From the
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.
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)
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.
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 pEMSVscribe
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 (
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.
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.
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.
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.
We thank Alan Michelson for helpful discussions.
(
)
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) .
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.
2 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.
pG
ECP3Con2X was derived from pG
ECAT 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.)
Hd). In each case, the mutant
proteins were unable to inhibit myogenic differentiation
().
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).
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.
Table:
Pax3 and PAX3/FKHR inhibit myosin expression by
cultured myoblasts
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.