cFacioscapulohumeral 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.
Facioscapulohumeral disease (FSHD) is an autosomal dominant muscular dystrophy characterized by progressive weakness and atrophy of specific muscle groups (1, 2). The genetics of FSHD are complex. The most common form of FSHD, FSHD1 (Online Mendelian Inheritance in Man [OMIM] entry 158900), is linked to contractions of a D4Z4 macrosatellite repeat array in the subtelomere of chromosome 4 at 4q35.2 (3-5). In the general healthy population, this repeat array varies between 11 and 100 D4Z4 repeats on both 4q chromosomes, whereas in FSHD1 patients the array is contracted to 1 to 10 repeats on one chromosome, with a requirement for at least one D4Z4 unit to develop disease. This telomeric region exists as two prominent alleles and a third rare allele distal to the array: 4qA, which contains a 6.2-kb -satellite region, and 4qB are equally represented in the population while 4qC is rare (6). Only contractions on specific diseasepermissive haplotypes of 4qA (the common 4qA161 variant and the rare 4qA159 and 4qA168 variants) are associated with FSHD1 (6-8). Less than 5% of cases (known as phenotypic FSHD or FSHD2; OMIM 158901) show no contraction of the D4Z4 repeats on chromosome 4 although these patients still carry at least one permissive 4qA161 allele (9-11).Both forms of FSHD are associated with epigenetic alterations indicative of chromatin relaxation in the D4Z4 region. In FSHD1, there is a general DNA hypomethylation and loss of heterochromatic histone marks in the contracted allele, consistent with a chromatin environment that is permissive for gene expre...