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INTRODUCTION |
Several genes, such as muscle creatine kinase, troponins,
caveolin-3,
-actin, and myosin, have been reported to be
predominantly expressed in skeletal muscle. A family of muscle-specific
transcription factors such as myoD, myogenin, myf-5, and
MRF-4/herculin/myf-6 that regulate muscle-specific gene expression has
also been cloned. These are phosphorylated nuclear proteins, containing
helix-loop-helix motifs required for dimerization and DNA binding, that
can determine a specific cellular differentiation program (1). The myoD
family of transcription factors has been shown to direct myogenesis, repress proliferation, activate differentiation, and induce contractile phenotypes. The introduction of any one of these into nonmyogenic cells
induces their differentiation into mature muscle cells (2). The MyoD
and myf-5 are expressed in proliferating myoblasts whereas myogenin and
MRF-4 are not expressed until myoblasts exit the cell cycle in response
to mitogen depletion. Therefore, myoD and myf-5 have been im-
plicated as having a role in proliferating myoblasts whereas myogenin
and MRF-4 have been shown to activate and maintain muscle gene
expression (3). In addition, the cell cycle regulatory proteins such as
RB (4, 5), p21 (6), cyclin D, cdk2, cdk4 (7), and p53 (8) have been
implicated in the muscle differentiation program. Recently, caveolin-3,
-dystroglycan, and DNA methyltransferase (9-11) have also been
assigned a positive role in myogenic differentiation.
While looking for genes involved in senescence and immortalization, we
fortuitously cloned a novel gene that is specifically expressed in fast
twitch skeletal muscles. The gene is named "striamin" because of its specific expression in striated muscle. Cloning of the
cDNA, expression analyses, subcellular localization, chromosomal assignment, its interactions with the tumor suppressor p53, and its
possible significance during muscle differentiation are reported herein.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Normal mouse embryonic fibroblasts from the
CD1-ICR strain of mouse (CMEF), an immortal clone (RS-4) established
from CMEF, and NIH 3T3 cells, initially used for comparison of proteins
and cloning studies, were cultured as described (12). C2 cells
originally isolated by Yaffe and Saxel (13) and subcloned by Blau
et al. (14) were grown in DMEM (Life Technologies, Inc.,
Melbourne, Australia) supplemented with 20% FCS (Commonwealth Serum
Labs, Melbourne, Australia) and 0.5% chick embryo extract (Flow
Laboratories, North Ryde, Australia). Cells were induced to
differentiate by replacing growth medium with mitogen-poor medium, DMEM
plus 2% horse serum. COS7 and Rat-1 cells used for transfection and
microinjection studies were cultured in DMEM supplemented with 10%
FCS.
cDNA Cloning and Sequencing--
A cDNA library from
RS-4 cells was constructed in the
ZAP II vector and was screened
with a polyclonal anti-p33 antibody raised against a protein identified
from P-100 fractions of NIH 3T3 cells (12). cDNA clones were
sequenced by the dideoxy chain termination method, and the reactions
were analyzed on an ABI 377 automated sequencing machine. Full sequence
of a 2.4-kb cDNA clone designated 336 was derived by generation of
nested deletions from the 3' end by exonuclease III (Deletion kit,
Takara, Tokyo, Japan) and primer walking. Subsequently, the 5' end of
the cDNA was obtained by 5' MarathonTM RACE polymerase chain
reaction (PCR) on mouse skeletal muscle cDNA by using three
antisense gene-specific primers SP1 (5'-TGT CAC TGC CAC GCC TTC TCG GTG
CGC AG -3'), SP2 (5'-TCC CGG CTG CCC TTT GGC CCA TCT TGT CCC -3') and
SP3 (5'-TGA GAA AGC GTT AGA CGC TCT CAG AGC CCT-3'). 5' MarathonTM RACE
PCR was performed as described (CLONTECH).
RNA Isolation--
Total RNA was prepared from C2C12 cultures
grown in DMEM supplemented with 20% FCS or 2% horse serum
(differentiation medium, for 24-96 h) using Trizol (Life Technologies,
Inc). Skeletal muscles were excised from B6D2 males (F1 progeny of
C57BL/6J female × DBA/2J male matings), frozen in liquid
N2, and homogenized in denaturant. Total cellular RNA was
isolated from all muscle samples using the Trizol reagent.
Northern Blot and RT-PCR Analyses--
Mouse and human multiple
tissue Northern blots containing 2 µg of poly(A)+ RNA per
lane were purchased from CLONTECH. Total cellular
RNA from C2C12 cultures and B6D2 muscles was denatured and size
fractionated on 1% agarose gels containing 2.2 M
formaldehyde and transferred to Hybond N membrane (Amersham Pharmacia
Biotech). A 1.4-kb 3'-untranslated region fragment obtained by
BamHI digestion of pBSSK-336 plasmid was used as a probe.
Hybridization was performed at 65 °C in SSC-Denhardt's-SDS buffer.
The membrane was washed for 10 min each in 2× SSC, 2× SSC and 0.1%
SDS, then washed in 1× SSC and 0.1% SDS twice, and autoradiographed.
RNA loading on the blots was determined by hybridization with 18 S
ribosomal probe. RT-PCR was performed on total RNA from mouse tissues
and C2C12 cells cultured in normal and mitogen poor medium using
primers from 5' and 3' of striamin open reading frame (ORF).
Control RT-PCR was performed with glyceraldehyde-3-phosphate dehydrogenase-specific primers.
Cellular Localization of striamin--
The striamin
ORF was ligated in frame C-terminal to the GFP-ORF in pEGFPC1 vector
(CLONTECH). The plasmid encoding the
GFP-striamin fusion protein was transfected into COS7 cells
growing on coverslips using LipofectAMINETM (Life Technologies, Inc.).
Coverslips were incubated with nuclear dye, Hoechst 33258 (Sigma)
(5-10 µg/ml in culture medium for 10 min before cell fixation), and
fixed with methanol:acetone (1:1). After three washings in
phosphate-buffered saline, the coverslips were mounted with Fluoromount
(Difco). The cells were examined using an Olympus BH-2 microscope with epifluorescence optics or 40× Plan-Neofluar objective on a Zeiss Axiophot microscope (Carl Zeiss, Germany) equipped with a CELLscan system (Scanalytics, Billerica, MA). Microinjections of the
pGFPC1/striamin, pEGFPC1/N striamin (N-terminal
75 amino acids), or pEGFPC1/C striamin (C-terminal 74 amino
acids) were performed directly into the nuclei of NIH 3T3 cells growing
on coverslips using an Eppendorf semiautomated microinjection system
mounted on an inverted Zeiss microscope. Cells were fixed and examined
for cellular localization of striamin as described above.
p53-mediated Reporter Assays--
p53
/
mouse embryonic
fibroblasts were transfected with a p53-responsive luciferase reporter
plasmid, PG-13luc (kindly provided by Dr. Bert Vogelstein). A
temperature-sensitive p53 expression plasmid, pMSVp53Val135 (a kind
gift from Dr. Paul Jackson) that results in wild-type p53 conformation
at 32.5 °C was used for exogenous p53 expression. Control expression
plasmid pLK444 (15) or its striamin, N striamin
(N-terminal 75 amino acids), or C striamin (C-terminal 74 amino acids) containing derivatives, pLK444/striamin, pLK444/N striamin, or pLK444/C
striamin, respectively, were cotransfected with the reporter
plasmid. Cotransfections of pRL-CMV were performed as an internal
control to determine the efficiency of transfections. Luciferase assays
(Dual-LuciferaseTM reporter assay system, Promega) were
performed 48 h after transfection. Luciferase values were
calculated per microgram of protein as determined by Bradford protein assay.
p53
/
mouse embryonic fibroblasts were microinjected with a mixture
of plasmids containing 0.1 µg/µl each of the pMSVp53Val135, p53-responsive
-gal reporter pRGC
fos-lacZ (a kind gift from Dr.
David Wynford-Thomas), and pLK444 or pLK444/striamin.
Control IgG was co-injected for the identification of the injected
cells. After overnight incubation at 32.5 °C, the cells were fixed
with 4% formaldehyde, permeabilized with phosphate-buffered saline containing 0.1% Triton X-100 for 5 min on ice, washed three times with
phosphate-buffered saline, and then stained with fluorescein isothiocyanate-conjugated secondary antibodies to detect injected IgG
and
-galactosidase expression using the
-gal staining kit (Roche
Molecular Biochemicals). Cells were viewed using a Zeiss microsope. All
cells showing any trace of blue staining were scored as positive for expression.
In Vivo Co-immunoprecipitations--
COS7 cells were used for
high transfection efficiencies. Lysates (400 µg) prepared from
pEGFPC1 vector-, pEGFPC1/striamin-, pEGFPC1/N
striamin-, or pEGFPC1/C striamin-transfected
cells after 48 h of transfection were incubated with anti-p53
antibody (CM-1, Novocastra Laboratories Ltd.) overnight at 4 °C.
Immunocomplexes were precipitated by incubating with protein
A/G-Sepharose (30 min at 4 °C) and were analyzed for the presence of
striamin by Western blotting with anti-GFP monoclonal
antibody. Precipitation of p53 was detected by Western blotting with
anti-p53 monoclonal antibody (Ab-1, Calbiochem).
Preparation of Recombinant striamin Protein--
The ORF of
striamin cDNA was amplified by PCR of
pBSSK/striamin clone with sense (5'- GGA TCC AAG AAA GGC CTG
GCT GGC GAG-3') and antisense (5'-AAG CTT TCA TGT CAC TGC CAC GCC
TTC-3') primers with BamHI and HindIII sites,
respectively. The PCR-amplified 0.5-kb fragment was first cloned in
pGEM-T vector, confirmed to be correct by DNA sequencing, excised with
BamHI-HindIII, and then cloned into pQE30 vector
(Qiagen) to yield His-tagged protein. The pQE30/striamin,
pQE30/N striamin, and pQE30/C striamin
constructs were used to transform M15 bacteria, and cells grown to
OD580 = 0.6 were induced with
isopropyl-1-thio-
-D-galactopyranoside (IPTG) (0.2 mM) at 37 °C for 5 h. The bacterial lysates
(induced and uninduced with IPTG) were analyzed by SDS-polyacrylamide
gel electrophoresis followed by Western blotting with anti-His (Qiagen) and anti-p33 antibodies.
In Vitro Pull-down Assay--
His-tagged recombinant
striamin, N striamin, and C striamin
were purified from E. coli transformed with the
pQE30/striamin, pQE30/N striamin, and pQE30/C
striamin construct, respectively. Cells were centrifuged,
and the pellets were resuspended in buffer A (10 mM
Tris-Cl, pH 7.5, 150 mM NaCl, 20 mM imidazole,
6 M urea, and 5 mM
-mercaptoethanol),
sonicated for 2 min on ice, and agitated for 30 min at room
temperature. The extract was centrifuged at 15,000 × g
for 20 min. 0.5 ml of nickel-NTA-agarose affinity resin (Qiagen) was
added to the supernatant, and the mixture was agitated at room
temperature for 2 h. The mixture was then loaded into a disposable
plastic column, followed by washing with 20 ml of 10 mM
Tris-Cl, pH 7.5, and 0.5 M NaCl (TBS). The recombinant
protein was eluted with 0.5 M imidazole in TBS followed by
desalting on a PD-10 column (Amersham Pharmacia Biotech) using TBS as
eluent. Aliquots of the purified protein were stored at
20 °C
until use. The purity of the preparations was examined by
SDS-polyacrylamide gel electrophoresis and Western analysis with
anti-His antibody. Purified proteins (2-5 µg) were incubated with
GST or GST-p53 (1 µg, Santa Cruz) in an Nonidet P-40-lysis buffer to
a final volume of 500 µl. glutathione-Sepharose beads (20 µl) were
added after 2 h and rotated at 4 °C for 1 h. Beads were
pelleted by centrifugation, washed three times with TBS, boiled in SDS
sample buffer, and analyzed by Western blotting with anti-His tag antibody.
Chromosomal Assignment--
A mouse P1 genomic clone was
obtained by PCR screening of a P1 bacteriophage mouse genomic library
with clone 336-specific primers (sense:
5'-TGGTATTCTTATATTGTTTGCAACTAACTA-3'; antisense, 5'-GGAAGGCCATGTGACCTAATGTTTCATGTCA-3'). The isolated P1 clone was seen
to hybridize with the 3'-untranslated region of the gene, following
which it was used for chromosomal localization by fluorescence in
situ hybridization (FISH). DNA from the mouse P1 clone was labeled
with digoxigenin-dUTP by nick translation. Labeled probe was combined
with sheared mouse DNA and hybridized to metaphase chromosomes derived
from mouse embryonic fibroblasts in a solution containing 50%
formamide, 10% dextran sulfate, and 2× SSC. Specific hybridization
signals were detected by incubating the hybridized slides with
fluoresceinated antidigoxigenin antibodies, followed by
counterstaining with 4',6'- diamidino-2-phenylindole.
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RESULTS |
Cloning and Characterization of striamin cDNA--
A
comparison of plasma membrane Triton X-100-insoluble fractions from
normal (CMEF) and immortal (NIH 3T3) murine cells revealed a protein of
nearly 33 kDa (called p33) present in NIH 3T3 but not in CMEF cells
(12). The protein was isolated from SDS-polyacrylamide gels and was
used for raising polyclonal antibody. The anti-p33 antibody thus raised
was used for cDNA cloning by immunoscreening of a RS-4 cDNA
library as described previously (16). Five cDNA clones were
obtained and were characterized by partial sequencing. Three clones
showed identity to known genes, namely FusCHOP, G-utrophin, and
dystrophin, while two clones had no matches in the DNA sequence data
bases. The in vitro translated products of these two novel clones could not be precipitated with the anti-p33 antibody, indicating that they were not related to the p33. The characterization of one of
these clones, 336, is described here.
The complete sequence of 336 (2.4 kb) contained one predicted ORF of
309 base pairs, which was located at the 5' end of the cloned cDNA.
5' RACE PCR performed using skeletal muscle cDNA generated an
overlapping clone, 5' 336. The additional 5' sequence extended the ORF
to 447 base pairs with an upstream in-frame stop codon (Fig.
1). The full-length cDNA sequence
thus obtained had no homology to any sequence in the DNA sequence data
banks. Analysis of the cDNA sequence by BLAST, PROSITE, GCG, and
PSORT programs revealed no known motifs that could predict its possible
function. The 5'-noncoding sequence of striamin contains
C/GAAAA repeats and the 3'-noncoding region contains GT repeats;
however, the functional significance of these repeats is not known. It
encodes a 149-amino acid protein (pI 10.2) that also has no significant matches in the protein data base. The ProtPram program predicted a
soluble protein with average hydrophobicity of 0.5 and aliphatic index
of 0.74. Two protein kinase C phosphorylation sites, SDR at 45-47
amino acid residues and SPK at 78-80 amino acid residues; one casein
kinase II phosphorylation site, SGLD at 12-15 amino acid residues; and
two myristoylation sites, GNYYCC at 111-116 and GTRWAK at 120-125
amino acid residues, are predicted by the ScanProsite program. Other
interesting features of the protein include a high positive charge; a
large number of serine, leucine, and proline residues; and the presence
of four cysteines. The protein could not be characterized as a member
of any known gene family based upon cDNA or protein sequence
analyses. In view of its expression in striated
muscle (see below), the gene has been named
striamin.

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Fig. 1.
Nucleotide and predicted amino acid sequence
of striamin. The sequence obtained by 5' RACE PCR
on mouse skeletal muscle cDNA is underlined up to the 5'
upstream in-frame stop codon.
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The predicted ORF was cloned in pBSSK, and in vitro
translation was performed to confirm the existence of the ORF in the
given sequence. A protein of approximately 18 kDa mass was detected (data not shown). The ORF was also cloned into the bacterial expression vector pQE30, and recombinant protein containing a 6×His tag at the
amino terminus was obtained. The induction of the promoter by IPTG led
to the synthesis of a protein of approximately 18 kDa (data not shown).
Expression Analyses--
Northern blotting of mouse and human
tissues showed a strong reactivity of the striamin probe to
a 3.0-kb transcript in mouse and human skeletal muscle and mouse heart
(Fig. 2, A and B).
It also reacted very weakly to transcripts of approximately 4.0 and 8.0 kb from human and 4.0 kb from mouse tissues. We therefore performed
RT-PCR on mouse tissues and myoblasts with striamin primers
(Fig. 2C). An expected size of DNA fragment was obtained from heart and myoblasts (Fig. 2C). This, together with the
Northern data, confirmed that the cloned cDNA corresponds to
striamin that is preferentially expressed in heart and
skeletal muscle. We next investigated whether striamin
exhibits muscle fiber type specificity. Four fiber phenotypes, namely
fast twitch fiber types 2A, 2B, and 2X and slow fiber type I, have been
defined on the basis of expression of the type of myosin heavy chain
isoform (17). striamin was predominantly expressed in fast
(quadriceps) versus slow fibers (soleus) (Fig.
3A). Northern analysis using
RNA isolated from mouse skeletal muscles of differing fast and slow
fiber content (quadriceps: 95% fast 2B, 4% fast 2X (18); extensor
digitorum longus: 60% fast 2B, 28% fast 2X, 12% fast 2A (19);
superficial gastrocnemius: 100% fast 2B (20); diaphragm: 57% fast 2X,
34% fast 2A, 7% slow (20); and soleus: 45% fast 2A, 55% slow (21)) revealed that striamin is expressed preferentially in fast
glycolytic (2B) fibers (Fig. 3B).

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Fig. 2.
Expression of striamin in
mouse and human tissues. A, mouse multiple tissue
Northern blot showing striamin expression in skeletal muscle
and heart (shown by arrow). The probe showed a weak
reactivity to a transcript of 4.0 kb in other tissues. B,
human multiple tissue Northern blot showing striamin
expression in skeletal muscle (shown by arrow). Probe
reacted weakly to transcripts of 8.0 and 4.0 kb in some other tissues.
RNA loading was assessed by hybridization with an 18 S ribosomal RNA
oligonucleotide probe. C, striamin RT-PCR was
performed using total RNA from mouse heart, kidney, testis, liver,
brain, spleen, and myoblasts cultured in serum-supplemented and
-deficient medium (lanes 1-8, respectively).
striamin expression was detected in heart and myoblasts, and
was down-regulated with differentiation (lanes 7 and
8).
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Fig. 3.
Expression in mouse skeletal muscle in
vivo and in vitro. A,
Northern analysis for striamin expression in different
muscle fiber phenotypes. Expression was detected in fast (quadriceps)
(lane 1) and absent in slow (soleus) twitch fibers
(lane 2). B, Northern analysis for striamin on
RNA from muscle fibers with varying composition of fast and slow
fibers. Lanes 1-5 contain RNA from quadriceps (95% fast
2B, 4% fast 2X), extensor digitorum longus (60% fast 2B, 28% fast
2X, 12% fast 2A), superficial gastrocnemius (100% fast 2B), diaphragm
(57% fast 2X, 34% fast 2A, 7% slow), and soleus (45% fast 2A, 55%
slow). striamin is expressed preferentially in fast
glycolytic (2B) fibers. The blots were hybridized with 18 S ribosomal
RNA oligonucleotide probe for loading controls. C,
striamin expression in C2C12 cells undergoing
differentiation in vitro. Lane 1 has RNA from dividing
myoblasts cultured in normal growth medium, and lanes 2-5
have RNA from cells cultured in differentiation medium for 24, 32, 40, and 88 h and showed approximately 10, 20, 30, and 85% myotube
formation, respectively. Absence of striamin is seen in the
differentiated C2C12 culture (lane 5). Ethidium
bromide-stained gel with 28 S and 18 S RNA bands is shown as a loading
control.
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We examined striamin expression during in vitro
myogenesis of C2C12 myoblasts. These cells were cultured in
differentiation medium, and RNA was isolated at various time points
representing gradual formation of myotubes as observed microscopically.
Interestingly, a 4-day culture that showed about 80% myotube formation
had negligible expression of striamin as compared with the
1- and 2-day cultures, which had about 10-30% myotube formation (Fig.
3C). Consistent with this Northern analysis,
striamin RT-PCR from C2C12 cells cultured in
serum-supplemented and -deficient medium for 60-72 h exhibited its
down-regulated expression in the latter (differentiated myoblasts)
(Fig. 2C, lanes 7 and
8).
Cellular Localization of striamin--
COS7 cells were transfected
with a plasmid, pEGFPC1-striamin, encoding a
GFP-striamin fusion protein. Transfected cells had distinct
green fluorescence (Fig. 4A)
in the nucleus that overlapped with the nuclear dye, Hoechst 33258, staining (Fig. 4B). pEGFPC1-striamin, pEGFPC1/N
striamin (N-terminal 75 residues), and pEGFPC1/C
striamin (C-terminal 74 residues) were microinjected into
the nucleus of NIH 3T3 cells. Whereas distinct green nuclear
fluorescence was detected for striamin as in the
transfection assays, N striamin and C striamin
were both retained in the cytoplasm (Fig. 4, a-c). Three
dimensional image scanning of cells revealed that the full striamin protein is localized in the nucleus and is clearly
excluded from nucleolar structures (Fig. 4a). N
striamin was concentrated around the nuclear membrane (Fig.
4b), and C striamin in addition to its
concentration around the nucleus was also distributed diffusely in the
cytoplasm (Fig. 4c). The present data and the fact that striamin does not contain any known nuclear localization
signal could suggest that the predicted high positive charge of the
native protein may be responsible for its nuclear localization.
Alternatively, striamin may translocate to the nucleus by
interacting with some nuclear localization signal-containing
protein.

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Fig. 4.
Intracellular localization of
striamin. Transfection of
pEGFPC1-striamin resulted in distinct green fluorescence of
GFP-striamin fusion protein in the nuclei (A) of
COS7 cells that overlapped with Hoechst dye staining (B)
shown by arrowheads. Microinjection of
pEGFPC1/striamin (a), pEGFPC1/N
striamin (b) and pEGFPC1/C striamin
(c) plasmids into the nucleus of NIH 3T3 cells resulted in
nuclear, perinuclear, and cytoplasmic green fluorescence, respectively,
as visualized by laser scanning microscopy.
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striamin Represses p53 Activity--
Wild type (wt) p53 has a role
during cell differentiation (reviewed in Ref. 22). Evidence in support
of this includes the following: (i) overexpression of exogenous wt p53
or endogenous wt p53 following cell irradiation can partially restore
differentiation of several tumor cells (23, 24), (ii) up-regulation of
p53 mRNA occurs during C2 differentiation (25), and (iii)
interference with endogenous wt p53 inhibits hematopoietic and muscle
cell differentiation, which is shown to be independent of its cell cycle activity (8). In view of these reports and characteristics of
striamin such as nuclear localization and down-regulation
with myogenic differentiation, we asked whether striamin can
interfere with p53 activity. p53
/
MEF cells were transfected with
the wt p53-responsive luciferase reporter plasmid (PG-13luc), a
temperature-sensitive p53 expression plasmid, pMSVp53Val135, and either
the pLK444 vector or pLK444/striamin. The presence of the
striamin expression construct caused a significant reduction
in p53 reporter gene activity in four independent experiments. This
result demonstrated that striamin can inhibit the
transcriptional activity of p53 (Fig.
5A). Furthermore, cotransfections of the antisense construct were seen to have a mild
positive effect on p53 activity (Fig. 5A). Similar results were obtained following microinjection of pMSVp53Val135, the
p53-responsive
-gal reporter, pRGC
fos-lacZ and the various
striamin expression plasmids. Injected cells were identified
by coinjection of rabbit IgG that was visualized by staining with
fluorescein isothiocyanate-conjugated secondary antibody.
-Gal
staining was observed in 86% and 88% of cells injected with control
and antisense striamin plasmid, respectively, but only in
5% of cells that were injected with striamin sense
construct (Fig. 5B). These data confirmed the repression of
p53 activity by striamin and were consistent with its
down-regulation observed during C2C12 differentiation. To further
characterize the specificity of striamin-p53 interactions,
we also performed p53 reporter assays in which expression plasmids
encoding N-terminal 75 (N striamin) or C-terminal 74 (C
striamin) amino acid residues were transfected. Whereas full
and C striamin were seen to repress p53 activity,
transfections of N striamin were neutral (Fig.
5A).

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Fig. 5.
Interaction of striamin and
p53. A, effect of striamin on
transcriptional activity of wild type p53. p53-expression
(pMSVp53Val135) and p53-responsive luciferase reporter (PG-13luc)
plasmids were transfected into p53 / mouse embryonic fibroblasts
(MEF) along with antisense (AS) or sense
(S) expression constructs encoding full, N-terminal 75 (N
striamin), and C-terminal 74 (C striamin) amino
acid residues. Numbers indicate the amount (µl) of each
plasmid (0.5 µg/µl) used. Error bars
represent standard deviation (n = 3). A 4.6-fold
repression of p53 activity was observed with cotransfection of the full
striamin sense construct, whereas the antisense construct
resulted in 1.6-fold activation. Co-transfections of C
striamin resulted in repression of p53 activity similar to
the full striamin protein. N striamin was
neutral. B, repression of p53 activity by microinjected
striamin. p53 / cells were microinjected with
p53-expression (pMSVp53Val135) and p53-responsive -gal reporter
(pRGC fos-lacZ) plasmids with or without striamin sense or
antisense expression constructs. Injected cells were visualized by
staining with fluorescein isothiocyanate-conjugated anti-rabbit IgG
(upper panel) and are shown by arrows in the
lower panel. Blue staining for -gal expression was
observed in cells coinjected with vector (a and
d) and the antisense construct (c and f).
Coinjection of striamin (b and e)
resulted in repression of p53 activity as demonstrated by the absence
of -gal staining (e). Note: dense blue stain in some
cells caused interference in visualization of fluorescein
isothiocyanate staining (e and f). C,
in vivo coimmunoprecipitation of striamin and
p53. p53 immunocomplexes from COS7 cells transfected with GFP vector or
the one encoding GFP-full striamin, GFP-N
striamin, or GFP-C striamin fusion protein were
analyzed by Western blotting with anti-GFP antibody. Protein fractions
of the cell lysates that were soluble in Nonidet P-40 lysis buffer and
the remaining that were solubilized by boiling in 0.5% SDS were used
for immunoprecipitaion. GFP-full striamin, -N
striamin, or -C striamin were found to
coprecipitate with p53 (right panel); no coprecipitation was
observed with control antibody (middle panel) or of the GFP
tag only with anti-p53 antibody (right panel, lanes 1 and
5). Input (left panel) shows 10% of the protein
used for immunoprecipitation. Co-immunoprecipitated p53 was detected by
Western blotting with anti-p53 antibody (Ab-1, upper right
panel). D, in vitro coimmunoprecipitation of
striamin and p53. His-tagged recombinant full
striamin, N-striamin or C-striamin
(5% of the input; lanes 1-3) were mixed with GST
(lanes 4-6) or GST-p53 (lanes 7-9) and were
precipitated by glutathione-Sepharose beads. GST-Complexes were
analyzed by Western blotting with anti-p53 (CM-1) and
anti-His tag antibody. The full striamin, N
striamin, and C striamin were seen to
coprecipitate with p53 (lanes 7-9).
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In Vivo and in Vitro Interactions of striamin and p53--
The
effect of striamin on the transcriptional activation
function of p53 prompted us to investigate a possible interaction between these two proteins. We used COS7 cells for their high amounts
of wt p53 and high transfection efficiencies. p53 immunocomplexes from
pEGFPC1- and pEGFPC1/striamin-transfected cells when
analyzed by Western blotting with anti-GFP antibody revealed the
presence of GFP-striamin (Fig. 5C). This
demonstrated that the striamin interacts with p53 in
vivo. Similar immunoprecipitations were also performed from cells
expressing GFP-N striamin and GFP-C striamin
fusion proteins. The full, N-, and C- striamin were found to
interact to p53 (Fig. 5C); no coimmunoprecipitation of GFP tag was detected in these experiments.
We next performed an in vitro pull-down assay for
striamin and p53. His-tagged recombinant striamin
was incubated with either GST alone (negative control) or with GST-p53.
Western analysis of glutathione-Sepharose-reacting complexes using an
anti-His tag antibody revealed the presence of His-tagged
striamin, demonstrating that striamin can
physically interact with p53 (Fig. 5D). Furthermore, as in
the in vivo coimmunoprecipitations, both N
striamin and C striamin were found to bind to p53
(Fig. 5D).
Chromosomal Localization--
A mouse genomic P1 clone containing
the striamin gene was obtained by PCR screening of a P1
library. In FISH analysis, this clone specifically hybridized to a
medium-sized chromosome, which appeared to be chromosome 12 on the
basis of 4',6'-diamidino-2-phenylindole staining. To confirm the
localization of striamin to mouse chromosome 12, FISH
analysis was repeated using the P1 clone and chromosome 12 centromere-specific probe. The striamin P1 and chromosome 12 probes localized to the same chromosome (Fig.
6). A total of 80 metaphase cells were
analyzed, out of which 71 exhibited specific labeling. Measurements of
specifically hybridized chromosome 12 in 10 metaphase spreads
demonstrated that striamin is located at a position that is
57% of the distance from the heterochromatic-euchromatic boundary to
the telomere of chromosome 12, an area that corresponds to band 12C3.
This region is syntenic to human chromosome 14q21-22.

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Fig. 6.
A, chromosomal localization of
striamin on mouse metaphase chromosomes. striamin
and chromosome 12-specific probes are seen as green fluorescence, and
are also marked by triangles and arrows,
respectively. B, diagrammatic representation of
striamin on mouse chromosome 12C3 region.
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DISCUSSION |
Here we report cloning and characterization of a novel gene,
striamin, whose expression is restricted to the striated
muscles. The expression pattern of striamin shares features
in common with a few other genes, but is most similar to that of MyoD.
Both are expressed in proliferating myoblasts, decline during
differentiation, and yet are present in adult skeletal muscle (26).
Furthermore, they appear to be preferentially expressed in fast
glycolytic muscle fibers in the adult mouse (27). In adult myofibers
myoD is thought to mediate innervation and thyroid hormone effects on
fiber type-specific gene expression (26) as well as repress slow
isoform gene function (28). Other genes that are specific to fast
glycolytic fiber include myosin heavy chain 2B (MyHC 2B) (reviewed in
Ref. 29) and a muscle-specific form of the glycolytic enzyme aldolase A
(M-aldA) (30). Therefore, striamin may function as a
mediator of extrinsic factors on gene expression in fast glycolytic
fibers, as a determinant of metabolism, or as a determinant of muscle
contractile activity.
Adult skeletal muscle can undergo regeneration, repair, and growth in
response to injury or various stresses (31, 32). These processes are
achieved by the activation of muscle precursor or satellite cells. In
normal skeletal muscle, satellite cells are mitotically quiescent,
mononucleated cells that are situated between the basement membrane and
the myofiber plasma membrane. Injury or stress results in the mitotic
activation of the satellite cells, which proliferate and fuse to repair
damaged fibers or increase the size of existing fibers. The progression
from proliferating to fusion competent satellite cells is marked by a
precise order of expression of myogenic regulatory factors and muscle
structural proteins. This includes, in order, MyoD, myogenin,
-smooth muscle actin, and sarcomeric myosin (33). Because
striamin is expressed in myoblasts in culture, it is a
candidate marker for activated satellite cells and may play a role in
the differentiation process in vivo.
striamin is expressed in mouse, but not in human, heart.
Differences exist between rodent and human cardiac myofibers in
contraction velocities and force production, which in large part
reflects the ATPase activity conferred by the MyHC isoform present
(34).
-MyHC, the predominant isoform in the rodent heart, confers a faster shortening velocity and low efficiency of force production. In
contrast,
-MyHC predominates in the human heart, which has a slower
shortening velocity and high efficiency of force production. Rodent and
human hearts also differ in the relative amounts of sarcomeric actins
present, cardiac and skeletal actin (35, 36), which most likely
reflects a difference in force development (37). The combinations of
MyHCs and sarcomeric actins in rodent versus human heart
results in a rodent heart that is more similar in contractile
properties to a fast-twitch skeletal muscle fiber, whereas the opposite
is true for the human heart. Therefore, the expression of
striamin in striated muscles and mouse heart is consistent
with a role in a fast contractile phenotype.
striamin Was Found to Interact with p53 in Vitro and in
Vivo--
Repression of p53 activity by striamin is
consistent with its down-regulation during in vitro
myogenesis when significant increase in p53 activity has been reported
(22). These data suggests that striamin may affect
myogenesis via a direct interaction with p53. Our data suggested that
both the N- and C-terminal halves of striamin protein can
bind to p53; however, it is the C terminus of striamin that
represses transcriptional activity of p53. This suggests that there are
more than one p53 binding sites in striamin protein and
vice versa. Characterization of these warrant further studies.
The myogenic differentiation program includes activation of myogenic
transcription factors, intercellular fusion of myoblasts, their
withdrawal from the cell cycle, and terminal differentiation to
myotubes. Besides the muscle-specific family of transcription factors,
myoD family, several adhesion molecules such as N-CAM, N-cadherin, very
late activation antigen 4, vascular cell adhesion molecule 1 (VCAM-1),
and meltrin-
have been implicated in this process (38-41). Bone
morphogenetic protein-12 and -13, TGF-
, and other members of the
TGF-
superfamily (42, 43), ERK-6, a mitogen-activated protein kinase
(44), and PAX3 (45) have been shown to interfere with or suppress
in vitro myogenesis of C2C12 myoblasts. Cyclin D1 is found
to be down regulated with myogenesis of C2C12, in contrast to cyclin
D2, which showed transient increase, and cyclin D3, which showed
20-fold increase (7, 46). striamin does not show any
structural homology to any of these proteins that have been implicated
in different aspects of muscle differentiation. Of particular interest
are its fast fiber specificity, nuclear localization, down-regulation
with myogenic differentiation, and functional interactions with the tumor suppressor p53, which may predict it to be an important gene in
the regulation of the myogenic differentiation program and warrant
further studies to elucidate its role in myogenesis and the fast
contractile phenotype.