Allele-specific DNA hypomethylation characterises FSHD1 and FSHD2.

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is associated with an epigenetic defect on 4qter. Two clinically indistinguishable forms of FSHD are known, FSHD1 and FSHD2. FSHD1 is caused by contraction of the highly polymorphic D4Z4 macrosatellite repeat array on chromosome 4q35. FSHD2 is caused by pathogenic mutations of the SMCHD1 gene.Both genetic defects lead to D4Z4 DNA hypomethylation. In the presence of a polymorphic polyadenylation signal (PAS), DNA hypomethylation leads to inappropriate expression of the D4Z4-encoded DUX4 transcription factor in skeletal muscle. Currently, hypomethylation is not diagnostic per se because of the interference of non-pathogenic arrays and the lack of information about the presence of DUX4-PAS. METHODS: We investigated, by bisulfite sequencing, the DNA methylation levels of the region distal to the D4Z4 array selectively in PAS-positive alleles. RESULTS: Comparison of FSHD1, FSHD2 and Control subjects showed a highly significant difference of methylation levels in all CpGs tested. Importantly, using a cohort of 112 samples, one of these CpGs (CpG6) is able to discriminate the affected individuals with a sensitivity of 0.95 supporting this assay potential for FSHD diagnosis. Moreover, our study showed a relationship between PAS-specific methylation and severity of the disease. CONCLUSIONS: These data point to the CpGs distal to the D4Z4 array as a critical region reflecting multiple factors affecting the epigenetics of FSHD. Additionally, methylation analysis of this region allows the establishment of a rapid and sensitive tool for FSHD diagnosis.

Highly efficient, in vivo optimized, archaeal endonuclease for controlled RNA splicing in mammalian cells.

ARCHAEA-ExPRESs is an mRNA modification technology that makes use of components derived from the Archaeon Methanocaldococcus jannaschii, namely the tRNA splicing endonuclease (MJ-EndA) and its natural substrate, the bulge-helix-bulge (BHB) structure (1). These components can perform both cis- and trans-splicing in cellular and animal models and may provide a convenient way to modulate gene expression using components independent of cellular regulatory networks. To use MJ-EndA in stable expression mammalian systems, we developed variants characterized by high efficiency and sustainable in vivo activity. The MJ-EndA variants were created by the introduction of proper localization signals followed by mutagenesis and direct selection in mammalian cells. Of note, enzyme selection used an in vivo selection method based on puromycin resistance conferred to cells by BHB-mediated intron splicing from an out-of-frame puromycin N-acetyl transferase (PAC) gene. This approach yielded several endonuclease variants, the best of which showed 40-fold higher activity compared to the parental enzyme and stable processing of 30% of the target mRNA. Notably, these variants showed complete compatibility with long-term expression in mammalian cells, suggesting that they may be usefully applied in functional genomics and genetically modified animal models.

Estrogens enhance myoblast differentiation in facioscapulohumeral muscular dystrophy by antagonizing DUX4 activity.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder that is characterized by extreme variability in symptoms, with females being less severely affected than males and presenting a higher proportion of asymptomatic carriers. The sex-related factors involved in the disease are not known. Here, we have utilized myoblasts isolated from FSHD patients (FSHD myoblasts) to investigate the effect of estrogens on muscle properties. Our results demonstrated that estrogens counteract the differentiation impairment of FSHD myoblasts without affecting cell proliferation or survival. Estrogen effects are mediated by estrogen receptor ß (ERß), which reduces chromatin occupancy and transcriptional activity of double homeobox 4 (DUX4), a protein whose aberrant expression has been implicated in FSHD pathogenesis. During myoblast differentiation, we observed that the levels and activity of DUX4 increased progressively and were associated with its enhanced recruitment in the nucleus. ERß interfered with this recruitment by relocalizing DUX4 in the cytoplasm. This work identifies estrogens as a potential disease modifier that underlie sex-related differences in FSHD by protecting against myoblast differentiation impairments in this disease.