Excision of Expanded GAA Repeats Alleviates the Molecular Phenotype of Friedreich's Ataxia

Li, Yanjie; Polak, Urszula; Bhalla, Angela D.; Rozwadowska, Natalia; Butler, Jill Sergesketter; Lynch, David R.; Dent, Sharon Y.R.; Napierala, Marek

doi:10.1038/mt.2015.41

Cited by 83 publications

(89 citation statements)

References 52 publications

(67 reference statements)

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“…Retroviral transgene expression in the established iPSC lines was analyzed exactly as described in Ref. [34]. Briefly, the expression levels of the OSKM transcription factors were determined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR).…”

Section: Human Ipsc Characterizationmentioning

confidence: 99%

“…Silencing of the retroviral transgenes was also confirmed in resulting iPSCs as described in Ref. [34].…”

Section: Modulation Of the Epigenetic Environment During Reprogramminmentioning

confidence: 99%

“…Reactions were performed as described in Refs. [34,36] using the FailSafe PCR System and mix D (cat. FS99100; Epicentre).…”

Section: Amplification Of the Gaa Repeat Regionmentioning

confidence: 99%

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Alleviating GAA Repeat Induced Transcriptional Silencing of the Friedreich's Ataxia Gene During Somatic Cell Reprogramming

Polak

Butler

et al. 2016

Stem Cells and Development

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Friedreich's ataxia (FRDA) is the most common autosomal recessive ataxia. This severe neurodegenerative disease is caused by an expansion of guanine-adenine-adenine (GAA) repeats located in the first intron of the frataxin (FXN) gene, which represses its transcription. Although transcriptional silencing is associated with heterochromatin-like changes in the vicinity of the expanded GAAs, the exact mechanism and pathways involved in transcriptional inhibition are largely unknown. As major remodeling of the epigenome is associated with somatic cell reprogramming, modulating chromatin modification pathways during the cellular transition from a somatic to a pluripotent state is likely to generate permanent changes to the epigenetic landscape. We hypothesize that the epigenetic modifications in the vicinity of the GAA repeats can be reversed by pharmacological modulation during somatic cell reprogramming. We reprogrammed FRDA fibroblasts into induced pluripotent stem cells (iPSCs) in the presence of various small molecules that target DNA methylation and histone acetylation and methylation. Treatment of FRDA iPSCs with two compounds, sodium butyrate (NaB) and Parnate, led to an increase in FXN expression and correction of repressive marks at the FXN locus, which persisted for several passages. However, prolonged culture of the epigenetically modified FRDA iPSCs led to progressive expansions of the GAA repeats and a corresponding decrease in FXN expression. Furthermore, we uncovered that differentiation of these iPSCs into neurons also results in resilencing of the FXN gene. Taken together, these results demonstrate that transcriptional repression caused by long GAA repeat tracts can be partially or transiently reversed by altering particular epigenetic modifications, thus revealing possibilities for detailed analyses of silencing mechanism and development of new therapeutic approaches for FRDA.

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Section: Human Ipsc Characterizationmentioning

confidence: 99%

“…Silencing of the retroviral transgenes was also confirmed in resulting iPSCs as described in Ref. [34].…”

Section: Modulation Of the Epigenetic Environment During Reprogramminmentioning

confidence: 99%

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Alleviating GAA Repeat Induced Transcriptional Silencing of the Friedreich's Ataxia Gene During Somatic Cell Reprogramming

Polak

Butler

et al. 2016

Stem Cells and Development

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“…The use of engineered nucleases enables the generation of disease models by conveniently introducing DNA repeats into a specific region [92], removing disease-causing expanded DNA repeats [93,94], or neutralizing the effect of expanded DNA repeats [95].…”

Section: Genome Editing Of Disease-related Trinucleotide Repeatsmentioning

confidence: 99%

“…For Friedreich's ataxia (FRDA), regions containing GAA repeats from frataxin (FXN) intron 1 were removed by expressing ZFNs targeting 334 bp and 896 bp upstream of GAA repeats in FRDA fibroblasts and lymphoblasts ( Figure 3B) [93]. The FRDA phenotype was reversed, with increased frataxin expression, when corrected FRDA fibroblasts were converted to iPSCs and differentiated into neurons.…”

Section: Genome Editing Of Disease-related Trinucleotide Repeatsmentioning

confidence: 99%

Genome Editing of Structural Variations: Modeling and Gene Correction

Park

Sung

Kim

2016

Trends in Biotechnology

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Deep sequencing of mitochondrial genomes reveals increased mutation load in Friedreich's ataxia

Bhalla

Khodadadi‐Jamayran

et al. 2016

Ann Clin Transl Neurol

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ObjectiveFriedreich's ataxia (FRDA) is an autosomal recessive trinucleotide repeat expansion disorder caused by epigenetic silencing of the frataxin gene (FXN). Current research suggests that damage and variation of mitochondrial DNA (mtDNA) contribute to the molecular pathogenesis of FRDA. We sought to establish the extent of the mutation burden across the mitochondrial genome in FRDA cells and investigate the molecular mechanisms connecting FXN downregulation and the acquisition of mtDNA damage.MethodsDamage and mutation load in mtDNA of a panel of FRDA and control fibroblasts were determined using qPCR and next‐generation MiSeq sequencing, respectively. The capacity of FRDA and control cells to repair oxidative lesions in their mtDNA was measured using a quantitative DNA damage assay. Comprehensive RNA sequencing gene expression analyses were conducted to assess the status of DNA repair and metabolism genes in FRDA cells.ResultsAcute or prolonged downregulation of FXN expression resulted in a significant increase in mtDNA damage that translated to a significant elevation of mutation load in mtDNA. The predominant mutations identified throughout the mtDNA were C>T, G>A transitions (P = 0.007). Low FXN expression reduced capacity to repair oxidative damage in mtDNA. Downregulation of FXN expression strongly correlated (r = 0.73) with decreased levels of base excision repair (BER) DNA glycosylase NTHL1.InterpretationDownregulation of FXN expression in FRDA cells elevates mtDNA damage, increases mutation load of the mitochondrial genome, and diminishes DNA repair capacity. Progressive accumulation of mtDNA mutations in vulnerable FRDA patient cells reduces mitochondrial fitness ultimately leading to cell death.

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Excision of Expanded GAA Repeats Alleviates the Molecular Phenotype of Friedreich's Ataxia

Cited by 83 publications

References 52 publications

Alleviating GAA Repeat Induced Transcriptional Silencing of the Friedreich's Ataxia Gene During Somatic Cell Reprogramming

Alleviating GAA Repeat Induced Transcriptional Silencing of the Friedreich's Ataxia Gene During Somatic Cell Reprogramming

Genome Editing of Structural Variations: Modeling and Gene Correction

Deep sequencing of mitochondrial genomes reveals increased mutation load in Friedreich's ataxia

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