Epigenetic promoter silencing in Friedreich ataxia is dependent on repeat length

Abstract

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.

Friedreich Ataxia: current status and future prospects

“…The latter is rendered transcriptionally non-permissive causing defective transcriptional initiation [73–75]. The repeat length directly influences the extent of promotor silencing and the deficiency of transcriptional initiation [76]. There is also evidence for moderately impaired transcriptional elongation [77].…”

Friedreich's ataxia – a case of aberrant transcription termination?

“…promoter is affected by spreading of the heterochromatin-like environment toward the transcription initiation region. 11,12 Other studies indicated that transcription elongation rather than initiation is critically affected by expanded GAAs. 10,13,14 RNA polymerase II (RNAP II) profiling data (both total and serine 2 phosphorylated RNAP II) showed that RNAP II recruitment and transcription initiation proceed with similar efficiency in both control as well as in FRDA cells.…”

Translating HDAC inhibitors in Friedreich’s ataxia

“…More recently, Bidichandani and colleagues have provided strong evidence that initiation of transcription at pathogenic FXN alleles is impaired by the presence of a nucleosome over the start-site both in patient cells [49] and in a mouse model [50]. Interestingly, the degree of promoter silencing and chromatin compaction is directly related to the length of the GAA•TTC repeats [51]. …”

“…While HDAC inhibitors 3 (N 1 -(4-aminobiphenyl-3-yl)-N 7 -phenylheptanediamide [68], Figure 3) and 233 (N-(2-amino-5- (2-thienyl)phenyl)-7-nicotinoylamino-heptanamide [71], Figure 3) are potent inhibitors of recombinant HDAC1 and in cells, these compounds are without effect on FXN gene expression either in lymphoblasts [68] or iPSC-derived neurons [31]. Also an HDAC3-selective inhibitor (with a fluorine at the 4- position of the benzamide ring; 966 ; ( E )-N-(2-amino-4-fluorophenyl)-3-(1-cinnamyl-1 H -pyrazol-4-yl)acrylamide; ~30-fold selectivity for HDAC3 over HDAC1/2 [72], Figure 3) is without effect on FXN mRNA levels in lymphoblasts [51] and in neuronal cells [31]. Potent inhibitors of class II and class III (sirtuin) HDACs also fail to activate FXN expression in the FRDA cells, although each of these compounds is active against known substrates [68].…”