DUX4, a candidate gene of facioscapulohumeral muscular dystrophy, encodes a transcriptional activator of PITX1

doi:10.1073/pnas.0708659104

DUX4, a candidate gene of facioscapulohumeral muscular dystrophy, encodes a transcriptional activator of PITX1

*Center for Genetic Medicine Research, Children's National Medical Center, Washington, DC 20010;
^†Laboratory of Molecular Biology, University of Mons-Hainaut, 7000 Mons, Belgium;
^‡Department of Biological Chemistry, University of California, Irvine, CA 92697;
^§Institut de Biologie et Médecine Moléculaires, Université Libre de Bruxelles, 6041 Gosselies, Belgium;
^¶Department of Neurology, University of Michigan, Ann Arbor, MI 34295;
^‖Institut National de la Santé et de la Recherche Médicale, ERI 25 Muscle et Pathologies, CHU A. de Villeneuve, University of Montpellier I, 34295 Montpellier, France; and
**Department of Pediatrics, George Washington University, Washington, DC 48109

Communicated by Shirley M. Tilghman, Princeton University, Princeton, NJ, September 14, 2007 (received for review May 16, 2007)

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder linked to contractions of the D4Z4 repeat array in the subtelomeric region of chromosome 4q. By comparing genome-wide gene expression data from muscle biopsies of patients with FSHD to those of 11 other neuromuscular disorders, paired-like homeodomain transcription factor 1 (PITX1) was found specifically up-regulated in patients with FSHD. In addition, we showed that the double homeobox 4 gene (DUX4) that maps within the D4Z4 repeat unit was up-regulated in patient myoblasts at both mRNA and protein level. We further showed that the DUX4 protein could activate transient expression of a luciferase reporter gene fused to the Pitx1 promoter as well as the endogenous Pitx1 gene in transfected C2C12 cells. In EMSAs, DUX4 specifically interacted with a 30-bp sequence 5′-CGGATGCTGTCTTCTAATTAGTTTGGACCC-3′ in the Pitx1 promoter. Mutations of the TAAT core affected Pitx1-LUC activation in C2C12 cells and DUX4 binding in vitro. Our results suggest that up-regulation of both DUX4 and PITX1 in FSHD muscles may play critical roles in the molecular mechanisms of the disease.

Footnotes

^††To whom correspondence may be addressed. E-mail: frederique.coppee{at}umn.ac.be or ychen{at}cnmresearch.org
Author contributions: M.D., E.A., and A.T. contributed equally to this work; M.D., E.A., A.T., S.W., R.S., H.Q., S.S., C.M., A.M.v.A., O.L., D.F., D.L.-C., A.B., F.C., and Y.-W.C. designed research; M.D., E.A., A.T., S.W., R.S., H.Q., S.S., C.M., A.M.v.A., M.B., D.L.-C., F.C., and Y.-W.C. performed research; A.T., A.M.v.A., O.L., and Y.-W.C. contributed new reagents/analytic tools; M.D., E.A., A.T., R.S., H.Q., O.L., D.L.-C., A.B., F.C., and Y.-W.C. analyzed data; and M.D., E.A., A.T., A.B., F.C., and Y.-W.C. wrote the paper.
The authors declare no conflict of interest.
Data deposition: The sequences reported in this paper have been deposited in the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov) (accession no. GSE9397) and Children's National Medical Center Public Expression Profiling Resources (http://pepr.cnmcresearch.org) databases.
This article contains supporting information online at www.pnas.org/cgi/content/full/0708659104/DC1.
Abbreviation:

FSHD,

facioscapulohumeral muscular dystrophy.