Friedreich ataxia patient tissues exhibit increased 5-hydroxymethylcytosine modification and decreased CTCF binding at the FXN locus.

Sahar Al-Mahdawi,Ricardo Mouro Pinto,Chiranjeevi Sandi,Mark A Pook

doi:10.1371/journal.pone.0074956

Sahar Al-Mahdawi, Ricardo Mouro Pinto + Show 2 more

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https://doi.org/10.1371/journal.pone.0074956

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Abstract

BackgroundFriedreich ataxia (FRDA) is caused by a homozygous GAA repeat expansion mutation within intron 1 of the FXN gene, which induces epigenetic changes and FXN gene silencing. Bisulfite sequencing studies have identified 5-methylcytosine (5mC) DNA methylation as one of the epigenetic changes that may be involved in this process. However, analysis of samples by bisulfite sequencing is a time-consuming procedure. In addition, it has recently been shown that 5-hydroxymethylcytosine (5hmC) is also present in mammalian DNA, and bisulfite sequencing cannot distinguish between 5hmC and 5mC.Methodology/Principal FindingsWe have developed specific MethylScreen restriction enzyme digestion and qPCR-based protocols to more rapidly quantify DNA methylation at four CpG sites in the FXN upstream GAA region. Increased DNA methylation was confirmed at all four CpG sites in both FRDA cerebellum and heart tissues. We have also analysed the DNA methylation status in FRDA cerebellum and heart tissues using an approach that enables distinction between 5hmC and 5mC. Our analysis reveals that the majority of DNA methylation in both FRDA and unaffected tissues actually comprises 5hmC rather than 5mC. We have also identified decreased occupancy of the chromatin insulator protein CTCF (CCCTC-binding factor) at the FXN 5’ UTR region in the same FRDA cerebellum tissues.Conclusions/SignificanceIncreased DNA methylation at the FXN upstream GAA region, primarily 5hmC rather than 5mC, and decreased CTCF occupancy at the FXN 5’ UTR are associated with FRDA disease-relevant human tissues. The role of such molecular mechanisms in FRDA pathogenesis has now to be determined.

Highlights

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative mitochondrial disorder caused primarily by a homozygous GAA repeat expansion mutation within intron 1 of the FXN gene [1]
Overall we show that increased 5hmC at the FXN upstream GAA region and decreased CTCF occupancy at the FXN 5’ UTR are both associated with FRDA disease-relevant human tissues
The MethylScreen method does not require bisulfite treatment of DNA, but involves digestion of genomic DNA with a methylation-sensitive restriction enzyme (MSRE), a methylation-dependent restriction enzyme (MDRE), and both a MSRE and a MDRE, followed by qPCR amplification of the residual non-digested DNA using an amplicon that spans the CpG site of interrogation

Summary

Introduction

FRDA is an autosomal recessive neurodegenerative mitochondrial disorder caused primarily by a homozygous GAA repeat expansion mutation within intron 1 of the FXN gene [1]. There is evidence that GAA repeat expansions can induce heterochromatin-mediated gene silencing effects [12] Consistent with the latter hypothesis, several FRDA-related. We have identified decreased occupancy of the chromatin insulator protein CTCF (CCCTC-binding factor) at the FXN 5’ UTR region in the same FRDA cerebellum tissues. Conclusions/Significance: Increased DNA methylation at the FXN upstream GAA region, primarily 5hmC rather than 5mC, and decreased CTCF occupancy at the FXN 5’ UTR are associated with FRDA disease-relevant human tissues. The role of such molecular mechanisms in FRDA pathogenesis has to be determined

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