Fig. 2

DNA hypermethylation at the expanded C9ORF72 promoter appears in a fraction of adult mice. Site-specific DNA methylation sensitive PCR assessment of the human C9ORF72 promoter in the cortex of C9-BAC mice at seven time points, indicated in weeks (wks) of age. Two HhaI restriction sites located at −215 and −109 base pairs from the transcriptional start site were interrogated; three hypermethylated animals are indicated by open shapes (17wks square, 30wks triangle and 36wks circle). Assay controls (grey circles on right) include DNA isolated from post mortem brain tissues of ALS patients with the hexanucleotide repeat expansion (C9+) with (me+) or without (me-) promoter hypermethylation, an unaffected healthy control (C9-) individual, and synthetic DNA enriched (CTL Me 100%) or depleted of 5mC (CTL Me 0%). Values are plotted relative to the synthetic high control, which is set to 100% (a). C9ORF72 promoter methylation assessment from brain cortex, cerebellum, blood and tail clippings of a 30 week old hypermethylated mouse using HhaI methylation sensitive PCR (b). Bisulfite pyrosequencing of brain cortex from 17, 30 and 36 weeks old C9-BAC mice (n = 1 per age group per methylation status) across 8 CpG dinucleotides within the human C9ORF72 promoter, positions relative to TSS are shown on the x-axis. Open symbols indicate samples from hypermethylated (me+) animals, filled symbols are samples from unmethylated (me-) animals (c). Glycine-Proline DPR assessment of whole brain tissue samples from three hypermethylated animals (open symbols) and representative unmethylated samples (filled symbols) from 17, 30 and 36 week old C9-BAC mice (n = 3 per age group) (d)