Friedreich Ataxia Patient Tissues Exhibit Increased 5-Hydroxymethylcytosine Modification and Decreased CTCF Binding at the FXN Locus

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.

Generation and Characterisation of Friedreich Ataxia YG8R Mouse Fibroblast and Neural Stem Cell Models

“…MethylScreen was used to analyse the two CpG sites, CpG3 and CpG6 [38], at FXN locus upstream of the GAA repeat. 1 µg of genomic DNA was digested with: (1) a MSRE, (2) MDRE, (3) both MSRE and MDRE (double digest, DD), and (4) neither MSRE or MDRE (mock control).…”

Playing TETris with DNA modifications

“…Upstream from the repeats lay CpGs that appear hypermethylated in patients suffering from the disease. In the studies concerned, however, the techniques used to map 5mC could not distinguish this mark from 5hmC (Campuzano et al , ; Saveliev et al , ; Al‐Mahdawi et al , ). In 2013, Al‐Mahdawi et al () addressed this issue, showing that nearly all the methylcytosine is in fact hydroxymethylcytosine in both FRDA and normal human cerebellar tissues, and that 5hmC is more abundant in the former than in the latter.…”

“…In the studies concerned, however, the techniques used to map 5mC could not distinguish this mark from 5hmC (Campuzano et al , ; Saveliev et al , ; Al‐Mahdawi et al , ). In 2013, Al‐Mahdawi et al () addressed this issue, showing that nearly all the methylcytosine is in fact hydroxymethylcytosine in both FRDA and normal human cerebellar tissues, and that 5hmC is more abundant in the former than in the latter. They further hypothesized that the observed 5hmC increase might enhance production of FAST‐1 antisense RNA, which could in turn mediate heterochromatin formation and FXN downregulation, causing the typical symptoms of the disease.…”

The emerging role of 5-hydroxymethylcytosine in neurodegenerative diseases

“…Therefore, there is good evidence that DNA methylation may have an, as yet unknown, role to play in the molecular mechanism of FRDA. With this in mind, our group have recently analyzed the 5hmC status of one of the FXN upstream GAA CpG sites in FRDA cerebellum and heart tissues using a restriction enzyme-based procedure that allowed distinction between 5hmC and 5mC and we found that the majority of the hypermethylated DNA at this CpG residue comprises 5hmC rather than 5mC (Al-Mahdawi et al, 2013 ) (Table 1 ). It is possible that raised 5hmC levels reflect an attempt to reverse GAA repeat-induced FXN gene silencing, which is marked by increased DNA methylation at the upstream GAA repeat region, rather than indicating any involvement in the cause of disease.…”