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Facioscapulohumeral Muscular Dystrophy (FSHD) Region Gene 1 (FRG1) Is a Dynamic Nuclear and Sarcomeric Protein

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Facioscapulohumeral Muscular Dystrophy (FSHD) Region Gene 1 (FRG1) Is a Dynamic Nuclear and Sarcomeric Protein Differentiation 81 (2011) 107–118 Contents lists available at ScienceDirect Differentiation journal homepage: www.elsevier.com/locate/diff Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) is a dynamic nuclear and sarcomeric protein Meredith L. Hanel a, Chia-Yun Jessica Sun a, Takako I. Jones a, Steven W. Long a, Simona Zanotti b, Derek Milner a,c, Peter L. Jones a,n a Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, 601 S. Goodwin Ave, B107 Chemical and Life Sciences Laboratory, Urbana, IL 61801, USA b Neuromuscular Diseases and Neuroimmunology Unit, Muscle Cell Biology Laboratory, Fondazione IRCS Istituto Neurologico ‘‘C. Besta’’, Via Temolo 4—20126 Milano, Italy c Regeneration Biology and Tissue Engineering Theme, Institute for Genomic Biology, University of Illinois at Urbana, Champaign, Urbana, IL 61801, USA article info abstract Article history: Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) is a candidate gene for Received 9 April 2010 mediating FSHD pathophysiology, however, very little is known about the endogenous FRG1 protein. Received in revised form This study uses immunocytochemistry (ICC) and histology to provide insight into FRG1’s role in 20 August 2010 vertebrate muscle development and address its potential involvement in FSHD pathophysiology. In cell Accepted 30 September 2010 culture, primary myoblast/myotube cultures, and mouse and human muscle sections, FRG1 showed distinct nuclear and cytoplasmic localizations and nuclear shuttling assays indicated the subcellular Keywords: pools of FRG1 are linked. During myoblast differentiation, FRG1’s subcellular distribution changed Facioscapulohumeral muscular dystrophy dramatically with FRG1 eventually associating with the matured Z-discs. This Z-disc localization was FRG1 confirmed using isolated mouse myofibers and found to be maintained in adult human skeletal muscle Muscle biopsies. Thus, FRG1 is not likely involved in the initial assembly and alignment of the Z-disc but may be Z-disc Sarcomere involved in sarcomere maintenance or signaling. Further analysis of human tissue showed FRG1 is strongly expressed in arteries, veins, and capillaries, the other prominently affected tissue in FSHD. Overall, we show that in mammalian cells, FRG1 is a dynamic nuclear and cytoplasmic protein, however in muscle, FRG1 is also a developmentally regulated sarcomeric protein suggesting FRG1 may perform a muscle-specific function. Thus, FRG1 is the only FSHD candidate protein linked to the muscle contractile machinery and may address why the musculature and vasculature are specifically susceptible in FSHD. & 2010 International Society of Differentiation. Published by Elsevier Ltd. All rights reserved. 1. Introduction large heterochromatic region alters the chromosome architecture as well as the epigenetic landscape of chromosome 4q35, and in Facioscapulohumeral muscular dystrophy (FSHD) is the most doing so presumably changes localized gene regulation that prevalent of the adult muscular dystrophies (incidence of 1:7500– ultimately leads to the pathology (de Greef et al., 2008). Multiple 1:14,000) and third most common overall (Lunt and Harper, candidate genes have been proposed to lead to FSHD pathology 1991; Prevalence of Rare Diseases, 2009), although its etiology is based in part on their proximity to the deletion (Wijmenga et al., still not clear. In addition to the muscular dystrophy, 50–75% of 1993; van Deutekom et al., 1996; Gabriels et al., 1999; Snider FSHD patients develop retinal vasculopathy (Gieron et al., 1985; et al., 2009), their differential expression patterns in FSHD patient Fitzsimons et al., 1987), highlighting the complex nature of FSHD versus unaffected controls (Bodega et al., 2009; Ansseau et al., pathophysiology. The genetic lesion for FSHD1A (OMIM 158900), 2009; Bosnakovski et al., 2008; Gabellini et al., 2006; Rijkers et al., the most common form of FSHD (98% of all cases), is a dominant 2004; Winokur et al., 2003), and overexpression phenotypes in contraction of the large D4Z4 tandem repeat array at chromosome animal models (Gabellini et al., 2006; Liu et al., 2010; Wuebbles 4q35 (Wijmenga et al., 1992; Lunt et al., 1995). Removing this et al., 2010; Hanel et al., 2009; Wuebbles et al., 2009). This study focuses on the FSHD candidate gene FRG1 (FSHD region gene 1) (Grewal and Todd, 1998), encoding a highly evolutionarily Abbreviations: FSHD, facioscapulohumeral muscular dystrophy; FRG1 FSHD, conserved protein of unknown cellular function (Fig. S1). region gene 1; HSMM, human skeletal muscle myoblast; ICC, immunocytochem- FRG1, located 125 kb centromeric to the FSHD1A deletion, was istry; MDSC, muscle-derived stem cell n one of the early candidate genes for FSHD (van Deutekom et al., Correspondence to: Boston Biomedical Research Institute, 64 Grove, St. Watertown, MA 02472, US. Tel.: +1 617 658 7745; fax: +1 617 972 1760. 1996). However, recent expression studies have failed to find E-mail addresses: [email protected], [email protected] (P.L. Jones). significant FRG1 misexpression in numerous FSHD patient-derived 0301-4681/$ - see front matter & 2010 International Society of Differentiation. Published by Elsevier Ltd. All rights reserved. Join the International Society for Differentiation (www.isdifferentiation.org) doi:10.1016/j.diff.2010.09.185 108 M.L. Hanel et al. / Differentiation 81 (2011) 107–118 muscle cells and biopsies casting doubt on its involvement in Proliferating primary human skeletal muscle myoblasts (HSMM) mediating FSHD pathology (Osborne et al., 2007; Arashiro et al., were obtained from Lonza (Walkersville, MD) and were seeded on 2009; Masny et al., 2010; Klooster et al., 2009). Complicating the 0.02% collagen-coated surfaces and maintained in SkBM-2 issue is the lack of understanding towards FRG1’s normal medium supplemented with SkGM-2 SingleQuots (Lonza) accord- spaciotemporal expression, distribution, and cellular function ing to the manufacturer’s instructions. For myotube formation, during normal human muscle development. Initial studies using HSMMs were seeded on 0.02% collagen-coated coverslips at Xenopus as a model for vertebrate development found frg1 was 1.5 Â 104/cm2 density for ICC analysis, and the following day widely expressed early and throughout development, showing were induced to form myotubes by adding fusion medium elevated levels in vascular tissues and developing muscles with (DMEM/F-12 50:50 supplemented with 2% horse serum). Murine preferential expression in the capillaries, veins, and arteries muscle derived stem cells (MDSC) were isolated and cultured as located between muscle fibers (Hanel et al., 2009; Wuebbles described (Qu-Petersen et al., 2002). For ICC analysis, MDSCs were et al., 2009). Knockdown and overexpression experiments seeded on 0.02% collagen-coated coverslips. confirmed a necessary role for frg1 in development of the musculature and vasculature. Interestingly, systemic increases in 2.2. Myofiber and myofibril isolation frg1 levels had specific effects on the developing musculature and vasculature, impairing myogenesis and muscle precursor cell Mice (C57/B6) were humanely euthanized in accordance with migration and causing spurious angiogenesis leading to a approved UIUC IACUC protocols. Adult mouse muscle fibers were tortuous vasculature (Hanel et al., 2009; Wuebbles et al., 2009). isolated from the flexor digitorum brevis muscle of 1–3 month old These phenotypes are consistent with the two major pathologies female mice. Isolated muscles were washed briefly in DMEM, then seen in FSHD patients (Gieron et al., 1985; Padberg, 1982). A incubated in DMEM with 0.2% collagenase type I (Worthington similar analysis of the Caenorhabditis elegans FRG1 homolog (FRG- Biochemical, Lakewood, NJ) for 4 h at 37 1C, with gentle agitation 1) showed the development, organization, and integrity of the every 15 min and changes into fresh collagenase solution every adult body wall musculature is unique in its susceptibility to hour. At the completion of digestion, excess tendon material was increased FRG-1 levels (Liu et al., 2010). Interestingly, FRG-1 had carefully dissected away, and fiber bundles were transferred to a to be overexpressed in the spaciotemporal pattern dictated by the dish of myofiber medium (DMEM supplemented with 10 mM FRG-1 promoter and there was no affect on the musculature when HEPES, 5% heat-inactivated horse serum, 1% penicillin– FRG-1 was overexpressed specifically in adult muscle from the streptomycin, and 0.1% amphotericin B). Individual fibers were myo-3 promoter. Although FRG1 may function in many tissues, liberated from the muscle mass by gentle tituration and agitation, the developing musculature and vasculature are uniquely and cultured overnight in myofiber medium at 37 1C, 5%CO2. The susceptible to systemic changes in FRG1 levels suggesting FRG1 following day, healthy, undamaged myofibers were plated on has tissue specific functions. Thus, in FSHD, small pathogenic glass coverslips coated with Geltrex (Invitrogen, Carlsbad, CA) and changes in FRG1 expression may be occurring early in muscle allowed to adhere for 1–2 h before fixation. Myofibrils were development or also involve non-myogenic cell lineages (Liu et al. isolated as described (Knight and Trinick, 1982) using skeletal 2010; Hanel et al., 2009; Wuebbles et al., 2009). muscle dissected from rectus femoris muscle. Purified myofibrils FRG1 is proposed to be involved in aspects of RNA biogenesis were plated on coverslips coated with poly-L-lysine and
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