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

Abstract

Background: Friedreich 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 (5 mC) 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 (5 hmC) is also present in mammalian DNA, and bisulfite sequencing cannot distinguish between 5 hmC and 5 mC.

Methodology/principal findings: We 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 5 hmC and 5 mC. Our analysis reveals that the majority of DNA methylation in both FRDA and unaffected tissues actually comprises 5 hmC rather than 5 mC. 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/significance: Increased DNA methylation at the FXN upstream GAA region, primarily 5 hmC rather than 5 mC, 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.

Figure 1. FXN region of analysis.

Schematic diagram of 2.5kb of the FXN exon 1 and intron 1 region showing the GAA repeat within an Alu sequence (light grey box), together with the regions of 5mC/5hmC MethylScreen (dark grey box) and 5hmC EpiMarkTM (black box) analysis. The numbers represent the CpG sites, which are numbered according to [13]. Asterisks indicate the positions of the three potential CTCF binding sites that were analysed by EMSA.

Figure 2. DNA methylation levels in cerebellum.

MethylScreen analysis of four CpG sites in the FXN upstream GAA repeat region of DNA from FRDA and control cerebellum tissues. UM = unmethylated, IM = intermediately methylated, DM = densely methylated. Error bars = s.e.m. n=4.

Figure 3. DNA methylation levels in heart.

MethylScreen analysis of four CpG sites in the FXN upstream GAA repeat region of DNA from FRDA and control heart tissues. UM = unmethylated, IM = intermediately methylated, DM = densely methylated. Error bars = s.e.m. n=4.

Figure 4. 5hmC levels in cerebellum and heart.

EpiMark^TM 5hmC and 5mC analysis of CpG site 12 in the FXN upstream GAA repeat region of DNA from FRDA and control cerebellum (A) and heart (B) tissues. G = glucosylated DNA. MspI values represent complete digestion control levels of DNA; MspI + G represents 5hmC levels, HpaII and HpaII + G represent combined 5mC and 5hmC levels; Mock and Mock +G represent undigested control levels of DNA. Error bars = s.e.m. n=4.

Figure 5. CTCF analysis.

(A) EMSA showing a mobility shift for the FXN 5’ UTR probe and CTCF-positive DM1 (1) probe [35], while there is no such shift detected for the FXN upstream GAA (Up) probe, FXN downstream GAA (Down) probe or CTCF-negative DM1 (3) probe [35]. (B) ChIP analysis showing the relative CTCF occupancy in the FXN 5’ UTR of DNA from FRDA and control cerebellum tissues. Error bars = s.e.m. n=3. * = P<0.05.