Methylated and unmethylated epialleles support variegated epigenetic silencing in Friedreich ataxia.

Friedreich ataxia (FRDA) is typically caused by homozygosity for an expanded GAA triplet-repeat in intron 1 of the FXN gene, which results in transcriptional deficiency via epigenetic silencing. Most patients are homozygous for alleles containing > 500 triplets, but a subset (~20%) have at least one expanded allele with < 500 triplets and a distinctly milder phenotype. We show that in FRDA DNA methylation spreads upstream from the expanded repeat, further than previously recognized, and establishes an FRDA-specific region of hypermethylation in intron 1 (~90% in FRDA versus < 10% in non-FRDA) as a novel epigenetic signature. The hypermethylation of this differentially methylated region (FRDA-DMR) was observed in a variety of patient-derived cells; it significantly correlated with FXN transcriptional deficiency and age of onset, and it reverted to the non-disease state in isogenically corrected induced pluripotent stem cell (iPSC)-derived neurons. Bisulfite deep sequencing of the FRDA-DMR in peripheral blood mononuclear cells from 73 FRDA patients revealed considerable intra-individual epiallelic variability, including fully methylated, partially methylated, and unmethylated epialleles. Although unmethylated epialleles were rare (median = 0.33%) in typical patients homozygous for long GAA alleles with > 500 triplets, a significantly higher prevalence of unmethylated epialleles (median = 9.8%) was observed in patients with at least one allele containing < 500 triplets, less severe FXN deficiency (>20%) and later onset (>15 years). The higher prevalence in mild FRDA of somatic FXN epialleles devoid of DNA methylation is consistent with variegated epigenetic silencing mediated by expanded triplet-repeats. The proportion of unsilenced somatic FXN genes is an unrecognized phenotypic determinant in FRDA and has implications for the deployment of effective therapies.

The Cerebellar Cognitive Affective Syndrome in Ataxia-Telangiectasia.

Ataxia-telangiectasia (AT) is an autosomal recessive, multisystem disease causing cerebellar ataxia, mucocutaneous telangiectasias, immunodeficiency, and malignancies. A pilot study reported cognitive and behavioral manifestations characteristic of the cerebellar cognitive affective / Schmahmann syndrome (CCAS). We set out to test and further define these observations because a more comprehensive understanding of the spectrum of impairments in AT is essential for optimal management. Twenty patients (12 males; 9.86 ± 5.5 years, range 4.3 to 23.2) were grouped by age: AT-I (toddlers and preschoolers, n = 7, 4.3-5.9 years), AT-II (school children, n = 7, 5.9-9.8 years), AT-III (adolescents/young adults, n = 6, 12.6-23.2 years). Standard and experimental tests investigated executive, linguistic, visual-spatial, and affective/social-cognitive domains. Results were compared to standard norms and healthy controls. Cognitive changes in AT-I were limited to mild visual-spatial disorganization. Spatial deficits were greater in AT-II, with low average scores on executive function (auditory working memory), expressive language (vocabulary), academic abilities (math, spelling, reading), social cognition (affect recognition from faces), and emotional/psychological processing. Full Scale IQ scores were low average to borderline impaired. AT-III patients had the greatest level of deficits which were evident particularly in spatial skills, executive function (auditory working memory, sequencing, word/color interference, set-shifting, categorization errors, perseveration), academic achievement, social cognition (affect recognition from faces), and behavioral control. Full Scale IQ scores in this group fell in the impaired range, while language was borderline impaired for comprehension, and low average for expression. Cognitive deficits in AT at a young age are mild and limited to visual-spatial functions. More widespread cognitive difficulties emerge with age and disease progression, impacting executive function, spatial skills, affect, and social cognition. Linguistic processing remains mildly affected. Recognition of the CCAS in children with AT may facilitate therapeutic interventions to improve quality of life.

Reversal of epigenetic promoter silencing in Friedreich ataxia by a class I histone deacetylase inhibitor.

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.

Altered nucleosome positioning at the transcription start site and deficient transcriptional initiation in Friedreich ataxia.

Most individuals with Friedreich ataxia (FRDA) are homozygous for an expanded GAA triplet repeat (GAA-TR) mutation in intron 1 of the FXN gene, which results in deficiency of FXN transcript. Consistent with the expanded GAA-TR sequence as a cause of variegated gene silencing, evidence for heterochromatin has been detected in intron 1 in the immediate vicinity of the expanded GAA-TR mutation in FRDA. Transcriptional deficiency in FRDA is thought to result from deficient elongation through the expanded GAA-TR sequence because of repeat-proximal heterochromatin and abnormal DNA structures adopted by the expanded repeat. There is also evidence for deficient transcriptional initiation in FRDA, but its relationship to the expanded GAA-TR mutation remains unclear. We show that repressive chromatin extends from the expanded GAA-TR in intron 1 to the upstream regions of the FXN gene, involving the FXN transcriptional start site. Using a chromatin accessibility assay and a high-resolution nucleosome occupancy assay, we found that the major FXN transcriptional start site, which is normally in a nucleosome-depleted region, is rendered inaccessible by altered nucleosome positioning in FRDA. Consistent with the altered epigenetic landscape the FXN gene promoter, a typical CpG island promoter, was found to be in a transcriptionally non-permissive state in FRDA. Both metabolic labeling of nascent transcripts and an unbiased whole transcriptome analysis revealed a severe deficiency of transcriptional initiation in FRDA. Deficient transcriptional initiation, and not elongation, is the major cause of FXN transcriptional deficiency in FRDA, and it is related to the spread of repressive chromatin from the expanded GAA-TR mutation.

Epigenetic promoter silencing in Friedreich ataxia is dependent on repeat length.

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 to 1,300 triplets, and phenotypic severity is significantly correlated with the length of the shorter of the 2 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 whether 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 (200-1,122 triplets) were evaluated for FXN transcript levels by quantitative reverse transcriptase polymerase chain reaction. 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 2 expanded alleles, which was noted both upstream (R(2) = 0.84, p = 0.014) and downstream (R(2) = 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 2 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 2 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.

Deficiency of RecA-dependent RecFOR and RecBCD pathways causes increased instability of the (GAA*TTC)n sequence when GAA is the lagging strand template.

The most common mutation in Friedreich ataxia is an expanded (GAA*TTC)n sequence, which is highly unstable in human somatic cells and in the germline. The mechanisms responsible for this genetic instability are poorly understood. We previously showed that cloned (GAA*TTC)n sequences replicated in Escherichia coli are more unstable when GAA is the lagging strand template, suggesting erroneous lagging strand synthesis as the likely mechanism for the genetic instability. Here we show that the increase in genetic instability when GAA serves as the lagging strand template is seen in RecA-deficient but not RecA-proficient strains. We also found the same orientation-dependent increase in instability in a RecA+ temperature-sensitive E. coli SSB mutant strain (ssb-1). Since stalling of replication is known to occur within the (GAA*TTC)n sequence when GAA is the lagging strand template, we hypothesized that genetic stability of the (GAA*TTC)n sequence may require efficient RecA-dependent recombinational restart of stalled replication forks. Consistent with this hypothesis, we noted significantly increased instability when GAA was the lagging strand template in strains that were deficient in components of the RecFOR and RecBCD pathways. Our data implicate defective processing of stalled replication forks as a mechanism for genetic instability of the (GAA*TTC)n sequence.

FXN Promoter Silencing in the Humanized Mouse Model of Friedreich Ataxia.

BACKGROUND: Friedreich 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 FINDINGS: The 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 / SIGNIFICANCE: Deficient 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.

Epigenetic silencing in Friedreich ataxia is associated with depletion of CTCF (CCCTC-binding factor) and antisense transcription.

BACKGROUND: Over 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 FINDINGS: We 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 (FXNAntisense 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/SIGNIFICANCE: CTCF 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.