Epigenetic variability is a modifier of facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD), the most prevalent myopathy afflicting both children and adults, is strongly associated with epigenetic changes of the 4q35-localized macrosatellite D4Z4 repeat. Recent studies propose that FSHD pathology is caused by the misexpression and missplicing of the DUX4 (double homeobox 4) gene, encoded within the repeat array, resulting in production of a pathogenic protein, DUX4-FL. We have analyzed DUX4 mRNA and protein expression in a large collection of myogenic cells and muscle biopsies derived from muscles of FSHD1 affected subjects and their unaffected first-degree relatives. We confirmed that stable DUX4-fl mRNA and protein were expressed in myogenic cells and muscle tissues derived from FSHD affected subjects, including several genetically diagnosed adults yet to show clinical manifestations of the disease; however, there was great individual and familial variation in the levels of DUX4-FL. In addition, we found DUX4-fl mRNA and protein expression in muscle biopsies and myogenic cells from genetically unaffected relatives of the FSHD subjects, although at a significantly lower frequency. These results establish that DUX4-fl expression per se is not sufficient for FSHD muscle pathology. To investigate if subtle differences in the epigenetic status of the 4q35 region could account for the wide variation in DUX4-fl expression among FSHD1 subjects and potentially the spurious expression in certain unaffected controls, family cohorts of myogenic cells from FSHD1 subjects were tested for their sensitivity to small molecules that can alter the chromatin state. We find that myogenic cells from FSHD1 subjects are overall epigenetically poised to express DUX4 compared with unaffected subjects; however, FSHD1 subjects show individual differences in their capacity to express DUX4-fl in response to DNA demethylation and blocking histone deacetylation. Therefor, individual differences in the epigenetic status likely impacts progression of disease pathology, variability in age of onset, disease severity, and asymmetry of affected muscles

Myogenic Enhancers Regulate Expression of the Facioscapulohumeral Muscular Dystrophy-Associated DUX4 Gene

Facioscapulohumeral muscular dystrophy (FSHD) is linked to epigenetic dysregulation of the chromosome 4q35 D4Z4 macrosatellite. However, this does not account for the tissue specificity of FSHD pathology, which requires stable expression of an alternative full-length mRNA splice form of DUX4 (DUX4-fl) from the D4Z4 array in skeletal muscle. Here, we describe the identification of two enhancers, DUX4 myogenic enhancer 1 (DME1) and DME2 which activate DUX4-fl expression in skeletal myocytes but not fibroblasts. Analysis of the chromatin revealed histone modifications and RNA polymerase II occupancy consistent with DME1 and DME2 being functional enhancers. Chromosome conformation capture analysis confirmed association of DME1 and DME2 with the DUX4 promoter in vivo. The strong interaction between DME2 and the DUX4 promoter in both FSHD and unaffected primary myocytes was greatly reduced in fibroblasts, suggesting a muscle-specific interaction. Nucleosome occupancy and methylome sequencing analysis indicated that in most FSHD myocytes, both enhancers are associated with nucleosomes but have hypomethylated DNA, consistent with a permissive transcriptional state, sporadic occupancy, and the observed DUX4 expression in rare myonuclei. Our data support a model in which these myogenic enhancers associate with the DUX4 promoter in skeletal myocytes and activate transcription when epigenetically derepressed in FSHD, resulting in the pathological misexpression of DUX4-fl