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

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.

Fig. 1.

PITX1 is specifically up-regulated in FSHD compared with 11 other neuromuscular diseases. The expression level of the gene with arbitrary unit was determined by either dCHIP or Affymetrix MAS5.0. The sample size of each disease was normal healthy muscle (NHM), n = 15; juvenile dermatomyositis (JDM), n = 25; human spastic paraplegia (HSP), n = 4; FSHD, unaffected, n = 5, affected, n = 9; fukutin-related protein deficiency (FKRD), n = 7; Emery–Dreifuss muscular dystrophy, lamin A/C deficiency) (ED-L), n = 4; Emery–Dreifuss muscular dystrophy, emerin deficiency (ED-E), n = 4; dysferlinopathy (DYSF), n = 10; Duchenne muscular dystrophy (DMD), n = 10; Calpain-3 deficiency (CALP), n = 10; Becker muscular dystrophy (BMD), n = 5; acute quadriplegic myopathy (AQM), n = 5; and amyotrophic lateral sclerosis (ALS), n = 9.

Fig. 2.

DUX4 activates the Pitx1 promoter in transient expression and specifically interacts with a cis element in the Pitx1 promoter region. (a) C2C12 cells were cotransfected with the pCIneo expression vector encoding DUX4, DUX4c, or DUX1, as indicated, and a Pitx1-LUC reporter vector where the Pitx1 promoter is either wild type (WT) or mutated (M; TAAT to TACC in the putative homeodomain-binding site). Luciferase activity was assayed on cells lysed 24 h after transfection. Data are provided as fold induction compared with luciferase activities obtained in cotransfection of the Pitx1-LUC vectors with the insertless pCIneo vector. (b) EMSA was performed with nuclear extract prepared from C2C12 cells at 16 h after transfection. Wild-type probe (lanes 1–3, 5, 6, 8, and 9) and mutated probes (lanes 4, 7, and 10) were end-labeled and incubated with nuclear extracts from cells transfected with pCIneo-DUX4 (lane 2–4), -DUX4c (lanes 5–7), or -DUX1 (lanes 8–10). Supershifts were determined by incubating probe and protein complexes with mAb 9A12 that recognizes DUX4 and DUX4c (lanes 3, 6, and 9). Small arrows in lanes 2 and 6 and arrowheads in lanes 3 and 6 indicate shifts and supershifts, respectively.

Fig. 3.

DUX4 induces expression of endogenous Pitx1 gene in C2C12 cells. C2C12 cells were transfected with the pCIneo-DUX4 expression vector (Left), the insertless pCIneo as a negative control (Center), or a Pitx1 expression vector as a positive control (Right). Double immunofluorescence was performed 24 h after transfection with mAb 9A12 and a secondary antibody coupled to FITC for DUX4 (green, Upper) and with a rabbit serum raised against a Pitx1-specific peptide and secondary antibodies coupled to Texas red (Lower). Arrows point to three nuclei coexpressing DUX4 and Pitx1. (Scale bar, 20 μm.)

Fig. 4.

Determination of the DUX4 mRNA 5′ and 3′ends. (a) Schematic representation of the DUX4 promoter with the putative CATT, GC, TACAA, and E boxes, the ATG translation start codon, and the primers used in 5′-RACE (arrows, nos. 68 and 73). The two 5′ ends found previously in total RNAs of C2C12 cells transfected with pGEM42 are indicated. One of these is identical to the single transcription start site determined here on total RNAs of FSHD and control primary myoblasts (*). (b Upper) Scheme of the EcoRI genomic fragment end as cloned in pGEM42 with the stop codon of the DUX4 ORF and the pLAM region. The KpnI and EcoRI restriction sites, the polyA addition signal (ATTAAA), and the primers used in 3′-RACE (arrows, nos. 94 and 95) are indicated. (Lower) Mapping of the ends obtained by 3′-RACE, with introns A and B.

Fig. 5.

Detection of the DUX4 mRNAs in FSHD primary myoblasts. (a) Schematic representation of the DUX4 RNAs with alternative 3′ ends and the primers used for RT (no. 407) and PCR (nos. 222 and 407). (b) For controls, RT-PCR was performed on 3–4 μg of total RNA extracted from C2C12 cells transfected with the pGEM7Z vector containing either no insert (lanes 2 and 3) or the 13.5-kb genomic fragment of a patient with 2 D4Z4 units (pGEM42, lanes 4 and 5). RT-PCR was similarly performed on total RNA of control (N036, 9719, and C20) and FSHD primary myoblasts (F22, 5 D4Z4 units; M038, 7 units) either in proliferation (lanes 6–9) or in differentiation (diff) (lanes 10–13). RNA samples were incubated (+) or not (−) with DNase I and RT, as indicated. As a positive control (lane 14), PCR was performed on the pGEM42 vector present in a control sample (as in lanes 4 and 5) not treated with DNase I.