Research Article 1423 FHL3 binds MyoD and negatively regulates myotube formation

Denny L. Cottle, Meagan J. McGrath, Belinda S. Cowling, Imogen D. Coghill, Susan Brown and Christina A. Mitchell* Department of Biochemistry and Molecular Biology, Monash University, Wellington Road, Clayton, 3800, Australia *Author for correspondence (e-mail: [email protected])

Accepted 14 February 2007 Journal of Cell Science 120, 1423-1435 Published by The Company of Biologists 2007 doi:10.1242/jcs.004739

Summary MyoD initiates muscle differentiation and promotes early during differentiation. We demonstrate that FHL3 is skeletal myogenesis by regulating temporal a MyoD-associated by direct binding assays, expression. MyoD-interacting induce regulatory colocalisation in the nucleus of myoblasts and GST pull- effects, and the identification of new MyoD-binding down studies. Moreover, we determined that FHL3 partners may provide mechanistic insights into the interacts with MyoD, functioning as its potent negative co- regulation of during myogenesis. FHL3 is transcriptional regulator. Ectopic expression of FHL3 in one of three members of the FHL protein family that are myoblasts impaired MyoD-mediated transcriptional expressed in skeletal muscle, but its function in myogenesis activity and muscle gene expression. By contrast, is unknown. Overexpression of human FHL3 in mouse siRNA-mediated FHL3 knockdown enhanced MyoD C2C12 cells retarded myotube formation and decreased the transcriptional activity in a dose-dependent manner. These expression of muscle-specific regulatory such as findings reveal that FHL3 association with MyoD may but not MyoD. By contrast, short interfering contribute to the regulation of MyoD-dependent RNA (siRNA)-mediated FHL3 protein knockdown transcription of muscle genes and thereby myogenesis. enhanced myoblast differentiation associated with increased myogenin, but not MyoD protein expression, Key words: MyoD, Myogenesis, FHL, FHL3, LIM

Introduction transcription and scaffold sarcomeric and signaling

Journal of Cell Science Postnatal skeletal muscle myogenesis is crucial in maintaining proteins. The LIM motif contains the consensus sequence muscle mass during aging and essential for muscle repair, (Cx2Cx16-23[C/H]x2-4[C/H/E]x2Cx2Cx14-21[C/H]x1-3[C/H/D/E]) particularly in muscular dystrophies. Myoblasts are muscle but does not directly bind DNA and, instead, mediates protein- precursor cells that are committed to the skeletal muscle protein interactions (Kadrmas and Beckerle, 2004). The four lineage and respond to external signals leading to activation of and a half LIM domain (FHL) proteins are a family of LIM- specific gene expression, which in turn promotes a only proteins, characterised by four complete LIM domains, differentiated phenotype. The myogenic basic helix-loop-helix preceded by an N-terminal half LIM motif. To date five (bHLH) family of proteins govern the activation of muscle- mammalian family members FHL1, FHL2, FHL3, FHL4 and specific gene expression and include Myf5, MyoD, myogenin ACT have been identified, which act as transcriptional and Myf6. These transcription factors direct the temporal regulators (Fimia et al., 2000) and/or play structural roles in regulation of myogenesis (Ludolph and Konieczny, 1995). the actin cytoskeleton (Coghill et al., 2003; McGrath et al., Myf5 and MyoD play significant roles in committing somite- 2003; Robinson et al., 2003; Samson et al., 2004; McGrath et derived cells to skeletal muscle fates (Braun et al., 1994; Cossu al., 2006). FHL1, FHL2 and FHL3 are all expressed in striated and Borello, 1999; Buckingham et al., 2003). In addition muscle, and FHL3 is more highly expressed in skeletal muscle MyoD also initiates the onset of differentiation and plays a relative to cardiac tissue (Morgan and Madgwick, 1996; Chu regulatory role in governing the temporal expression of specific et al., 2000). genes such as myogenin (Montarras et al., 2000). In turn The function of FHL1 and FHL2, but not FHL3, in skeletal myogenin governs myofibril formation and Myf6 regulates myogenesis has been explored. FHL1 is significantly muscle maintenance and function (Bober et al., 1991; upregulated during cardiac and skeletal muscle hypertrophy Buckingham et al., 2003). These four myogenic bHLH (Morgan et al., 1995; Lim et al., 2001) and its overexpression proteins, collectively called muscle regulatory factors (MRFs), induces ␣5␤1-integrin-dependent hyper-elongation of form heterodimers with E proteins including E12 or E47 and myocytes and hypertrophic myosacs during differentiation of bind to specific DNA sites called E-boxes found in many C2C12 myoblasts (McGrath et al., 2003; Robinson et al., 2003; muscle-specific gene promoters thereby converting non- McGrath et al., 2006). FHL1 localises at the I-band and M-line myogenic, mesenchymal cells into myoblasts (Davis et al., where it binds myosin-binding protein C (MyBP-C) to regulate 1987; Choi et al., 1990; Lassar et al., 1991). myosin filament formation and sarcomere assembly (McGrath LIM proteins contain LIM motifs and regulate gene et al., 2006). FHL2 is the most extensively characterised of the 1424 Journal of Cell Science 120 (8)

FHL proteins (Johannessen et al., 2006) and forms a transcriptional co-activator, co-repressor, or competitor, dependent on tissue type and promoter context. FHL2 interacts with transcription factors, such as the androgen (Muller et al., 2002), p300, ␤-catenin (Labalette et al., 2004), FOS, JUN (Morlon and Sassone-Corsi, 2003) and FOXO1 (Yang et al., 2005). FHL2 regulates cellular events including apoptosis (Scholl et al., 2000), survival (Stilo et al., 2002), proliferation (Chen et al., 2003) and differentiation (Du et al., 2002; Martin et al., 2002; Bai et al., 2005; Lai et al., 2006). Fhl2 knockout mice exhibit an exaggerated hypertrophic cardiomyopathy in response to ␤-adrenergic stimulation (Kong et al., 2001) and develop osteopenia as a consequence of reduced osteoblast and increased osteoclast differentiation and function (Bai et al., 2005; Gunther et al., 2005; Lai et al., 2006). In skeletal muscle, FHL2 enhances C2C12 myogenesis via the Wnt signalling pathway mediated by its interaction with ␤-catenin (Martin et al., 2002). FHL3 also possesses intrinsic transactivation and repressor activity in a cell-type and gene-specific manner. FHL3 interacts with transcription factors, such as CREB (Fimia et al., 2000), BKLF/KLF3 and CtBP2 (Turner et al., 2003), PLZF (McLoughlin et al., 2002), and MZF-1 (Takahashi et al., 2005). FHL3 also forms a complex with CDC25B2 phosphatase (Mils et al., 2003) and ERK2 (Purcell et al., 2004). In myoblasts the only known function of FHL3 is to destabilise actin bundles by preventing ␣-actinin crosslinking (Coghill et al., 2003); however, its transcriptional targets in skeletal muscle have not been reported. Fig. 1. FHL3 overexpression retards C2C12 cell differentiation. (A) C2C12 Journal of Cell Science The function of FHL3 in skeletal myogenesis is currently unknown and forms the basis of this study. myoblast cell lines that stably express hemagglutinin (HA) vector [Vector (1) and Vector (2)] or HA-FHL3 [HA-FHL3 (1) and HA-FHL3 (2)] were generated Using FHL3 overexpression and siRNA-mediated in duplicate. Lysates were immunoblotted for FHL3 or total (pan) actin. protein knockdown during C2C12 myogenesis, we Immunoblots are representative of four independent experiments. (B) The mean demonstrate that FHL3 negatively regulates C2C12 level of FHL3 protein expression in cell lines stably expressing HA-FHL3 myotube formation. FHL3 forms a complex with versus those expressing Vector was quantified by densitometry and standardised MyoD inhibiting its transcriptional activity and to total actin (n=4, *P<0.05, **P<0.01). FHL3 protein levels standardised to regulates the expression of genes such as muscle actin were expressed relative to vector levels that were arbitrarily set at 1. creatine kinase (Ckm, also known as and hereafter (C) Lysates from differentiating C2C12 myoblasts stably expressing vector or referred to as MCK) and myogenin. These studies HA-FHL3 were immunoblotted using anti-HA or anti-actin antibodies. have identified FHL3 as a new regulator of MyoD- Immunoblots are representative of three independent experiments. (D) C2C12 dependent myoblast differentiation. myoblasts stably expressing vector or HA-FHL3 were differentiated, stained with anti-MHC antibodies (green) and propidium iodide (red), and then imaged by confocal microscopy. Bar, 40 ␮m. Shown are representative images following Results 120 hours of differentiation. Immunofluorescence images are representative of FHL3 overexpression retards C2C12 cell three experiments for all cell lines. (E) Cells were co-stained with anti-MHC differentiation antibodies and propidium iodide. The differentiation index was determined by FHL3 is most highly expressed in skeletal muscle, scoring the proportion of nuclei, of the total number of nuclei within MHC- however, its function in myogenesis is unknown. positive cells (myocytes and myotubes). A minimum of 116 nuclei were scored C2C12 cells are a well-defined model for studying per stable cell line per time point for each of three independent experiments myogenic differentiation because, when cultured (*P<0.05). Dashed line represents combined results of Vector (1) and (2) lines under conditions that promote differentiation, they and black line shows combined results from HA-FHL3 (1) and (2) cell lines. fuse to form mature myotubes (Yaffe and Saxel, (F) Cells were co-stained with anti-MHC antibodies and propidium iodide. The degree of cell fusion was quantified by determining the average number of 1977). To determine whether FHL3 overexpression nuclei per MHC-positive cell. A minimum of 42 and 69 MHC-positive cells affected myoblast differentiation, C2C12 myoblast were scored following 96 and 120 hours differentiation respectively, for each of cell lines that were transfected to stably overexpress three independent experiments, (*P<0.05, **P<0.01). Dashed line shows hemagglutinin-tagged FHL3 (HA-FHL3) or vector at combined results of Vector (1) and (2) lines, and black line represents HA-FHL3 low levels were generated in duplicate by pooling (1) and (2) cell lines combined results. Error bars represent ± s.e.m. FHL3 regulates myogenesis 1425

G418-resistant clones (pool 1 or 2, respectively). FHL3 myotubes were identified by MHC-positive staining, and immunoblot analysis revealed a 33 kDa immunoreactive nuclei by propidium-iodide staining. HA-FHL3 C2C12 polypeptide, whose expression levels were greater (~1.4-fold) myotubes exhibited a significantly lower differentiation index in cell lines overexpressing HA-FHL3 relative to Vector (Fig. (Fig. 1E), than Vector myotubes at 96 hours and 120 hours of 1A,B). HA-immunoblot analysis revealed that the HA-FHL3 differentiation, but not prior to these time points. Myocyte cell construct was expressed at all times during C2C12 cell fusion was calculated as the average number of nuclei per differentiation (Fig. 1C). Significantly, the myotubes formed in MHC-positive cell (Sabourin et al., 1999). At 96-120 hours of HA-FHL3 C2C12 cell lines appeared consistently and differentiation, HA-FHL3 cell lines exhibited decreased cell reproducibly smaller and thinner, with less nuclei per myotube fusion relative to cells transfected with Vector (Fig. 1F). relative to Vector cell lines after 120 hours of differentiation Myoblast differentiation was also assessed by analysis of the (Fig. 1D). The pan actin antibody, which recognises all forms temporal expression of the muscle-specific proteins MyoD and of actin was used as a protein loading control throughout this myogenin, standardised to total actin levels as a protein loading study. Whereas ␣-skeletal and ␣-cardiac actin levels may control. HA-FHL3 myotubes consistently showed a ~50% increase, and ␤-actin and ␥-actin levels decrease during C2C12 decrease in myogenin protein at 24 hours of differentiation cell differentiation (Bains et al., 1984), the total actin protein relative to Vector cells, as assessed by anti-myogenin pool remains a constant with ~18% of total cellular protein for immunoblot analysis, however, MyoD levels appeared confluent C2C12 cells and ~17% for differentiating C2C12 relatively unaffected (Fig. 2A,B). In addition, the proportion of cells (Babcock and Rubenstein, 1993). myogenin-positive nuclei in HA-FHL3 myocytes was reduced To provide a quantitative analysis of C2C12 myoblast by ~50% at 24 hours of differentiation relative to Vector cell differentiation into multinuclear myotubes, specific aspects of lines (Fig. 2C,D). Collectively, these studies reveal that FHL3 myoblast differentiation using well-defined parameters, expression delays myogenin expression, and retards myoblast including the differentiation and fusion indices, were assessed. differentiation and cell fusion into multinucleated myotubes, At the onset of differentiation, ~50% of C2C12 myoblasts whereas expression of MyoD is relatively unaffected. express myogenin and myosin heavy chain (MHC) proteins (Miller, 1990; Conejo and Lorenzo, 2001; Conejo et al., 2001), FHL3 is expressed throughout differentiation of C2C12 and form myocytes, which subsequently fuse into multi- cells and its siRNA-mediated knockdown accelerates nucleated myotubes. The remaining myoblasts form myogenin C2C12 cell differentiation and MHC-negative reserve cells (Yoshida et al., 1998). The Since we had observed that FHL3 gain-of-function inhibits differentiation index is a measure of the proportion of all myoblast differentiation and fusion, we next determined the nuclei, that are localised within myocytes and myotubes, effect of extinguishing the expression of endogenous FHL3 providing a score of myoblasts differentiating into myofibres using siRNA technology. Expression of either of two siRNA (Sabourin et al., 1999; Erbay et al., 2003). Normally, this does oligonucleotides (#2 and #3) targeting the sequence of not exceed ~50% owing to the presence of reserve cells GenBank entry NM_010213 (mouse Fhl3 mRNA), (Yoshida et al., 1998). For these experiments myocytes and significantly reduced FHL3 protein levels at all times during Journal of Cell Science Fig. 2. FHL3 overexpression retards myogenin induction following 24 hours differentiation. (A) Lysates from differentiating C2C12 myoblasts stably expressing Vector or HA-FHL3 were immunoblotted for MyoD, myogenin or total actin using specific antibodies. Immunoblots are representative of three independent experiments. (B) The mean relative level of myogenin protein in C2C12 myoblasts stably expressing Vector or HA-FHL3 after 24 hours of differentiation was quantified by densitometry and presented as the combined data from Vector or HA- FHL3 (1) and (2) cell lines (n=3, **P<0.01). The myogenin protein levels in vector cell lines standardised to actin were arbitrarily set at 1 and myogenin levels in HA-FHL3 lines expressed relative to vector cell lines. Error bars represent ± s.e.m. (C) C2C12 myoblasts were differentiated for 24 hours, stained with anti-myogenin antibodies (blue), and propidium iodide (red) and imaged by confocal microscopy. Bar, 20 ␮m. Yellow arrowheads indicate myogenin-positive nucleus. White arrowheads indicate myogenin-negative nucleus. Shown are representative images for Vector (2) and HA- FHL3 (2) cell lines. Vector (1) and HA-FHL3 (1) cell lines showed equivalent results (data not shown). Results shown are representative of three independent experiments. (D) The mean percentage of nuclei expressing myogenin was quantified following 24 hours of differentiation and is presented as the combined data from Vector and HA-FHL3 (1) and (2) cell lines. A minimum of 116 nuclei were scored for each stable cell line per time point for each of three independent experiments (**P<0.01). Error bars represent ± s.e.m. 1426 Journal of Cell Science 120 (8)

Fig. 3. FHL3 siRNA-knockdown accelerates C2C12 cell differentiation. C2C12 myoblasts were transfected with siRNA oligonucleotides, Journal of Cell Science grown for 48 hours and then differentiated for up to 72 hours. In B,D,E, control siRNA-transfected cells are represented by dashed lines, siRNA FHL3 #2 by black solid lines and siRNA FHL3 #3 by grey solid lines, error bars represent ± s.e.m. *P<0.05, ***P<0.001. (A) Cell lysates from siRNA transfected C2C12 cells differentiated for up to 72 hours were immunoblotted for FHL3 or total actin. (B) FHL3 protein levels were quantified by densitometry and standardised to total actin (n=3). (C) siRNA-transfected C2C12 cells were differentiated, stained with anti- MHC antibodies (green) and propidium iodide (red) and imaged by confocal microscopy. Bars, 40 ␮m. Shown are representative images following 72 hours of differentiation. (D) The differentiation index was quantified using a minimum of 320 nuclei scored per transfected siRNA oligonucleotide species, per time point, for each of four independent experiments. (E) The average number of nuclei per MHC-positive cell was determined for siRNA-transfected cells, a minimum of 56 and 40 MHC-positive cells were scored following 48 and 72 hours differentiation, respectively, per transfected siRNA oligonucleotide species (n=4).

differentiation in C2C12 myoblasts compared with a non- assess cell fusion, the average number of nuclei per MHC- silencing control siRNA, as assessed by FHL3 immunoblot positive cell was determined (Sabourin et al., 1999). analysis (Fig. 3A,B). In control-siRNA-transfected cultures, Significantly, after 72 hours of differentiation cells transfected anti-FHL3 western blots revealed that endogenous FHL3 with the FHL3 siRNAs exhibited a more than threefold protein was expressed at all stages of C2C12 cell increase in cell fusion compared with control-siRNA- differentiation, with levels increasing threefold at 48 hours of transfected cells (Fig. 3E). Hence, siRNA-mediated differentiation (Fig. 3B). FHL3-siRNA-transfected cells knockdown of FHL3 accelerates myoblast differentiation and exhibited an early increase in myocyte numbers after 24 hours myotube formation, the opposite phenotype to FHL3 of differentiation, correlating with the accelerated formation of overexpression. visibly larger myotubes containing increased numbers of No consistent differences in MyoD protein levels nuclei at 48-72 hours (Fig. 3C, images shown are at 72 hours (standardised to total actin) were detected following siRNA- of differentiation). We also noticed an increased differentiation mediated FHL3 knockdown compared with control siRNA index. For example, cultures transfected with the FHL3 during C2C12 cell differentiation (Fig. 4A). The expression of siRNAs #2 and #3 had already reached a differentiation index MyoD in the nucleus of C2C12 myoblasts transfected with of ~50%, whereas cells transfected with control siRNA FHL3 siRNA oligonucleotides was also examined by indirect achieved ~32% at 72 hours of differentiation (Fig. 3D). To immunofluorescence, with no significant differences observed FHL3 regulates myogenesis 1427

(data not shown). siRNA-mediated FHL3-knockdown- accelerated myogenesis was associated with a 2.7-fold increase in myogenin protein levels at 24 hours of differentiation (Fig. 4A,B), levels not observed until 48 hours of differentiation in control siRNA cultures. Beyond 24 hours of differentiation, myogenin levels were more variable and, as such, no significant reproducible differences were observed for later time points. In addition, the proportion of myogenin-positive nuclei in cells transfected with FHL3 siRNA was increased after 24 hours of differentiation by 1.7-fold compared with that detected in control siRNA transfected cells (Fig. 4C,D). Given FHL3 was reported to bind actin and destabilise actin filaments (Coghill et al., 2003), sarcomere formation was also evaluated in cells with altered FHL3 expression. FHL3- overexpressing C2C12 cells demonstrated a reduction in myotubes displaying visible Z-line formation relative to Vector cell lines at 120 hours of differentiation (data not shown). siRNA-mediated FHL3 knockdown induced a temporal increase in the numbers of myotubes displaying Z-line formation (data not shown). However, in FHL3-overexpressing cell lines, no disruptions were observed in the F-actin cytoskeleton, as evaluated by phalloidin staining. Furthermore, FHL3 did not colocalise to actin-rich sites of Z- and M-line myofibrillogenesis in wild-type C2C12 cell myotubes (data not shown), suggesting the myogenic phenotypes induced by FHL3-altered expression are distinct from its actin regulatory effects.

Ectopic FHL3 overexpression rescues FHL3 siRNA- mediated phenotype siRNA may induce non-specific off-target gene effects unrelated to the downregulation of the gene of interest (Jackson and Linsley, 2004). To confirm the phenotypes induced by FHL3 siRNA were specific to FHL3, a rescue of the siRNA

Journal of Cell Science FHL3 phenotypes was attempted by overexpression of HA- FHL3. The FHL3 siRNA oligonucleotides used in this study were designed to target mouse mRNA and exhibit several base- pair differences with the human FHL3 sequence; therefore, human HA-FHL3 should be resistant to degradation by mouse FHL3 siRNA oligonucleotides. Stable cell lines overexpressing human HA-FHL3 were transfected with mouse FHL3 siRNA oligonucleotides. FHL3 protein expression as assessed by FHL3 immunoblot analysis in the stable cell lines was significantly reduced by FHL3 siRNA transfection, relative to control siRNA-transfected cells (Fig. 5A). By contrast, anti- HA immunoblot analysis revealed that expression of 33 kDa Fig. 4. FHL3 siRNA knockdown accelerates myogenin protein recombinant human HA-FHL3 was not affected by mouse expression during differentiation. (A) Lysates from differentiating FHL3 siRNA transfection (Fig. 5A). C2C12 cells stably siRNA-transfected C2C12 cells were immunoblotted for MyoD, overexpressing Vector or HA-FHL3, were transfected with myogenin or actin. (B) Myogenin protein levels in siRNA- FHL3 or control siRNA oligonucleotides, differentiated for 72 transfected C2C12 cells were quantified after 24 hours of hours and stained with anti-MHC antibody and propidium differentiation by densitometry of immunoblots standardised to actin iodide. HA-FHL3 cells transfected with control siRNA as a loading control and vector levels arbitrarily set at 1 (n=4, demonstrated a more immature myofibre phenotype, with *P<0.05, **P<0.01). Error bars represent ± s.e.m. (C) siRNA- thinner myofibres showing decreased multi-nucleation relative transfected C2C12 cells were differentiated for 24 hours, stained to Vector-transfected myofibres. By contrast, siRNA FHL3 #2 with anti-myogenin antibodies (blue) and propidium iodide (red) and imaged by confocal microscopy. Bar, 20 ␮m. Yellow arrowheads or #3 transfection in Vector cell lines resulted in accelerated indicate myogenin-positive nucleus. White arrowheads indicate myotube formation as described above. However, HA-FHL3 myogenin-negative nucleus. (D) The mean percentage of nuclei overexpressing cells transfected with siRNA FHL3 #2 or #3 expressing myogenin was quantified following 24 hours of exhibited myotube formation at similar rates and levels to differentiation, a minimum of 320 nuclei were scored per transfected control siRNA transfected cells (Fig. 5B). The relative level of siRNA oligonucleotide species, per time point (n=4, *P<0.05, myogenin protein after 24 hours of differentiation, as assessed **P<0.01). Error bars represent ± s.e.m. 1428 Journal of Cell Science 120 (8)

by anti-myogenin immunoblot analysis, revealed rescue of the elevated myogenin protein levels in FHL3 siRNA cells following HA-FHL3 overexpression (Fig. 5C,D). In addition, the number of myogenin-positive nuclei following 24 hours of differentiation was also decreased (data not shown). By this analysis overexpression of HA-FHL3 rescued the accelerated myotube development mediated by siRNA FHL3 #2 or #3. Therefore, HA-FHL3 expression antagonises the FHL3- siRNA-induced phenotype and FHL3 negatively regulates myogenin protein expression early in differentiation.

FHL3 forms complexes with MyoD and other bHLH proteins These results prompted us to ask whether FHL3 inhibits myogenesis by regulating muscle gene expression. Given that we demonstrated MyoD protein expression was unchanged by FHL3 overexpression or siRNA-mediated knockdown, although expression of its downstream target gene myogenin was altered, we investigated whether FHL3 forms a complex with MyoD, thereby regulating the transcription of its target genes and, hence, myoblast differentiation. FHL proteins exhibit varying levels of intrinsic activation potential (Fimia et al., 2000). Evidence of in vivo complex formation between FHL3 and MyoD was initially demonstrated by mammalian two-hybrid analysis. The interaction of MyoD and FHL3 was quantified by the activation of a luciferase reporter containing Gal4-binding sites in the promoter. To this end a Gal4- promoter luciferase reporter was transactivated by Gal4DBD- FHL3 and the effect of MyoD overexpression determined. Under these conditions the intrinsic transactivation domain of MyoD was used (Fig. 6A). These results confirmed that MyoD and FHL3 interact in vivo, because the luciferase activity from cells expressing both proteins was increased ~eightfold compared with cells expressing Gal4DBD-FHL3 protein plus

Journal of Cell Science empty vector. In control studies all recombinant proteins used in these assays were expressed intact, as determined by immunoblot analysis (Fig. 6B,C). Do FHL3 and MyoD interact directly, without the cooperation or requirement for other muscle-specific proteins, or do they form part of a multi-protein complex and interact indirectly? To address this question, we evaluated whether purified recombinant GST-FHL3 directly interacts with His- Fig. 5. Rescue of FHL3 siRNA knockdown phenotype. (A) siRNA- tagged MyoD. Recombinant His-MyoD and GST-FHL3 were transfected C2C12 Vector (2) or HA-FHL3 (2) cell lines were coexpressed in Escherichia coli, and bacterial lysates were harvested 48 hours post transfection (0 hours differentiation) and incubated with glutathione Sepharose and washed extensively. lysates immunoblotted for FHL3, HA or actin. Western blots are representative of four experiments. (B) siRNA-transfected C2C12 In control studies, the muscle LIM protein (MLP) GST fusion Vector (2) or HA-FHL3 (2) cell lines were differentiated for 72 protein (GST-MLP) was coexpressed with His-MyoD, because hours and stained with anti-MHC antibodies (green) and propidium previous studies have shown that these species interact directly iodide (red), and imaged by confocal microscopy. Images are (Kong et al., 1997). GST-FHL3 did bind full-length His-MyoD representative of four independent experiments. Bar, 20 ␮m. as well as His-tagged C-terminally truncated MyoD breakdown (C) siRNA-transfected C2C12 Vector (2) and HA-FHL3 (2) cell fragments, however, the interaction between GST-FHL3 and lines were harvested following 24 hours of differentiation and MyoD was consistently less, under these in vitro binding lysates immunoblotted for myogenin or actin. Western blots are conditions, than the complex between GST-MLP and His- representative of three experiments. (D) The mean relative level of MyoD (Fig. 6D). myogenin protein during siRNA-mediated knockdown of To further confirm an interaction between FHL3 and MyoD, endogenous FHL3 in Vector (2) or HA-FHL3 (2) cell lines was quantified by densitometry expressed relative to actin loading and a GST pull-down assay was undertaken using purified GST or standardised to vector levels set at 1 arbitrarily. C2C12 cells GST-FHL3 protein produced in E. coli and partially purified transfected with Vector (2) are represented by white bars, HA- by coupling to glutathione Sepharose beads. C2C12 cells were FHL3 (2) by black bars, error bars represent ± s.e.m. (n=3, differentiated for 24 hours and cell lysates mixed with GST- *P<0.05, **P<0.01). siRNA transfected is shown below figure as FHL3-coupled or GST-coupled beads. A 24-hour time point indicated. was selected because differentiation-dependent transactivation FHL3 regulates myogenesis 1429

of the myogenin gene occurs by then (Shimokawa et al., 1998). or E47, and bind to specific E-box DNA sites in many muscle- Bound, supernatant and lysate input fractions were specific gene promoters (Lassar et al., 1991). The GST pull- immunoblotted with rabbit anti-MyoD or anti-GST antibodies down assay outlined above was repeated and samples were as a loading control. A 40 kDa polypeptide corresponding to immunoblotted with antibodies against other related bHLH endogenous MyoD was specifically pulled down by GST- proteins, such as Myf5, myogenin and E47. All these proteins FHL3 but not GST (Fig. 6E). This polypeptide was not were detected in complex with purified recombinant GST- detected by immunoblot analysis using non-immune rabbit FHL3 but not GST (Fig. 6F), suggesting that FHL3 either binds antibody, purified GST or GST-FHL3 probed with anti-MyoD a common structural motif in these proteins or a common antibody, which had not been incubated with C2C12 cell adapter molecule; however, it might bind MyoD itself, which lysates (data not shown). Hence, endogenous MyoD protein in turn associates with E47 as previously reported (Lassar et specifically associates with recombinant GST-FHL3 protein, al., 1991). confirming an interaction between these proteins. To further determine whether endogenous FHL3 and MyoD bHLH transcription factors that are upregulated in response indeed interact in vivo, we performed a series of colocalization to myogenic signals include MyoD, myogenin, Myf5, and studies. Endogenous FHL3 colocalised in the nucleus of MRF4. MRFs form heterodimers with E proteins, such as E12 myoblasts with endogenous MyoD but was also observed in

Fig. 6. FHL3 forms a complex with MyoD and other bHLH proteins. (A) C2C12 cells were co-transfected with various combinations of expression vectors for Flag-vector, MyoD-Flag, Gal4DBD and/or Gal4DBD-FHL3, together with Gal4-luciferase and Renilla luciferase reporters. Transfected myoblasts were maintained in growth media for 24 hours then differentiated for 48 hours and assayed for luciferase activity. Luciferase values were corrected for background luminescence, normalised to Renilla luciferase activity to adjust for transfection efficiency and standardised relative to Gal4DBD expressing samples (arbitrarily defined as 1 relative luciferase unit RLU). Error bars represent ± s.e.m. (n=4, ***P<0.001). White boxes represent GAL4DBD-FHL3 and black- Journal of Cell Science line just above baseline represents GAL4DBD. (B) Lysates from C2C12 myoblasts transfected with pCMX- Gal4DBD or pCMX-Gal4DBD-FHL3 for 24 hours were immunoblotted to confirm recombinant protein expression. (C) Lysates from C2C12 myoblasts transfected with pEFBOS-Flag or pEFBOS-MyoD-Flag for 24 hours were immunoblotted to confirm recombinant protein expression. (D) GST-FHL3 and His-MyoD were coexpressed in E. coli. GST-proteins and associated proteins were bound to glutathione Sepharose, washed extensively and eluted with reduced glutathione. Eluted proteins (bound) and whole-cell extracts (input fractions) were immunoblotted for recombinant GST, or His protein expression. Western blots are representative of three experiments. (E) GST or GST-FHL3 captured on glutathione-Sepharose was incubated for 4 hours, with cell lysates from C2C12 cells differentiated (24 hours), washed extensively and immunoblotted for endogenous MyoD, or recombinant GST and/or GST-FHL3 using antibodies as indicated. Results shown are representative of three similar experiments. S/N and input fractions represent unbound lysate and C2C12 cell lysates prior to incubation with GST or GST-FHL3 respectively. (F) GST or GST-FHL3 pull-downs described above in (E) were also immunoblotted for endogenous Myf5, myogenin, and E47 using indicated antibodies. Western blots are representative of three experiments. (G) C2C12 myoblasts were stained with anti-FHL3 (green), anti-MyoD antibodies (red), and with the nuclear stain To-Pro-3 (blue) and imaged by confocal microscopy. Bar, 20 ␮m. 1430 Journal of Cell Science 120 (8)

the cytoplasm (Fig. 6G). The relative level of nuclear FHL3 compared with cytoskeletal FHL3 varied in C2C12 cells – with predominant nuclear FHL3 staining observed in sub-confluent growing myoblasts – but was absent from the nuclei of myotubes, suggesting the nuclear localization of FHL3 is regulated by cell density, serum growth factors and/or the degree of differentiation (data not shown). Therefore, our data support the contention that FHL3 and MyoD interact in the nucleus of myoblasts to regulate the onset of differentiation.

FHL3 suppresses MyoD transcriptional activation As we had demonstrated that FHL3 and MyoD form a complex, and FHL3 overexpression or siRNA-mediated depletion was associated with altered expression of a MyoD-dependent gene, myogenin, we postulated that FHL3 regulates MyoD-dependent gene transcription. To address this issue a series of luciferase assays were carried out. In the first assay the effect of FHL3 overexpression on the transcriptional activity of a Gal4DBD- MyoD fusion protein on a Gal4-promoter was investigated. C2C12 myoblasts were seeded at a sub-confluent density and co-transfected with various combinations of plasmids expressing Gal4DBD, Gal4DBD-MyoD, Vector (HA-vector) or HA-FHL3. Following transfection, myoblasts were maintained in growth media for 48 hours and luciferase activity was assayed. Gal4DBD-MyoD coexpression with Vector transactivated the luciferase reporter gene approximately 18- fold and this activity was specifically repressed approximately threefold by coexpression of HA-FHL3 (Fig. 7A), consistent with the contention that FHL3 suppresses MyoD transcriptional activity. As FHL3 regulates C2C12 cell differentiation, these Fig. 7. FHL3 overexpression reduces MyoD transcription of assays were repeated in C2C12 cells differentiated for 48 hours. luciferase reporter genes. (A) C2C12 cells were co-transfected with Gal4DBD-MyoD, coexpressed with Vector, activated the various combinations of expression vectors for Vector (HA-vector), luciferase reporter gene sixfold (compared with Gal4DBD), and HA-FHL3, Gal4DBD and/or Gal4DBD-MyoD, together with a coexpression of HA-FHL3 significantly inhibited MyoD Gal4-luciferase and Renilla luciferase reporters. Transfected myoblasts were maintained in growth media for 48 hours and Journal of Cell Science reporter gene activity by approximately twofold (Fig. 7B). Expression of intact recombinant proteins from pCMX- assayed for luciferase activity. Luciferase values were corrected for background luminescence, normalised to Renilla luciferase activity Gal4DBD and pCGN constructs was verified in C2C12 to adjust for transfection efficiency and standardised relative to myoblasts by immunoblot analysis (Fig. 7C,D). Gal4DBD-expressing samples (arbitrarily defined as 1 relative MyoD binds the E-boxes (CANNTG) of the gene encoding luciferase unit- RLU). Gal4DBD activity is represented by grey MCK with high affinity to activate gene transcription bars, Gal4DBD-MyoD by white bars, error bars represent ± s.e.m. (Chakraborty et al., 1991). The ability of FHL3 to regulate (n=6, *P<0.05). Co-transfection with Vector or HA-FHL3 is shown MyoD-dependent transcription of the MCK-promoter below graph. (B) C2C12 cells were co-transfected as above. luciferase reporter pGL3-MCK (Novitch et al., 1999) was Transfected myoblasts were maintained in growth media for 24 investigated. The basal reporter activity – i.e. 1 relative hours and switched to differentiation media for 48 hours, and then luciferase unit, was arbitrarily defined as 1 RLU – in Flag- assayed. Gal4DBD activity is represented by grey bars, Gal4DBD- vector transfected cells was decreased by approximately MyoD by white bars, error bars represent ± s.e.m. (n=7, *P<0.05). Co-transfection with Vector or HA-FHL3 is indicated below figure. threefold by expression of HA-FHL3, relative to Vector. This (C) Lysates from C2C12 myoblasts transfected with pCMX- change in the basal level of reporter activity is probably due to Gal4DBD or pCMX-Gal4DBD-MyoD for 24 hours, were FHL3 regulation of endogenous MyoD, although FHL3 immunoblotted to confirm recombinant protein expression. regulation of other transcription factors cannot be excluded, (D) Lysates from C2C12 myoblasts transfected with pCGN or because the full-length MCK promoter (in pGL3-MCK) pCGN-FHL3 for 24 hours, were immunoblotted to confirm contains many binding elements, including (Amacher et recombinant protein expression. (E) C2C12 cells were co- al., 1993). The MCK-luciferase reporter gene was further transfected with various combinations of expression vectors for transactivated by expression of MyoD-Flag with Vector and Vector, HA-FHL3, Flag-vector and/or MyoD-Flag, with a pGL3- decreased significantly following coexpression with HA-FHL3 MCK (muscle creatine kinase luciferase reporter) and renilla (Fig. 7E). Therefore, FHL3 inhibits MyoD-dependent luciferase. Assays were performed under differentiation conditions as outlined above except values were standardised relative to Flag- transcription of target genes. vector and Vector coexpressing samples (arbitrarily defined as 1 RLU). Flag-vector activity is represented by grey bars, MyoD-Flag siRNA-mediated FHL3 knockdown increases MyoD by white bars, error bars represent ± s.e.m. (n=3, *P<0.05, transcription of luciferase reporter genes **P<0.01). Co-transfection with Vector or HA-FHL3 is shown To further verify that FHL3 regulates MyoD transcriptional below the graph. FHL3 regulates myogenesis 1431

activity, the Gal4-based luciferase assay outlined above was and control siRNA co-transfected, arbitrarily defined as 1 repeated in C2C12 cells undergoing differentiation under RLU) was significantly increased approximately threefold and conditions of siRNA-mediated FHL3 knockdown. Gal4DBD- tenfold fold in cells transfected with siRNA FHL3 #2 or #3, MyoD activity increased 1.5-fold and 2.7-fold by siRNA respectively. This change in the basal level of reporter activity FHL3 #2 or #3, respectively (Fig. 8A). FHL3 immunoblot is probably due to the regulation of endogenous MyoD by analysis was performed on lysates to confirm FHL3 protein FHL3, although the regulation of FHL3 on other transcription knockdown (Fig. 8B). The level of FHL3 protein significantly factors can, again, not be excluded. Basal MCK promoter decreased with transfection of FHL3 siRNA, relative to activity was also increased sixfold with MyoD-Flag expression control siRNA and negatively correlated with the increased and was further significantly increased twofold and sixfold Gal4DBD-MyoD transcriptional activity, suggesting FHL3 upon transfection with siRNA FHL3 #2 and #3 regulates MyoD-dependent gene transcription in a dose- oligonucleotides, respectively (Fig. 8C). The relative level of dependent manner. FHL3 siRNA protein knockdown negatively correlated with To verify whether FHL3 regulates MyoD transcriptional the magnitude of MyoD (recombinant or endogenous) activity during C2C12 cell differentiation on native gene transcriptional activity, suggesting that FHL3 dose dependently targets, the MCK promoter reporter was used as a reporter regulates MyoD transcriptional activity (Fig. 8D). construct under conditions of FHL3 knockdown mediated by transfection of siRNA. The basal reporter activity (Flag-vector Discussion We have demonstrated here that FHL3, a relatively uncharacterised member of the FHL protein family, inhibits MyoD transcriptional activity, providing new insights into the molecular mechanisms governing gene expression during myogenesis. We have shown that FHL3 functions as a new regulator of myofibre formation. Evidence from FHL3 overexpression and siRNA protein knockdown has revealed that altered expression of this LIM protein affects both the differentiation and fusion of myoblasts. Other C2C12 cell sub- populations, such as reserve cells and/or myoblasts, were assessed using specific markers for CD34, Pax7, MyoD and PCNA, which showed no differences in the levels and/or localization of these markers following altered FHL3 expression (data not shown). These results suggest that FHL3 functions in myoblast differentiation along the myofibre lineage. We propose that the altered sarcomere assembly detected in myotubes following FHL3 overexpression or

Journal of Cell Science siRNA-mediated depletion was a consequence of altered gene transcription, as indicated by temporal changes in myogenin

Fig. 8. siRNA-mediated FHL3 knockdown increases MyoD transcription of luciferase reporter genes. (A) C2C12 cells were co- transfected with various combinations of expression vectors for Gal4DBD or Gal4DBD-MyoD and siRNA oligonucleotides as indicated, with a Gal4-luciferase and Renilla luciferase reporters. Assays were performed under differentiation conditions. Gal4DBD activity is represented by grey bars, Gal4DBD-MyoD by white bars, error bars represent ± s.e.m. (n=4 *P<0.05, **P<0.01). Co- transfection with specific siRNAs is shown below the graph. (B) Cellular extracts (20 ␮l) prepared from C2C12 cells transfected with the indicated constructs and used for the luciferase assay shown in A), were also immunoblotted with the anti-FHL3 antibody to confirm siRNA-mediated FHL3 knockdown and with total actin antibody as a protein loading control. (C) C2C12 cells were co- transfected with various combinations of expression vectors for Flag- tagged vector or MyoD-Flag and siRNA oligonucleotides as indicated with a pGL3-MCK and Renilla luciferase reporters. Assays were performed under differentiation conditions. Flag-tagged vector activity is represented by grey bars, MyoD-Flag by white bars, error bars represent ± s.e.m. (n=8, *P<0.05, ***P<0.001). Co-transfection with specific siRNAs is shown below the graph. (D) Cellular extracts (20 ␮l) prepared from C2C12 cells transfected with the indicated constructs and used for the luciferase assay shown in C), were also immunoblotted with the anti-FHL3 antibody to confirm siRNA- mediated knockdown of FHL3 protein and with total actin antibody as a protein loading control. 1432 Journal of Cell Science 120 (8)

protein levels. We were unable to detect any changes in MyoD protein expression following FHL3 overexpression or siRNA- mediated depletion; however, induction of myogenin protein following 24 hours of differentiation was altered and negatively correlated with the FHL3 protein levels. MyoD and myogenin are essential to skeletal myogenesis. MyoD-null primary myoblasts demonstrate delayed differentiation in vitro (Yablonka-Reuveni et al., 1999) and myogenin is required for cell fusion and myotube formation (Hasty et al., 1993; Fig. 9. Model for FHL3 regulation of MyoD-dependent myogenesis. Nabeshima et al., 1993; Hashimoto and Ogashiwa, 1997). FHL3 physically interacts with MyoD to negatively regulate MyoD- dependent transcription of differentiation genes such as myogenin, Downregulation of either MyoD or myogenin during C2C12 retarding myotube formation and myogenesis. cell myogenesis causes differentiation and fusion defects, a similar phenotype to the defects observed in cells overexpressing FHL3 (Hashimoto and Ogashiwa, 1997; overexpression promotes myogenesis (Arber et al., 1994; Kong Dedieu et al., 2002). Owing to the experimental limitations of et al., 1997). the C2C12 skeletal muscle cell line, it was impossible to Very large transcriptional complexes that involve FHL3 have determine whether FHL3 overexpression completely blocked been previously reported (Takahashi et al., 2005), suggesting or merely altered the rate of differentiation – the latter that additional proteins facilitate or participate in MyoD:FHL3 phenotype is exemplified by MyoD gene-targeted deletion. For complex. Some candidate adapter proteins based on published example, MyoD gene deletion delays myogenesis of branchial FHL3 and other FHL family member binding partners may arches, tongue, limbs and diaphragm during embryogenesis, include histone-associated proteins, such as NFY or HDACS and the timing of trunk and abdominal wall musculature (Yang et al., 2005; Takahashi et al., 2006), p300 (Labalette et formation. However, these MyoD-null mice display only minor al., 2004) and CREB (Fimia et al., 2000). CREB is of particular skeletal muscle changes in adult life (Kablar et al., 1998). significance because it forms a complex with MyoD and is also FHL3 has been shown previously to repress gene an FHL3-binding partner in other cells; however, this has not transcription in non-muscle cell types (Turner et al., 2003; been shown in muscle (Fimia et al., 2000; Magenta et al., 2003; Takahashi et al., 2005). As described here in myoblasts, FHL3 Kim et al., 2005). negatively regulates MyoD-dependent gene transcription, We propose a model whereby FHL3 in the nucleus of whereas siRNA knockdown of FHL3 enhanced MyoD myoblasts complexes with MyoD to repress differentiation transcriptional activity. The FHL3-mediated effects (Fig. 9). The nuclear localization of FHL3 is regulated by demonstrated by luciferase assays are most likely the integrin engagement, Rho activity, serum, cell density and consequence of a transcriptional complex between FHL3 and cytoskeletal dynamics (Muller et al., 2002; Coghill et al., MyoD, as shown by GST pull-down, direct protein-protein 2003), suggesting FHL3 connects cell signalling through these binding and colocalization studies. Both MLP and the FHL3- pathways, directly to a master control of myogenesis, MyoD.

Journal of Cell Science family member FHL2 bind bHLH proteins, and alter bHLH Once appropriate differentiation conditions are met, FHL3 is heterodimerization with E proteins to regulate their actively excluded from the nucleus and, thus, is unable to transcriptional activity. MLP binds MyoD and myogenin to interact with MyoD and regulate its transcriptional activity. enhance heterodimerization with co-activator E proteins and Consistent with this model, FHL3 is absent from the nucleus promote C2C12 cell myogenesis, whereas FHL2 retards of developing myotubes and FHL3 localises to the Z-line of HAND1 transcriptional activity in cardiac tissue by repressing mature skeletal muscle (Li et al., 2001; Coghill et al., 2003). heterodimerization between Hand1 and E proteins (Arber et al., In summary, FHL3 protein knockdown and overexpression 1994; Kong et al., 1997; Hill and Riley, 2004; Lu et al., 2004). during C2C12 cell myogenesis have demonstrated a functional The FHL3 overexpression phenotype was reminiscent of the role for FHL3 in negatively regulating myotube formation. The phenotype induced by Id overexpression which blocks MyoD selective recruitment of interacting proteins such as FHL3, and E12 heterodimerization and, thereby, retards MyoD which negatively regulates MyoD transcriptional activity, may transcriptional activity (Jen et al., 1992). Hence, it is likely that represent a mechanism by which MyoD temporally regulates FHL3 also regulates the heterodimerization and/or activity of gene-specific activation and, thereby, myogenesis. MyoD-bHLH dimers. In this regard it is noteworthy that FHL3 forms a complex with other bHLH proteins, such as Myf5, Materials and Methods myogenin and E47. However, the muscle-differentiation Materials phenotype mediated by FHL3 in retarding myotube formation Restriction and DNA-modifying enzymes were obtained from Promega (Australia), is unique and different to that mediated by other FHL proteins MBI Fermentas or New England Biolabs. DNA oligonucleotides were purchased from Geneworks (Australia) or Micromon (Australia). E. coli TOP10 were or unrelated LIM proteins, such as MLP. For example, FHL1 purchased from Invitrogen (Australia). Big Dye Version 3.1 sequencing terminators overexpression results in myosac formation, whereas its were supplied by PE applied systems (Australia). All other reagents were purchased siRNA-mediated depletion impairs the formation of myosin from Sigma-Aldrich or BDH Chemicals unless otherwise stated. thick filaments associated with reduced incorporation of Plasmids and cloning myosin-binding protein C into the sarcomere (McGrath et al., pCGN (Tanaka and Herr, 1990) was a kind gift from Tony Tiganis (Monash 2006). FHL2 overexpression in C2C12 mouse myoblasts University, Melbourne, Australia). pGEX-5x1 was purchased from Amersham results in increased myogenic differentiation and accelerated Biosciences. pCGN-FHL3, pEGFP-C2-FHL3, pGEX-5x1-FHL3 have been ␤ described previously (Coghill et al., 2003). pTrio12R-HGN has been described myotube formation by binding to -catenin to reduce the previously (McGrath et al., 2006) and pTrio12R-HA-FHL3-GN was generated by expression of LEF/TCF (Martin et al., 2002). MLP cloning FHL3 cDNA from pCGN-FHL3 into the XbaI site downstream of the HA- FHL3 regulates myogenesis 1433

tag. pCMX-Gal4DBD and G5E1b-LUC (Muller et al., 2000) were kind gifts from Antibodies Roland Schule (University of Freiberg, Freiberg, Germany). pHRLTK was Antibodies include anti-pan actin Ab-5 clone ACTN05 (1:3333 dilution western purchased from Promega. pGL3-MCK (Novitch et al., 1999) was a kind gift of blotting-WB, NeoMarkers anti-E47 N-649 (1:100 WB, Santa Cruz Biotechnology), Andrew B. Lassar (Harvard Medical School, Boston, MA). The complete mouse anti-FHL3 (1:30 immunofluorescence- IF and 1:200 WB) (Coghill et al., 2003), MyoD sequence was amplified with flanking AscI sites from pEMSV-MyoD (Davis anti-Flag polyclonal (1:500 WB, Sigma-Aldrich), anti-Gal4(DBD) clone RK5C1 et al., 1987), a gift from Edna Hardeman (Children’s Medical Research Institute, (1:100 WB, Santa Cruz Biotechnology), anti-GST polyclonal (1:1000 WB, Sydney, Australia), with the primers 5Ј-cctggcgcgccagatggagcttctatcgccg and 3Ј- Amersham Biosciences), anti-HA.11 clone 16B12 (1:5000 WB, Covance), anti- cctggcgcgccataagcacctgataaatcgc, based on gene bank entry NM_010866 polyHistidine Clone HIS-1 (1:3000 WB, Sigma-Aldrich), anti-MHC MF20 (1:20 nucleotides 192-209 and 1128-1145 respectively, and cloned via pCR-Blunt IF, University of Iowa, Developmental Studies Hybridoma Bank), anti-Myf5 C-20 (Invitrogen) into the pEFBOS-Flag vector (Mizushima and Nagata, 1990), a gift (1:100 WB, Santa Cruz Biotechnology), anti-myogenin F5D (1:100 WB, Santa Cruz from Tracey Wilson (Walter and Eliza Hall Institute of Medical Research, Biotechnology), anti-myogenin M-225 (1:100 IF and WB, Santa Cruz Melbourne, Australia). The MyoD coding sequence was cloned from pEFBOS- Biotechnology), anti-MyoD 5.8A (1:50 IF, Imgenex Corporation), anti-MyoD C-20 MyoD-Flag into the EcoRI site of pCMX-Gal4DBD to generate pCMX-Gal4DBD- (1:100 WB, Santa Cruz Biotechnology), anti-MyoD M-318 (1:100 WB, Santa Cruz MyoD. The FHL3 sequence was cloned from pEGFP-C2-FHL3 into pCMX- Biotechnology), donkey anti-goat-HRP 1:10,000 WB (Chemicon), donkey anti- Gal4DBD (EcoRI site), to generate pCMX-Gal4DBD-FHL3. pET-30a(+) was mouse (H+L)-Alexa Fluor-594 (1:600 IF, Molecular Probes), donkey anti-rabbit purchased from Novagen, and pET-30a(+)-MyoD was generated by shuttling the (H+L)-Alexa Fluor-647 (1:600 IF, Molecular Probes), sheep anti-mouse (H+L)- MyoD sequence from pCR-Blunt using in frame flanking EagI sites in the cloning FITC (1:400 IF, Chemicon), sheep anti-mouse-HRP (1:10,000 WB, Chemicon), vector into the NotI site of pET-30a(+). The identity and fidelity of the PCR products sheep anti-rabbit (H+L)-FITC (1:400 IF, Chemicon), sheep anti-rabbit-HRP were confirmed by sequencing (Micromon). (1:10,000 WB, Chemicon),

Culturing and differentiation of C2C12 myoblasts Direct FHL3:MyoD protein interaction Murine C2C12 myoblasts (ATCC, Manassas, VA) were maintained in growth The E. coli strain BL21 DE3 Gold Codon Plus RP (Stratagene) were co-transformed media (DMEM supplemented with 20% foetal calf serum, 2 mM L-glutamine, with pGEX-5x1 derivatives (Amersham Biosciences) and pET-30a(+) derivatives 100 units/ml penicillin and 0.1% streptomycin). To induce differentiation (Novagen), grown to an OD600 of ~0.4 at 37°C in LB supplemented with 50 ␮g/ml experiments, myoblasts at 100% confluency were incubated in DMEM ampicillin, 50 ␮g/ml chloramphenicol, 25 ␮g/ml kanamycin and 10 ␮M ZnSO4. supplemented with 2% horse serum, 2 mM L-glutamine, 100 units/ml penicillin, Recombinant protein production was induced with 0.1 mM IPTG for 2 hours at and 0.1% streptomycin for up to 120 hours. Differentiation media was replaced 33°C. Cell pellets were collected and resuspended in 2.5 ml of media and lysed with every 48 hours. For all siRNA transfections, myoblasts were seeded, grown and 0.25 ml of PopCulture lysis reagent, 2.5 ␮l of Lysonase bioprocessing reagent differentiated in media without antibiotics. Growth media containing 1.5 mg/ml (Novagen), and 5 ␮l of protease inhibitors P8849 (Sigma Aldrich). GST fusion G418-sulphate (AG Scientific) was used for C2C12 cell lines stably expressing proteins were bound to glutathione-Sepharose 4B resin (Amersham Biosciences) Vector (HA-vector) and HA-FHL3. G418-sulphate was removed during followed by extensive washing in Tris-Saline pH 8 containing 1% Triton X-100, differentiation experiments. then Tris-saline pH 7.4. GST proteins were eluted with 10 mM reduced glutathione in 50 mM Tris.Cl pH 8, and immediately stored at 4°C in SDS reducing-buffer prior Transfection of plasmid DNA and/or siRNA oligonucleotides to immunoblotting. Transfection-quality plasmid DNA was prepared using Qiagen (Germany) midiprep kits and transfected using Lipofectamine (Invitrogen) according to the GST pull-down from mammalian cell lysates manufacturer’s protocol. HP GenomeWide siRNA Oligonucleotides: Recombinant GST and GST-FHL3 protein were produced by auto-induction of E. Mm_FHL3_2_HP siRNA (SI01002890), Mm_FHL3_3_HP siRNA (SI01002897), coli BL21 DE3 pLysS (Novagen) transformed with pGEX-5x1 (Amersham and control (non-silencing) siRNA (1022076) oligonucleotides (Qiagen) were Biosciences) or pGEX-5x1-FHL3 in Overnight Express Media (Novagen), transfected into C2C12 myoblasts as previously described (McGrath et al., 2006). supplemented with 100 ␮g/ml ampicillin, and purified using 0.1 ml per 1 ml of Plasmid and siRNA co-transfection was performed using Lipofectamine 2000 culture of PopCulture lysis reagent, 1 ␮l per 1 ml of culture of Lysonase (Invitrogen) according to the manufacturer’s protocol. bioprocessing reagent (Novagen), 2 ␮l per 1 ml of culture of protease inhibitors P8849 (Sigma Aldrich) and glutathione-Sepharose 4B beads (Amersham Stable overexpression of HA-vector C2C12 myoblast cell lines Biosciences) according to manufacturer’s protocols. Bound recombinant GST and Journal of Cell Science and HA-FHL3 in C2C12 cells GST-FHL3 protein were washed and stored at 4°C as a 1:5 resin slurry in Tris-saline pTrio12R-HGN and its derivatives autonomously replicate and express a tri- pH 7.4 containing Roche complete mini cocktail inhibitor tablets and P8849 liquid cistronic mRNA encoding HA or HA-fusion proteins, low amounts of glutathione inhibitors. The concentration of bound GST or GST-FHL3 was quantified by S transferase (GST) and neomycin resistance in mammalian cells (McGrath et al., Coomassie-stained SDS-PAGE. C2C12 myoblasts were differentiated for 24 hours 2006). pTrio12R-HGN (HA-vector) and pTrio12R-HA-FHL3-GN (HA-FHL3) and lysates prepared in HEPES lysis buffer with 0.2% or 1% NP-40, then pre- constructs were stably transfected into C2C12 myoblasts and selected in 1.5 mg/ml cleared for 2 hours with glutathione-Sepharose 4B resin. 5 ␮g of GST or GST-FHL3 G418-sulphate for 3 weeks. Individual G418 resistant myoblasts for each transfected were incubated for 2-4 hours, rocking at room temperature with C2C12 cell lysates. construct were not isolated, but rather were pooled as a heterogeneous population Resin-bound GST or GST-FHL3 pellets were then washed six times in Tris-saline (which equalise upon cell fusion), to limit the background variability of individual pH 7.4 or Tris-saline pH 7.4 including 1% Triton X-100 and immunoblotted. clones and to increase the passaging life-time of the C2C12 cell lines, as previously reported (Jen et al., 1992). C2C12 myoblast cell lines stably expressing Vector (HA- Luciferase assays vector) or HA-FHL3 were created in duplicate (Pool 1 and Pool 2) and designated C2C12 myoblasts were seeded in 12-well plates at 2.5ϫ104 cells per well for growth as either Vector (1) and Vector (2) or HA-FHL3 (1) and HA-FHL3 (2), respectively. condition assays or 1ϫ105 cells per well for differentiation assays. Cells were cultured for 24 hours in growth media, and transfected with the following plasmids Immunofluorescent labeling of C2C12 cells and/or siRNA oligonucleotides in various combinations at the amount indicated. Immunofluorescence staining of C2C12 cells has been previously described Mammalian two hybrid: pCMX-Gal4DBD or pCMX-Gal4DBD-FHL3 (50 ng) (McGrath et al., 2003). Briefly, 2ϫ105 stably transfected C2C12 myoblasts were and pEFBOS-Flag or pEFBOS-MyoD-Flag (500ng), with G5E1b-LUC firefly seeded onto 22ϫ22 mm fibronectin-coated coverslips in six-well plates, cultured luciferase reporter vector (500 ng) and pHRLTK renilla luciferase reporter (50 ng). for 24 hours in growth media, then switched to differentiation media. Alternatively, Gal4 FHL3 Overexpression: pCMX-Gal4DBD or pCMX-Gal4DBD-MyoD (50 ng) 5ϫ104 C2C12 myoblasts were seeded on fibronectin-coated coverslips in growth and pCGN or pCGN-FHL3 (500 ng) with G5E1b-LUC firefly luciferase reporter media, transfected the following day with siRNA oligonucleotides, cultured for 48 vector (500ng) and pHRLTK renilla luciferase reporter (50 ng). MCK FHL3 hours in growth media, then switched to differentiation media. Cells grown on Overexpression: pEFBOS-Flag or pEFBOS-MyoD-Flag (50 ng) and pCGN or coverslips were fixed, permeabilised, blocked and incubated with propidium iodide pCGN-FHL3 (1 ␮g), along with pGL3-MCK (50 ng) and pHRLTK renilla luciferase at ~25 ␮g/well (Sigma-Aldrich) or To-Pro-3 at a dilution of 1:3000 (Molecular reporter (50 ng). Gal4 FHL3 knockdown: pCMX-Gal4DBD or pCMX-Gal4DBD- Probes) and antibodies. MyoD (50 ng) and control siRNA, siRNA FHL3 #2 or siRNA FHL3 #3 (12.5 pmol), with G5E1b-LUC firefly luciferase reporter vector (500 ng) and pHRLTK Renilla Western blot analysis luciferase reporter (50 ng). MCK FHL3 knockdown: pEFBOS-Flag or pEFBOS- C2C12 cells were washed twice in PBS and scraped into HEPES lysis buffer (10 MyoD-Flag (50 ng) and control siRNA, siRNA FHL3 #2 or siRNA FHL3 #3 (12.5 mM HEPES pH 8, 10 mM KCl, 0.1 mM EDTA, 0.2% NP-40, Roche complete mini pmol), with pGL3-MCK (50 ng) and pHRLTK renilla luciferase reporter (50 ng). protease cocktail inhibitor tablet). Samples were rocked for 1 hour at 4°C, then For growth conditions, transfected myoblasts were maintained in growth media for pelleted for 10 minutes at 16,000 g at 4°C. Alternatively, extracts were prepared 48 hours post transfection and harvested. For differentiation conditions, transfected using Passive Lysis Buffer as part of the Dual Luciferase Reporter Assay Kit myoblasts were maintained in growth media for 24 hours, switched to differentiation (Promega). Protein concentration was determined using BioRad DC protein assay media for 48 hours, then harvested. Lysates were prepared and assayed using the kit. 25-50 ␮g of lysate was analysed by SDS-PAGE and western blotting. ‘dual luciferase reporter assay kit’ (Promega) according to manufacturer’s protocol 1434 Journal of Cell Science 120 (8)

and analysed on a BMG Labtech Fluostar Optima plate reader. Luciferase values transcription in skeletal myogenesis is controlled by mTOR and nutrients. J. Cell Biol. were adjusted for background luminescence and normalised to Renilla luciferase 163, 931-936. activity to adjust for transfection efficiency. Relative luciferase units were Fimia, G. M., De Cesare, D. and Sassone-Corsi, P. (2000). A family of LIM-only standardised as a fold relative to either Gal4DBD, Vector (HA-vector) and Flag- transcriptional coactivators: tissue-specific expression and selective activation of vector, or control siRNA and Flag-vector expressing samples, as indicated. CREB and CREM. Mol. Cell. Biol. 20, 8613-8622. Gunther, T., Poli, C., Muller, J. M., Catala-Lehnen, P., Schinke, T., Yin, N., Vomstein, Image and statistical analysis S., Amling, M. and Schule, R. (2005). Fhl2 deficiency results in osteopenia due to decreased activity of osteoblasts. EMBO J. 24, 3049-3056. For confocal image based cell counts, a minimum of 4 random fields were scanned Hashimoto, N. and Ogashiwa, M. (1997). Isolation of a differentiation-defective for each slide and measured/counted using the public domain ImageJ software myoblastic cell line, INC-2, expressing muscle LIM protein under differentiation- (version 1.34 NIH) (Abramoff et al., 2004). For quantitative western blot analysis inducing conditions. Dev. Growth Differ. 39, 363-372. films were scanned and the band signal intensities determined using ImageJ Hasty, P., Bradley, A., Morris, J. H., Edmondson, D. G., Venuti, J. M., Olson, E. N. software. The densitometry values were expressed as a fold level relative to the and Klein, W. H. (1993). Muscle deficiency and neonatal death in mice with a targeted control, and standardised to corresponding total actin densitometry values obtained mutation in the myogenin gene. Nature 364, 501-506. from the same sample. 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