Figure 9: DNMT3a is required for the nerve injury-induced increase in Kcna2 DNA methylation in rat injured DRG.
(a) Two regions (R1, −663/−389; R2, −491/−199), but not other regions (R3, −237/+42; R4, +20/+278; R5, +185/+464; R6: +411/+649; R7: +680/+928), from the Kcna2 gene were immunoprecipitated by rabbit anti-DNMT3a (not by rabbit normal serum) in rat lumbar DRG. Input, total purified fragments. M, ladder marker. n=3 repeats. (b) DNMT3a binding to R1 and R2 regions within the Dnmt3a gene in the ipsilateral L5 DRG of rats on day 7 post-SNL or sham surgery. Left: the binding of Dnmt3a to the R1 region of Kcna2 gene. Right: the quantitative analysis of the binding. n=3 repeats (3 rats/repeat)/group. **P<0.01 versus the corresponding sham group by two-tailed unpaired Student’s t-test. (c,d) The increases in the levels of DNA methylation at −457, −444, −440, and −374 CpG sites by bisulfite clone-sequencing assay (c) and at −482, −457, and −444 CpG sites by bisulfite pyro-sequencing assay (d) from −540 to 500 bp sites of Kcna2 gene consisted of 65 CpG sites in the ipsilateral L5 DRG of rats on day 7 post-SNL. n=3 repeats (6 rats/repeat)/group. **P<0.01 versus the corresponding sham group by two-tailed unpaired Student’s t-test. (e) The percentages of methylation at −457 and −444 CpG sites of the Kcna2 gene in the ipsilateral L5 DRG on day 7 post-SNL or sham surgery from five groups as indicated. GFP: AAV5-GFP. scram: AAV5-Dnmt3a scrambled shRNA. shRNA: AAV5-Dnmt3a shRNA. Left: Representation of a single cloned allele per group at the -457 CpG site. Right: Statistical analysis at −457 and −444 CpG sites. n=3 repeats (3 rats/repeat)/group. One-way ANOVA (methylation versus. group) followed by post hoc Tukey test, Fgroup (3, 19)=15.8 for −457 site and Fgroup (3, 19)=6.4 for −440 site. *P<0.05 versus the corresponding PBS plus sham group. #P<0.05 versus the corresponding GFP plus SNL group.
