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.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by an unusual deletion with neomorphic activity. This deletion derepresses genes in cis ; however which candidate gene causes the FSHD phenotype, and through what mechanism, is unknown. We describe a novel genetic tool, inducible cassette exchange, enabling rapid generation of isogenetically modified cells with conditional and variable transgene expression. We compare the effects of expressing variable levels of each FSHD candidate gene on myoblasts. This screen identified only one gene with overt toxicity: DUX4 (double homeobox, chromosome 4), a protein with two homeodomains, each similar in sequence to Pax3 and Pax7. DUX4 expression recapitulates key features of the FSHD molecular phenotype, including repression of MyoD and its target genes, diminished myogenic differentiation, repression of glutathione redox pathway components, and sensitivity to oxidative stress. We further demonstrate competition between DUX4 and Pax3/Pax7: when either Pax3 or Pax7 is expressed at high levels, DUX4 is no longer toxic. We propose a hypothesis for FSHD in which DUX4 expression interferes with Pax7 in satellite cells, and inappropriately regulates Pax targets, including myogenic regulatory factors, during regeneration.

The ontogeny of expression of the alpha-fetoprotein (AFP) and albumin genes was examined in livers from late prenatal to 1-month-postpartum C3H/He mice. A parallel accumulation of both AFP and albumin mRNAs before birth, followed by a selective nonreciprocal decrease in AFP mRNA after birth, was observed. The decrease in AFP mRNA was the result of a decrease in transcription of the AFP gene, as measured by an in vitro nuclear transcription assay. We suggest a model for hepatic expression of the AFP and albumin gene cluster in which transcription of the two genes is activated simultaneously during differentiation and each gene is thereafter modulated independently in committed cells.