Expanded CTG repeat demarcates a boundary for abnormal CpG methylation in myotonic dystrophy patient tissues

Abstract

Myotonic dystrophy (DM1) affects multiple organs, shows age-dependent progression and is caused by CTG expansions at the DM1 locus. We determined the DM1 CpG methylation profile and CTG length in tissues from DM1 foetuses, DM1 adults, non-affected individuals and transgenic DM1 mice. Analysis included CTCF binding sites upstream and downstream of the CTG tract, as methylation-sensitive CTCF binding affects chromatinization and transcription of the DM1 locus. In humans, in a given foetus, expansions were largest in heart and smallest in liver, differing by 40-400 repeats; in adults, the largest expansions were in heart and cerebral cortex and smallest in cerebellum, differing by up to 5770 repeats in the same individual. Abnormal methylation was specific to the mutant allele. In DM1 adults, heart, liver and cortex showed high-to-moderate methylation levels, whereas cerebellum, kidney and skeletal muscle were devoid of methylation. Methylation decreased between foetuses and adults. Contrary to previous findings, methylation was not restricted to individuals with congenital DM1. The expanded repeat demarcates an abrupt boundary of methylation. Upstream sequences, including the CTCF site, were methylated, whereas the repeat itself and downstream sequences were not. In DM1 mice, expansion-, tissue- and age-specific methylation patterns were similar but not identical to those in DM1 individuals; notably in mice, methylation was present up- and downstream of the repeat, but greater upstream. Thus, in humans, the CpG-free expanded CTG repeat appears to maintain a highly polarized pattern of CpG methylation at the DM1 locus, which varies markedly with age and tissues.

Figure 1.

Detailed genomic and sequence location of the DNA regions analysed for CpG methylation. (A) View of the gene-enriched region in human chromosome 19 where the DMPK gene and the CTG repeat tract are enclosed. Pink shadow shows the human region (∼45 kb) single integrated in the DM1 transgenic mice genomes. (B) A more detailed view of the ∼45 kb region that includes the DMPK gene (20 or more than 300 CTG repeats in mice DM20-949 and DM300-328, respectively) and neighbouring genes. CpG islands through this region are marked in green, as derived from the UCSC Genome Browser (104). The regions previously analysed for methylation (black bars) (53,54) and the DNAase hypersensitive site detected upstream (yellow triangle) (27), coincident with the anti-sense transcription initiation reported in the DM1 locus (11), are also indicated. (C) The DMPK regions analysed by bisulphite sequencing PCR, located proximal and distal to the CTG repeat tract, are shown, as well as the CTCF binding sites 1 and 2 (grey shadows) (9). (D) DMPK sequence analysed by bisulphite sequencing PCR. PCR primers location, CTCF binding sites, CpG sites and HpaII, HhaI and SacII methylation-sensitive sites are indicated. For all sections, the CTG repeat is shown in red. Ctcf1 and ctcf2 primer sets for hemi-nested PCR amplification after DNA bisulphite treatment are shown.

Figure 2.

Tissue-specific CTG repeat sizing of the expanded allele in human DM1 samples. Representative LNA-Southerns (46) of some (A) foetal and (B) adult DM1 individuals. The range of CTG lengths in a given tissue was considerable and difficult to assess precisely as many were evident as smears (Table 1). As previously noted by others (7), the sensitivity to detect the range of smeared products depended on the amount of DNA loaded.

Figure 3.

CpG methylation profiles from unborn individuals. Top panel: non-DM1 foetus analysed (muscle, liver, kidney and heart); bottom panel: DM1 foetuses analysed including different tissue panels depending on the individual. DMPK alleles CTG repeat lengths (inside red box) and CTCF binding sites location (grey shadows) are indicated. White dots, CpG sites devoid of methylation; black dots, CpG sites with methylation presence.

Figure 4.

CpG methylation profiles from adult individuals. Top panel: non-DM1 adults analysed (cerebellum, skeletal muscle, cortex liver and heart); bottom panel: DM1 adults analysed including different tissue panels depending on the individual. DMPK alleles CTG repeat lengths (inside red box) and CTCF binding sites location (grey shadows) are indicated. White dots, CpG sites devoid of methylation; black dots, CpG sites with methylation presence.

Figure 5.

Assessment of non-CpG methylation through DM1-expanded alleles. DC-FEDM2 brain and heart samples were used by the strong difference between upstream (high methylated) and downstream (unmethylated) levels of CpG methylation (Fig. 3). DNA digestion with the non-methylation-sensitive enzymes AluI and Sau3AI (to free the repeat) and Fnu4HI (sensitive to CpNpG methylation) (top panel) was combined with LNA-Southern detection (46). No presence of high levels of methylation was detected (lanes 4, 6, 8 and 10, bottom panel). DNA fragments of 800 CTG repeats (methylated with the M.CviPI GpC methylase) and 500 CTG repeats (unmethylated) were included as controls for the methyl-sensitive Fnu4HI digestion, either alone (lanes 1 and 2) or with the DC-FEDM2 genomic DNA (lanes 7–10).

Figure 6.

Assessment of methylation allele specificity. (A) SacII methylation-sensitive digestion combined with multiplex PCR amplification was performed, using two upstream primers and one downstream primer to produce two PCR products from only the non-expanded CTG allele, that either did or did not encompass the methylation-sensitive SacII site present upstream of the CTG tract. Following SacII digestion, the elimination of only the PCR product encompassing the SacII site revealed that this site in the non-expanded allele was not methylated in the DC-FEDM1 (heart) and the UR-ADM5 (cortex) samples, whichby bisulphite analysis showed high levels of upstream methylation (Figs 3 and 4, respectively). Thus, the high levels of CpG methylation detected by bisulphite sequencing were exclusively present on the expanded DM1 allele.

Figure 7.

CpG methylation levels from other non-affected and DM1 samples. Methylation analysis was performed over some other samples, including fibroblast cells (DMPK493 and DMRB993, from a non-affected and DM1 individual, respectively), tissues from a DM1 individual with a short CTG repeat expansion (UR-ADM3) and blood samples from rare individuals with both alleles in the range of disease-associated lengths. More details of all these samples are given in Table 1. DMPK alleles, CTG repeat lengths (inside red box) and CTCF binding sites location (grey shadows) are indicated. White dots, CpG sites devoid of methylation; black dots, CpG sites with methylation presence. No data from the upstream region in the 06-2095 sample.

Figure 8.

DM1 transgenic mice CpG methylation profiles. Top panel: methylation from DM20-949 (20 repeats) mouse tissues; bottom panel: methylation from DM300-328 (more than 300 repeats) mice tissues. The analysis included 15 CpG sites upstream and 11 CpG sites downstream from the CTG repeat tract. White dots, CpG sites devoid of methylation; black dots, CpG sites with methylation presence; grey shadows, CTCF binding sites location. A pool of three to eight mice was analysed.

Figure 9.

Age-specific methylation in DM300-328 mice tissues. Methylation was assessed in 2 and 6 months DM300-328 mice in the presence of more than 300 CTG repeat tracts in the DMPK gene. Changes in methylation levels are indicated for upstream and downstream regions using +, − and =, for increases, decreases and minimal overall changes based on the number of CpG sites methylated.

Figure 10.

Tissue CTG repeat heterogeneity and CpG methylation levels in DM300-328 mice. (A) Methylation quantification (based on CpG sites’ height peaks in the chromatogram trace files using 4Peaks software, see Supplementary Material, Fig. S4). CpG sites devoid of methylation (white dots), with less than 50% methylation (grey dots) and from 50 to 100% methylation (black dots) CTCF binding sites location (grey shadows). (B) Somatic repeat heterogeneity profiles between different DM300-328 tissues in old mice (19 months) (99). *Unstable somatic tissues. (C) Methylation levels and repeats heterogeneity association in the tissues analysed from DM300-328 mice.