The G-Rich Repeats in FMR1 and C9orf72 are Hotspots for DNA Unpairing

Pathological mutations involving noncoding microsatellite repeats, including fragile X syndrome (FMR1) and C9-related ALS/FTD (C9orf72), are typically located near promoters in CpG islands, and are coupled with extensive repeat instability when sufficiently long. How these regions are prone to repeat instability is not fully understood. It is generally accepted that instability results from the induction of unusual structures in the DNA by the repeats as a consequence of mispairing between complementary strands. In addition, there is some evidence to show that repeat instability is mediated by RNA transcription through the formation of three-stranded nucleic structures, composed of persistent DNA:RNA hybrids concomitant with single strand DNA displacements (R-loops).

Using human embryonic stem cells with wild type and repeat expanded alleles in the FMR1 (CGGs) and C9orf72 (GGGGCCs) genes, we show that these loci constitute hotspots for DNA unpairing. When R-loops are formed, DNA unpairing is more extensive, and is coupled with the interruptions of double strand structures by the non-transcribing (G-rich) DNA strand. These interruptions are likely to reflect unusual structures in the DNA that drive repeat instability when the G-rich repeats reach disease range. Further, we demonstrate that when the CGGs in FMR1 are hypermethylated and transcriptionally inactive, local DNA unpairing is abolished. Our study thus takes one more step towards the identification of dynamic, unconventional DNA structures across the G-rich repeats at the FMR1 and C9orf72 disease-associated loci.