Figure 5. Regulation of alternative splicing and isoform-specific expression by intragenic DNA methylation.

Intragenic modifications of DNA methylation arising during cocaine self-administration may contribute to the differential regulation of select splice variants. The isoforms selected include the major protein-coding isoform of the select gene and isoforms transcribed from the genomic region proximal to the DMR of interest. (a) There was a trend towards the overall decreased expression of Cdh13, F_3,26 =2.53, p=0.07. (b) However, this is likely due to the regulation of non-coding transcripts of Cdh13, as the expression of the protein-coding transcript was not significantly altered following cocaine self-administration; F_3,26 = 1.35, not significant. (c). Subsequent to self-administration, overall Cpeb4 expression increased in all treatment groups; F_3,26 = 5.96, p<0.01. (d) Nevertheless, the expression of the common isoform, Cpeb4-001, displayed a different pattern of expression relative to that in naïve animals, with the sole significant difference in expression being between animals subject to relapse testing at 21 days of abstinence compared to those that were not (F_3,26 = 4.32, p<0.01, IVSA 21 R vs. IVSA 21 NR, p<0.01, Holm-Sidak post hoc test). (e) When the collective expression of all protein-coding isoforms of Mctp1 was explored, the sole significant difference was again between animals subject to relapse testing at 21 days and those that were not (F_3,26 = 3.62, p<0.05, Holm-Sidak post hoc, IVSA 21 R vs. IVSA 21 NR, p<0.05). (f) However, when explored individually, the expression of one protein-coding isoform (Mctp1-001, Ensembl 37) was persistently decreased at all time points relative to naïve animals; F_3,26= 4.07, p<0.05. Data are displayed as mean ± SEM; all p-values are derived from Holm-Sidak post hoc tests relative to naïve animals except where indicated, * p<0.05, ** p<0.01.