Oncogene (2013) 32, 651 --662 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc

ORIGINAL ARTICLE Alveolar rhabdomyosarcoma-associated PAX3/FOXO1A and PAX7/FOXO1A suppress the transcriptional activity of MyoD-target in muscle stem cells

F Calhabeu1, S Hayashi2, JE Morgan3, F Relaix2 and PS Zammit1

Rhabdomyosarcoma (RMS) is the commonest soft-tissue sarcoma in childhood and is characterized by expression of myogenic proteins, including the transcription factors MyoD and . There are two main subgroups, embryonal RMS and alveolar RMS (ARMS). Most ARMS are associated with chromosomal translocations that have breakpoints in introns of either PAX3 or PAX7, and FOXO1A. These translocations create chimeric transcription factors termed PAX3/FOXO1A and PAX7/FOXO1A respectively. Upon ectopic PAX3/FOXO1A expression, together with other genetic manipulation in mice, both differentiating myoblasts and satellite cells (the resident stem cells of postnatal muscle) can give rise to tumours with ARMS characteristics. As PAX3 and PAX7 are part of transcriptional networks that regulate muscle stem cell function in utero and during early postnatal life, PAX3/FOXO1A and PAX7/FOXO1A may subvert normal PAX3 and PAX7 functions. Here we examined how PAX3/FOXO1A and PAX7/FOXO1A affect in satellite cells. PAX3/FOXO1A or PAX7/FOXO1A inhibited myogenin expression and prevented terminal differentiation in murine satellite cells: the same effect as dominant-negative (DN) Pax3 or Pax7 constructs. The transcription of MyoD-target genes myogenin and muscle creatine kinase were suppressed by PAX3/FOXO1A or PAX7/ FOXO1A in C2C12 myogenic cells again as seen with Pax3/7DN. PAX3/FOXO1A or PAX7/FOXO1A did not inhibit the transcriptional activity of MyoD by perturbing MyoD expression, localization, phosphorylation or interaction with E-proteins. Chromatin immunoprecipitation on the myogenin promoter showed that PAX3/FOXO1A or PAX7/FOXO1A did not prevent MyoD from binding. However, PAX3/FOXO1A or PAX7/FOXO1A reduced occupation of the myogenin promoter by RNA polymerase II and decreased acetylation of histone H4, but did not directly bind to the myogenin promoter. Together, these observations reveal that PAX3/FOXO1A and PAX7/FOXO1A act to prevent myogenic differentiation via suppression of the transcriptional activation of MyoD-target genes.

Oncogene (2013) 32, 651--662; doi:10.1038/onc.2012.73; published online 18 June 2012 Keywords: alveolar rhabdomyosarcoma; PAX3/FOXO1A; PAX7/FOXO1A; MyoD; myogenin; satellite cell

INTRODUCTION Both Pax3 and Pax7 are part of the transcriptional networks Rhabdomyosarcoma (RMS) is the commonest soft-tissue sarcoma that control activation of the myogenic programme during 8 in childhood, characterized by expression of myogenic genes. skeletal muscle development. Pax3-null mice have reduced/ There are two main subgroups, embryonal RMS and alveolar RMS absent trunk musculature, whereas the limb and diaphragm 8 (ARMS), which are associated with distinct morphological, clinical muscles completely fail to form, and deletion of both Pax3 and and genetic characteristics. ARMS often contain t(2;13)(q35;q14) or Pax7 cause a loss of muscle progenitors and arrest of develop- 9 t(1;13)(p36;q14) chromosomal translocations that encode a mental myogenesis. Conditional Pax7 inactivation shows that it is chimeric under the control of upstream regulatory required for satellite cell function during early postnatal muscle 10 elements and the promoter of the paired-box (Pax) transcription growth. Myogenic progression in satellite cells is controlled by factors PAX3 or PAX7, containing the 50-untranslated region and the muscle-specific MyoD, which can be 11,12 first seven exons of PAX3/7, fused to the last two exons and 30- directly regulated by Pax3 or Pax7, whereas myogenin is an untranslated region of another transcription factor---FOXO1A. The early marker of differentiation, whose expression occurs con- 11,13 resulting hybrid transcription factors PAX3/FOXO1A and PAX7/ comitantly with downregulation of Pax7. Consistent with FOXO1A (collectively called PAX/FOXO1A here) thus have the these observations MyoD is upregulated in embryos in which 14 DNA-binding homeo- and paired-domains, and the octapeptide PAX3/FOXO1A has been targeted to a Pax3 allele, and PAX3/ 15 motif that mediates protein--protein interactions, of PAX3/7, fused FOXO1A directly binds the MyoD distal regulatory region. to the potent transactivation domain of FOXO1A.1 PAX/FOXO1A However, although MyoD is expressed in ARMS, it is transcrip- 16,17 have enhanced protein stability, abundance, transcriptional tionally inactive. activity and additional target sequences,1,2 and may subvert These connections to the myogenic lineage implicate muscle normal functions of PAX3/7 protein by deregulating the PAX3/7- stem cells as a potential cellular source of ARMS. Furthermore, downstream target genes.3--7 ARMS often occurs in the vicinity of skeletal muscle in the

1King’s College London, Randall Division of Cell and Molecular Biophysics, London, UK; 2INSERM UMR S 787-Myology Group, Avenir Team Mouse Molecular Genetics, UPMC- Faculte´ de Me´decine Pitie´-Salpeˆtrie`re, Institut de Myologie, 105 Bd de l’Hoˆpital, Paris, France and 3Dubowitz Neuromuscular Centre, UCL Institute of Child Health, London, UK. Correspondence: Professor PS Zammit, King’s College London, Randall Division of Cell and Molecular Biophysics, New Hunt’s House, Guy’s Campus, London SE1 1UL, UK. E-mail: [email protected] Received 24 February 2011; revised 13 December 2011; accepted 8 January 2012; published online 18 June 2012 PAX3/FOXO1A and PAX7/FOXO1A inhibit MyoD-target genes F Calhabeu et al 652 extremities and trunk of adolescents and young adults.18 --21 Myogenic differentiation is inhibited by PAX3/FOXO1A or PAX7/ Work in mice indicates that upon ectopic expression of PAX3/ FOXO1A FOXO1A, together with other genetic manipulations, both We used retroviral-mediated constitutive expression to examine presumed differentiating myoblasts (Mrf4/myf6-directed Cre) the effects of PAX3/FOXO1A and PAX7/FOXO1A on myogenic and Pax7-expressing (Pax7-directed Cre) cells can generate progression in satellite cells from a muscle with few satellite cells tumours with an ARMS phenotype,22,23 although they are unlikely with Pax3 activity (EDL), compared with one with many to be the exclusive source.24 As Pax7 is expressed in muscle satellite cells with Pax3 locus activity (biceps). Cultured wildtype satellite cells,25 the resident stem cells of adult muscle, satellite myofibres were exposed to pMSCV-IRES-PAX3/FOXO1A-eGFP cells are a good candidate for transformation into oncogenic cells (PAX3F-RV), pMSCV-IRES-PAX7/FOXO1A-eGFP (PAX7F-RV) or in ARMS. pMSCV-IRES-eGFP (control-RV) and fixed 48 h later. In myogenic cells, PAX3/FOXO1A generally inhibits differentia- Infected myofibre-associated satellite cells were identified by tion, but it is unclear at which point it intervenes, or how.2,26 eGFP, and endogenous Pax7 was assayed using an antibody Importantly, MyoD is able to convert many non- against the C-terminus (epitopes 352--523), not present in PAX7/ types to the myogenic lineage27,28 and PAX3/FOXO1A can activate FOXO1A. Co-immunostaining for eGFP and Pax7 (Figures 2a MyoD in fibroblasts, but the degree of subsequent myogenic and b) showed that more myofibre-associated cells expressing differentiation varies between studies from none, through to PAX3/FOXO1A (EDL 58.0±8%; biceps 64±10.5%) or PAX7/ fusion into large multinucleated myotubes.29 --31 PAX3/FOXO1A FOXO1A (EDL 45.8±13.7%; biceps 42.8±8.2%) contained Pax7, can also inhibit this MyoD-mediated conversion,26,29 but this effect compared with control-RV (EDL 24.4±0.1%; biceps 14.3±3.2% - is MyoD-specific, as PAX3/FOXO1A does not have substantial Figure 2c). effects on myogenin-dependent myogenic conversion.26 Co-immunostaining for either eGFP/MyoD or eGFP/myogenin Here we investigated and directly compared the effects of showed that while PAX3F-RV- or PAX7F-RV-infected satellite PAX3/FOXO1A and PAX7/FOXO1A on satellite cell function, and cells maintained MyoD (Figure 3a), induction of myogenin was how this effect is mediated. Constitutive PAX3/FOXO1A or inhibited (Figure 3b) [PAX3/FOXO1A (EDL 7.6±4.6%, biceps PAX7/FOXO1A in activated satellite cells was compatible with 6.9±3.6%) and PAX7/FOXO1A (EDL 13.2±1.5%, biceps MyoD expression, but myogenin was not induced and differentia- 17±5.8%) compared with control-RV (EDL 60.7±0.5%; biceps tion was blocked. The activity of the MyoD-target reporters, 69.6±0.05%; Figures 3c--d)]. can co-operate with MyoD to including myogenin, muscle creatine kinase (MCK) and p21, were activate MyoD-target genes, such a myogenin, during differentia- all suppressed in C2 myoblasts, indicating that PAX/FOXO1A can tion,34,35 but also failed to be upregulated in satellite cells inhibit the ability of MyoD to activate its transcriptional target expressing PAX3/FOXO1A or PAX7/FOXO1A (Supplementary- genes. A similar suppression of reporter activity occurs with Figure S2). dominant-negative (DN) versions of Pax3 and Pax7. Various post- Plating satellite cells allows the later stages of myogenic translational mechanisms control the transcriptional activity of differentiation to be examined, such as fusion into multi-nucleated MyoD,32 but PAX/FOXO1A did not affect MyoD localization, myotubes. To better understand how PAX/FOXO1A affects phosphorylation, or interaction with E-proteins. However, chro- satellite cells, we directly compared PAX/FOXO1A with wild-type matin immunoprecipitation (ChIP) revealed that although PAX3/ Pax3/7, together with Pax3DN and Pax7DN constructs, in which FOXO1A or PAX7/FOXO1A did not affect MyoD binding to the the transactivation domain has been replaced with the myogenin promoter, it did reduce acetylation of histone H4 and repressor domain.36 Plated EDL and biceps-derived satellite cells occupation of the myogenin promoter by RNA polymerase II. were infected with PAX3F-RV, PAX7F-RV, Pax7-RV, Pax3-RV, Together, these observations show that PAX3/FOXO1A and PAX7/ Pax3DN-RV, Pax7DN-RV or control-RV, and switched to differentia- FOXO1A suppress the transcriptional activation of MyoD-target tion medium 24 h later for 4 days, before fixing and genes to inhibit myogenic differentiation. co-immunostaining for eGFP together with Pax7, MyoD, myogenin or troponinT. Again, MyoD was present in infected cells (eGFP þ ) expressing PAX3/FOXO1A or PAX7/FOXO1A, but few cells co-expressed eGFP and myogenin (Supplementary-Figures S3/ RESULTS S4A). Few cells expressing PAX3/FOXO1A or PAX7/FOXO1A also The Pax3 locus is active in a subset of quiescent satellite cells contained troponinT (Figures 4a and b; À13.9±0.9 and We have previously shown that the Pax3 locus is active in a subset 23.4±4.7%, respectively, compared with 94±1% for control-RV), of adult skeletal muscles.11,33 Here we examined the precise with most cells remaining unfused (Figures 4b and c; PAX3/ expression of the Pax3 locus and Pax7 in individual satellite cells. FOXO1AB83% and PAX7/FOXO1AB73% compared with B6% Myofibres were isolated from extensor digitorum longus (EDL), for control-RV). As expected, Pax3 or Pax7 expression was biceps, soleus, masseter and diaphragm of adult Pax3GFP/ þ mice, compatible with terminal differentiation,12,36 although the fusion and their associated satellite cells co-immunostained for index was reduced (Figure 4c). The Pax3DN and Pax7DN also enhanced green fluorescent protein (eGFP) (Pax3) and Pax7.13 inhibited myogenic differentiation (Figures 4a--c). Suppression of The Pax7-expressing satellite cells from the masseter and soleus differentiation by PAX3/FOXO1A, PAX7/FOXO1A, Pax3DN and did not express Pax3, whereas 7% of those from the EDL did. Pax7DN was correlated with maintenance of Pax7 expression By contrast, 49% of satellite cells from biceps and 64% (Supplementary-Figure S4B). from diaphragm co-expressed eGFP (Pax3) and Pax7, with the vast majority of the reminder only expressing Pax7, as cells with just Pax3 were rare (Figures 1a and b; Supplementary PAX3/FOXO1A or PAX7/FOXO1A suppress transcriptional targets Figure S1A/B). Regarding their distribution, the majority of of MyoD myofibres from biceps and diaphragm had associated satellite To confirm that PAX3/7/FOXO1A is able to activate transcription cells that co-immunostained for Pax7 and eGFP (Pax3) (Supple- through binding to the same consensus sequences as wild-type mentary Figure S1B). Myofibres were cultured to activate the Pax3/7, we used the p34 reporter construct in which Tk-nlacZ is associated satellite cells, but EDL-derived MyoD þ activated controlled by multimerized Pax3/7 binding sites.12,37 C2C12 cells satellite cells remained eGFP-negative, showing that the Pax3 were infected with retroviruses encoding PAX3/FOXO1A, PAX7/ locus was not induced upon activation (Figures 1c--d). Even in FOXO1A, Pax3, Pax7, Pax3DN or Pax7DN, and then transiently activated satellite cells from the biceps, most cells did not express transfected with p34 and Renilla-luciferase as control (Figure 5a). eGFP (Figures 1c and e). Using the panel of Pax3/7 constructs allows direct comparison,

Oncogene (2013) 651 --662 & 2013 Macmillan Publishers Limited PAX3/FOXO1A and PAX7/FOXO1A inhibit MyoD-target genes F Calhabeu et al 653

Figure 1. The Pax3 locus is active in a sub-population of satellite cells. Myofibres from the EDL, biceps, soleus, masseter and diaphragm were isolated from Pax3GFP/ þ mice. (a and b) Co-immunostaining of freshly isolated myofibres (T0) for eGFP (green) and Pax7 (red) showed that the Pax3 locus was active (eGFP þ ) in many quiescent satellite cells of biceps and diaphragm, but few in EDL and virtually none in masseter and soleus. Counterstaining with DAPI (4,6-diamidino-2-phenylindole) was used to identify all the nuclei present. (c and d) Co-immunostaining for eGFP and MyoD of cultured myofibre-associated satellite cells from the EDL showed that the Pax3 locus was not induced in activated satellite cells. (c and e) Similarly, the Pax3 locus was not universally induced in activated satellite cells from the biceps. Scale bar equals 50 mm. Values are population means±s.e.m. from at least 60 myofibres from three independent experiments. and we found that PAX3/FOXO1A activated p34 more than Pax3, FOXO1A or PAX7/FOXO1A, to a similar extent as observed with whereas PAX7/FOXO1A and Pax7 resulted in a similar increase Pax3DN or Pax7DN, whereas no reduction was measured with compared with control-RV. Pax3DN or Pax7DN repressed reporter either Pax3 or Pax7 (Figures 5b and c). A control dominant- activity, as expected.11 negative MyoD construct (MyoDDN)39 inhibited the MCK-reporter Pax3 and Pax7 are upstream of MyoD in the genetic hierarchy as expected (Figure 5c). that regulates myogenesis in embryo and adult.11,12,14,21,38 Thus, MyoD also induces the cyclin-dependent kinase inhibitor PAX/FOXO1A may act by interfering with the ability of MyoD to p21CIP1/WAF1, a critical regulatory event for establishment of the activate its transcriptional target genes, and so, we also examined post-mitotic state in myotubes.40 --42 PAX3/FOXO1A or PAX7/ effects of PAX3/FOXO1A or PAX7/FOXO1A on several MyoD- FOXO1A inhibited p21-luciferase reporter activity (Figure 5d). Thus, responsive reporters. C2C12 cells were infected, then transiently PAX3/FOXO1A and PAX7/FOXO1A suppress the activity of at least transfected with the reporter construct and maintained in three MyoD-responsive genes. differentiation medium for 4 days. Activity of myogenin-luciferase Direct effects of PAX3/FOXO1A or PAX7/FOXO1A on the MyoD- or MCK-luciferase reporters were clearly suppressed by PAX3/ target reporters in the absence of MyoD was assayed in C3H10T1/2

& 2013 Macmillan Publishers Limited Oncogene (2013) 651 --662 PAX3/FOXO1A and PAX7/FOXO1A inhibit MyoD-target genes F Calhabeu et al 654

Figure 2. PAX3/FOXO1A or PAX7/FOXO1A maintain Pax7 expression in satellite cells. Myofibres from the EDL and biceps of C57BL/10 wildtype mice were cultured in suspension, and the associated satellite cells exposed to retrovirus (RV) at 24 h, before co-immunostaining for eGFP (green) and Pax7 (red) 48 h later. Infected satellite cells were identified by eGFP from the IRES-eGFP of the RV, and counterstaining with DAPI (4,6- diamidino-2-phenylindole) was used to identify all nuclei present. (a-- c) Constitutive RV-mediated expression of PAX3/FOXO1A or PAX7/FOXO1A resulted in more infected cells (eGFP þ ) containing Pax7 in both EDL (a) and biceps (b), compared with control-RV-infected cells (quantified in c). Values for each RV infection are population means±s.e.m. from 411 to 640 EDL- or biceps-derived-satellite cells from three independent experiments. An asterisk denotes data significantly different from control-RV-infected cultures, where Po0.05. Scale bar equals 50 mm.

fibroblasts. Cells were infected, then transiently transfected peptide linker joins MyoD and E-protein together39 was used to with the reporter constructs and maintained in proliferation determine whether this could overcome PAX3/FOXO1A or PAX7/ medium for 24 h before luciferase activity was measured. PAX3/ FOXO1A. Stable C2C12 clones expressing MyoDBE were infected FOXO1A, PAX7/FOXO1A, Pax3 or Pax7 did not affect the basal with PAX3F-RV or PAX7F-RV, and switched 24 h later to activity of the myogenin-luciferase reporter in C3H10T1/2 fibro- differentiation medium for 4 days. Co-immunostaining for eGFP blasts (Figure 5e). and troponinT (Figure 6c) revealed that cells expressing PAX3/ FOXO1A or PAX7/FOXO1A generally did not fuse into multi- PAX3/FOXO1A or PAX7/FOXO1A do not alter MyoD phosphorylation nucleated myotubes. Furthermore, in the presence of PAX3/ or interaction with E-proteins FOXO1A or PAX7/FOXO1A, MyoDBE was unable to activate The ability of MyoD to regulate its transcriptional targets is myogenin- and MCK-luciferase reporters to the same levels as in regulated in multiple ways, including MyoD localization, control-RV infected cells (Figures 6d and e). phosphorylation or interaction with E-proteins,43,34 processes with which PAX3/FOXO1A or PAX7/FOXO1A could interfere. However, PAX3/FOXO1A or PAX7/FOXO1A decrease acetylation of histone MyoD remained located in the nucleus in cells expressing H4 and RNA polymerase II occupation at the myogenin promoter PAX3/FOXO1A or PAX7/FOXO1A (Supplementary Figure S2), and To examine occupancy of the myogenin promoter in the presence western blot analysis did not reveal changes to MyoD phosphor- of PAX3/FOXO1A or PAX7/FOXO1A, C2 myoblasts were infected ylation (Figure 6a). Dimerization of MyoD with E-protein (E12/47) is with PAX3F-RV, PAX7F-RV or control retroviruses, and incubated in a required step to activate transcription and initiate muscle differentiation medium for 24 h once confluent. This point was differentiation, and immunoblotting analysis showed that MyoD chosen, as MyoD and myogenin transcript levels were high in retained its ability to associate with E12/47protein in the presence control cells. Although MyoD was unchanged in PAX3-7/FOXO1A- of PAX3/FOXO1A or PAX7/FOXO1A (Figure 6b). expressing differentiating cells (Figure 7a), myogenin transcription MyoD transcriptional activity can be repressed by inhibitory was inhibited (Figure 7b). proteins that physically interact with MyoD, whereas others act by To understand how PAX3/FOXO1A or PAX7/FOXO1A inhibits the indirect mechanisms, such as displacement of E-protein and activity of MyoD target genes, we examined whether they directly competition for DNA.34,35 A forced dimer (MyoDBE) in which a bind to the myogenin promoter using ChIP, as has recently been

Oncogene (2013) 651 --662 & 2013 Macmillan Publishers Limited PAX3/FOXO1A and PAX7/FOXO1A inhibit MyoD-target genes F Calhabeu et al 655

Figure 3. PAX3/FOXO1A or PAX7/FOXO1A inhibit myogenic differentiation. Myofibres from the EDL and biceps of C57BL/10 wildtype mice were cultured in suspension, and the associated satellite cells exposed to retrovirus (RV) at 24 h, before co-immunostaining for eGFP (green) and either MyoD (a) or myogenin (b), and counterstaining with DAPI (4,6-diamidino-2-phenylindole). (c) MyoD expression in cells infected with PAX3F-RV or PAX7F-RV was not significantly reduced. (d) By contrast, although many cells infected with control-RV expressed myogenin, the majority infected with PAX3F-RV or PAX7F-RV did not, indicating that PAX3/FOXO1A or PAX7/FOXO1A inhibit the onset of myogenic differentiation. Values for each retrovirus infection are population means±s.e.m. from 590 to 1075 EDL- or biceps-derived satellite cells from three independent experiments. An asterisk denotes data significantly different from control-RV infected cells, where Po0.05. Scale bar equals 50 mm.

proposed.44 Chromatin was prepared from C2 myoblasts after 24 h function, in a cell that may ultimately undergo transformation to in differentiation medium. Neither PAX3/FOXO1A nor PAX7/FOXO1A establish an oncogenic phenotype. were enriched at the myogenin locus (Figure 7c). The verified Satellite cells are heterogeneous, for example, in their binding site of the 145-bp regulatory element at À57.5 kb from proliferation rates, clonogenic ability and myogenicity,47 and so, Myf545,46 was used as a positive control and showed binding of both certain subsets may be more pre-disposed to oncogenic PAX3/FOXO1A and PAX7/FOXO1A (Figure 7d). transformation than others. Of relevance to ARMS, although all Chromatin modification and recruitment of transcriptional satellite cells express Pax7,25 the Pax3 locus is only active in regulatory proteins to the proximal myogenin promoter was also particular skeletal muscles in adult mouse.11,33 We found that even assessed by ChIP, to investigate if PAX3/FOXO1A or PAX7/FOXO1A within these muscles though, the Pax3 locus is only active in a altered chromatin remodelling and protein--DNA interaction at a subset of their satellite cells. Although Pax3 and Pax7 distribution MyoD-target gene. Binding of MyoD at the myogenin promoter has not yet been characterized in detail in man, heterogeneity in was not affected by PAX3/FOXO1A or PAX7/FOXO1A (Figure 7e). Pax3 and Pax7 expression may have implications for oncogenic By contrast, the level of acetylation of core histone H4 was transformation. Therefore, the effects of PAX3/FOXO1A and PAX7/ reduced (Figure 7f), as was binding of RNA polymerase II to the FOXO1A were tested on populations of satellite cells with high myogenin promoter (Figure 7g). (biceps) and low (EDL) numbers of satellite cells with clear Pax3 locus activity. Satellite cells from limb muscles were also used, as a high proportion of ARMS tumours (B39%) in man originate in the DISCUSSION extremities.19 Neither PAX3/FOXO1A nor PAX7/FOXO1A decreased The cellular source of ARMS is unclear, but recent work indicates expression of MyoD, but both drastically reduced the expression that mutations/chromosomal translocations in myogenic cells of myogenin, showing that the early steps of differentiation were could be responsible in some cases.22,23 We therefore wished to inhibited, consistent with the general lack of myogenic differ- understand the early effects of PAX/FOXO1A on satellite cell entiation seen in ARMS. However, the effects of PAX3/FOXO1A or

& 2013 Macmillan Publishers Limited Oncogene (2013) 651 --662 PAX3/FOXO1A and PAX7/FOXO1A inhibit MyoD-target genes F Calhabeu et al 656 a b eGFP+ve TroponinT / eGFP+ve eGFP (RV) TroponinT / DAPI

100 ****** 6,0 18,8 80 43,2

60 76,6 76,9 77,3 86,1 94,0 Control-RV 40 81,2 positive cells 56,8 20 Percentage of eGFP Percentage 23,4 23,1 22,7 0 13,9

PAX3F-RV c Control-RV 100 eGFP+ve 80 60 TroponinT / eGFP+ve cells 40 PAX7F-RV 20 % of eGFP+ve 0 U 2-4 5-9 ≥10

PAX3F-RV PAX7F-RV 100 100 80 80 60 60 Pax3DN-RV 40 40 20 20 0 0 U 2-4 5-9 ≥10 U 2-4 5-9 ≥10

Pax3DN-RV Pax7DN-RV 100 100 Pax7DN-RV 80 80 60 60 % of eGFP+ve cells 40 40 20 20 0 0 U 2-4 5-9 ≥10 U 2-4 5-9 ≥10

Pax3-RV Pax7-RV Pax3-RV 100 100 80 80 60 60 40 40 cells 20 20 0 0 % of eGFP+ve U 2-4 5-9 ≥10 U 2-4 5-9 ≥10 200 μm Pax7-RV Number of nuclei in eGFP+ve myotubes

Figure 4. PAX3/FOXO1A or PAX7/FOXO1A inhibit fusion into multi-nucleated myotubes. Plated EDL-or biceps derived satellite cells from two C57BL/10 wildtype mice were infected with retrovirus (RV) while proliferating, then cultured in differentiation medium for 4 days and fixed before co-immunostaining for eGFP (green) and troponinT (red), and counterstained with DAPI (4,6-diamidino-2-phenylindole). (a-- c) Cells infected with control-RV fused into large multi-nucleated myotubes (B49% of myotubes with more than 10 nuclei (c)). The vast majority of eGFP þ cells expressing PAX3/FOXO1A (PAX3F-RV) or PAX7/FOXO1A (PAX7F-RV) failed to express troponinT (a and b) and most remained unfused [u] (c), as also occurred in the presence of Pax3DN-RV or Pax7DN-RV. Constitutive expression of Pax3 or Pax7 was generally compatible with differentiation, but myotubes were generally smaller. Values are from five random fields and are expressed as population means±s.e.m. from two independent infections of EDL and two independent infections of bicipital satellite cells. Scale bar equals 200 mm.

PAX7/FOXO1A were the same in satellite cells from both EDL and Other studies in plated myogenic cells report either expression biceps, so the levels of Pax3 locus activity did not overtly influence of myosin heavy chain but no fusion in perinatal mouse the outcome, although endogenous Pax3 levels in adult mouse myoblasts,2 or a drastic inhibition of myosin expression in muscle are negligible.36,48 C2 myoblasts.26 In plated EDL or bicipital-derived satellite cells

Oncogene (2013) 651 --662 & 2013 Macmillan Publishers Limited PAX3/FOXO1A and PAX7/FOXO1A inhibit MyoD-target genes F Calhabeu et al 657

Figure 5. PAX3/FOXO1A or PAX7/FOXO1A suppress activity of MyoD-target genes. C2C12 cells were infected with the indicated retrovirus (RV), then transfected with the reporter construct and Renilla luciferase plasmid pRL (Promega) as a control, using Lipofectamine LTX. The p34 reporter has Tk-nlacZ controlled by multimerized Pax3/7-binding sites, so it measures the transcriptional activity of Pax3/7. The p34 activity, measured using the Beta-Glo Assay (Promega), was increased in proliferating C2 myoblasts infected with PAX3F-RV, PAX7F-RV, Pax3 or Pax7 2 days previously. By contrast, constitutive expression of Pax3DN or Pax7DN resulted in a significant decrease in p34 activity (a). To evaluate activity of MyoD-target genes, C2C12 cells were infected with RV, transfected with a MyoD-responsive reporter, and stimulated to differentiate for 4 days before measuring luciferase activity, using the dual-Luciferase reporter Assay (Promega) (b-- d). Constitutive expression of Pax3 or Pax7 did not decrease the activity of the myogenin (b)orMCK (c) reporters, while PAX3F-RV- or PAX7F-RV-infected cells had reduced luciferase activity, as did Pax3DN-RV- or Pax7DN-RV-infected cells. The MCK reporter was also suppressed when control-RV-infected cells were trans- fected with a dominant-negative MyoD construct (MyoD DN). Activity of the p21 reporter was also decreased by PAX3/FOXO1A (PAX3F-RV) or PAX7/FOXO1A (PAX7F-RV) in differentiated cells. Myogenin-luciferase (e) levels were unaltered in C3H10T1/2 fibroblasts expressing PAX3F-RV, PAX7F-RV, Pax3 or Pax7. Results are mean firefly luciferase or b-galactosidase activity normalized to control Renilla luciferase activity±s.e.m. from at least five transfections. Values are expressed as fold activation relative to the reporter level in transfected control-RV infected cells where an asterisk denotes significantly different (Po0.05) from reporter levels in control-RV-infected cells.

from adult mouse, we found that PAX/FOXO1A was compatible the level of the ability of MyoD to activate its transcriptional target with MyoD expression, but inhibited both early (myogenin) and genes, of which myogenin is one. Therefore, we assayed the effects late (troponinT) markers of myogenic differentiation, as well as cell of PAX/FOXO1A on the activity of well-characterized MyoD-target fusion into multinucleated myotubes. Differentiation was also gene reporter constructs in direct comparison with native Pax3/7 prevented by dominant-negative Pax3/7DN constructs, but not by and Pax3/7DN. PAX/FOXO1A suppressed the activity of myogenin, native Pax3/7 (although myotubes were generally smaller36). and MCK reporters to a similar extent as Pax3/7DN, whereas Pax3/ As MyoD was unaffected by PAX/FOXO1A, yet, myogenin was 7 did not inhibit their activity. As PAX/FOXO1A and Pax3/7DN both clearly suppressed; the block on differentiation appears to be at inhibit myogenic differentiation, which is normally associated with

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Figure 6. PAX3/FOXO1A or PAX7/FOXO1A does not alter MyoD localization, phosphorylation or interaction with E12/47 proteins. C2C12 cells were infected and then stimulated to differentiate for 72 h before total protein was extracted, and either immunoblotted or immunoprecipitated with a MyoD antibody (a). Immunoblot analysis showed that MyoD phosphorylation was not affected by PAX3/7-- FOXO1A. Total protein immunoprecipitated with MyoD antibody and analysed by western blot for E12/47 protein showed that MyoD still co-immunoprecipitated with E-proteins in myoblasts expressing PAX3/FOXO1A or PAX7/FOXO1A (b). ‘Negative’ represents the negative control where an equal volume of total protein extract was directly incubated with the beads without being immunoprecipitated with MyoD antibody. Stable C2C12 clones expressing a forced active heterodimer of MyoDBE12 were infected with retrovirus and then cultured in differentiation medium for 4 days, fixed and co-immunostained for eGFP (green) and troponinT (red), and counterstained with DAPI (4,6- diamidino-2-phenylindole). MyoDBE12 stable cells infected with PAX3F-RV or PAX7F-RV failed to express troponinT and remained unfused (c). Scale bar represents 50 mm. MyoDBE12 stable C2C12 cells were infected with retrovirus, transfected with a MyoD-responsive reporter and stimulated to differentiate for 4 days before measuring luciferase activity (d and e). PAX3/FOXO1A or PAX7/FOXO1A suppressed myogenin (d) and MCK (e) luciferase-reporter activity even in the presence of MyoDBE12. Results are mean firefly luciferase activity normalized to control Renilla luciferase activity±s.e.m. from at least 8 transfections. Values are expressed as fold activation relative to the reporter level in transfected control-RV infected cells, where an asterisk denotes significantly different (Po0.05) from control-RV infection.

an increase in the transcriptional activity of MyoD, the effects on FOXO1A, compared with those with PAX7/FOXO1A.49 Transcrip- these MyoD-responsive reporter constructs could be an indirect tional activation by PAX3/FOXO1A is required to prevent myoblast consequence of lack of differentiation. However, in non-myogenic differentiation, as mutant species that are unable to bind DNA are fibroblast cell lines that lack MyoD, reporter activity remained compatible with differentiation.26 Furthermore, PAX3/FOXO1A is unaltered by all Pax constructs. Interestingly, PAX3/FOXO1A can more abundant and stable than Pax31,2 and localizes exclusively to activate MyoD in fibroblastic cell lines, but the degree of the nucleus,50 which may also add to its transcriptional potency. subsequent myogenic differentiation varies between studies from However, although both PAX/FOXO1A and Pax3/7 can augment none, through to fusion into large multinucleated myotubes.29 --31 MyoD levels,12,14 only PAX/FOXO1A inhibits differentiation. Gene PAX3/FOXO1A can also inhibit MyoD-mediated myogenic conver- targets of PAX3/FOXO1A and PAX3 are not identical;5,7 thus, sion of fibroblasts,26,29 but this effect is MyoD-specific, as PAX3/ repression of MyoD transcriptional activity likely occurs through FOXO1A does not have substantial effects on myogenin- regulation of a largely different cohort of gene targets not highly dependent myogenic conversion.26 activated/recognized by native PAX3 or PAX7. The overall effect of PAX3/FOXO1A is a more potent activator of a Pax3/7 reporter PAX/FOXO1A in preventing myogenic differentiation though, is construct (p34) than PAX7/FOXO1A, but although PAX3/FOXO1A more akin to that of the Pax3/7DN constructs. However, the is more potent than Pax3,3 PAX7/FOXO1A and Pax7 induced p34 mechanism whereby differentiation is inhibited is different, as to a similar level. These differences in transcriptional potency may Pax3/7DN versions suppress MyoD expression, whereas PAX/ be related to the poorer prognosis of ARMS containing PAX3/ FOXO1A acts downstream of MyoD.

Oncogene (2013) 651 --662 & 2013 Macmillan Publishers Limited PAX3/FOXO1A and PAX7/FOXO1A inhibit MyoD-target genes F Calhabeu et al 659

Figure 7. PAX3/FOXO1A or PAX7/FOXO1A reduces histone H4 acetylation and RNA polymerase II recruitment at the myogenin promoter. C2C12 cells were infected with indicated retroviruses (RV) and then stimulated to differentiate for 24 h. Total RNA was extracted from infected cells and the levels of MyoD (a) and myogenin (b) transcripts measured by real-time PCR, with values normalized to the reference house- keeping gene Gapdh. MyoD levels were enhanced by PAX3/FOXO1A (PAX3F-RV) or PAX7/FOXO1A (PAX7F-RV) in proliferating myoblasts. However, PAX3/FOXO1A or PAX7/FOXO1A reduced myogenin expression after the induction of differentiation (Po0.05). (b). Chromatin was prepared from C2 myoblasts after 24 h in differentiation medium, when myogenin transcript levels are high in control cells (b). Neither PAX3/ FOXO1A nor PAX7/FOXO1A were enriched at the myogenin locus (c). The verified binding site of the 145-bp regulatory element at À57.5 kb from Myf545 was used as a positive control and showed binding of both PAX3/FOXO1A and PAX7/FOXO1A (d). Binding of MyoD at the myogenin promoter was not affected by PAX3/FOXO1A or PAX7/FOXO1A (e). By contrast, the level of acetylation of core histone H4 was reduced (f), as was binding of RNA polymerase II to the myogenin promoter (g). Representative results from multiple DNA preparations from one of two independent experiments are shown for c-- g.

MyoD transcriptional activity is regulated by various post- MyoD to activate its target genes. However, these effects on Mef2 translational events.32 However, PAX/FOXO1A did not affect the levels may be an indirect consequence of the general lack of localization or phosphorylation of MyoD. The MyoD promoter differentiation. (along with those of myogenin and Mrf4) contains MEF2-binding Dimerization of MyoD with E-protein (E12/47) is also a required sites that amplify and maintain their expression, and augment the step to activate transcription targets and initiate muscle myogenic activity of MyoD.32,51 Moreover, a DN Mef2A inhibits differentiation,32 but MyoD retained its ability to associate with myogenin expression and prevents terminal differentiation.52 We E-protein in myoblasts expressing PAX/FOXO1A. This is consistent found that Mef2 is expressed in many cells during the early stages with previous studies showing MyoD interaction with E-proteins of myogenic differentiation in satellite cells (likely Mef2a since the in several RMS cell lines,16 although co-expression of MyoD and antibody strongly recognises this isoform), but PAX/FOXO1A E-proteins does not induce myogenic differentiation in ARMS.32 inhibits this Mef2 induction, which would affect the ability of We found that even a forced MyoDBE heterodimer could

& 2013 Macmillan Publishers Limited Oncogene (2013) 651 --662 PAX3/FOXO1A and PAX7/FOXO1A inhibit MyoD-target genes F Calhabeu et al 660 not overcome PAX/FOXO1A-dependent inhibition of myogenic Retroviral vectors and reporter plasmids differentiation. Interestingly, MyoDBE heterodimer expression Human PAX3/FOXO1A and PAX7/FOXO1A6 were cloned into pMSCV-IRES- in embryonal RMS cell lines strongly induces myogenic eGFP12 to generate pMSCV-PAX3/FOXO1A-IRES-eGFP and pMSCV-PAX3/ 53 differentiation, and as these lines lack the chromosomal FOXO1A-IRES-eGFP, producing PAX3/FOXO1A or PAX7/FOXO1A as a translocation that generate PAX/FOXO1A, it again highlights that bisitronic message with eGFP. Retroviral constructs encoding wild-type PAX/FOXO1A acts to inhibit the ability of MyoD to activate its and dominant-negative forms of murine Pax3 and Pax7, and retroviral targets genes. preparation have been described previously.11,36 Satellite cells (5 Â 104)or The ability of MyoD to initiate the myogenic programme in non- C2C12 cells (8 Â 104) were incubated with retrovirus-containing medium muscle cells is associated with chromatin remodelling at the with 4 mg/ml polybrene. Cells were harvested after 10--12 h and re-plated 54 regulatory regions of target genes. We performed ChIP on the in Matrigel-coated LAB-TEKH chamber slides. Satellite cells associated with myogenin promoter to determine if these epigenetic changes, myofibres were exposed to a 1:10 dilution of supernatant after 24 h in consistent with active gene expression, were in place and if the culture, and cells analysed 48 h later. transcriptional machinery was assembled. However, although pclBabe-MyoDBE12 forced-dimmer, pCS2-MCK-luciferase reporter,53 MyoD was present, there was reduced acetylation of histone H4 human p21WAF1/CIP1-luciferase reporter,57 myogenin-luciferase reporter58 and decreased occupation of the myogenin promoter by RNA and Pax3/7 reporter (p34)14 have been described previously. polymerase II. Although PAX/FOXO1A is able to bind defined Pax3/7 consensus sequences in Myf5 regulatory elements,45,46 Immunostaining PAX/FOXO1A did not bind at the myogenin promoter, although direct binding via a newly described motif has been reported.44 Cultures or myofibres were fixed in 4% paraformaldehyde/phosphate- MyoD directly interacts with the acetyl-transferase proteins p300 buffered saline for 10 min and permeabilized with 0.5%(v/v) Triton X-100/ and pCAF,55 leading to acetylation of histones H3 and H4, phosphate-buffered saline. Cells were blocked with 10% goat-serum and facilitating recruitment of RNA polymerase II transcriptional 10% swine-serum/phosphate-buffered saline for 1 h and incubated over- complex.56 Thus, PAX/FOXO1A inhibits recruitment of histone night at 4 1C with primary antibodies: monoclonal mouse anti-Pax7 (DSHB), acetyl-transferases to target genes, likely via interfering with MyoD anti-MyoD (clone 5.8A, DakoCytomation---Ely, Cambridgeshire, UK), anti- function. myogenin clone F5D (DSHB), anti-troponinT (JLT12, Sigma-Aldrich), rabbit In summary, MyoD normally activates target genes that polyclonal anti-eGFP (Molecular Probes, Invitrogen Life Technologies, Paisley, initiate a cascade that leads to cell cycle exit and myogenic UK) and rabbit polyclonal anti-Mef2 (C2, sc313, mainly recognizes Mef2a). differentiation.32 PAX/FOXO1A represses the ability of MyoD Primary antibodies were visualized with fluorochrome-conjugated second- to activate some of these target genes, so preventing differentia- ary antibodies (Molecular Probes, Invitrogen Life Technologies, Paisley, UK) tion. PAX/FOXO1A acts in part, by preventing epigenetic changes before mounting in DakoCytomation Faramount fluorescent mounting and assembly of the transcriptional complex at MyoD-target medium containing 100 ng/ml 4,6-diamidino-2-phenylindole. Digital images genes. were acquired on a Zeiss Axiophot 200 M using Plan-Neofluar lenses and a Zeiss AxioCam HRm Charge-Coupled Device with AxioVision software (version 4.4, Zeiss, Hertfordshire, UK), optimized globally and assembled into MATERIAL AND METHODS figures. Differences between conditions were tested using Student’s t-test. Myofibre isolation Experimental procedures were carried out under the Animals (Scientific Immunoprecipitation and western blot analysis Procedures) Act 1986. Adult (8--12 weeks old) C57BL/10 or Pax3GFP/ þ mice Infected C2C12 myoblasts were maintained in differentiation medium for 3 (in which eGFP replaces the coding sequence in exon 1---Hayashi et al., days and lysed for 20 min on ice in NonidetP-40 buffer (50 mM HEPES (4-(2- manuscript in preparation) were used. Mice were killed by cervical hydroxyethyl)-1-piperazineethanesulfonic acid), pH7.4, 150 mM NaCl, 5 mM dislocation before the EDL, soleus, biceps, masseter and diaphragm were EDTA, pH8.0, 1% Nonidet P-40) containing complete mini protease dissected, and digested in 0.2% collagenase type 1, with myofibres isolated inhibitor cocktail (Roche Diagnostics Ltd, Burgess Hill, UK), 50 mM NaF, 47 as previously described. 1mM sodium orthovanadate and 10 mM sodium pyrophosphate. Lysates were collected, sonicated for 20 s and centrifuged at 15 000 g for 10 min. Cell culture Equal volumes of extracts were incubated with 4 mg of monoclonal anti- MyoD (clone 5.8A, DakoCytomation) or E12/47 (BD Biosciences, Oxford, UK) Myofibres were incubated in plating medium (Dulbecco’s modified Eagle’s antibody for 3 h at 4 1C. A volume of 70 ml of protein A-agarose was then medium with L-glutamine (400 mM) or GlutaMax (Sigma-Aldrich, Dorset, added and incubated overnight. Beads were washed with HEPES buffer, UK) and 10% horse-serum (PAA Laboratories, Somerset, UK), 0.5%. resuspended in SDS--polyacrylamide gel electrophoresis sample buffer, chicken-embryonic-extract (MP Biomedicals, Illkirch, France) and 1% and processed for western blot using either rabbit polyclonal anti-MyoD 1 penicillin--streptomycin (Sigma)) at 37 Cin5%CO2. For adherent cultures, Santa Cruz Biotechnology INC., Heidelberg, Germany or mouse monoclonal myofibres were plated in 6-well plates (Nunc A/S, Roskilde, Denmark) anti-E12/47 antibody. coated with 1 mg/ml Matrigel (BD Biosciences, Bedford, MA, USA) Immunocomplexes were detected by chemiluminescence (ECL, maintained for 3 days in growth medium (Dulbecco’s modified Eagle’s GE Healthcare Life Sciences, Buckinghamshire, UK). Primary antibodies medium with L-glutamine (400 mM) or GlutaMax, 30% fetal bovine serum, used: rabbit polyclonal anti-MyoD (Santa Cruz) and monoclonal mouse 1% chicken-embryo-extract, 10 ng/ml basic fibroblast growth factor and anti-b-tubulin (cloneE7, DSHB). 1% penicillin--streptomycin), at 37 1Cin5%CO2. Myofibres were removed and cells re-plated on Matrigel at low density and maintained in proliferation medium. For differentiation, cells were plated at high density Reporter assays in Matrigel-coated LAB-TEK eight-well chamber slides (Nunc) and cultured Transduced C2C12 myoblasts and C3H-10T1/2 fibroblasts were transfected in differentiation medium for 4 days (Dulbecco’s modified Eagle’s medium with firefly luciferase or nlacZ reporter plasmids, using Lipofectamine LTX, containing 5% fetal bovine serum). with Renilla luciferase plasmid pRL (Promega, Corporation, Madison, WI, C2C12 cells were maintained in growth medium (Dulbecco’s modified USA) as a control. After 48 or 96 h transfection, the dual-Luciferase Eagle’s medium with L-glutamine (400 mM) or GlutaMax, 10% fetal bovine reporter Assay (Promega, corporation) or the Beta-Glo Assay (Promega serum and 1% penicillin--streptomycin). Induction of myogenic differentia- Corporation) combined with the Renilla luciferase Assay (Promega tion was by incubation in differentiation medium for 3--4 days at 37 1C. For Corporation) was performed and measured with an Anthos Lucy3 the stable MyoDBE12 clone, cells were infected with pclBabe-MyoDBE12 (JENCONS-PLS, West Sussex, UK). Differences between conditions were retrovirus and selected for 5 weeks with puromycin (3 mg/ml). tested using Student’s t-test.

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Common musculoskeletal tumors of childhood and independent experiments. Data are presented as percentage of input, adolescence. N Engl J Med 1999; 341: 342 --352. and the values were calculated using the formula 100 Â 2^ [Ct(Input)ÀCt(Ab)], 19 Gurney JG, Young JL, Roffers SD, Smith MA, Bunin GR. Soft Tissue Sarcomas. In: where ‘Ct’ is the threshold cycles, ‘Ab’ is the antibody, and ‘Input’ is the Ries LAG SM, Gurney JG, Linet M, Tamra T, Young JL, Bunin GR (eds). Cancer adjusted input genomic DNA (Ct Input--6.644). Incidence and Survival Among Children and Adolescents: United States SEER Program 1975 --1995. National Cancer Institute: Bethesda, MD, 1999, pp 111 --123. 20 Cui S, Hano H, Harada T, Takai S, Masui F, Ushigome S. Evaluation of new monoclonal anti-MyoD1 and anti-myogenin antibodies for the diagnosis of CONFLICT OF INTEREST rhabdomyosarcoma. Pathol Int 1999; 49: 62 --68. The authors declare no conflict of interest. 21 Maroto M, Reshef R, Munsterberg AE, Koester S, Goulding M, Lassar AB. Ectopic Pax-3 activates MyoD and Myf-5 expression in embryonic mesoderm and neural tissue. Cell 1997; 89: 139 --148. 22 Keller C, Hansen MS, Coffin CM, Capecchi MR. Pax3:Fkhr interferes with embryonic ACKNOWLEDGEMENTS Pax3 and Pax7 function: implications for alveolar rhabdomyosarcoma cell of We thank Frederic Barr for PAX3-FOXO1A and PAX7-FOXO1A, and Stephen Tapscott origin. Genes Dev 2004; 18: 2608 --2613. for pclBabe-MyoDBE12 and pCS2-MCK-luciferase. We acknowledge colleagues who 23 Nishijo K, Hosoyama T, Bjornson CR, Schaffer BS, Prajapati SI, Bahadur AN et al. shared antibodies through the Developmental Studies Hybridoma Bank maintained Biomarker system for studying muscle, stem cells, and cancer in vivo. FASEB J by the University of Iowa. FC was funded by the Association of International Cancer 2009; 23: 2681 --2690. Research (07-0151). The laboratory of PSZ is also supported by The Wellcome Trust 24 Hettmer S, Wagers AJ. Muscling in: uncovering the origins of rhabdomyosarcoma. (085137/Z/08/Z), The Medical Research Council and OPTISTEM (Grant 223098) from Nat Med 2010; 16: 171 --173. the European Commission FP7. JEM is supported by a Wellcome Trust University 25 Gnocchi VF, White RB, Ono Y, Ellis JA, Zammit PS. Further characterisation of the Award. The laboratory of FR is supported by the INSERM Avenir Program, AFM, INCa, molecular signature of quiescent and activated mouse muscle satellite cells. PLoS LNCC, ARC and FP7 ENDOSTEM (Grant 241440). ONE 2009; 4: e5205.

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