DUX4c, an FSHD candidate gene, interferes with myogenic regulators and abolishes myoblast differentiation
Introduction
Facioscapulohumeral muscular dystrophy (FSHD) is a dominant inherited neuromuscular disease with a prevalence of 1 in 20,000, which makes it the third most common myopathy. It is characterized by uneven and progressive weakness and atrophy of facial, shoulder and upper arm muscle as well as in some cases with retinovasculopathy, mental retardation and epilepsy. The underlying molecular mechanism of the disease is still unknown but 95% of patients carry a subtelomeric deletion on chromosome 4q35 (Wijmenga et al., 1992). This region is mainly composed of a tandem repeat array consisting of 3.3 kb units (D4Z4). Each D4Z4 unit is GC rich and contains a repetitive element related to the heterochromatic LSau sequence and binding sites for a transcriptional repressor complex containing YY1, HMGB2, and nucleolin (Gabellini et al., 2002). In the unaffected population, the D4Z4 repeat array is polymorphic and may contain between 11 and over 100 U. Due to internal deletions, FSHD patients carry only 1 to 10 U (Hewitt et al., 1994).
One of the prevailing models for the mechanism of FSHD is that the normally large D4Z4 array nucleates a repressive chromatin and contractions of this array disable this repression, resulting in upregulation or derepression of local genes. Consistent with this model, Gabellini et al. observed that 2 nearby genes (FRG1 and FRG2) and one gene from 5 Mb upstream (ANT1) were upregulated in FSHD muscle (Gabellini et al., 2002). Furthermore, over expression of FRG1 from the human skeletal actin promoter was later shown to be deleterious, leading to muscular dystrophy phenotype in transgenic mice (Gabellini et al., 2006). However, the relevance of this transgenic model, which has very high-level expression of FRG1 to FSHD pathology, has been debated as similar levels of FRG1 have not been demonstrated in FSHD muscle.
The D4Z4 repeats contain an open reading frame with two homeoboxes (DUX4) (Gabriels et al., 1999). Homeodomain proteins regulate many important developmental processes including muscle development (Buckingham et al., 2003). Remarkably the homeodomains of DUX4 have a high degree of similarity with Pax3 and Pax7, the critical homeodomain regulators of muscle development and regeneration. Recently, expression of DUX4 RNA and protein have been selectively detected in primary myoblasts from FSHD patients suggesting its involvement in FSHD (Kowaljow et al., 2007, Dixit et al., 2007). Furthermore over expression of DUX4 in different cell lines induced apoptosis (Kowaljow et al., 2007). A truncated and inverted D4Z4 repeat is located approximately 42 kb centromeric from the tandem D4Z4 repeats and just proximal of FRG2 (Fig. 1A) (Wright et al., 1993). This repeat also contains an ORF, referred to as DUX4c, which encodes a protein identical to DUX4 from the N-terminus through the homeodomains, but the last 82 amino acids have been substituted for an unrelated 32 amino acid sequence. Preliminary data suggests that DUX4c may be expressed in FSHD samples (Ansseau et al., 2006).
In this study, using a conditional, regulated gene expression system, we have analyzed the effect of DUX4c expression on C2C12 myoblast differentiation. We demonstrate that DUX4c down-regulates expression of myogenic transcriptional factors MyoD, Myf5 and myogenin. This interaction with myogenic regulators correlates with diminished myoblast fusion and terminal myotube differentiation even at low levels of DUX4c expression. Overexpression of MyoD or Myf5 in DUX4c-expressing cells rescued their ability to differentiate suggesting that they are the key downstream genes that mediate this effect. Our results predict that DUX4c expression would have a deleterious effect on muscle regeneration, and could therefore play a role in FSHD pathogenesis.
Section snippets
DUX4c-inducible myoblasts
We modified mouse C2C12 myoblasts to enable conditional DUX4c expression by inserting the cDNA for DUX4c into the inducible locus of iC2C12, a C2C12 subclone that expresses rtTA and carries a tetracycline-inducible locus that can be targeted by cre–lox recombination (manuscript submitted). The parent iC2C12 cell line has a cassette exchange recombination locus which allows efficient gene targeting. The integration site of this locus was selected for absence of leakiness and minimal silencing.
Conclusion
In summary, we have shown that DUX4c does not have toxic effects on myoblasts while proliferating, but that it interferes with the expression of myogenic regulators and inhibits differentiation of myoblasts, even at very low levels of expression. Diminished differentiation is in large part due to down-regulation of the crucial myogenic transcription factors MyoD and Myf5. As myogenesis is necessary for homeostatic maintenance of muscle, if DUX4c were expressed as satellite cells undergo cycles
Cell culture
C2C12 cells were cultured in proliferation medium consisting of high glucose Dulbecco's Modified Eagle Media (DMEM) supplemented with l-glutamine and sodium pyruvate (Gibco), penicillin and streptomycin (P/S, Gibco) and 20% fetal bovine serum (FBS, Atlanta Biologicals) at 37 °C in 5% O2/5% CO2. For myotube formation, C2C12 cells were cultured on gelatin-coated dishes in proliferation medium until confluence, and then washed with serum-free DMEM and differentiated with DMEM supplemented with 2%
Acknowledgments
We thank the Dr. Bob and Jean Smith Foundation for their generous support. This work was also supported in part by a Muscular Dystrophy Association Fellowship (to D.B.), the Facioscapulohumeral Muscular Dystrophy Society, and a U.T. Southwestern S.U.R.F. Award (to S.L.).
References (20)
- et al.
Inappropriate gene activation in FSHD: a repressor complex binds a chromosomal repeat deleted in dystrophic muscle
Cell
(2002) - et al.
Nucleotide sequence of the partially deleted D4Z4 locus in a patient with FSHD identifies a putative gene within each 3.3 kb element
Gene
(1999) - et al.
The DUX4 gene at the FSHD1A locus encodes a pro-apoptotic protein
Neuromuscul. Disord.
(2007) - et al.
MyoD or Myf-5 is required for the formation of skeletal muscle
Cell
(1993) - et al.
Redefining the genetic hierarchies controlling skeletal myogenesis: Pax-3 and Myf-5 act upstream of MyoD
Cell
(1997) - et al.
Facioscapulohumeral muscular dystrophy (FSHD) myoblasts demonstrate increased susceptibility to oxidative stress
Neuromuscul. Disord.
(2003) - et al.
Characterization of the DUX4c gene located within a repeated element close to the FSHD locus
Neuromuscul. Disord.
(2006) - et al.
A novel genetic hierarchy functions during hypaxial myogenesis: Pax3 directly activates Myf5 in muscle progenitor cells in the limb
Genes Dev.
(2006) - et al.
The formation of skeletal muscle: from somite to limb
J. Anat.
(2003) - et al.
Parallel protein and transcript profiles of FSHD patient muscles correlate to the D4Z4 arrangement and reveal a common impairment of slow to fast fibre differentiation and a general deregulation of MyoD-dependent genes
Proteomics
(2006)
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