DNA Methylation Analysis of the Macrosatellite Repeat Associated with FSHD Muscular Dystrophy at Single Nucleotide Level

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common inherited diseases of the skeletal muscle. It is characterized by asymmetric muscle weakness and variable penetrance. FSHD is linked to a reduction in copy number of the D4Z4 3.3 kb macrosatellite repeat, located in 4q35. This causes the epigenetic de-repression of FSHD candidate genes leading to disease. Nevertheless, the molecular mechanism responsible for silencing of FSHD candidate genes in healthy subjects is not fully understood. While a role for DNA methylation has been suggested, so far there is limited information regarding the methylation status of the 325 CpGs contained in each D4Z4 unit. Using a human/rodent monochromosomal hybrid cell line containing a single human chromosome 4, we performed an in depth analysis of DNA methylation for the majority of the CpGs inside D4Z4 at single nucleotide level. We found that D4Z4 is not uniformly methylated and that the level of DNA methylation does not correlate with the density of CpG dinucleotides. Moreover, in several D4Z4 regions characterized by near complete methylation, we found specific unmethylated CpGs. These elements are enriched in transcription factor binding sites that could be involved in muscle-specific D4Z4 activity. Our approach also detected differential methylation among different D4Z4 units, suggesting that the D4Z4 array is a mosaic of euchromatic and heterochromatic domains. Finally, we found that DNA methylation and histone de-acetylation are required to maintain FSHD candidate genes repressed. Taken together, our data underscore new players involved in the epigenetic regulation of the FSHD locus that could be targeted for therapeutic purposes.

Protein kinase A activation inhibits DUX4 gene expression in myotubes from patients with facioscapulohumeral muscular dystrophy

“…FSHD1, which accounts for 95% of FSHD cases, is an autosomal dominant disease caused mainly by a DNA contraction of the D4Z4 macrosatellite repeat elements found on human chromosome 4, specifically 4q35 (8,11,12). This D4Z4 repeat reduction results in epigenetic changes at the locus, such as DNA hypomethylation within the D4Z4 repeats and the surrounding locus (13)(14)(15) as well as the loss of repressive histone methyl marks (16). It was postulated that the change in chromosomal conformation from heterochromatin to euchromatin facilitates access to DUX4 by as of yet unidentified transcriptional activators.…”

Does DNA Methylation Matter in FSHD?

“…It is noteworthy that YY1 binds DNA only when it is not methylated [105]. The region encompassing the DBE element is hypermethylated in FSHD and control subjects, except from the CpGs neighboring the YY1 consensus site, which show variable levels of methylation (Tupler, personal observation and [106]). Conversely, CTCF binding is disrupted by CpG methylation [107].…”

“…Conversely, CTCF binding is disrupted by CpG methylation [107]. Two CTCF binding sites are located within the 5 region of D4Z4 repeat, which is reported to display hypomethylation in the presence of a DRA [74,101,106,108]. CTCF binding to 4q35 displays an inverse correlation to D4Z4 copy number and function as a Lamin A/C-dependent chromatin insulator, protecting D4Z4 from epigenetic silencing by surrounding heterochromatic regions, therefore keeping the D4Z4 chromatin open primarily in a DRA context [102].…”

Reduced representation optical methylation mapping (R2OM2)

“…Commonly, healthy individuals carry an array of more than 10 repeats. However, even long arrays result in FSHD symptoms when losing their methylation (termed FSHD2) while carriers of short but highly methylated repeat arrays, do not manifest the disease 15,16 . The multiple combinations of copy number and methylation level span a broad range of possible variations which are correlated with the disease severity and manifestation 16,17 .…”

“…In this context, it has been shown that methylation levels of repetitive DNA can regulate repeat-related genetic diseases 12,13 , and are correlated with various types of cancer and their severity 14 . One striking example of a repeat array, addressed in this work, is facioscapulohumeral muscular dystrophy (FSHD), one of the most commons form of muscular dystrophy, affecting approximately 1 in 7,000-20,000 individuals [15][16][17][18] .…”