Genome-Wide DNA Methylation Profiling Reveals Epigenetic Changes in the Rat Nucleus Accumbens Associated With Cross-Generational Effects of Adolescent THC Exposure.

Global features of CpGs associated with cross-generational effects of THC exposure. (a) Density of CpGs plotted against the estimated mean methylation values between 0 and 100% (calculated across all THC and VEH animals) from both the background CpG list (black; n=567,306), and the hypermethylated (red) and hypomethylated (blue) DMRs; CpGs within DMRs have higher methylation levels on average. (b) Density of significant CpGs (q<0.01) from hyper- and hypomethylated DMRs (n=3,758) plotted against the difference in mean methylation values observed between THC and VEH groups, with hypermethylated changes (between-group difference>0%) shown in red, and hypomethylated changes shown in blue. DMRs, differentially methylated regions; THC, tetrahydrocannabinol; VEH, control vehicle

Plots demonstrating consistent differences in mean methylation between THC and VEH groups across significant CpGs within: (a) a hypermethylated DMR within an intron of Mta1, showing only the three significant hypermethylated CpGs within the DMR; (b) a hypermethylated DMR identified spanning exon 3 of Cdh15, showing only the three significant hypermethylated CpGs within the region; and (c) a hypomethylated DMR residing in exon 1 of Grin2a, ~2.5 kbp downstream of the transcript start site. In each panel, a schematic of Refseq gene annotations for each loci are shown, with exons depicted as purple blocks. A scale bar and bp positions in the rat rn4 genome assembly are provided. The locations of each hyper- and hypomethylated DMRs are indicated under each gene by red and blue boxes, respectively. DMR plots are shown below each gene. Within each plot, clusters of colored triangles represent methylation values at a given CpG across all individuals (THC, green; VEH, black). The mean methylation values for THC and VEH groups are represented by solid green (THC) and black (VEH) lines, connecting each CpG position. Approximate CpG bp positions (rn4) are indicated on the x axis. DMR, differentially methylated region; THC, tetrahydrocannabinol; VEH, control vehicle.

A circos plot showing the positions of all CpGs (n=5611) within significant DMRs across autosomes (chr1–20). Absolute mean methylation differences between THC and VEH groups for each hypo- and hypermethylated CpG are shown in the inner most panel (scale, 0–12%) as blue and red dots, respectively. The locations and names of 18 Refseq genes overlapped by the top 30 DMRs (Table 1) are indicated. The plot was generated using Rcircos (Zhang et al, 2013). DMR, differentially methylated region; THC, tetrahydrocannabinol; VEH, control vehicle.

Localization of CpGs with respect to exon/intron, promoter/TSS, and CpG island annotations in the rn4 genome. (a) The proportions of CpGs in hyper- and hypomethylated DMR (‘Hyper', ‘Hypo' n=5,611) and background sets (‘All' n=567,306) overlapping Refseq gene (left panel) and CpG island annotations (right panel). (b) Comparison of genomic distances (bp) from Refseq gene TSSs of promoter-associated CpGs within DMRs (n=612) and the background set (n=235 524), represented by red/blue and black lines; DMR-CpGs were preferentially located downstream of TSSs.DMR, differentially methylated region; TSS, transcription start site.

DMR-associated genes and genes involved in synaptic plasticity and synaptic transmission interact with common PPIhubs. (a) Heatmap showing Fisher's exact test P-values from PPIhub enrichment analyses (calculated by Lists2Networks (Lachmann and Ma'ayan, 2010)) using three gene sets: DMR-associated genes (n=492), and genes in GO categories GO:0048167 (‘regulation of synaptic plasticity' n=146) and GO:0035249 (‘glutamatergic synaptic transmission' n=79). Only PPIhubs for which a significant enrichment (P<0.05) was found for the DMR gene set are shown. (b) Protein interaction network including proteins corresponding to genes from DMR, GO:0048167, and GO:0035249 sets that interact with Dlg4, based on known interactions from rat, mouse, and human interaction databases (Berger et al, 2007; Lachmann and Ma'ayan, 2010). Proteins are colored according to whether they belong to one or more of the three gene sets. DMR, differentially methylated region.