The Notch coactivator, MAML1,
functions as a novel coactivator
for MEF2C-mediated transcription and
is required for normal myogenesis
Huangxuan Shen,1Abigail S. McElhinny,2Yang Cao,1Ping Gao,1Jingxuan Liu,1Roderick Bronson,3
James D. Griffin,1and Lizi Wu1,4
1Department of Medical Oncology, Dana-Farber Cancer Institute and Departments of Medicine, Brigham and Women’s
Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA;2Department of Cell Biology and Anatomy,
University of Arizona, Tucson, Arizona 85724, USA;3Rodent Histopathology Core, Harvard Medical School,
Boston, Massachusetts 02115, USA
The MAML (mastermind-like) proteins are a family of three cotranscriptional regulators that are essential for
Notch signaling, a pathway critical for cell fate determination. Though the functions of MAML proteins in
normal development remain unresolved, their distinct tissue distributions and differential activities in
cooperating with various Notch receptors suggest that they have unique roles. Here we show that mice with a
targeted disruption of the Maml1 gene have severe muscular dystrophy. In vitro, Maml1-null embryonic
fibroblasts failed to undergo MyoD-induced myogenic differentiation, further suggesting that Maml1 is
required for muscle development. Interestingly, overexpression of MAML1 in C2C12 cells dramatically
enhanced myotube formation and increased the expression of muscle-specific genes, while RNA interference
(RNAi)-mediated MAML1 knockdown abrogated differentiation. Moreover, we determined that MAML1
interacts with MEF2C (myocyte enhancer factor 2C), functioning as its potent cotranscriptional regulator.
Surprisingly, however, MAML1’s promyogenic effects were completely blocked upon activation of Notch
signaling, which was associated with recruitment of MAML1 away from MEF2C to the Notch transcriptional
complex. Our study thus reveals novel and nonredundant functions for MAML1: It acts as a coactivator for
MEF2C transcription and is essential for proper muscle development. Mechanistically, MAML1 appears to
mediate cross-talk between Notch and MEF2 to influence myogenic differentiation.
[Keywords: MAML1; MEF2C; Notch; muscular dystrophy; myogenesis; transcriptional coactivator]
Supplemental material is available at http://www.genesdev.org.
Received October 12, 2005; revised version accepted January 18, 2006.
Myogenesis is a carefully orchestrated process that is
essential not only for muscle development, but also for
the regeneration of old and injured muscle fibers. The
myogenic program is initiated when mononucleated
muscle progenitor cells (myoblasts) expand and exit the
cell cycle in response to specific extrinsic signals. The
myoblasts fuse, elongate, and develop into multinucle-
ated myotubes, which form mature skeletal muscle. Bio-
chemically, myogenic differentiation is characterized by
the expression of muscle-specific genes including des-
min, creatine kinase, and muscle myosin (Bailey et al.
2001; Parker et al. 2003).
Two distinct classes of transcription factors regulate
the myogenic expression program, myogenic regulatory
factors (MRFs) and myocyte enhancer factor 2 proteins
(MEF2) (Black and Olson 1998; Berkes and Tapscott
2005). MRFs are a family of basic helix–loop–helix
(bHLH) transcription factors up-regulated in response to
myogenic signals and include MyoD, myogenin, Myf5,
and MRF4. They form heterodimers with E proteins such
as E12 or E47, and bind to specific DNA sites called
E-boxes found within many muscle-specific gene pro-
moters. The MRFs have myogenic activities and are able
to convert nonmyogenic, mesenchymal cells into myo-
blasts. On the other hand, the MEF2 proteins are ex-
pressed in many different tissues and belong to the
MADS box family that includes MEF2A, MEF2B,
MEF2C, and MEF2D. The MEF2 family shares conserved
MADS and adjacent MEF2 domains that mediate DNA
binding and dimerization. They bind as homodimers or
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heterodimers with the MRFs to the AT-rich consensus
sequences that are often associated with regions of
muscle-specific gene control. Although MEF2 proteins
themselves are not myogenic, they interact with MRFs
to potentiate MRF-mediated myogenic activities (Mol-
kentin et al. 1995).
The exact molecular mechanisms dictating myogen-
esis remain unclear. However, it is well established that
this process involves multiple complex signaling path-
ways that modulate both MRF and MEF2 functions (Dias
et al. 1994; McKinsey et al. 2001; Lazaro et al. 2002; Liu
et al. 2004). One signaling pathway with an emerging
and seemingly critical role in muscle stem cell activa-
tion and myogenesis is the Notch pathway: an evolu-
tionarily conserved mechanism in which cell–cell inter-
actions influence distinct cellular fates (Artavanis-Tsa-
konas et al. 1999; Luo et al. 2005). The four mammalian
Notch receptors (Notch1–Notch4) are transmembrane
proteins that are activated by ligands expressed on the
surfaces of neighboring cells. Upon activation, the recep-
tors undergo proteolytic processing events, resulting in
the release of the intracellular domain of Notch (ICN).
The ICN then translocates to the nucleus and activates
the CSL family of DNA-binding transcription factors.
Importantly, the cellular effects and target genes of the
Notch pathway are highly cell-context dependent. The
target genes of Notch include the well-studied bHLH
HES gene family.
Notch signaling is involved in pattern formation and
cyclic gene expression during somitogenesis, indicating
its complicated and tightly regulated role in muscle de-
velopment (Weinmaster and Kintner 2003; Rida et al.
2004). During postnatal regeneration of skeletal muscle,
Notch signaling appears to play distinct roles at different
stages. For instance, aged muscles have an impaired abil-
ity to produce sufficient myoblasts necessary for regen-
eration due to the decreased induction of the Notch
ligand Delta following injury (Conboy et al. 2003).
Forced activation of Notch restored regenerative poten-
tials, suggesting that Notch signaling is required for
muscle stem cell activation and proliferation. In con-
trast, several studies have determined that the Notch
pathway must be inhibited for subsequent myoblast fu-
sion and muscle differentiation events (Kopan et al.
1994; Lindsell et al. 1995; Conboy and Rando 2002).
Notch signaling is thought to inhibit myogenesis be-
cause it activates the transcription of target genes in-
cluding HES1, which, in turn, inhibit the expression and
activities of MyoD. This process requires the transcrip-
tion factor for the Notch pathway, CSL. Interestingly,
however, some aspects of Notch’s inhibitory effect on
myogenesis appear to be mediated by CSL-independent
mechanisms (Shawber et al. 1996; Nofziger et al. 1999).
For example, a mutant Notch receptor that is unable to
bind to CSL and activate transcription still prevents
C2C12 myoblast differentiation. One potential mecha-
nism for the CSL-independent Notch activity is the find-
ing that Notch ICN1 may interact directly with the
muscle transcription factor MEF2C, thereby inhibiting
MEF2C’s transcriptional activity (Wilson-Rawls et al.
1999). Therefore, Notch-mediated inhibition of myogen-
esis is both CSL dependent and independent, and Notch
signaling must be tightly controlled during different
Previously, three homologous mammalian master-
mind-like (MAML) proteins were discovered as tran-
scriptional coactivators for Notch receptors: They form
ternary complexes with Notch ICN and CSL in the
nucleus and are essential for Notch-mediated transcrip-
tion of target genes (Lin et al. 2002; Wu et al. 2002).
Strikingly, the three MAML members exhibit distinct
expression patterns and also cooperate differentially
with various Notch receptors in the activation of target
genes. Thus, it appears that the MAML family members
are not functionally redundant and may contribute to
the distinct biological effects of the Notch pathway that
occur in different tissues. Recently, MAML family mem-
bers were implicated in tumorigenesis (Tonon et al.
2003). However, their roles in normal developmental
processes, including myogenesis, are unknown. It also
remains unclear whether MAML family members func-
tion independently of Notch receptors.
In this study, we generated a mouse strain deficient in
the Maml1 gene. Maml1 knockout (KO) mice died
within the perinatal period, and histological analyses in-
dicated that they exhibit muscular dystrophy. Indeed,
their embryonic fibroblasts are unable to undergo myo-
genic events when transduced with MyoD, and further
studies in C2C12 cells revealed Maml1’s critical role in
myogenesis. Mechanistically, MAML1 functions as a
novel coregulator for MEF2C during myogenesis, but can
be recruited away to act as a coregulator for Notch upon
activation of the Notch pathway.
Knockout of the Maml1 gene in mice results
in muscular dystrophy and perinatal lethality
Previously, we cloned the murine Maml1 gene and char-
acterized its early developmental expression pattern via
whole-mount in situ hybridization studies (Wu et al.
2004). Maml1 gene expression is temporally and spa-
tially dynamic and detectable in many tissues. Rela-
tively high expression levels were detected in the devel-
oping brain, spinal cords, lung, limb buds, and somites.
Previous Northern blotting also revealed that Maml1 is
expressed in many adult tissues including skeletal
muscle (Wu et al. 2000). To determine the in vivo func-
tion of this gene, we generated a targeting vector to de-
lete the first exon and the 5? promoter region as a gene
inactivation strategy (Fig. 1A). The inactivation of the
Maml1 gene and consequent loss of the expression of the
mRNA transcript and protein was verified by Southern
blot analysis (Fig. 1B), Northern blot analysis (Fig. 1C),
and Western blot analysis (Fig. 1D), respectively. Mice
heterozygous for the Maml1 gene exhibited no discern-
ible phenotype in comparison with wild-type mice.
However, mice withthe
(Maml1−/−), were delayed in their growth at birth, re-
Shen et al.
676GENES & DEVELOPMENT
mained significantly smaller than control mice in the
perinatal period (Fig. 1E,F), and died within ∼10 d of
We performed histological analyses on hematoxylin
and eosin (H&E)-stained paraffin sections from both con-
trol (wild-type or heterozygous) (Fig. 2A, panels 1,3) and
homozygous null littermates (Fig. 2A, panels 2,4,5). The
most striking observation was that ablation of the
Maml1 gene resulted in severe perturbations in skeletal
muscle structure. Muscle sections from the Maml1−/−
mice exhibited pathology consistent with muscular dys-
trophy, including variable myofiber size (both atrophic
and hypertrophic), with myofibrillar degeneration and
necrosis/loss. In addition, many muscle sections (par-
ticularly from the appendicular skeletal muscles) con-
tained a significant number of vacuoles, also consistent
with muscle degeneration (Fig. 2A, panels 4,5). This phe-
notype was mosaic (i.e., some fibers from the null mice
appeared normal, while others were severely affected),
but the penetrance was 100%. The skeletal muscle le-
sions indicate that the Maml1 gene is critical for proper
Maml1 deficiency blocks MyoD-stimulated
myogenic conversion in murine embryonic fibroblasts
(MEFs), while exogenous MAML1 expression rescues
To investigate the molecular mechanisms by which
Maml1 affects myogensis, we undertook a series of cel-
lular and biochemical studies. First, MEFs were gener-
ated from wild-type and Maml1 KO littermates. Such
cells are known to be capable of differentiating into a
variety of cell types. For example, ectopic MyoD expres-
sion converts MEFs into cells that express muscle-spe-
cific genes and have a distinct myotube morphology (No-
vitch et al. 1996). We infected self-immortalized MEFs
from wild-type and Maml1 KO littermate embryos (em-
bryonic day 12.5 [E12.5]) with a retroviral construct en-
coding MyoD. After puromycin selection, cells were in-
duced to differentiate. We found that the majority of
wild-type MEFs infected with MyoD exhibited differen-
tiation into myotubes, while the MyoD-transduced
Maml1 KO MEFs remained in typical fibroblast mor-
phology (Fig. 2B). Moreover, expression of the muscle
construct. Black boxes denote exons of the mouse Maml1 gene. Exon 1 was replaced with a neo gene in the indicated targeting vector.
When genomic DNA is digested with XmnI, it is predicted that, using the denoted 5?-flanking probe, 11.7- and 23.3-kb bands would
be detected for the wild-type (WT) and KO alleles, respectively. (B) Southern blotting indicated successful targeting of the Maml1 gene.
Genomic DNA was isolated from embryos, digested with XmnI, blotted, and hybridized with the 5?-flanking probe. The predicted
23.3-kb band was detected in both the heterozygous and homozygous KO progeny, while the wild type and hetertozygotes contained
the 11.7-kb fragment, as expected. (C) Northern blotting showed a loss of Maml1 transcripts in embryonic fibroblasts derived from a
Maml1 KO embryo. Note that heterozygotes have reduced levels of Maml1 transcripts compared with wild-type transcripts. The size
of the Maml1 transcript is ∼6 kb. Relative loading is indicated by 28S rRNA. (D) Western blotting confirmed a loss of MAML1 protein
in embryonic fibroblasts derived from a Maml1 KO embryo and a reduction of the protein in the heterozygotes. MAML1 expression
(∼140 kDa) was analyzed by immunoprecipitation with anti-MAML1 antibodies, followed by Western blotting. (E) Maml1 KO neonate
(right) is growth retarded, as compared with its wild-type littermates (left), and die soon after birth (within 10 d). Mice are shown at
P5. (F) A representative growth curve reveals that Maml1 KO neonates (black diamonds) fail to thrive, compared with wild-type (WT)
littermates (gray squares). Data is presented as weight in grams at successive days after birth.
Generation of Maml1-null mice reveals that it is required for normal development. (A) Diagram of the Maml1 targeting
MAML1 function in myogenesis
GENES & DEVELOPMENT677
marker, myosin was significantly less in MyoD-trans-
duced Maml1 KO MEFs, compared with wild-type MEFs
infected with MyoD that robustly expressed myosin (Fig.
2C). These data suggest that Maml1 KO mice are defec-
tive in myogenic events regulated by MyoD, and so we
next tested whether the phenotype could be rescued by
exogenous MAML1 expression. Indeed, upon reintroduc-
tion of MAML1 expression in the MyoD-transduced
Maml1 KO MEFs, the cells exhibited a significantly aug-
mented degree of myogenic conversion, as indicated by
an increased number of myotube-like cells (Fig. 2D). Fur-
thermore, the rescued cells had significantly higher ex-
pression levels of myosin (Fig. 2E) compared with the
MEFs infected with MyoD, but lacking exogenous
MAML1 expression. These data indicate that Maml1 in-
deed is directly essential for MyoD-induced myogenic
Maml1 is expressed throughout C2C12 myoblast
differentiation and its overexpression enhances
We next performed experiments using the murine myo-
blast C2C12 cell line. These cells provide a valuable
model for studying myogenic differentiation because,
when cultured under conditions to promote differentia-
tion, they fuse, express muscle-specific proteins, and
form mature myotubes. First, we analyzed the expres-
sion of the Maml1 gene in proliferating and differentiat-
ing C2C12 cells by Western blotting. Our data indicate
that the MAML1 protein is expressed at similar levels
during the myoblast-to-myotube transition (Supplemen-
tary Fig. S1). Thus, Maml1 is expressed throughout
C2C12 myoblast differentiation.
We next determined whether ectopic expression of the
rescued upon exogenous Maml1 expression. (A) Whole neonatal mice were sectioned and stained with H&E. (Panel 1) Hip muscles of
a P6 heterozygous mouse shows normal fiber size and distribution. No differences were detected between heterozygotes and wild-type
muscles (data not shown). (Panel 2) Hip muscles from a P6 Maml1 KO littermate have marked variation in muscle fiber size and the
distribution of fibers is atypical. (Panel 3) Shoulder muscles of a P1 wild-type mouse show normal fiber size and distribution. (Panels
4,5) Advanced dystrophic phenotype in the shoulder muscles of a P1 Maml1 KO littermate showing many hypertrophic and atrophic
muscle fibers, and, notably, sarcoplasmic vacuolization. The KO muscles also exhibit a noticeable increase in connective tissue. (B)
Maml1 deficiency led to defects in MyoD-induced myogenic convesion in MEFs. Wild-type (right) and Maml1 KO (left) MEFs were
infected with MyoD and cultured under differentiation medium for 3 d. (C) Wild-type (WT) MEFs have higher levels of myosin
expression than the Maml1 KO MEFs. (− and +) Cells infected with retroviral vectors or viruses expressing MyoD. (D) Exogenous
expression of MAML1 in KO MEFs rescues MyoD-induced myotube formation. Stably transduced MyoD KO MEF cells were infected
with Flag-tagged MAML1 viruses or controls, and cells were induced for differentiation. (E) Expression of exogenous MAML1, MyoD,
and Myosin were determined by Western blot analysis.
Maml1 deficiency results in muscular dystrophy and defects in MyoD-induced myogenic conversion in MEFs that is
Shen et al.
678 GENES & DEVELOPMENT
MAML1 gene affected myoblast differentiation. C2C12
cells were infected with MAML1 viruses (pLXSN based)
or vector control viruses (see Fig. 3A for schematic of the
viral constructs used in these studies). After G418 selec-
tion, the expression of exogenous MAML1 was con-
firmed by Western blot analysis (Fig. 3B, lane 2 is full-
length [FL] MAML1). The cells were induced to differen-
tiate, and myotube formation and muscle-specific gene
expression were examined. As shown in Figure 3C (panel
2), ectopic MAML1 expression led to the formation of
strikingly large, robust myotubes, compared with cells
infected with control virus (panel 1). Consistent with
these data, MAML1-infected myotubes exhibited en-
hanced expression of muscle myosin within 2–6 d of dif-
ferentiation, while MyoD and myogenin levels appeared
unaffected (Fig. 3D, cf. lanes 2 and 1).
The results prompted us to ask whether MAML1 en-
hances myogenesis by regulating muscle gene expres-
sion. Thus, we examined the effect of MAML1 expres-
sion on the promoter activity of another muscle-specific
gene, muscle creatine kinase (MCK). C2C12 cells were
transfected with a MCK promoter reporter in the pres-
ence of increasing amounts of the MAML1 expression
vector. We found that MAML1 expression activated the
MCK promoter reporter in a dose-dependent fashion (Fig.
3E). These data suggest that MAML1 is involved in regu-
lation of myogenesis by enhancing muscle gene expres-
sion, including myosin and MCK.
To determine the specific domain of the MAML1 pro-
tein that is involved in the enhancement of myogenesis,
we also generated stably transduced C2C12 cells ex-
MAML1 mutants (Fig. 3B, lanes 3,4). Both mutants,
MAML1(124–1016) (construct 3, defective in Notch
binding) and MAML1(1–302) (construct 4, defective in
transcriptional activation), exert dominant-negative ef-
fects on Notch signaling (Wu et al. 2000). We found that
C2C12 cells expressing MAML1(124–1016) showed sub-
stantially reduced myotube formation (Fig. 3C, panel 3)
and myosin expression (Fig. 3D, lanes 3), while cells ex-
pressing MAML1(1–302) did not have apparent effects
(Fig. 3C [panel 4], D [lanes 4]). In addition, MAML1(124–
for this effect. (A) Schematic diagram of FL and truncated MAML1 constructs in the pLXSN viral vectors used in this study. (B)
Expression of exogenous MAML1 proteins in C2C12 cells. C2C12 cells were infected with pLXSN-based MAML1 viruses, and selected
with G418. Expression of MAML1 proteins was confirmed by blotting with an anti-HA antibody. (C) Overexpression of MAML1 in
C2C12 cells dramatically increases myotube formation and the N-terminal region of MAML1 is required for myogenic enhancement.
Stably infected C2C12 Cells were induced to differentiate, and photographed at day 6. (D) Overexpression of MAML1 enhances myosin
expression during myogenesis. Whole-cell lysates from infected C2C12 cells were prepared at days 0, 2, 4, and 6 after culturing in DM,
and expression levels of muscle proteins were analyzed by Western blot analyses. (E) Expression of MAML1 enhances the activation
of the promoter driving MCK. C2C12 cells were transfected with 10 ng of Renilla luciferase plasmid, 0.5 µg of pMCK-luc, and
increasing amounts of expression plasmids encoding Flag-tagged MAML1. After 24 h transfection, cells were cultured in DM overnight
and lysates were harvested. MCK reporter firefly luciferase activity, corrected for Renilla luciferase, is expressed as fold activation
relative to cells not expressing MAML1.
Exogenous MAML1 expression enhances myogenic differentiation of C2C12 cells, and its N-terminal region is required
MAML1 function in myogenesis
GENES & DEVELOPMENT 679
1016) appeared to inhibit MyoD and myogenin induction
(Fig. 3D, lanes 3). Consistent with these data, the two
truncated mutants did not significantly activate the
MCK promoter compared with FL MAML1 (Fig. 3E).
Taken together, these data indicate that MAML1 en-
hances myogenesis; specifically, it promotes myotube
formation and enhances muscle-specific gene expres-
sion, including myosin and MCK. Furthermore, the N-
terminal region of MAML1, a domain previously found
to interact with Notch, is essential for these effects.
RNA interference (RNAi)-mediated knockdown of
Maml1 expression inhibits muscle cell differentiation
To determine whether Maml1 is required for myoblast
differentiation, we tested several small interfering RNA
(siRNA) targeted to different regions of the mouse
Maml1 gene, and found that two independent siRNA
duplexes, V and VII, significantly knocked down endog-
enous MAML1 expression. C2C12 cells were transfected
with these two Maml1-targeted or control siRNA du-
plexes, or water-only controls, and cultured under con-
ditions to promote differentiation 48 h after transfection.
Lysates were harvested at different culture times and the
expression levels of MAML1 and muscle-specific genes
were determined by Western blot analyses.
As shown in Figure 4A, transfection with Maml1
RNAi V and RNAi VII resulted in >90% and 80% de-
crease, respectively, in endogenous MAML1 protein ex-
MAML1 expression decreased subsequent myosin ex-
pression and myotube formation compared with controls
when the cells were induced to differentiate (Fig. 4A,B).
Interestingly, this occurred despite the observation that
the expression of MAML1 protein appeared to return to
nearly normal levels within 2 and 4 d of culture. In ad-
dition, the induction of MyoD and myogenin expression
was delayed in the Maml1 RNAi-treated cells (Fig. 4A).
In fact, the level of MyoD expression was significantly
lower after Maml1 RNAi treatment, suggesting that
MAML1 may regulate MyoD expression. Overall, these
data demonstrated that MAML1 is required for the in-
duction of muscle-specific genes and the normal differ-
entiation of myoblasts into myotubes in the C2C12
MAML1 enhances MEF2-mediated transcription
We next investigated the molecular mechanisms by
which MAML1 functions in myogenesis. Since MAML1
functions as a transcriptional coactivator for the Notch
signaling pathway (Wu et al. 2000, 2004), we hypoth-
esized that MAML1 may also act as a coactivator for
muscle-specific transcription factors. Because expression
of exogenous MAML1 up-regulated the MCK promoter
reporter, which contains MyoD and MEF2-binding sites,
we asked whether MAML1 also coactivates MyoD or
whether MAML1 is a coactivator for MyoD, C2C12 cells
were cotransfected with increasing amounts of MAML1
and MyoD, and the activity of the MCK promoter re-
porter was measured. As shown in Figure 5A, we found
individual expression of exogenous MAML1 or MyoD
up-regulated the MCK promoter reporter, but coexpres-
sion of MAML1 and MyoD did not have any cooperative
effects. These data indicate that MAML1 is unable to
function as a coactivator for MyoD.
Next, we examined whether MAML1 coactivates
MEF2-mediated transcription. Here, C2C12 cells were
cotransfected with an artificial MEF2 reporter (contain-
knockdown results in decreased expression of muscle proteins in C2C12 cells. C2C12 were transfected with two Maml1-specific
siRNAs (V and VII), control siRNAs (Ctl), and water on two consecutive days, and cultured in DM at 48 h after the first transfection
for 0, 2, or 4 d. Lysates were harvested for Western blotting. Note that MAML1 expression (shown at top, the upper band) is
significantly (>80%–90%) decreased at day 0, but then returns to normal levels by day 2. However, myosin levels are significantly
decreased in the cells, while myogenin and MyoD levels appear reduced as well. (B) Phase microscopy reveals that C2C12 cells with
knocked down levels of MAML1 fail to form myotubes (panels 2,3), while cells transfected with control siRNAs or water (panels 1,4)
form large, mature myotubes within 4 d of culture.
RNAi-mediated knockdown of Maml1 expression inhibits myogenesis in C2C12 cells. (A) RNAi-mediated Maml1
Shen et al.
680 GENES & DEVELOPMENT
ing three copies of MEF2-responsive elements: 3xMEF2),
increasing amounts of MAML1 and/or MEF2 (including
MEF2A, MEF2C, or MEF2D) constructs. Indeed, we
found that MAML1 activated the MEF2 reporter in a
dose-dependent fashion (Fig. 5B), and also cooperated
with MEF2C in the reporter activation (Fig. 5C).
MAML1’s coregulatory function is apparently specific
for MEF2C, as we did not observe any cooperative effects
between MAML1 and other MEF2 members, MEF2A or
MEF2D (Supplementary Fig. S2). The specific, synergis-
tic effect between MEF2C and MAML1 on the MEF2-
responsive promoter raised the possibility that MAML1
and MEF2C interact.
MEF2C and MAML1 interact in vivo
To determine whether MAML1 and MEF2C indeed in-
teract, we first performed colocalization studies. We ex-
pressed constructs encoding GFP-tagged MEF2C and
Flag-tagged MAML1 in U20S osteosarcoma cells. We
found that GFP-tagged MEF2C is localized diffusely in
the nucleus when expressed ectopically and alone (Fig.
6A, top). However, when coexpressed with MAML1,
GFP-MEF2C redistributed into a nuclear dot pattern and
colocalized with MAML1 (Fig. 6A, bottom). Similar
staining patterns were found when C2C12 cells were
used (data not shown). These data are consistent with
MAML1 previously reported in nuclear dots (Wu et al.
2000), In contrast, we found that MAML1 does not co-
localize with another MEF2 member, MEF2A (Supple-
mentary Fig. S3). Therefore, our data support the idea
that MAML1 and MEF2C specifically interact.
We also performed Western blot assays on lysates from
C2C12 cells coexpressing MAML1 and MEF2C. Interest-
ingly, we found that their coexpression caused a mobil-
ity shift of MEF2C (Fig. 6B), likely because MAML1 in-
duced a post-translational modification of MEF2C. The
mobility shift was reduced by incubation of the MEF2C
immunoprecipitates with calf intestinal alkaline phos-
phatase (data not shown), suggesting that the modifica-
tion of MEF2C was due to phosphorylation.
To confirm the in vivo binding of MEF2C and MAML1
and map their respective binding sites, we performed
mammalian two-hybrid assays. FL or truncated MEF2C
constructs were expressed as fusion proteins with a
GAL4 DNA-binding domain (DB) (see Fig. 6C for sche-
matic of constructs), and FL MAML1 was expressed as a
fusion protein with the activation domain (AD) in
C2C12 cells. The interaction of MEF2C and MAML1
was quantified by the activation of a luciferase reporter
containing GAL4-binding sites in the promoter. Our re-
sults confirmed that MEF2C and MAML1 interact in
vivo; the luciferase activity from cells expressing both
proteins was increased ∼150-fold compared with cells ex-
pressing either protein plus empty vector. Furthermore,
the MEF2C mutant with the deletion of its MADS and
MEF domains still retained its ability to bind to
MAML1. In addition, the 1–177 amino acids of the N-
terminal region of MEF2C was sufficient to bind to
MAML1 (Fig. 6C). Thus, the binding site for MAML1 is
located within the 87–177-amino-acid region of the
Using similar assays with a series of deletion mutants
of MAML1 (Fig. 6D), we found that MEF2C failed to
interact with the MAML1 mutant missing its N-termi-
nal 123 amino acids (MAML1 124–1016), but was still
able to bind to the mutant missing the internal 71–300
amino acids. These data show that MEF2C interacts
within the 1–70-amino-acid region of MAML1. The im-
portant function of this MAML1 N-terminal region is
supported by our earlier studies, revealing that it is es-
sential for activating muscle-specific gene expression in
C2C12 cells (Fig. 3C–E). It should be noted that the in-
teraction between MAML1 and MEF2C might be rela-
tively weak and possibly indirect, as we were not able to
to activate a MEF2-dependent reporter. (A) MAML1
is not a coactivator for MyoD. C2C12 cells were
transfected with 10 ng of Renilla luciferase plasmid,
0.5µg ofpMCK-luc reporter,
amounts of expression plasmids encoding Flag-
tagged MAML1 and/or MyoD. After 24 h transfec-
tion, cells were switched to DM, cultured overnight,
and then lysed to prepare cell extracts for luciferase
assay. The pMCK-luc reporter activities were ex-
pressed as fold activation relative to cells not ex-
pressing MAML1 and MyoD. (B) MAML1 activates a
MEF2-responsive promoter in a dose-dependent
manner. Assays were performed as described above,
except the 3xMEF2-luc reporter is used. (C) MAML1
cooperates with MEF2C to activate MEF2-respon-
sive promoter. Assays were performed as described
above, except various amount of MEF2C expression
vectors were cotransfected with MAML1.
MAML1 and MEF2C act synergistically
MAML1 function in myogenesis
GENES & DEVELOPMENT681
detect a large amount of MEF2C in MAML1 immuno-
precipitates (data not shown). Nonetheless, our results
indicate a potentially important interaction between
MEF2C and MAML1, which is supported by changes in
the nuclear localization and post-translational modifica-
tion of MEF2C upon coexpression with MAML1, confir-
mation and mapping of the interaction sites by mamma-
lian two-hybrid assays, and their cooperative activation
of a MEF2-responsive promoter. Thus, MAML1 likely
coactivates MEF2-mediated gene transcription via its N-
terminal region, the same domain previously found to
interact with Notch (Wu et al. 2000).
Activation of Notch abrogates MAML1-enhanced
Next, we sought to investigate MAML1 function in the
context of Notch signaling during myogenesis. MAML1
is a known component of the active Notch transcrip-
tional complex, and is required for Notch-induced tran-
scriptional activation of downstream targets. It was
shown previously that activation of Notch signaling in-
hibits myogenesis in C2C12 cells (Kopan et al. 1994;
Lindsell et al. 1995). However, our data reveal that
MAML1 expression enhances myogenesis, while its ab-
the GFP-tagged MEF2C alone (top) or with Flag-tagged MAML1 (bottom), and stained with an anti-Flag antibody to detect MAML1
expression. (BG) Background staining. DAPI staining was performed to label the nuclei. Upon coexpression, MEF2C changed its
localization from a diffuse nuclear pattern to colocalize with MAML1 in nuclear dots (merged image at bottom). (B) Coexpression of
MAML1 and MEF2C results in post-translational modification of MEF2C. C2C12 cells were transfected with Flag-tagged MEF2C, with
or without cotransfection of Flag-tagged MAML1. The expression of both MAML1 and MEF2C were detected by Western blot analysis
with anti-Flag antibodies. Note that when the two proteins are coexpressed, the mobility of the MEF2C band shifts. (C) MAML1
interacts with the 87–177-amino-acid region of MEF2C by mammalian two-hybrid assays. C2C12 cells were cotransfected with a
firefly luciferase reporter containing GAL4-binding sites (pSG5-luc) along with two expression vectors; i.e., one encoding DB or DB
fused to FL or truncated MEF2C, and the other encoding AD or AD fused to MAML1. A total of 0.5 µg of each construct was used for
transfection. Cellular lysates were harvested 44 h post-transfection. Firefly luciferase activity, normalized with Renilla luciferase
expressed from the DB vector, was expressed as fold activation relative to the background level of firefly luciferase expression in the
presence of empty DB and AD vectors. (D) MEF2C interacts with the N-terminal region of the MAML1, 1–70 amino acids by
mammalian two-hybrid assays. Similar assays were carried out as described. FL MEF2C was expressed as DB fusion, while FL or
truncated MAML1 expressed as AD fusion.
MEF2C and MAML1 interact in vivo. (A) MEF2C and MAML1 colocalize in the nucleus. U20S cells were transfected with
Shen et al.
682 GENES & DEVELOPMENT
sence inhibits development, and that it functions as
a coactivator for MEF2C. Therefore, we designed ex-
periments to examine how MAML1 affects muscle dif-
ferentiation once the Notch signaling pathway is acti-
C2C12 cells stably transduced with MAML1, or with
empty vector, were incubated in culture wells coated
with IgG (as a negative control) or the Notch ligand,
Delta-Extra–IgG (an IgG fusion with the extracellular do-
main of Delta ligand), and allowed to differentiate for 6 d.
Delta-Extra–IgG, when immobilized on culture wells,
activates Notch signaling and blocks differentiation in
C2C12 myoblasts (Varnum-Finney et al. 2000). We found
that myoblasts overexpressing MAML1 and cultured in
control IgG-coated wells differentiated into large, robust
myotubes compared with myoblasts transduced with
vector alone (Fig. 7A, top). These data are consistent
with our previous findings that MAML1 enhances myo-
genesis (as in Fig. 3C). However, when cultured with
immobilized Delta ligand, both control and MAML1-
overexpressing myoblasts exhibited a dramatic, morpho-
logical block in differentiation (Fig. 7A, bottom). In
agreement with this observation, the expression of the
constitutively activated form of Notch1, ICN1, inhibited
the MAML1-mediated activation of the MCK promoter
in C2C12 cells in a dose-dependent manner (Fig. 7B).
Thus, activation of the Notch signaling pathway abro-
gated MAML1-enhanced myogenesis.
Activation of Notch signaling recruits MAML1 to the
Notch transcriptional complex and inhibits
Maml1-enhanced MEF2C-dependent transcription
To investigate the possible mechanisms by which Notch
activation overrides MAML1-enhanced myogenesis, we
aimed to assess the interactions of MEF2C, MAML1, and
Notch ICN1 in the C2C12 myoblasts. By transfecting
C2C12 cells with various combinations of these expres-
abrogated MAML1-mediated enhancement of myotube formation. C2C12 cells stably transduced with pLXSN based MAML1 viruses
or vector control viruses were plated on culture wells coated with either control IgG or ligand Delta-Extra–IgG, and cultured in DM
for 6 d. (B) Expression of a constitutively activated form of Notch1 (ICN1) inhibited MAML1-induced activation of MCK promoter.
MAML1-transduced C2C12 cells or controls were transfected with 0.5 µg of pMCK-luc, 10 ng of Renilla luciferase plasmid, and various
amounts of Notch ICN1, and luciferase assays were performed at 44–48 h after transfection. (C) Notch ICN1–MAML1–CSL complex
formed in C2C12 cells. C2C12 cells were cotransfected with different combinations of expression plasmids encoding Flag-tagged
MAML1, HA-tagged ICN1, Myc-tagged CSL, and Flag-tagged MEF2C as indicated. Whole-cell lysates (WCL) or anti-HA immunopre-
cipitates (IP) were blotted with anti-Flag or anti-HA, or anti-Myc antibodies. (D) Expression of ICN1 interfered with the coregulatory
function of Maml1 on MEF2C-mediated transcription. C2C12 cells were transfected with 0.5 µg of a firefly luciferase reporter
containing GAL4-binding sites (pSG5-luc), 0.5 µg of the plasmid encoding DB fused to MEF2C, and 0.5 µg of expression construct
expressing Flag-tagged MAML1 in the presence of various amounts of HA-tagged ICN1 expression constructs (0, 0.25, and 0.5 µg).
pSG5-luc firefly luciferase activity, corrected for Renilla luciferase activity, is expressed as fold activation relative to cells not
expressing MAML1 and ICN1.
Activation of the Notch signaling pathway overrides MAML1-enhanced myogenesis. (A) Notch ligand stimulation
MAML1 function in myogenesis
GENES & DEVELOPMENT683
sion plasmids, we detected MAML1 and the Notch-spe-
cific transcription factor, CSL, in Notch ICN1 immuno-
precipitates (Fig. 7C, lanes 3,4), indicating the formation
of the MAML1–ICN1–CSL complex in the C2C12 myo-
blasts; this complex previously was determined to be
essential for the transcription of Notch target genes (Wu
et al. 2000). Previously, it was reported that the Notch
ICN1 interacts with MEF2C, leading to the inhibition of
MEF2C DNA-binding activity (Wilson-Rawls et al.
1999), suggesting that this is one of the CSL-independent
mechanisms for Notch-induced inhibition of myogen-
esis. However, we could not detect MEF2C in the ICN1
immunoprecipates under the conditions we used (Fig.
7C, lane 2), indicating that the Notch ICN1–MAML1–
CSL complex may be more stable than the ICN1–MEF2C
complex. These data suggest that CSL-dependent mecha-
nisms—i.e., transcription of Notch target genes—may be
one of the major mechanisms for Notch effects. Interest-
ingly, we found that expression of the combination of
MAML1 and ICN1, but not individually, decreased ex-
ogenous MEF2C expression (Fig. 7C, cf. lanes 5,6,8), sug-
gesting that MEF2C expression might be regulated by
both MAML1 and ICN1. Overall, our data suggest that
MAML1 might predominantly function as a coactivator
for Notch receptors instead of for MEF2C when Notch
signaling is activated. Indeed, we found that expression
of ICN1 inhibits MAML1-enhanced MEF2C-mediated
transcription in a dose-dependent fashion in luciferase
assays, indicating that MAML1’s coactivator function
for MEF2C is greatly reduced in the context of Notch
activation (Fig. 7D).
Our studies thus reveal that once the Notch pathway
is activated, MAML1 is recruited from its role as a co-
activator for MEF2-mediated transcription to that of a
Notch transcriptional coactivator. The consequence of
this role switching of MAML1 is expected to result in an
expression of Notch targets and an inhibition of myo-
genesis once the Notch signaling pathway is activated.
Transcriptional coactivators are proteins that, along
with transcription factors, exist in multiprotein com-
plexes and mediate transcriptional events. However, co-
activators are not solely components of the basic tran-
scriptional machinery, since recent studies implicate
them as primary targets of developmental and physi-
ological signals in many diverse biological processes
(Spiegelman and Heinrich 2004). Our present study re-
veals a novel role for MAML1, a known coactivator for
Notch. Mice with a targeted deletion of the Maml1 gene
were found to have dystrophic skeletal muscles, and em-
bryonic fibroblasts from these mice failed to undergo
muscle differentiation in vitro. Interestingly, exogenous
MAML1 expression in C2C12 cells promoted signifi-
cantly enhanced myotube formation and muscle gene
MAML1 abrogated muscle differentiation, suggesting
that at least some of the effects of MAML1 on muscle
development could be Notch independent. In support of
this notion, MAML1 was found to bind to MEF2C and to
function as a potent coactivator. However, activated
Notch blocked the myogenic effects of MAML1 and
MEF2C, possibly by recruiting MAML1 into the Notch
transcriptional complex. Thus, MAML1 is essential for
proper muscle development and may function both to
promote and inhibit muscle differentiation, depending
Maml1 is essential for myogenic differentiation in
vivo and in vitro
Maml1 is one of three mammalian homologs of Dro-
sophila mastermind, a gene that was genetically linked
to the Notch signaling pathway in flies (Xu et al. 1990).
Previously, biochemical studies determined that all
three mammalian MAML proteins exhibited differential
transcriptional coactivities for different Notch receptors
(Wu et al. 2002), and some MAML family members have
been implicated in cancer (Tonon et al. 2003). In addi-
tion, a dominant-negative mutant of MAML1 interfered
with Notch-mediated T/B-cell fate decisions in mouse
bone marrow (Maillard et al. 2004), indicating an impor-
tant role for MAML1 in mediating Notch signaling.
However, the exact in vivo roles of the MAML family of
transcriptional coactivators have not been established.
Here, we generated a Maml1 KO mouse and found that
an absence of Maml1 results in death within the perina-
tal period. The exact cause(s) of death are currently un-
known and being investigated. Strikingly, the Maml1-
null mice exhibit severe skeletal muscle defects typical
of a muscular dystrophy. While defective Notch signal-
ing could contribute to abnormal muscle development,
we found that MEFs from Maml1-null mice failed to
undergo MyoD-induced myogenic differentiation in
vitro, suggesting that there were intrinsic defects in
muscle differentiation. Indeed, our data using C2C12
cells further indicate that the muscular dystrophy in the
Maml1-null mice is most likely a result of a severe per-
turbation in myogenesis, with a disruption in key inter-
actions between MAML1–MEF2C- and MAML1–Notch-
mediated pathways. However, it must be noted that, al-
though the Maml1-null muscles are severely perturbed
in their structure, they are “functional” enough to allow
the mice to live beyond the embryonic stage. This may
be due to expression of the two other members of the
Maml family, Maml2 and Maml3, also found expressed
in human skeletal muscles (Wu et al. 2002), or a result of
compensatory effects from these genes. Future studies
on the expression and functional properties of these two
members will help in understanding the muscle defects
in the KO mice, and the potential roles of other MAML
family members in muscle development.
MAML1 coactivates MEF2C to promote the
transcription of muscle-specific genes
MEF2C is one of four MEF2 gene family members in
mammals encoding key transcription factors that medi-
Shen et al.
684 GENES & DEVELOPMENT
ate gene expression in myocytes (Black and Olson 1998).
Mice lacking Mef2c die at E9.5 due to defects in heart
looping (Lin et al. 1997), and MEF2C is also known to
regulate skeletal muscle-specific genes including MCK,
desmin, and myogenin. One recent study showed that
the loss of SrpK3 kinase, one of the MEF2C downstream
targets, resulted in centronuclear myopathy in mice (Na-
kagawa et al. 2005). In addition, MEF2C is able to inter-
act with MRFs to potentiate their transcriptional activi-
Previously, it was reported that a number of coactiva-
tors including GRIP1 (Chen et al. 2000), CARM1 (Chen
et al. 2002), PGC1 (Michael et al. 2001), and p300 (Sar-
torelli et al. 1997) enhance MEF2C-dependent transcrip-
tion. In this regard, diverse signaling pathways regulate
MEF2C activities by interfering with its interactions
with coactivators, some of which are known to perturb
the myogenic program. For instance, cyclin D–cdc4 ki-
nase prevents the activation of MEF2C by blocking its
interaction with the coactivator GRIP1, thereby sup-
pressing the muscle differentiation program in prolifer-
ating myoblasts (Lazaro et al. 2002). Transforming
growth factor ? (TGF-?) suppresses myogenic differen-
tiation via its effector, Smad3, which is in part capable of
disrupting MEF2C and GRIP1 association (Liu et al.
2004). This evidence suggests a critical, functional sig-
nificance in MEF2C-mediated transcription in myogen-
esis. Indeed, here we identified another mechanism by
which MEF2C is regulated in myogenesis, via its coac-
The respective interaction domains of MAML1 and
MEF2C were mapped to the N-terminal domain of the
MAML1 (which also interacts with Notch receptors),
and the region just adjacent to the MADS-MEF2 domains
of MEF2C (a binding site that differs from the reported
GRIP1-binding site) by mammalian two-hybrid assays. It
should be noted that the interaction of MAML1 and
MEF2C might be relatively weak based on our coimmu-
noprecipitation studies, and it is unclear whether such
interaction is direct in vivo. Nevertheless, MAML1 and
MEF2C interaction sufficiently activates MEF2-medi-
ated promoters. We found that MAML1 is a potent co-
activator of MEF2C, especially in comparison with one
of the MEF2C’s previously described coactivators,
GRIP1 (data not shown). Combining our data with other
studies, we can propose at least two mechanisms by
which MAML1 is able to coactivate MEF2C transcrip-
tional events. First, we found that in our overexpression
studies, the MAML1–MEF2C interaction resulted in
post-translational modifications of MEF2C, likely phos-
phorylation, which could contribute to MAML1-medi-
ated potentiation of MEF2C transcription. Consistently
with this observation, MAP kinases, both p38 and extra-
cellular signal-regulated kinase (ERK), enhance the tran-
scriptional activities of MEF2C via phosphorylation
(Yang et al. 1999). Since the endogenous levels of
MAML1 in the cells are low, it remains unclear regard-
ing the amount of physiological MEF2C that is phos-
phorylated by endogenous MAML1 and the functional
impact of such modification. Thus, further studies in-
vestigating potential phosphorylation sites, the respon-
sible kinase(s), and whether phosphorylation regulates
the DNA-binding and/or transcriptional activities of
MEF2C, are warranted. Second, based on mammalian
two-hybrid assays, MAML1 and MEF2C physically in-
teract, bringing additional transcriptional regulators to
MEF2C through MAML1, including p300 (Fryer et al.
2002) and likely other currently unknown proteins.
Finally, an important future question is to understand
exactly how the MEF2C and MAML1 interaction regu-
lates the myogenic program, and the exact target genes
that are controlled by the complex. In this regard, we
found that RNAi-mediated down-regulation of MAML1
expression, as well as the expression of a N-terminal
truncated mutant of MAML1 in C2C12 myoblasts, both
resulted in a delayed induction of the key myogenic
regulators, MyoD, and myogenin. Since a key proximal
MEF2 site in the mouse myogenin promoter regulates
myogenin expression levels (Cheng et al. 1993), it is
likely that the loss of MAML1/MEF2C interaction ac-
counts for the delayed induction of myogenin in Maml1-
siRNA-treated cells. The absence of the MAML1–
MEF2C interaction in Maml1-null mice may lead to de-
creased myogenin expression, contributing to muscle
Maml1 is involved in cross-stalk between Notch
signaling and MEF2C signaling
We initially speculated that MAML1, as an essential co-
activator for Notch signaling, might contribute to the
Notch-mediated inhibition of myogenesis. Surprisingly,
however, we discovered a novel promyogenic differentia-
tion function for the Maml1 gene. These seeming para-
doxical results prompted us to examine how MAML1
affects muscle differentiation in the context of Notch
activation. In fact, upon Notch activation, the MAML1-
mediated enhancement of myogenesis was abrogated.
Therefore, it appears that MAML1’s role of a coactivator
for the Notch signaling pathway dominates over its role
in coactivating MEF2C-mediated muscle gene transcrip-
tion. Mechanistically, Notch activation led to a recruit-
ment of MAML1 away from MEF2C to the Notch/CSL
Taken together, our data allow us to propose a model
for the role of MAML1 in myogenesis that may at least
partially explain the Notch-induced inhibition of myo-
genesis. (1) In the absence of the activated Notch path-
way (i.e., during events that occur after myoblast prolif-
eration and recruitment), MAML1 serves as a transcrip-
tional coactivator for the muscle-specific transcription
factor MEF2C. Together, they regulate muscle gene ex-
pression, leading to enhanced myogenic events. (2) Upon
activation of Notch receptors (e.g., induction of Notch
ligand delta after muscle injury) MAML1 switches its
role from a transcriptional coactivator of MEF2C to that
of a transcriptional coactivator for Notch–CSL, resulting
in the expression of specific Notch target genes. Thus,
MAML1 plays dual roles as transcriptional coactivators
for muscle-specific genes as well as for target genes for
MAML1 function in myogenesis
GENES & DEVELOPMENT685
the Notch pathway. The “functional switching” of
MAML1 in response to signals to activate Notch recep-
tors likely contributes to the CSL-dependent inhibition
Therefore, with our current knowledge about the regu-
lated role of Notch in different stages of myogenesis, the
loss of MAML1 expression in the Maml1 KO mice may
have both Notch-dependent and Notch-independent
myogenic defects that contribute to muscular dystrophy.
For example, the loss of the MAML1–Notch interaction
may result in decreased Notch signaling that is required
for proliferation and expansion of muscle stem cells, and
thus affect muscle regeneration. Currently, the extent of
Notch signaling affected in the mutant muscles is still
unknown, because Notch targets in the muscles are not
well defined. However, we found that the expression lev-
els of a Notch target gene, HES1, seemed to be slightly
decreased in the postnatal day 1 (P1) hind-limb muscles
of the maml1 KO mice as compared with the wild-type
mice (Supplementary Fig. S4). On the other hand, the
loss of MAML1–MEF2C might lead to impaired myo-
genic differentiation. Future research is required to as-
sess the functional contributions of these two potential
aspects of MAML1 functions in understanding the mus-
cular dystrophy in Maml1 KO mice.
Finally, aspects of the model proposed in this study
may contribute to understanding how the Notch signal-
ing pathway regulates multiple biological processes,
with very different cellular outcomes. Our previous
studies suggest that the MAML family, with its differ-
ential expression patterns and distinct interactions with
Notch receptors, could mediate Notch pathway effects
via coactivation of transcription. The present study sug-
gests the exciting possibility that the MAML family
serves as coactivators for other transcription factors and
mediates cross-talks between Notch and different bio-
logical signaling pathways.
Materials and methods
Generation of Maml1 KO mice
The gene structure of murine Maml1 has been previously de-
scribed (Wu et al. 2004). Screening of a BAC Mouse II Hybrid-
ization library was performed (Incyte Genomics, Inc.) using a
324-bp probe derived from the 5? end of the murine Maml1
cDNA (encoding 1–97 amino acids). One positive clone con-
tained the entire genomic sequence for the Maml1 gene. A
Maml1 targeting vector was subsequently made with the Neo
gene replacing the 5? promoter region and exon 1. The vector
was linearized with NotI and electroporated into J129 mouse
embryonic stem (ES) cells. ES cell clones that survived repeated
selection were chosen for Southern blot analyses. The genomic
probe used for Southern blotting was external to the genomic
DNA used in targeting vector, and XmnI digest yielded a 11.7-
kb band in the wild-type allele and a 23.3-kb band in the tar-
geted allele. Two of the ES clones containing the correctly tar-
geted allele were individually injected into C57BL/6 blasto-
cysts, one of which gave rise to the chimeric mice that transmit
the targeted allele to the germline. PCR genotyping of mice was
performed with the primers for the Neo gene (Top, 5?-ATT
GAACAAGATGGATTGCAC-3?, and bottom, 5?-TCTTCGTC
CAGATCATCCT-3?) to detect the targeted allele, and with the
primers for the deleted region of the Maml1 (top, 5?-GC
CACTCCCGCCACCAAAAAC-3?, and bottom, 5?-TTTCC
GACCTCATTCTTTACA-3?) to detect the wild-type allele.
For routine histological analysis, embryos or tissue samples
were fixed in Bouin’s solution and embedded in paraffin for
sectioning. Tissue sections were stained with H&E. These pro-
cedures were performed at the Rodent Histopathology Core at
Harvard Medical School.
Establishment of wild-type and Maml1 KO embryonic
fibroblast cell lines, and myogenesis assay
Primary wild-type and Maml1 KO MEFs were isolated from
E12.5 littermate embryos and cultured in DMEM plus 10% fetal
bovine serum (FBS). Early passage (less than five) MEFs were
plated at a density of 2.7 × 106cells per 10-cm dish, and replated
at the same density every 3 d (Todaro and Green 1963). Cells
were immortalized after 20–30 passages. For myogenic conver-
sion assays, MEFs were infected with pMSCV-based MyoD and/
or pLXSN-based MAML1 viruses, and selected in growth me-
dium (DMEM plus 20% FBS) with 2 µg/mL of puromycin or 250
µg/mL of G418. Cells were grown to 80%–90% confluence and
switched to differentiation medium (DMEM containing 2%
horse serum and 10 µg/mL insulin) for 3 d.
Cell culture, retroviral transduction, transfection,
and differentiation assay
C2C12 cells (ATCC) were cultured in DMEM with 20% inac-
tivated fetal calf serum (FCS). U2OS cells were cultured in
DMEM with 10% FCS. Cells were infected with pLXSN-based
retroviruses, and selected with G418 at 250 µg/mL. Transfec-
tions with Maml1 siRNA (to a final concentration of 100 nM)
were carried out in the six-well plates using Lipofectamine Plus
Reagent (Invitrogen) for two consecutive days. Two Maml1-spe-
cific siGenome duplexes V (D-059179-05) and VII (D-059179-
07), and control nontargeting siRNA pool were ordered from
Dharmarcon. For the myogenesis assay, C2C12 cells were
grown to 80%–90% confluence, and induced for differentiation
by switching from growth medium to differentiation medium
(DM: DMEM containing 2% horse serum).
Northern and Western blot analyses
Total RNA isolated by TRIZOL reagent (Invitrogen) was sub-
jected to Northern blot analysis as described previously (Wu et
al. 2000). Whole-cell protein extracts were prepared for Western
blot and immunoprecipitation studies as described previously
(Wu et al. 2000). The following antibodies were used: the late
muscle marker, myosin heavy chain (Clone MY32), Flag (M2),
Myc (9E10), and ?-actin from Sigma; Myogenin and MyoD
(Clone 5.8A) from PharMingen; HA from BabCo; and anti-HA
affinity matrix from Roche. Rabbit Maml1 antibodies (BL1237
and BL1239) were provided by Bethyl Laboratories, Inc.
FL MAML1 and two truncated mutants, MAML1(1–302) and
MAML1(124–1016), were cloned into the pLXSN vector (Clon-
tech). MAML1(303–1016) and MAML1(?71–300) were cloned
into the pFlag-CMV2 vectors (Sigma). MEF2C was recloned
from pCMX-MEF2C-Flag plasmid (Lazaro et al. 2002) to the
Shen et al.
686 GENES & DEVELOPMENT
pEGFP C3 vector (Clontech) to express GFP-MEF2C fusion. FL
MEF2C, ?MADS containing MEF2C 61–466 amino acids,
?MADS?MEF containing MEF2C 87–466 amino acids, and
177N containing MEF2C 1–177 amino acids were cloned into
pBIND vector (Promega). 3xMEF2-luc containing three copies of
MEF2-responsive sites in pGL3 vector and MEF2A, MEF2B, and
MEF2C in pcDNA vector were obtained from Eric Olson (Uni-
versity of Texas Southwestern Medical Center at Dallas, Dallas,
TX); MyoD in pMSCV puro vector were from Robert Roeder
(The Rockefeller University, New York, NY); HA-tagged
HA-p300 in pCMV were from David Livingston (Dana-Farber
Cancer Institute, Boston, MA); HA-tagged GRIP1 in pSG5
vector were from Michael Stallcup (University of Southern Cali-
fornia, Los Angeles, CA); and pMCK-luc were from Andrew
Lassar (Harvard Medical School, Boston, MA). FL MAML1,
MAML1(1–302), and MAML1(124–1016) in pFlag-CMV2; Myc-
tagged CSL and HA-tagged ICN1 in pcDNA vector; and pSG5-
luc (Promega) have all been described previously (Wu et al.
C2C12 cells were cultured at 1 × 105cells/well in six-well
plates and transfected the following day with cDNA constructs
as indicated in the figure legends using Superfect Transfection
reagent (Qiagen). Luciferase-based reporter assays were per-
formed as described previously (Wu et al. 2000).
Staining was performed as described previously (Wu et al. 2002).
We thank Irwin Bernstein, Andrew Lassar, David Livingston,
Eric Olson, Robert Roeder, and Michael Stallcup for the re-
agents; Carol Gregorio and Parker Antin for critical reading of
our manuscript; David Besselsen for tissue section analysis; and
Makoto Nakamura for technical help. This work was supported
in part by NIH RO1 CA036167 to J.D.G., NIH RO1 CA097148
to L.W., and an American Heart Association grant 0435316N to
A.S.M. H.S. (ZOC) is also supported by Natural Science Foun-
dation of China (30270530 and 30470677) and Guangdong Natu-
ral Science Foundation (021825).
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