Background: Friedreich ataxia (FRDA) is caused by an expanded GAA triplet repeat (GAA-TR) mutation that results in FXN transcriptional deficiency.Results: Repressive chromatin spreads from the expanded GAA-TR mutation, switching off the FXN gene promoter.Conclusion: Reduced transcriptional initiation is the major cause of FXN transcriptional deficiency in FRDA.Significance: Reactivation of FXN transcriptional initiation is a potential therapeutic strategy in FRDA.

Objective Friedreich ataxia (FRDA) is caused by an expanded GAA triplet-repeat (GAA-TR) mutation in the FXN gene. Patients are typically homozygous for expanded alleles containing 100–1300 triplets, and phenotypic severity is significantly correlated with the length of the shorter of the two expanded alleles. Patients have a severe deficiency of FXN transcript, which is predominantly caused by epigenetic silencing of the FXN promoter. We sought to determine if the severity of FXN promoter silencing is related to the length of the expanded GAA-TR mutation in FRDA. Methods Patient-derived lymphoblastoid cell lines bearing a range of expanded alleles (200–1122 triplets) were evaluated for FXN transcript levels by quantitative RT-PCR. FXN promoter function was directly measured by quantitative analysis of transcriptional initiation via metabolic labeling of newly synthesized transcripts in living cells. Results FXN transcriptional deficiency was significantly correlated with the length of the shorter of the two expanded alleles, which was noted both upstream (R2=0.84; p=0.014) and downstream (R2=0.89; p=0.002) of the expanded GAA-TR mutation, suggesting that FXN promoter silencing in FRDA is related to repeat length. A bilinear regression model revealed that length-dependence was strongest when the shorter of the two expanded alleles contained <400 triplets. Direct measurement of FXN promoter activity in patients with expanded alleles containing <400 versus >400 triplets in the shorter of the two expanded alleles revealed a significantly greater deficiency in individuals with longer GAA-TR alleles (p<0.05). Interpretation FXN promoter silencing in FRDA is dependent on the length of the expanded GAA-TR mutation.

No abstract

Friedreich ataxia, the most prevalent inherited ataxia, is caused by an expanded GAA triplet-repeat sequence in intron 1 of the FXN gene. Repressive chromatin spreads from the expanded GAA triplet-repeat sequence to cause epigenetic silencing of the FXN promoter via altered nucleosomal positioning and reduced chromatin accessibility. Indeed, deficient transcriptional initiation is the predominant cause of transcriptional deficiency in Friedreich ataxia. Treatment with 109, a class I histone deacetylase (HDAC) inhibitor, resulted in increased level of FXN transcript both upstream and downstream of the expanded GAA triplet-repeat sequence, without any change in transcript stability, suggesting that it acts via improvement of transcriptional initiation. Quantitative analysis of transcriptional initiation via metabolic labeling of nascent transcripts in patient-derived cells revealed a >3-fold increase (P < 0.05) in FXN promoter function. A concomitant 3-fold improvement (P < 0.001) in FXN promoter structure and chromatin accessibility was observed via Nucleosome Occupancy and Methylome Sequencing, a high-resolution in vivo footprint assay for detecting nucleosome occupancy in individual chromatin fibers. No such improvement in FXN promoter function or structure was observed upon treatment with a chemically-related inactive compound (966). Thus epigenetic promoter silencing in Friedreich ataxia is reversible, and the results implicate class I HDACs in repeat-mediated promoter silencing.

BackgroundFriedreich ataxia is caused by an expanded GAA triplet-repeat sequence in intron 1 of the FXN gene that results in epigenetic silencing of the FXN promoter. This silencing mechanism is seen in patient-derived lymphoblastoid cells but it remains unknown if it is a widespread phenomenon affecting multiple cell types and tissues.Methodology / Principal FindingsThe humanized mouse model of Friedreich ataxia (YG8sR), which carries a single transgenic insert of the human FXN gene with an expanded GAA triplet-repeat in intron 1, is deficient for FXN transcript when compared to an isogenic transgenic mouse lacking the expanded repeat (Y47R). We found that in YG8sR the deficiency of FXN transcript extended both upstream and downstream of the expanded GAA triplet-repeat, suggestive of deficient transcriptional initiation. This pattern of deficiency was seen in all tissues tested, irrespective of whether they are known to be affected or spared in disease pathogenesis, in both neuronal and non-neuronal tissues, and in cultured primary fibroblasts. FXN promoter function was directly measured via metabolic labeling of newly synthesized transcripts in fibroblasts, which revealed that the YG8sR mouse was significantly deficient in transcriptional initiation compared to the Y47R mouse.Conclusions / SignificanceDeficient transcriptional initiation accounts for FXN transcriptional deficiency in the humanized mouse model of Friedreich ataxia, similar to patient-derived cells, and the mechanism underlying promoter silencing in Friedreich ataxia is widespread across multiple cell types and tissues.