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

Abstract

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.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00149-14.

ACKNOWLEDGMENTS

This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Disease of the National Institutes of Health (grants R01AR062587 and R01AR055877 to P.L.J. and grant R01AR060328 to J.B.M.), the Association Francaise contre les Myopathies (grant AFM15700 to P.L.J. and J.B.M.), and by grants from the Thoracic Foundation, Boston, MA (to S.H. and P.L.J.). T.I.J. is supported by Muscular Dystrophy Association grant MDA216652.

We thank the participating families, the FSH Society for patient outreach, and members of the Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center for FSHD directed by Charles P. Emerson, Jr., for deriving the original cultures of myogenic cells; we thank Kathryn Wagner and Genila Bibat (Kennedy-Krieger Institute and Johns Hopkins School of Medicine) for obtaining the initial biopsy specimens, Jennifer C. J. Chen and Kendal Hanger (University of Massachusetts Medical School) for initial preparation of myogenic cells from biopsy specimens, and Charles P. Emerson, Jr., for helpful comments on the manuscript. We also thank Alexandra Belayew for providing pGEM/42 and Stephen J. Tapscott and Linda N. Geng (Fred Hutchinson Cancer Center) for generously providing initial samples of DUX4-FL MAbs. We are grateful to the Hawaii fund and the Thoracic Foundation, Boston, MA, for their continued support.