Elsevier

Experimental Neurology

Volume 214, Issue 1, November 2008, Pages 87-96
Experimental Neurology

DUX4c, an FSHD candidate gene, interferes with myogenic regulators and abolishes myoblast differentiation

https://doi.org/10.1016/j.expneurol.2008.07.022Get rights and content

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disease. It maps to the D4Z4 repeat array at 4q35, and correlates with a repeat contraction which derepresses transcription of local genes. Which, if any, of these genes is pathogenic to muscle, and through what molecular mechanism is unknown. The present study investigates the function of one candidate gene, DUX4c, encoded by a truncated inverted D4Z4 element located 42 kb proximal to the D4Z4 repeats. Using a gain of function approach we tested DUX4c for toxicity and effects on differentiation in C2C12 myoblasts. DUX4c-expressing myoblasts appear morphologically normal but have reduced expression of myogenic regulators, including MyoD and Myf5. Consistent with this, DUX4c-expressing myoblasts are unable to differentiate into myotubes. Furthermore, overexpression of Myf5 or MyoD rescued myoblast differentiation, suggesting that reductions in expression of these regulators are the relevant DUX4c-induced physiological changes that inhibit differentiation. Our results suggest that upregulation of DUX4c can have a deleterious effect on muscle homeostasis and regeneration, and point to a possible role for DUX4c in the pathology of FSHD.

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.).

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