©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Pax3 Inhibits Myogenic Differentiation of Cultured Myoblast Cells (*)

Jonathan A. Epstein (1) (2)(§), Paula Lam (3), Lisa Jepeal (2), Richard L. Maas (2), David N. Shapiro (3)

From the (1) Divisions of Cardiology and (2) Genetics, Brigham and Women's Hospital and Harvard Medical School, Howard Hughes Medical Institute, Boston, Massachusetts 02115 and the (3) Department of Experimental Oncology, Saint Jude Children's Research Hospital, Memphis, Tennessee 38101

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Pax3 is an evolutionarily conserved transcription factor expressed in the lateral dermomyotome, a region that gives rise to limb muscle progenitors. Mutations in Pax-3 account for the mouse mutant Splotch which develops without limb musculature. We demonstrate that Pax3 can inhibit myogenic differentiation of C2C12 myoblasts normally induced by exposure to low serum. Specific missense mutations that affect the DNA binding characteristics of the two distinct DNA binding domains of Pax3 abolish this effect. Furthermore, we show that Pax3 can inhibit myogenic differentiation of 10T1/2 fibroblasts transfected with MyoD, but not of 10T1/2 cells transfected with myogenin. This anti-myogenic property is shared by a PAX3-forkhead fusion protein resulting from a t(2;13) chromosomal translocation found in pediatric alveolar rhabdomyosarcomas. These results suggest that Pax3 may suppress the terminal differentiation of migrating limb myoblasts and that the PAX3-forkhead fusion may contribute to the phenotype of alveolar rhabdomyosarcoma by preventing terminal differentiation.


INTRODUCTION

The molecular events leading to myogenic differentiation have been the target of intensive recent investigation leading to the discovery of the myogenic basic helix-loop-helix (bHLH)() family of transcription factors, including MyoD and myogenin. These gene products are capable of inducing some non-muscle cultured cells to form myotubes (1) and are expressed in vivo during myogenesis. Nevertheless, additional factors involved in the myogenic pathway have been implicated, especially during the differentiation of certain muscle cell types. For example, homozygous Splotch mice develop without limb musculature, although axial muscles, which are of distinct embryological origin, are normal (2) . None of the known myogenic bHLH factors are expressed during development in the limbs of these mutant animals (3) . The genetic defect in Splotch is a mutated or deleted Pax-3 gene (4) , and Pax-3 is normally expressed in the region of the somite that will give rise to limb myoblasts (3) . In man, PAX3 mutations are responsible for Waardenburg's Syndrome, a dominant disorder characterized by abnormalities of neural crest derivatives and sometimes also by abnormalities of limb musculature (5, 6, 7) .

The involvement of Pax3 in normal myogenesis has also been implicated by the recent discovery of a specific t(2;13) chromosomal translocation responsible for some alveolar rhabdomyosarcomas in human patients (8) . This translocation results in the fusion of the DNA binding domains of Pax3 to the transcriptional activating domain of a novel forkhead family member (9, 10) . Other forms of rhabdomyosarcoma express high levels of Pax3 (or a closely related family member, Pax7) compared with normal human myoblasts (11) .

Pax-3 is a member of a gene family defined by the presence of a conserved 128 amino acid DNA binding domain termed the paired box (12) . Some Pax genes also contain a second DNA binding domain, a paired type homeodomain, located C-terminal to the paired domain. Finally, a C-terminal transactivating domain has been demonstrated for several Pax gene products (13, 14, 15) . Pax-3 is first expressed at embryonic day 8.5 in the mouse and is identified in the dorsal neural tube and in the region that gives rise to the neural crest, as well as in the lateral dermomyotome of the condensing somites (16). Pax-3 expression is seen in the mouse and chick along the migratory pathways followed by limb myogenic progenitor cells and is extinguished by embryonic day 14.5 (3, 17, 18) .

These observations suggest that Pax3 might repress myogenic differentiation during limb myoblast migration and that deregulated Pax3 activity, as might be caused by chromosomal translocation, could contribute to the phenotype of some forms of rhabdomyosarcoma. We provide evidence to support this hypothesis by demonstrating that Pax3, or the PAX3/FKHR fusion protein produced by the t(2;13) translocation, can prevent myogenic differentiation of a cultured myoblast cell line and can prevent the myogenic differentiation normally induced by MyoD, but not by the downstream bHLH regulator myogenin.


MATERIALS AND METHODS

Plasmids and Retroviral Constructs

The murine Pax-3 cDNA, pBH3.2, was kindly provided by Dr. P. Gruss. A BamHI-EcoRI fragment encompassing the entire coding region was subcloned into pCMV (Clontech) by the addition of NotI linkers and replacement of the -galactosidase coding region. This NotI insert was subcloned into pcDNA3 (Invitrogen) and engineered by PCR to contain three copies of a seven amino acid C-terminal hemagglutinin (HA) epitope (YPYDVPDYA). PCR-derived sequences were confirmed by sequencing. pCMV-Pax2 and pCMV-Pax6 have been described (19) . Specific missense mutations or deletions were engineered in pCMV-Pax3 and pCMV-PAX3/FKHR using the Unique Site Elimination mutagenesis kit (Pharmacia Biotech Inc.) or by overlap extension PCR (20) . Retroviral vectors were constructed by inserting cDNA inserts into the retroviral vector pSR(HindIII) (21) . Replication-deficient retroviral stocks were created by transiently transfecting COS cells with C-terminal HA epitope-tagged PAX3/FKHR constructs together with a 2 packaging plasmid. Conditioned medium containing virus was harvested and used to infect puromycin-resistant 10T1/2 cells (see below). PAX3/FKHR-un-1 represents a glycine to serine mutation at position 48 of PAX3 mimicking the paired domain mutation in Pax1 responsible for the mouse mutant undulated(22) . PAX3/FKHR-Hd lacks the PAX3 homeodomain (amino acids 219-289).

Transfection, Infection, and Myogenesis Assay

C2C12 myoblasts and 10T1/2 fibroblasts were obtained from the ATCC and maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (Life Technologies, Inc.). C2C12 cells were transfected in 60-mm dishes at 50% confluence with 5 µg of purified expression vector DNA (Quiagen) in 1.5 ml of serum-free medium with 12.5 µl of Transfectam (Promega) for 6 h, and the medium was then replaced with serum-containing growth medium. When pCMV based vectors were used, molar ratio of pMC1neo (Stratagene) was included. After 48 h, the cells were trypsinized and replated at a 1:10 dilution in medium containing 500 µg/ml (active concentration) of G418 (Life Technologies, Inc.). After 10-14 days of selection, individual colonies were identified and cloned with the use of cloning rings or tested for the ability to form myotubes by changing the medium to differentiation medium (Dulbecco's modified Eagle's medium with 2% heat inactivated horse serum) for 48 h prior to fixation in AFA (1) and staining with anti-myosin monoclonal antibody (MF20, kindly provided by Dr. Jay Schneider) and a biotin-conjugated secondary antibody kit (Vectastain, Vector Laboratories). Positively staining colonies were defined as those with more than one positively staining cell within a colony of 100 or more cells and were identified by scanning the plate under low power light microscopy.

10T1/2 cells were cotransfected by the calcium phosphate method with MyoD or myogenin cDNAs (kindly provided by Drs. H. Weintraub and E. Olson) inserted in the mammalian expression vector pEMSVscribe2 together with molar ratio of the puromycin resistance vector pPUR (Clontech) (1) . Individual puromycin-resistant clones exhibiting a high percentage of myosin-expressing cells upon exposure to differentiation medium were selected for infection by PAX3/FKHR retrovirus. Clones doubly selected in puromycin (5 µg/ml) and G418 (600 µg/ml) for 2 weeks were tested for the capacity to undergo myogenic differentiation.

RNase Protection Assay

RNA was isolated from cultured cells with the use of RNAzol B (Biotecx). RNase protection assays were performed as described (23) . The Pax-3-specific [-P]UTP-labeled RNA probe protects nucleotides 91-345 (GenBank number X59358). The MyoD probe protects nucleotides 1201-1787 (GenBank number M18779). The myogenin probe protects nucleotides 168-556 (GenBank number X15784).

Western Blotting

Whole cell lysates were electrophoresed on 10% polyacrylamide gels, transferred to nitrocellulose, and incubated in Blotto (5% powdered milk in phosphate-buffered saline) at 4 °C overnight, followed by incubation for 2 h at room temperature with 1:200 dilution of HA.11 antiserum (Babco) in Blotto, washing, and incubation with an alkaline phosphatase-conjugated secondary antibody for 1 h (Pierce, 1:5000 dilution). Developing was performed using ImmunoPure nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate kit (Pierce).

Electrophoretic Mobility Shift Assays (EMSA)

EMSA were performed as described (24) using glutathione S-transferase fusion proteins expressed in bacteria from pGEX2T (Pharmacia)-derived vectors encoding wild type or mutated Pax3 homeodomain protein (amino acids 190-297) or a larger construct encompassing the paired domain as well (amino acids 34-297). The proteins were purified by affinity to glutathione-agarose beads (Sigma) and aliquots stored at -80 °C. EMSA reactions included 250 µg/ml poly(dI-dC) and 10 cpm of gel-purified [-P]dCTP-labeled DNA probe. Probes e5 (5`-TCGGGCAGCACCGCACGATTAGCACCGTTCCGCTCAGGCTCGG (25) ) and P2 (5`-GATCCTGAGTCTAATTGATTACTGTACAGGATC (26) ) have been described. Nf3` (5`-CTAGTGTGTGTCACGCTTATTTTCCTGTACTTATTGCTAG) was identified as a high affinity Pax3 paired domain binding site by homology to a consensus derived from in vitro binding site selection assays, using the isolated Pax3 paired domain as bait, similar to those performed using the Pax6 and Pax2 paired domains (24).()

CAT Assay

P19 embryonal carcinoma cells were transfected in 60-mm dishes by calcium phosphate coprecipitation with 2 µg of reporter plasmid pGECP3Con2X (containing tandem Nf3` binding sites), 1 µg of expression plasmid, 0.5 µg of MAP1-SEAP (27), and 16.5 µg of sheared calf thymus DNA. After 48 h, the supernatant was assayed for secreted alkaline phosphatase activity (SEAP) and the cells lysed in 0.25 M Tris (pH 7.8) by three consecutive freeze-thaw cycles for determination of CAT activity by thin layer chromatography; percent acetylation was quantitated using a PhosphorImager. Transfection efficiency was normalized to SEAP. pGECP3Con2X was derived from pGECAT which contains five GAL4 binding sites upstream of CAT (28) . Cotransfection of pBXG2, which encodes the GAL4 DNA binding domain and activation region II, provided a positive control (29) . pBXG1, which lacks an activation domain, served as a negative control.

RESULTS AND DISCUSSION

In order to test the ability of Pax3 to prevent myogenic differentiation, we transfected C2C12 myoblasts with expression vectors encoding murine Pax3 and neomycin resistance and selected for stable antibiotic resistant colonies. C2C12 cells can be induced to differentiate into myosin-producing cells that fuse to form multinucleated myotubes by growth in medium containing low serum. Differentiation can be readily observed after 48 h and can be objectively assessed by staining for myosin expression with a monoclonal myosin antibody. Eighty-seven percent of G418-resistant C2C12 colonies transfected with the expression vector alone expressed detectable myosin after exposure to low serum for 48 h (). However, transfection with an expression vector encoding Pax3 resulted in only 43% of colonies able to express myosin. In addition, we tested the ability of PAX3/FKHR to inhibit myogenic differentiation. This fusion protein contains the intact paired domain and homeodomain of human PAX3, fused to a truncated forkhead DNA binding domain followed by an intact forkhead transactivating domain. After transfection with PAX3/FKHR, only 27% of G418-resistant colonies expressed myosin after exposure to low serum (). Hence, both Pax3 and the PAX3/FKHR fusion protein can significantly inhibit myogenic differentiation of C2C12 myoblasts. Deletion of the truncated forkhead DNA binding domain did not alter this affect (205/823 (25%) of colonies stained positive for myosin.)

In order to assess the specificity of this anti-myogenic effect, we tested the activity of Pax2 and Pax6 in this assay. These paired box family members are closely related to Pax3 and are able in vitro to bind some of the same target DNA sequences, though not others (24, 30) . Pax2 does not contain an intact homeodomain, and Pax6 does not contain the conserved octapeptide motif located between the paired and homeodomains of other Pax family members. Neither Pax2 nor Pax6 was capable of inhibiting myogenic differentiation in our assay (223/282 (79%) and 210/261 (80%) myosin-positive colonies, respectively).

Further experiments indicated that both the intact paired domain and homeodomain of Pax3 are required for the anti-myogenic effect. Mutations were engineered either in the paired domain or in the homeodomain, and the ability of the mutated proteins to inhibit myogenic differentiation was assessed. We tested several missense mutations within the paired domain, either in the context of Pax3 (BU26) or PAX3/FKHR (BU35, un-1), and we tested a missense mutation in the homeodomain of Pax3 (S268A) or deletion of the Pax3 homeodomain of PAX3/FKHR (Hd). In each case, the mutant proteins were unable to inhibit myogenic differentiation ().

The BU-26 mutation in the paired domain results in a proline to leucine replacement of amino acid 50, a mutation responsible for Waardenburg's Syndrome in at least one family (6) . This mutation results in the inability of the isolated paired domain to bind DNA sequences normally recognized with high affinity (31) and alters an amino acid seen to contact DNA in the crystal structure of the Drosophilapaired protein bound to an optimized binding site (32) . We show here (Fig. 1A) that a glutathione S-transferase-fusion protein consisting of a portion of Pax3 that includes the paired domain and the homeodomain is able to bind Pax3 binding sites (e5 and Nf3`) forming two shifted complexes. This is consistent with previous reports demonstrating that truncated and full-length Pax3 proteins can dimerize upon binding DNA (11, 31) . e5 contains both paired and homeodomain binding sites (25) and the BU26 paired domain mutation greatly reduces binding. Residual binding might be due to homeodomain/DNA interaction. The Nf3` sequence contains only a paired domain binding site, and the BU26 mutation eliminates binding to this site.


Figure 1: Missense mutations alter DNA binding and transactivating properties of Pax3 and PAX3/FKHR. A, EMSA using a Pax3 paired domain-specific probe, e5 or Nf3`, with a Pax3 peptide encompassing the paired and homeodomains (P3PdHd) or with a missense mutation in the paired domain (P3PdHd-BU26) or in the homeodomain (P3PdHd-S268A). With P3PdHd, two shifted complexes are seen (C1 and C2). When the isolated Pax3 paired domain is used, only one shifted complex is seen (data not shown). B, EMSA using a palindromic homeodomain binding site, P2, with a Pax3 peptide, including the homeodomain but not the paired domain (P3Hd), or with a missense mutation in the homeodomain (P3Hd-S268A). C, CAT assays indicate that Pax3 and PAX3/FKHR stimulate transcription from a reporter containing tandem Pax3 binding sites (see ``Materials and Methods''). Pax3-BU26, PAX3/FKHR-un-1, and PAX3/FKHR-BU35, all contain point mutations in the paired domain and fail to transactivate this reporter. The reporter also contains GAL4 binding sites providing a positive control when cotransfected with a GAL4-encoding expression vector (see ``Materials and Methods''). All values are normalized to cotransfected SEAP activity and are expressed as percent maximal activation seen with GAL4.



Pax3 is able to activate CAT activity from an appropriate reporter construct when the Nf3` paired domain binding site is placed upstream of CAT, and the BU-26 mutation eliminates this activity (Fig. 1C), consistent with its effect on DNA binding. PAX3/FKHR is a somewhat more potent activator of transcription from this reporter than Pax3 (whether or not the truncated forkhead DNA binding domain is retained; data not shown). This is consistent with its more potent anti-myogenic effect. Other missense mutations (BU-35 and un-1) that would be expected to reduce the DNA binding activity of the paired domain (5, 22) and also fail to inhibit myogenesis (), reduce the ability of the PAX3/FKHR fusion to transactivate from this reporter (Fig. 1C). Results using a reporter containing the e5 sequence were similar (data not shown).

The mutation we have introduced in the homeodomain results in a serine to alanine replacement at position 9 of the third ``recognition'' helix of the homeodomain (residue 268). Mutations in this position are known to alter sequence-specific DNA binding by paired-type (33) and other classes of homeodomains (34) and result in altered dimerization of the isolated Drosophilapaired protein homeodomain when binding a palindromic binding site (26) . The S268A mutation that we engineered in Pax3 has not been previously reported, and we demonstrate here that this alteration results in diminished dimerization of the isolated Pax3 homeodomain upon binding a palindromic binding site (Fig. 1B). Interestingly, this mutation also seems to reduce dimerization of the paired domain and homeodomain containing glutathione S-transferase-fusion protein when binding to e5 and Nf3` (Fig. 1A) and results in diminished activation of CAT activity from a reporter construct containing Nf3`, a paired domain binding site (Fig. 1C).

The ability of C2C12 cells to express the proteins encoded by the Pax3 expression vectors was verified after both transient and stable transfections. Protein levels after transient transfection of wild type or mutant constructs were similar (Fig. 2A). After selection with G418, numerous clonal Pax3-transfected cell lines expressed detectable levels of Pax3 protein (examples shown in Fig. 2B). None of the Pax3-positive cell lines tested was able to efficiently form myotubes after exposure to low serum.


Figure 2: Western blots. A, Expression of Pax3, Pax3-BU26, and Pax3-S268A proteins is detected by Western blot analysis of transiently transfected C2C12 myoblasts. Whole cell lysates were analyzed 48 h after liposome-mediated transfection of DNA using a polyclonal antibody (HA.11) directed against a C-terminal hemagglutinin epitope tag. The arrow indicates the expected 48-kDa band. B, Western blot analysis of five individual G418-resistant clones isolated after transfection with Pax-3 indicates stable Pax3 expression in at least three of these clones.



In order to assess the ability of Pax3 to overcome the muscle-inducing effect of the myogenic bHLH factors, we tested the ability of Pax3 to inhibit myogenic differentiation of fibroblast cells (10T1/2 cells) stably transfected with MyoD or myogenin. C2C12 cells express MyoD under normal growth conditions and up-regulate myogenin expression only after exposure to low serum (35, 36) (Fig. 3). In vivo, MyoD or myf-5 is thought to precede myogenin expression, and myogenin appears requisite for terminal differentiation (37, 38, 39) . Both Pax3 and PAX3/FKHR were able to inhibit myogenic differentiation of MyoD-expressing 10T1/2 cells after exposure to low serum. In contrast, 10T1/2 cells expressing myogenin were relatively resistant to the anti-myogenic effect of Pax3 and PAX3/FKHR (). This result was obtained with two independently derived 10T1/2 myogenin clonal lines. It is possible that the difference in anti-myogenic effect of Pax3 and PAX3/FKHR in MyoD- versus myogenin-expressing cells was due to elevated myogenic protein activity in the myogenin cell lines tested compared with the MyoD cell lines. However, these results suggested to us that the block in myogenesis imposed by Pax3 and PAX3/FKHR occurs upstream of myogenin.


Figure 3: RNase protection assay using Pax3-, MyoD-, and myogenin-specific RNA probes. Twenty µg of total RNA from vector control or Pax3-transfected C2C12 cells before and after 48 h of exposure to differentiation medium or 20 µg of yeast tRNA (negative control) was assayed. Actin mRNA was assayed to control for loading.



Support for this hypothesis comes from analysis of RNA expression of control and Pax3-transfected C2C12 cells before or after exposure to low serum. Fig. 3shows that stable expression of Pax3 RNA is not altered by exposure to low serum and is present only in Pax3-transfected cells. MyoD RNA levels are similar in control and Pax3-transfected cells and remain constant or slightly increased with low serum exposure, as has been demonstrated previously (35) . Myogenin RNA levels are greatly up-regulated in control cells upon exposure to low serum (36) , but rise not at all (Fig. 3) or only slightly (as in other experiments not shown) in Pax3-transfected cells exposed to low serum.

Thus, we have demonstrated that Pax3 expression in cultured myoblasts inhibits myogenesis normally induced by growth in low serum containing medium. This property is abrogated by a Waardenburg's Syndrome mutation in the paired domain that alters specific DNA binding and prevents transactivation of a reporter construct, including Pax3 binding sites. A homeodomain mutation that affects DNA binding and dimerization also destroys the anti-myogenic property. Taken together with the spatial and temporal expression patterns of Pax3 and the myogenic bHLH factors during development, these results suggest that Pax3 may suppress myogenic differentiation of migrating limb muscle progenitors. This inhibition occurs independent or downstream of MyoD and is overcome by forced expression of the downstream bHLH factor myogenin. It will be of interest to further specify the location of Pax3 in the molecular cascade of myogenesis as our understanding of this pathway continues to emerge.

A fusion protein formed by the t(2;13) chromosomal translocation resulting in alveolar rhabdomyosarcoma shares DNA binding properties with Pax3, since it includes the intact Pax3 paired and homeodomains. Compared with Pax3, it is a more potent activator of transcription and inhibitor of myogenic differentiation in our in vitro assays. We propose that deregulated Pax3-like activity prevents the terminal differentiation of muscle progenitor cells contributing to the rhabdomyosarcoma phenotype. This suggests that forms of Pax3 (and perhaps Pax7) deregulation other than chromosomal translocation will be identified as causes of rhabdomyosarcoma and/or abnormalities of limb muscle development.

  
Table: Pax3 and PAX3/FKHR inhibit myosin expression by cultured myoblasts

C2C12 cells were stably transfected with the indicated expresson plasmids (see text for explanation of specific paired and homeodomain mutations). After selection for G418 resistance and induction of differentiation, plates were stained for myosin expression. The anti-myogenic effect of transfecting Pax3 with or without a C-terminal hemagglutinin epitope tag was indistinguishable, and the results of both forms of Pax3 transfections are combined. 10T1/2 cells stably expressing MyoD or myogenin were infected with retrovirus encoding Pax3, PAX3/FKHR, or derivatives as indicated, induced to differentiate, and myosin staining colonies were counted. Each entry represents the sum of at least two independent transfections.



FOOTNOTES

*
This work was supported by the Howard Hughes Medical Institute, National Institutes of Health Grant CA-23099, Cancer Center Core Grant CA-21765, and the American Lebanese Syrian Associated Charities. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Thorn 910, HHMI, Brigham and Women's Hospital, 20 Shattuck St., Boston, MA 02115. Tel.: 617-732-5979; Fax: 617-738-5575.

The abbreviations used are: bHLH, basic helix-loop-helix; HA, hemagglutinin; EMSA, electrophoretic mobility shift assays; CAT, chloramphenicol acetyltransferase; SEAP, secreted alkaline phosphatase.

J. A. Epstein and R. L. Maas, unpublished results.


ACKNOWLEDGEMENTS

We thank Alan Michelson for helpful discussions.


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