Facioscapulohumeral muscular dystrophy region gene-1 (FRG-1) is an actin-bundling protein associated with muscle-attachment sites

Abstract

SummaryIn vertebrates, overexpression of facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) recapitulates the pathophysiology exhibited by FSHD patients, although the role of FRG1 in FSHD remains controversial and no precise function for FRG1 has been described in any organism. To gain insight into the function and potential role of FRG1 in FSHD, we analyzed the highly conserved Caenorhabditis elegans ortholog, frg-1. C. elegans body-wall muscles contain two distinct subcellular pools of FRG-1: nuclear FRG-1, concentrated in the nucleoli; and cytoplasmic FRG-1, associated with the Z-disk and costamere-like structures known as dense bodies. Functionally, we demonstrate that FRG-1 is an F-actin-bundling protein, consistent with its localization to dense bodies; this activity is conserved in human FRG1. This is particularly intriguing because it places FRG-1 along side the list of densebody components whose vertebrate orthologs are involved in the myriad myopathies associated with disrupted costameres and Z-disks. Interestingly, overexpressed FRG-1 preferentially accumulates in the nucleus and, when overexpressed specifically from the frg-1 promoter, disrupts the adult ventral muscle structure and organization. Together, these data further support a role for FRG1 overexpression in FSHD pathophysiology and reveal the previously unsuspected direct involvement of FRG-1 in muscle structure and integrity. Key words: FSHD, Dense body, Muscle, FRG1, Muscular dystrophy, ActinJournal of Cell Science 1117 FRG-1 is a dense-body actin bundler analyzed the C. elegans ortholog of FRG1, frg-1 (ZK1010.3), with respect to biological activity, expression and subcellular distribution in body-wall muscle and the effects of FRG-1 overexpression on the adult body-wall musculature. Results FRG-1 is a conserved actin-bundling proteinFRG1 is highly evolutionarily conserved among vertebrates and invertebrates (Grewal et al., 1998), with human FRG1 sharing 42% identity (58% similarity) with C. elegans FRG-1 over the full length of the protein (Fig. 1); however, its function has remained elusive. Analysis of the predicted amino acid sequence for the presence of conserved domains (Pfam 23.0, http://pfam.sanger.ac.uk/) revealed that FRG-1, as with all other FRG1 orthologs, contains a single fascin-like domain (PF06220), indicative of an actin-binding protein (Adams, 2004;Edwards and Bryan, 1995). To determine whether FRG-1 could, in fact, bind F-actin, high-speed actin cosedimentation assays were performed ( Fig. 2A). Recombinant FRG-1 (supplementary material Fig. S1A) was incubated with Factin for 2 hours and then centrifuged at 100,000ϫg to pellet all F-actin and F-actin-bound FRG-1. The amount of FRG-1 in the pellets (F-actin-FRG-1) and supernatants (free FRG-1) was determined by gel electrophoresis. Saturable F-actin binding was reproducibly achieved by the co-sedimentation of increasing concentrations of recombinant FRG-1 with a fixed amount of Factin. Nonlinear regression analysis of the binding data from four independent ...

Facioscapulohumeral muscular dystrophy (FSHD): an enigma unravelled?

“…Consistent with a putative role in FSHD, several functional studies of the FRG1 protein have shown it to have a role in both angiogenesis and muscle development by utilising quite different biochemical functions, i.e. alternative RNA splicing and actin bundling (Gabellini et al 2006;van Koningsbruggen et al 2007;Hanel et al 2009;Wuebbles et al 2009;Liu et al 2010;Sun et al 2011). Furthermore, a recent analysis of myoblasts isolated from affected muscle of a transgenic mouse overexpressing FRG1 reported a significant loss of cell proliferation and an increased doubling time not observed in unaffected muscle .…”

Facioscapulohumeral muscular dystrophy region gene 1 over-expression causes primary defects of myogenic stem cells

“…Therefore, the functional consequence of D4Z4 deletion, like overexpression of an FSHD candidate gene, is the only disease aspect that can be modeled in mice. FRG1 overexpression in mouse, frogs and worms causes an FSHD-like phenotype (Gabellini et al, 2006;Hanel et al, 2009;Liu et al, 2010;Wuebbles et al, 2009). Importantly, we also found a remarkable similarity of the expression profile of FRG1 mice to one of the FSHD patients.…”

“…On the contrary, mice overexpressing FRG1 (FRG1 mice), selectively in the skeletal muscle, display reduced muscle size and develop a muscular dystrophy resembling FSHD (Gabellini et al, 2006). Moreover, studies conducted in Xenopus laevis and Caenorhabditis elegans demonstrated that frg1 is required for normal muscle development and its overexpression causes muscular defects and vascular abnormalities correlated with the clinical findings from FSHD patients Liu et al, 2010;Wuebbles et al, 2009). Collectively, these results suggest that FRG1 is important for muscle function and its aberrant expression could contribute to the FSHD pathogenesis.…”

Facioscapulohumeral Muscular Dystrophy: From Clinical Data to Molecular Genetics and Return

“…In C. elegans, frg1 protein localized both in nuclei and in the dense bodies that are homologous to vertebrate Z-disk. Interestingly frg1 overexpression in this invertebrate model disrupts the body-wall musculature and the muscular organization (Liu et al, 2010). In Xenopus both knock down and overexpression of frg1 resulted in defective growth and morphogenesis of the myotome indicating that precise levels of frg1 must be maintained for normal muscle morphology (Hanel et al, 2009).…”

“…Recent studies show the crucial role of FRG1 in maintaining proper muscle structure and function (Hanel et al, 2011;Hanel et al, 2009;Liu et al, 2010). In C. elegans, frg1 protein localized both in nuclei and in the dense bodies that are homologous to vertebrate Z-disk.…”