Fig. 5.

DNA methylation is acquired independently of RNA-DNA hybrid formation at the C9ORF72 locus. Relative quantification of all three C9ORF72 transcript variants in iPSC-derived motor neurons stably expressing a C9ORF72-specifc shRNA (shC9) or a scrambled CTL (shCTL) (a). DNA-RNA immunoprecipitation at the C9ORF72 promoter of shC9 and shCTL motor neurons, relative quantification was measured using two sets of primers, designed upstream (b) and downstream (c) of the repeat expansion, RNase H treatment was performed prior to pull-down as a negative control. DNA methylation levels at the C9ORF72 promoter were assessed using bisulfite pyrosequencing across 16 CpG dinucleotides; positions relative to the transcription start site are indicated on the x-axis (d). Fragile X patient-derived iPSC-neurons (FXS) were used as a negative control. All experiments were performed in duplicates (N = 2 from a single biological sample for each iPSC line examined). Significance is indicated by p < 0.05 * and p < 0.01 **