Cause of chromosome 9p21-linked ALS and frontotemporal dementia (FTD) [1, 2]. The discovery places ALS and FTD among a large family of nearly 40 repeat expansion disorders, many of which specifically affect neurons [3]. The C9ORF72 HRE is hypothesized to confer cytotoxicity, in part, via RNA gain of function whereby sense and antisense transcripts harboring the repeat sequester RNA binding proteins resulting in ribonucleoprotein foci [4, 5], preventing the proteins from carrying out their normal function. In addition, expanded RNAs and dipeptiderepeat proteins that arise from aberrant translation of mutant RNAs disrupt nucleocytoplasmic transport [6?]. Given that mutant RNAs and the DPRs derived from them are the primary source of pathology in C9-ALS and epigenetic mechanisms regulate their production, it follows that epigenetic regulation of expanded C9ORF72 alleles is of particular significance. Epigenetic alterations occur in many repeat expansion disorders and there is now definitive evidence that epigenetic perturbations associated with the C9ORF72 HRE contribute to C9-ALS pathophysiology [9?1]. Expanded C9ORF72 alleles have reduced transcription rates, are depleted of active histone marks with concomitant enrichment of repressive epigenetic marks, including DNA hypermethylation in some cases. Specifically, the levels of all three C9ORF72 transcript variants are reduced in C9-ALS, including variant 2 which does not contain the PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28045099 repeat sequence due to alternative transcription start site (TSS) utilization [1, 5, 12]. Enriched repressive epigenetic marks include tri-methylation of histone 3 at lysine positions 9 and 27 (H3K9me3, H3K27me3) and histone 4 lysine 20 (H4K20me3) [12]. In addition, DNA methylation of cytosine residues AZD3759MedChemExpress AZD3759 within CpG islands near the C9ORF72 promoter occurs in approximately 30 of patients [13?5]. Notably, promoter hypermethylation is theorized to be protective due to decreased production of toxic products in patient cells [16] leading to reduced neuronal loss in the brain [17]. Mechanistically, DNA hypermethylation is counteracted by active DNA demethylation [18] while repeat instability is conferred by highly stable GC-rich RNA-DNA duplex formation across the HRE [19]. In addition to repeat instability, RNA-DNA hybrid structures, or R-loops, may also contribute to C9ORF72 hypermethylation. Indeed, R-loops are known to regulate DNA methylation at CpG islands of gene promoters including those affected by repeat expansion mutations [20, 21]. Taken together, these observations indicate that the C9ORF72 HRE alters the local epigenetic environment such that the rate of transcription, DNAmethylation, histone modification and R-loop formation are all perturbed by the expansion mutation. Two independent groups were first to develop transgenic mouse models of C9-ALS carrying the pathogenic C9ORF72 HRE [22, 23]. The human repeat expansion sequence was introduced into the mouse genome using a bacterial artificial chromosome (BAC). The C9-BAC mice displayed typical histopathological gain-offfunction features of C9-ALS including RNA foci and DPR aggregates, yet no motor or cognitive phenotypes were observed. Subsequently, two additional groups generated C9-BAC mice that exhibit reduced survival, motor deficits and cognitive dysfunction [24, 25]. While these previous reports focused on the production of toxic HRE products, none have described epigenetic features of the C9ORF72 transgene. Notably, the number of toxic mRNA foc.
