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.

Repeat instability during DNA repair: Insights from model systems

“…In a yeast study, cells heterozygous for Fen1/Rad27 maintained (CAG) 70-130 repeat lengths; expansions increased only when the tract reached 155 repeats, and the frequency was still less than in fen1 Δ cells (Yang & Freudenreich, 2007). In a mouse model of DM1, a hypomorphic mutation of DNA ligase 1 (46BR), reduced intergenerational repeat expansions and increased contractions but only when the expansion prone allele was maternally transmitted (Lopez Castel et al ., 2011). However, the same mutation had no effect on intergenerational and somatic expansions in the FX mouse model (Entezam et al ., 2010).…”

Aberrant splicing and expression of the non muscle myosin heavy-chain gene MYH14 in DM1 muscle tissues

“…The expression of the NMHC II-C inserted isoform is therefore also lowered by the concomitant reduction of the MYH14 transcript and protein product in DM1 tissues. We suggest that this deleterious effect could be mediated by the CUGcontaining RNA expansions through a transcriptional mechanism driven by direct binding of basic transcription factors (TFs), by epigenetic modifications of the DM1 locus (Ebralidze et al, 2004;López Castel et al, 2011), or may be due to a reduced stability of the MYH14 transcript in DM1 tissues. Although there is considerable information concerning the role of myosins in cytokinesis (Krendel & Mooseker, 2005;Sellers, 2000), little is known about the individual role of each of the NMHC isoforms in this process.…”

Epigenetic modifications in trinucleotide repeat diseases

“…In tissues isolated postmortem from adults with DM1, upstream of the TNR was methylated, but at a lower level in comparison to the fetus samples, with no methylation observed in the cerebellum and skeletal muscle. Both DM1 fetus and adult samples were not methylated downstream of the TNR [85]. Earlier reports show conflicting data, but these studies used small sample cohorts and different tissues [86][87][88].…”

“…This destabilization of repeat number by DNMT inhibitors supports a role for epigenetic processes in regulating the TNR expansion length and therefore modulation of disease phenotype. Altered DNA methylation of DMPK has been identified in DM1 individuals where the expanded TNR demarcated differential methylation -upstream elements were highly methylated, whereas downstream elements were not methylated [85]. In two DM1 fetuses, high levels of methylation upstream of the expanded CTG were observed in the liver, heart, and brain.…”