Abstract
BackgroundOver 15 inherited diseases are caused by expansion of triplet-repeats. Friedreich ataxia (FRDA) patients are homozygous for an expanded GAA triplet-repeat sequence in intron 1 of the FXN gene. The expanded GAA triplet-repeat results in deficiency of FXN gene transcription, which is reversed via administration of histone deacetylase inhibitors indicating that transcriptional silencing is at least partially due to an epigenetic abnormality.Methodology/Principal FindingsWe found a severe depletion of the chromatin insulator protein CTCF (CCCTC-binding factor) in the 5′UTR of the FXN gene in FRDA, and coincident heterochromatin formation involving the +1 nucleosome via enrichment of H3K9me3 and recruitment of heterochromatin protein 1. We identified FAST-1 (FXN Antisense Transcript – 1), a novel antisense transcript that overlaps the CTCF binding site in the 5′UTR, which was expressed at higher levels in FRDA. The reciprocal relationship of deficient FXN transcript and higher levels of FAST-1 seen in FRDA was reproduced in normal cells via knockdown of CTCF.Conclusions/SignificanceCTCF depletion constitutes an epigenetic switch that results in increased antisense transcription, heterochromatin formation and transcriptional deficiency in FRDA. These findings provide a mechanistic basis for the transcriptional silencing of the FXN gene in FRDA, and broaden our understanding of disease pathogenesis in triplet-repeat diseases.
Highlights
Friedreich ataxia (FRDA), the most common inherited ataxia, is an autosomal recessive disease characterized by progressive sensory ataxia, cardiomyopathy, diabetes, and premature death [1]
The same Chromatin immunoprecipitation (ChIP) assay performed with fibroblast cell lines from two FRDA patients, who were homozygous for expanded GAA tripletrepeat sequences in intron 1 of the FXN gene, showed four-fold reduced occupancy of CTCF in the 59 untranslated sequence (59UTR) (Fig. 1D)
Electrophoretic mobility shift assay (EMSA) performed with HeLa nuclear extract and an in vitro methylated 59UTR probe showed the same major complex obtained with the unmethylated probe, which was competed away by excess cold unmethylated probe (Fig. S2), indicating that altered DNA methylation is unlikely to be the reason for CTCF depletion in the FXN 59UTR in FRDA
Summary
Friedreich ataxia (FRDA), the most common inherited ataxia, is an autosomal recessive disease characterized by progressive sensory ataxia, cardiomyopathy, diabetes, and premature death [1]. In unrelated transgenic mouse experiments, the expanded GAA triplet-repeat sequence was found to be a source of position effect variegation (PEV) i.e., a source of heterochromatin spreading into adjacent euchromatin [5]. Histone deacetylase (HDAC) inhibitors resulted in partial reversal of the gene silencing in patient-derived cells [6], indicating that heterochromatin formation is an important underlying mechanism for the transcriptional deficiency in FRDA. The expanded GAA triplet-repeat results in deficiency of FXN gene transcription, which is reversed via administration of histone deacetylase inhibitors indicating that transcriptional silencing is at least partially due to an epigenetic abnormality
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