Bio-Rad).…”

“…Conventionally, DNMT3a and DNMT3b function as de novo DNMTs to revers-ibly methylate unmethylated DNA, whereas DNMT1 is classified as the primary DNMT to maintain DNA methylation that has been established at the genome (Jeltsch, 2006;Siedlecki and Zielenkiewicz, 2006). The contribution of de novo DNMT3atriggered DNA methylation to neuropathic pain genesis was reported Shao et al, 2017;Sun et al, 2017;Zhao et al, 2017;Mo et al, 2018;Mao et al, 2019;Yuan et al, 2019). Expression of DNMT3a, but not DNMT3b, is increased in the injured DRG following peripheral nerve injury .…”