codependent activators direct myoblast-specific myod ... · developmental cell article codependent...
Post on 05-Jun-2020
5 Views
Preview:
TRANSCRIPT
Developmental Cell
Article
Codependent Activators DirectMyoblast-Specific MyoD TranscriptionPing Hu,1 Kenneth G. Geles,1,6 Ji-Hye Paik,2,3 Ronald A. DePinho,2,3,4,5 and Robert Tjian1,*1Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA2Department of Medical Oncology, Dana-Farber Cancer Institute3Department of Medicine4Department of Genetics5Center for Applied Cancer Science, Belfer Foundation Institute for Innovative Cancer Science, Dana-Farber Cancer Institute
Harvard Medical School, Boston, MA 02115, USA6Present address: Wyeth Research, Discovery Oncology, 401 N. Middletown Rd., Pearl River, NY 10965, USA*Correspondence: jmlim@berkeley.edu
DOI 10.1016/j.devcel.2008.08.018
SUMMARY
Although FoxO and Pax proteins represent two impor-tant families of transcription factors in determiningcell fate, they had not been functionally or physicallylinked together in mediating regulation of a commontarget gene during normal cellular transcription pro-grams. Here, we identify MyoD, a key regulator ofmyogenesis, as a direct target of FoxO3 and Pax3/7in myoblasts. Our cell-based assays and in vitro stud-ies reveal a tight codependent partnership betweenFoxO3 and Pax3/7 to coordinately recruit RNA poly-merase II and form a preinitiation complex (PIC) toactivate MyoD transcription in myoblasts. The role ofFoxO3 in regulating muscle differentiation is con-firmed in vivo by observed defects in muscle regener-ation caused by MyoD downregulation in FoxO3 nullmice. These data establish a mutual interdependenceand functional link between two families of transcrip-tion activators serving as potential signaling sensorsand regulators of cell fate commitment in directingtissue specific MyoD transcription.
INTRODUCTION
Embryonic differentiation, postnatal maintenance, and regener-
ation of skeletal muscle in vertebrates are governed by a complex
transcriptional regulatory circuit (Buckingham et al., 2006;
Charge and Rudnicki, 2004). A key player in myogenesis is the
transcription factor MyoD, which is sufficient to transdifferentiate
many types of cells to muscle cells (Choi et al., 1990; Morosetti
et al., 2006; Weintraub et al., 1989, 1991), and is sometimes
referred to as a ‘‘master regulator’’ of skeletal muscle differenti-
ation (Berkes and Tapscott, 2005; Tapscott, 2005). A key chal-
lenge has been to determine what regulates this ‘‘master regula-
tor’’ at the transcriptional level. Distal DNA elements have been
shown to regulate myod transcription during embryonic muscle
differentiation by genetic studies (Asakura et al., 1995; Chen
et al., 2002; Goldhamer et al., 1995; Kucharczuk et al., 1999).
However, the molecular mechanism and key transcription fac-
534 Developmental Cell 15, 534–546, October 14, 2008 ª2008 Else
tors directing the activity of these enhancers remains largely
unknown. Although many transcription factors including SRF,
Sp1, YY1, and p300/CBP have been implicated in affecting
myod transcription (Gauthier-Rouviere et al., 1996; L’Honore
et al., 2003; Roth et al., 2003; Wilson and Rotwein, 2006), none
of these are thought to determine the cell type-specific expres-
sion of MyoD.
FoxOs belong to the large forkhead family of transcription
factors that function to integrate growth signals into diverse tran-
scriptional networks governing a wide range of physiological
processes including proliferation, differentiation, survival, and
metabolism (Accili and Arden, 2004; Carter and Brunet, 2007).
Like all forkhead factors, FoxOs share a conserved DNA binding
domain responsible for recognizing consensus forkhead regula-
tory elements (FRE). FoxOs are also thought to interact with
various activator/repressor partners to regulate their activities
(Gomis et al., 2006). Invertebrates typically encode only one
FoxO, while mammals employ multiple FoxO paralogs: FoxO1,
FoxO3, FoxO4, and FoxO6 (Jacobs et al., 2003; Lam et al.,
2006; van der Heide et al., 2005). These FoxOs all recognize
and bind similar DNA sequences (Furuyama et al., 2000) and
are thus subject to functional redundancy under certain circum-
stances (Paik et al., 2007; Tothova et al., 2007). However, null
mutations in FoxO1, 3, and 4 produce distinct phenotypes in
mice (Castrillon et al., 2003; Hosaka et al., 2004; Jonsson
et al., 2005; Lin et al., 2004), suggesting specific functions of
FoxOs during development. Even for FoxOs involved in the
same cellular process, there seems to be selective utilization of
individual factors under physiological conditions (Paik et al.,
2007). Thus, diversified functions for individual FoxOs and their
mechanisms of specificity remain challenging but important
questions to address. Although some FoxOs have been impli-
cated in muscle differentiation and maintenance (Bois and Gros-
veld, 2003; Hribal et al., 2003; Kitamura et al., 2007; Li et al.,
2007; Liu et al., 2005; Machida et al., 2003; Mammucari et al.,
2007; Sandri et al., 2004), whether this process is regulated by
one particular FoxO or several in combination remains unclear.
Another class of transcription factors, the Pax proteins (paired
and homeodomain containing), is essential for regulating embry-
onic organogenesis and differentiation in metazoans (Lang et al.,
2007). The closely related Pax3 and Pax7 are specifically ex-
pressed in the central nervous system as well as skeletal muscle,
vier Inc.
Developmental Cell
Activators Direct MyoD Transcription
and share some overlapping functions (Buckingham and Relaix,
2007). During embryonic myogenesis, Pax3 and 7 are expressed
exclusively in cells destined to become skeletal muscle (Kassar-
Duchossoy et al., 2005) and have important roles in regulating
expression of myogenic transcription factors (Bajard et al.,
2006; McKinnell et al., 2008). Genetic studies suggest that
Pax3 and Pax7 are potential upstream regulators of myod during
both embryonic and postnatal myogenesis (Bober et al., 1994;
Goulding et al., 1994; Maroto et al., 1997; Relaix et al., 2005;
Seale et al., 2000). However, the direct activation of myod
Figure 1. MyoD Is a Potential FoxO3 Target
in Myoblasts
(A) Schematic representation of FoxO1, 3, and 4.
The conserved forkhead DNA-binding domain is
indicated in yellow. The red bars illustrate the po-
sitions of the shRNA targets. The numbers below
the sequence indicate percentage of identities be-
tween FoxO1, 3, and 4 in CLUSTALW.
(B) Whole-cell lysates of C2C12 cells treated with
FoxO1, 3, or 4 shRNA were analyzed by immuno-
blot with antibodies specific to individual FoxOs.
Immunoblot against GAPDH serves as the loading
control. The percentages of mRNA and proteins
knocked down are indicated at right.
(C) The examples of genes suppressed by FoxO
RNAi. Logarithm of fold reduction in shRNA
treated cells is illustrated in the x axis.
transcription by Pax3 and Pax7 has not
been demonstrated. The importance
of a possible link between FoxOs and
Pax3/7 is underscored by the finding
of a naturally occurring chromosomal
translocation between FoxO1 and Pax3/7
that results in a Pax3/7-FKHR fusion pro-
tein in human alveolar rhabdomyosarco-
mas (Mercado and Barr, 2007). However,
neither a physical nor functional link
between FoxOs and Pax3/Pax7 in non-
transformed cells has been demon-
strated.
Employing a combination of in vitro
biochemistry, molecular genetic loss/
gain of function, and in vivo muscle re-
generation experiments, we have tested
the hypothesis that specific FoxO factors
play a direct role in regulating myogene-
sis. We also identify tissue-specific Pax
activator partners that work in conjunc-
tion with FoxO to serve as key regulators
required to synergistically activate myod
transcription in myoblasts. The identifica-
tion of FoxO3 as a myod transcription ac-
tivator reveals a potential link between
signaling cascades and transcription reg-
ulation of myod in adult muscles. This
connection between FoxO and Pax tran-
scription factors provides muscle cells
with a powerful mechanism to utilize
codependent activators expressed in multiple tissues to direct
cell type-specific transcription.
RESULTS
Expression of MyoD Is Regulated by FoxO3In C2C12 myoblasts, the presence of three FoxO members FoxO1,
FoxO3, and FoxO4 can be detected by RT-PCR and immunoblots.
All three FoxOs share the conserved Forkhead DNA binding do-
main, but each bears different transactivation domains (Figure 1A).
Developmental Cell 15, 534–546, October 14, 2008 ª2008 Elsevier Inc. 535
Developmental Cell
Activators Direct MyoD Transcription
Small hairpin RNA (shRNA) against each specific foxo gene (Fig-
ure 1A, red bars) was incorporated into C2C12 cells to generate
three distinct stable RNA interference (RNAi) lines. Each targeted
FoxO protein was efficiently depleted as determined by immuno-
blots and qRT-PCR (Figure 1B), while expression levels of control
genes (GAPDH) and other nontargeted FoxOs remained largely
unchanged afterFoxOshRNAtreatment (Figure 1B; seeFigureS1
available online).
In order to identify putative target genes for each FoxO protein
in an unbiased manner, we compared the gene expression pro-
files of control RNAi and FoxO1, 3, or 4 RNAi lines by microarray
analysis. RNA prepared from each of the FoxO RNAi lines or
scrambled control RNAi cells were utilized as probe sets for
hybridization. Potential target genes activated by FoxOs, with
more than 2-fold decrease in expression levels were analyzed
(GSE12582). In addition to genes activated by multiple FoxOs,
potential target genes selectively activated by individual FoxOs
were also found. Representative FoxO activated genes are
shown in Figure 1C, and repressed genes are shown in Table
S1. Among these putative target genes, we confirmed previously
identified mammalian FoxO targets such as p21 and insulin
receptor substrate (Bois and Grosveld, 2003; Puig et al., 2003;
Seoane et al., 2004), mammalian homologs of known Drosophila
FoxO targets, such as 4EBP (Marr et al., 2007; Puig et al., 2003),
and putative FoxO targets identified from a computational
analysis of FREs, such as SCAND1 (Xuan and Zhang, 2005). In-
terestingly, a series of genes involved in myogenic differentiation
including myod, myogenin, tropomyosin, and creatine kinase
were effectively downregulated by FoxO RNAi knockdown,
suggesting that FoxOs may play important, but previously unap-
preciated direct roles in muscle differentiation. An important
early marker in myogenic differentiation, MyoD, was specifically
downregulated by FoxO3 depletion (7.9-fold), but not by FoxO1
or FoxO4 RNAi. Among these myogenic regulatory genes, MyoD
is thought to be one of the most upstream regulators in the differ-
entiation cascade, raising the intriguing possibility that this early
marker is a bona fide direct target of FoxO3.
FoxO3 Binds the myod Promoter and ActivatesIts TranscriptionFoxOs are known to be phosphorylated by various kinases and
subsequently translocated into the cytoplasm (Arden, 2006;
Huang andTindall, 2007; Lam etal., 2006). We thereforeexamined
the subcellular localization of FoxO3 in C2C12 myoblasts. Both
immunofluorescent staining of cells and immunoblots of cell ex-
tracts showed predominantly nuclear localization of FoxO3 in
myoblasts (Figure S2). These results suggest that FoxO3 is likely
to be in its active form and regulating transcription of target genes
in vivo.
To confirm the microarray analysis results, we performed
quantitative RT-PCR (qRT-PCR) to measure the relative MyoD
mRNA abundance using U6 small RNA as an internal control in
C2C12 cells treated with either scrambled control or specific
shRNA against each of the three foxo genes. MyoD mRNA levels
were greatly reduced (�85%) in FoxO3 shRNA-treated cells, but
not in scrambled shRNA or FoxO1 shRNA-treated cells, and only
modestly reduced (�30%) in FoxO4 shRNA-treated cells
(Figure 2B). When a CMV-based rescue system was used to ex-
press the coding sequence of a FoxO3 construct that is resistant
536 Developmental Cell 15, 534–546, October 14, 2008 ª2008 Elsev
to shRNA targeted to the 30UTR, nearly normal MyoD mRNA
levels were restored. In contrast, introducing empty vector
or FoxO1 rescue construct into RNAi cells depleted of FoxO3
failed to restore expression of MyoD. Introducing a FoxO4 res-
cue construct to FoxO3 RNAi cells only partially restored
MyoD expression (Figure 2B). These results indicate that the
loss of FoxO3 leads to strong downregulation of MyoD and
that re-expression of FoxO3 and to a lesser extent FoxO4 can
rescue MyoD expression.
We next tested whether overexpression of functional FoxO3
can activate MyoD expression. In order to increase active
FoxO3 concentration in nuclei, we utilized the constitutively ac-
tive FoxO3A3 mutant wherein three Ser/Thr residues have
been changed to Ala thus circumventing the phosphorylation-
dependent cytoplasmic sequestration. When transiently trans-
fected into C2C12 cells, the FoxO3A3 constitutive mutant
strongly stimulated MyoD expression (25-fold), while empty
vector and FoxO1A3 showed no stimulation of MyoD expression
as expected. The FoxO4A3 mutant displayed weak upregulation
of myod transcription (Figure 2A). These gain-of-function studies
taken together with our loss-of-function assays strongly point to
FoxO3 as a primary potent activator of MyoD expression, while
FoxO4 functions partly overlap FoxO3 and modestly activates
myod transcription.
Consistent with this notion, sequence analysis revealed four
potential FRE sites in a 6 kb region upstream of the myod tran-
scription initiation site (Figure 2B), which are between the DRR
and PRR elements important for MyoD regulation during embry-
onic muscle development (Asakura et al., 1995). We next per-
formed ChIP to determine promoter occupancy of FoxOs at
the myod gene using antibodies specifically directed against
each of the three proteins. FoxO3 was efficiently detected at
the myod promoter regions overlapping putative FRE �940
and �1598, but not at �1928 and �2351 (Figure 2B). In marked
contrast to FoxO3, neither FoxO1 nor FoxO4 was detected sig-
nificantly above background at the four potential FREs in C2C12
cells (Figure 2B). As a control, the same FoxO1 and FoxO4 anti-
bodies were used to successfully detect their occupancy at the
p21 promoter (Figure S3A), which is known to be regulated by
these FoxOs (Figure 1C; Seoane et al., 2004). Indeed, it appears
that FoxO3 selectively binds two of the putative FREs at the
myod promoter in myoblasts, suggesting that FoxO3 binding
may correspond to one of the important steps regulating MyoD
expression. RNA polymerase II was also found at the myod
promoter by ChIP (Figure S3B), further confirming the correlation
between FoxO3 promoter occupancy and transcription activa-
tion. Although FoxO4 appeared to have low transcriptional activ-
ity, little, if any, FoxO4 was detected at the myod promoter
by ChIP in myoblasts (Figure 2B). Taken together, these results
suggest that at least FoxO3 is likely part of the active transcrip-
tion machinery directly recruited to the myod promoter in
myoblasts.
Since FoxO3 binds to putative FRE �940 and FRE �1598 of
the myod promoter in C2C12 cells, we next examined whether
FoxO3 is also able to recognize and bind these putative FREs
in vitro by electrophoretic mobility shift assays (EMSA). Purified
recombinant FoxO3 efficiently bound to both the FRE �940
and FRE �1598 probes, but not to mutant probes (Figure 2E),
confirming that FoxO3 can bind to both FRE sequences
ier Inc.
Developmental Cell
Activators Direct MyoD Transcription
specifically in vitro. Consistent with our ChIP results, in luciferase
reporter assays, mutations in the FREs at �940 or �1598 signif-
icantly debilitated myod transcription, while mutations in
FRE�1928 and FRE�2351 had little or no effect on transcription
activity (Figure S4). These results suggest that FRE �940
and �1598 bound by FoxO3 are critical for myod transcription
activation. Taken together, these assays indicate that FoxO3
can specifically bind two FREs in the myod promoter and acti-
vate transcription.
Figure 2. FoxO3 Binds the myod Promoter
and Activates Transcription
(A) qRT-PCR analysis of MyoD mRNA level in cells
treated with shRNA against each FoxO protein as
indicated below each yellow column. The pink col-
umns indicate qRT-PCR analysis of MyoD mRNA
levels in FoxO3 RNAi cells rescued by CMV driven
FoxO constructs as indicated below each column.
The purple columns represent qRT-PCR analysis
of MyoD mRNA levels in cells overexpressing
constitutively active FoxOs as indicated below
each column. The error bars represent values
from standard deviation calculations.
(B) The upper panel is a schematic diagram of the
myod promoter structure. Primers utilized for ChIP
are indicated by orange arrows. ChIP results with
antibodies against each FoxO protein are in the
lower panel.
(C) EMSAs were performed with probe encom-
passing either wild-type or mutant FRE. Recombi-
nant FoxO3 expressed in insect cells binds specif-
ically to the wild-type probe.
FoxO3 and MyoD Can RescueDifferentiation Defectsin FoxO3-Depleted CellsBased on the above results, we proposed
that FoxO3 is responsible for activating
MyoD expression under physiologically
relevant conditions. If this hypothesis
has merit, we might expect that depletion
of FoxO3 will disrupt myotube formation.
Indeed, FoxO3 RNAi cells failed to differ-
entiate into myotubes, whereas the
scrambled control and FoxO1 RNAi cells
differentiated normally (Figure 3A). When
a CMV-driven FoxO1, 3, or 4 construct
was introduced into the FoxO3-depleted
cells, only FoxO3 significantly rescued
the differentiation defect (Figure 3B). If
the FoxO3 rescue was achieved in large
measure by restoring MyoD expression,
re-expressing MyoD in these FoxO3
depleted cells should also at least partly
rescue the differentiation defects. As
expected, expression of CMV-driven
MyoD in FoxO3 RNAi cells partially res-
cued myotube formation (Figure 3B).
This partial rescue may be in part due to
inappropriate and unregulated levels of
FoxO3 and MyoD ectopically driven by
the highly active CMV promoter. Taken together, these observa-
tions establish that FoxO3, but not FoxO1 or 4, plays an impor-
tant role in activating myod transcription in cells.
Pax3/7 Binds the myod Promoter and ActivatesTranscriptionAlthough we have identified FoxO3 as an important activator of
myod transcription, FoxO3 is ubiquitously expressed in most
cell types (Anderson et al., 1998; Biggs et al., 2001), while
Developmental Cell 15, 534–546, October 14, 2008 ª2008 Elsevier Inc. 537
Developmental Cell
Activators Direct MyoD Transcription
MyoD expression is limited to muscle cells. This suggests that
tissue specific factors in addition to FoxO3 may be required
to activate myod transcription. Therefore, we performed a series
of chromatographic separations to identify potential partners
that may confer tissue selectivity. C2C12 nuclear extracts
were subjected to ammonium sulfate precipitation followed
by monoQ chromatography and DNA affinity chromatography
with synthetic DNA containing three tandem repeats of
FRE�940 linked to three tandem repeats of FRE�1598. Protein
complexes eluted from the DNA affinity resin were subsequently
analyzed by liquid chromatography tandem mass spectrometry
(LC MS/MS) (Figure 4A). The mass spec results revealed Pax3 or
Pax7 as transcription factors present in the DNA affinity column
eluents in addition to FoxO3. Based on the peptides identified
by LC MS/MS, we could not discriminate Pax3 from the closely
related Pax7, which shares 77% sequence identity to Pax3.
To test whether Pax3/7 has any effect on MyoD expression
in C2C12 cells, we carried out qRT-PCR in cells either lacking
or overexpressing Pax3/7. In cells treated with shRNAs against
Pax3 and Pax7, nearly 95% of the Pax3 and 87% of the
Pax7 mRNA was depleted, indicating an efficient knockdown.
In these same Pax3/7-depleted cells, MyoD mRNA was concom-
itantly downregulated more than 80% (Figure 4B, upper panel).
By contrast, in cells transiently overexpressing Pax3 or Pax7,
production of MyoD was specifically upregulated, while in
both cases FoxO3 mRNA levels remained unaffected (Figure 4B).
Previous mouse genetic studies had implicated Pax3 and
Pax7 as potential upstream activators of MyoD expression
(Bober et al., 1994; Goulding et al., 1994; Seale et al., 2000),
but it had not been shown whether these factors directly regulate
Figure 3. FoxO3 and MyoD Can Rescue
Differentiation Defects in FoxO3-Depleted
C2C12 Cells
(A) Phase contract images of C2C12 cells treated
with control or FoxO3 shRNA before and after
differentiation.
(B) Phase contract images of FoxO3 depleted
C2C12 cells rescued by CMV driven FoxO1, 3, 4,
or MyoD before and after differentiation. The red
arrows indicate myotubes. Bar, 330 mm.
myod transcription. Consistent with
these genetic data, our loss-of-function
and gain-of-function assays targeting
myod transcription suggest that Pax3/7
together with FoxO3 contributes to the
transcription activation of myod in myo-
blasts. Promoter DNA sequence analysis
revealed two potential paired boxes lo-
cated between FRE �940 and FRE
�1598 (Figure 4C). To test for direct
Pax3/7 binding to these sites in C2C12
cells, we performed ChIP experiments
with an antibody recognizing both Pax
proteins. Pax3/7 was significantly en-
riched at the myod promoter region
containing paired box �1502, but not at
the region containing paired box �989
(Figure 4C). The pattern of a Pax recognition site (paired box or
homeobox) lying between two FREs are conserved from rodent
to human (Figure S5). As expected, control (c2) primers mapping
to a region 50 kb upstream of the myod transcription initiation
site did not yield any amplification products above background
(Figure 4C). These data suggest that Pax3/7 binds to paired
box �1502 of the myod promoter in C2C12 cells.
To further examine the binding of Pax3/7 protein to this putative
paired box at �1502, we carried out EMSA. Recombinant Pax3
and Pax7 were found to bind the paired-box probe, but not a mu-
tant probe, although Pax3 binds with significantly higher affinity
than Pax7 at least in vitro (Figure S6A). Consistent with our
ChIP and RNAi results, paired-box mutant promoters displayed
lower activities in luciferase reporter assays (Figure S6B), sug-
gesting that the paired box is required for myod transcription
activation. Together, these cell-based and in vitro studies sug-
gest that Pax3/7 may function as a FoxO3 partner in regulating
myod transcription in myoblasts.
To test for the possible Pax3/7 and FoxO3 co-occupancy at
the endogenous myod promoter, sequential ChIP assays were
performed (Figure 4E). Consistent with the data shown in
Figure 2B, FoxO3 was found at the myod promoter after the first
anti-FoxO3 IP. Intriguingly, Pax3/7 was detected at the same
promoter region containing FRE �1928 and paired box �1502
after the second anti-Pax3/7 IP (Figure 4E), indicating that
FoxO3 and Pax3/7 are able to occupy the myod promoter simul-
taneously. Consistent with these sequential ChIP results, FoxO3
binds Pax3 and Pax7 with higher affinity than FoxO1 and FoxO4
in GST pull-down experiments (Figure S7A), suggesting a spe-
cific protein-protein interaction between FoxO3 and Pax3/7.
538 Developmental Cell 15, 534–546, October 14, 2008 ª2008 Elsevier Inc.
Developmental Cell
Activators Direct MyoD Transcription
FoxO3 and Pax3/7 Activate myod TranscriptionCooperatively and Are Mutually DependentIn order to directly test the functions of FoxO3 and Pax3/7 in
myod transcription, we next set up an in vitro transcription sys-
tem programmed with a DNA template containing the CAT
gene driven by a fragment encompassing �1 to �2000 region
of the myod promoter. Low levels of transcription from this tem-
plate were detected with crude nuclear extracts derived from
proliferating C2C12 cells. We next supplemented these in vitro
transcription reactions with purified recombinant activators. Ad-
dition of recombinant FoxO3 to the in vitro myod transcription
system potently activated transcription (4.7- and 8.7-fold; Fig-
ure 5A). Likewise, Pax3 alone modestly activated myod tran-
scription (2.3- and 4-fold; Figure 5A). Surprisingly, although
Pax7 showed lower affinity for the paired-box DNA than Pax3
in EMSA, it nevertheless showed stronger transcription activa-
tion properties compared to that of Pax3 in the in vitro transcrip-
tion system (6.8- and 14.9-fold; Figure 5A). When combined,
FoxO3 and Pax3 displayed a high degree of synergy for activat-
ing myod transcription in vitro (8- and 21-fold; Figure 5A). Simi-
larly, FoxO3 and Pax7 activated myod transcription coopera-
Figure 4. Pax3/7 Binds the myod Promoter
and Activates Transcription
(A) Purification scheme of FoxO3-associated
activator partners.
(B) The upper panel illustrates qRT-PCR analysis
of Pax3, Pax7, MyoD, and FoxO3 mRNA levels in
C2C12 cells treated with control shRNA (yellow
columns) or shRNA against Pax3 and Pax7 (purple
columns) as indicated below each group of col-
umns. Lower panel illustrates qRT-PCR analysis
of mRNA levels of MyoD and FoxO3 in C2C12 cells
transiently overexpressing vector (green col-
umns), Pax3 (orange columns), or Pax7 (red
columns) as indicated below each group of col-
umns. The error bars represent values from stan-
dard deviation calculations.
(C) The two putative paired-box elements in the
myod promoter are indicated in the upper panel.
Primer pairs utilized in the anti-Pax3/7 ChIP
assays are indicated as yellow arrows. p1 and p2
map paired-box elements as indicated. c1 map
a region 50 kb upstream of the myod transcription
initiation site.
(D) The left panel shows the scheme of the FoxO3-
Pax3/7 sequential ChIP. The results are illustrated
in the right panel.
tively (24-fold; Figure 5A, lanes 10–16).
Consistent with our in vitro transcription
results, FoxO3 and Pax7 display cooper-
ative binding in EMSA on the myod pro-
moter (Figure S7B). Thus, only FoxO3 in
combination with Pax3/7 behave as effi-
cient activators that can synergistically
potentiate myod transcription in vitro.
It has been reported that Pax3-FKHR,
but not Pax3, strongly induced many
myogenic genes including myod upon
transfection into NIH 3T3 cells (Khan
et al., 1999). Recent studies in mouse ES cells also suggest
that Pax3 is not sufficient for full MyoD activation. As expected,
when transfected into NIH 3T3 cells or D3 ES cells, the combina-
tion of Pax3/7 and FoxO3 activated myod transcription robustly,
while Pax3 or FoxO3 alone barely activated myod transcription
(Figure S8). These results are consistent with the observation
from our in vitro studies suggesting cooperative activation of
myod transcription by Pax3/7 and FoxO3.
We next used a double-template transcription system to fur-
ther confirm the functions of FoxO3 and Pax3/7. In these reac-
tions, both wild-type and mutant myod promoters are present
in the same transcription reaction. First, we examined transcrip-
tion from the myod promoter containing a mutant paired box. As
expected, supplementing the system with recombinant Pax3/7
failed to activate transcription from the paired-box mutant tem-
plate. Surprisingly, addition of FoxO3 was also insufficient to
activate transcription from the paired-box mutant promoter.
However, transcription directed by the wild-type myod promoter
in the same reaction was not affected (Figure 5B). These obser-
vations suggest that Pax proteins are important, probably
obligate, partners for FoxO3 to direct myod transcriptional
Developmental Cell 15, 534–546, October 14, 2008 ª2008 Elsevier Inc. 539
Developmental Cell
Activators Direct MyoD Transcription
Figure 5. FoxO3 and Pax3/7 Activate myod Transcription Cooperatively and Mutually Depend on Each Other
(A) In vitro transcription from myod promoter driven CAT template was performed with nuclear extracts from proliferating C2C12 myoblasts supplemented with
recombinant FoxO3, Pax7, or Pax3. The amount of proteins utilized in each reaction is indicated above each lane. In order to see cooperative activation by FoxO3
and Pax7, less protein was used as indicated in lanes 11–16. The fold of transcription activation by the activators is illustrated under each lane.
(B) Codependence between FoxO3 and Pax3/7 to activate myod transcription. Templates containing both wild-type and mutant myod promoter were supple-
mented with proliferating C2C12 myoblast nuclear extracts. The transcription product from the wild-type promoter is 15 nt longer than the one from the mutant
promoter. The transcripts from the mutant template are indicated in the upper panel. The transcripts from the wild-type promoter are indicated in the lower panel.
Lanes 1–7 indicate in vitro transcription directed by paired-box mutant promoter. Lanes 8–14 indicate in vitro transcription directed by myod promoter with both
FRE �940 and �1598 mutated.
(C) qPCR analysis of enrichment of the myod or b-actin promoter regions in anti-Pax3/7 or RNA polymerase II phospho-Ser5 ChIP as indicated below each group
of columns in cells treated with FoxO3 or control shRNAi as indicated by blue or yellow columns, respectively. The error bars represent values from standard
deviation calculations.
(D) qPCR analysis of enrichment of the myod or b-actin promoter in anti-FoxO3 or RNA polymerase II phospho-Ser5 ChIP as indicated below each group of
columns in cells treated with Pax3/7 or control shRNAi as indicated by purple or yellow columns, respectively. The error bars represent values from standard
deviation calculations.
activation. We also tested transcription from a myod promoter
containing mutant FREs in the double template system. As ex-
pected, FoxO3 failed to activate transcription from this mutant
FRE template. Interestingly, both Pax3 and Pax7 also failed to
activate transcription from the mutant FRE promoter. FoxO3
and Pax3/7 dependent transcription from the wild-type promoter
in the same reaction was not affected (Figure 5B). These in vitro
studies taken en toto suggest that FoxO3 is necessary for Pax3/7
dependent myod transcription activation.
Having established that FoxO3 and Pax3/7 can activate tran-
scription of myod in vitro, we next investigated potential inter-
540 Developmental Cell 15, 534–546, October 14, 2008 ª2008 Else
actions between FoxO3 and Pax3/7 in C2C12 cells. First, we
asked what happens to Pax3/7 binding at the myod promoter
in the absence of FoxO3. Indeed, ChIP experiments performed
in FoxO3-depleted cells revealed that neither Pax3/7 nor active
RNA polymerase II can be detected at the myod promoter in
the absence of FoxO3. As a control, we confirmed that active
RNA polymerase II is detected at the non-FoxO3-regulated
b-actin promoter (Figure 5C). Next, we carried out the converse
experiments using C2C12 cells depleted of both Pax3 and
Pax7. In this case, the amount of FoxO3 detected at FRE
�1598 and �940 was reduced to about 50% compared to
vier Inc.
Developmental Cell
Activators Direct MyoD Transcription
that in control RNAi cells. Curiously, we also detected some
FoxO4 in addition to FoxO3 at FRE �1598 in the absence of
Pax3/7, while FRE �940 was only recognized by FoxO3. Impor-
tantly, little, if any, RNA polymerase II was recruited to the
myod promoter, whereas its recruitment to the b-actin gene
was not affected (Figure 5D). Consistent with our in vitro tran-
scription results (Figure 5B), these ChIP experiments suggest
a mutual codependence between FoxO3 and Pax3/7 for estab-
lishing an active PIC. Apparently, only when both activators are
present is RNA polymerase II recruited and myod transcription
activated.
FoxO3 and Pax3/7 Both Bind the myod Promoterin Satellite CellsThe observations described thus far established FoxO3 and
Pax3/7 as codependent activators for myod transcription in
C2C12 cells. To determine whether this situation also occurs in
primary cells, we next carried out experiments using satellite
Figure 6. FoxO3 and Pax3/7 Bind the myod
Promoter in Satellite Cells
(A) Phase contract images of undifferentiated and
differentiated satellite cells. Bar, 80 mm.
(B) Immunofluorescent staining images of FoxO3
and Pax7 in undifferentiated satellite cells and
fibroblasts isolated from the same mice. Bar,
10 mm.
(C) ChIP performed in satellite cells and primary fi-
broblasts isolated from the same mice. F1 primers
(Figure 2B) were used to map FRE�940. F2 primers
(Figure 2B) were used to map FRE �1598. p1
primers (Figure 4C) were used to map the paired
box �1502. The PCR reactions are performed
with 32P-labeled primers.
cells (muscle stem cells), which are
responsible for postnatal growth and
muscle regeneration, isolated from new-
born mouse skeletal muscle (Collins,
2006; Conboy et al., 2005; Wagers and
Conboy, 2005), wherein MyoD is highly
expressed to confirm the presence of
FoxO3 and Pax3/7 at the myod promoter.
In vitro differentiation experiments con-
firmed that over 90% of the cells isolated
were indeed satellite cells (Figure 6A).
FoxO3 and Pax3/7 were both easily de-
tected in the nuclei of these cells. In
marked contrast, FoxO3 was diffusely
distributed in both nuclei and cytoplasm
of primary fibroblasts isolated from the
same mice, while Pax3/7 was not de-
tected in these nonmuscle cells (Fig-
ure 6B). ChIP experiments confirmed
the presence of FoxO3 at FRE �1598
and �940, while Pax3/7 occupied the
paired box at the myod promoter in satel-
lite cells, but not in primary fibroblasts
(Figure 6C). These experiments establish
that FoxO3 and Pax3/7 can specifically
target the myod promoter in primary cells and suggest that
FoxO3 and Pax3/7 likely operate together to activate myod
transcription in vivo.
FoxO3 Null Mice Display Muscle Regeneration DefectsThe results above suggest that FoxO3 and Pax3/7 directly acti-
vate myod transcription in both C2C12 and primary cells. If this
hypothesis has merit, we might expect that both Pax3/7 and
FoxO3 knockout mice will have lower MyoD expression levels
and display some degree of muscle defects. Consistent with
our hypothesis, satellite cells from Pax7 null mice showed
reduced differentiation potential (Seale et al., 2000) while Pax3
mutant mice lacked skeletal muscle in limbs (Bober et al.,
1994; Goulding et al., 1994). However, it was not known whether
FoxO3 knockout mice display any muscle defects. We anticipate
that if FoxO3�/� animals show any muscle defects, they might
resemble MyoD�/� mice wherein knockout mice do not display
any dramatic muscle phenotypes largely due to compensatory
Developmental Cell 15, 534–546, October 14, 2008 ª2008 Elsevier Inc. 541
Developmental Cell
Activators Direct MyoD Transcription
effects by Myf5 (Rudnicki et al., 1993). However, satellite cells
isolated from these knockout mice showed distinctly reduced
potential to form myotubes, thus revealing a muscle regenera-
tion defect after injury (Cornelison et al., 2000; Sabourin et al.,
1999; Yablonka-Reuveni et al., 1999).
We therefore set out to characterize the differentiation poten-
tial of satellite cells isolated from FoxO3 null mice (Castrillon
et al., 2003). Based on immunostaining of satellite cell markers
c-Met and Pax7, over 85% of the cells isolated from FoxO3
+/+, +/�, and�/�mice were satellite cells (Figure S9). The mor-
phologies of the satellite cells from all three sources appeared to
be similar in proliferation medium, suggesting self-renewal of
these cells remained largely unaffected. After differentiation,
myosin expression and multinucleate myotubes were easily de-
tected in wild-type cells. By contrast, only a small percentage of
FoxO3 heterozygous or homozygous satellite cells expressed
myosin and these cells formed much smaller myotubes (mea-
sured by the number of nuclei per cell) upon differentiation
(Figure 7A). These observations suggest a reduced differentia-
tion potential of satellite cells isolated from FoxO3 deficient
mice. We next examined the expression level of MyoD and
myogenin in these cells. RNA isolated from similar numbers of
FoxO3 +/+, +/�, or �/� cells were subjected to qRT-PCR anal-
ysis. Compared to wild-type cells, MyoD expression levels were
significantly reduced in both heterozygous (27% of wild-type)
and homozygous (13.6% of wild-type) KO satellite cells, while
the transcript levels of pax7, pax3, foxo1, and foxo4 were largely
unaffected in all littermates (Figure 7B).
To further investigate the contribution of FoxO3 in regulating
MyoD in vivo, muscle injury and regeneration studies were car-
ried out by intramuscular injection of cardiotoxin (CTX). Two
slightly different methods of CTX injection were utilized in paral-
lel. One group of mice was injected with 25 ml of 42 mM CTX, the
other group with 100 ml of 10 mM CTX (Yan et al., 2003). Hind leg
skeletal muscle sections from each time point taken during the
muscle regeneration process in both groups were stained with
anti-laminin and DAPI. In order to obtain a representative regen-
eration profile for wild-type mice, the numbers of cells containing
center located nuclei were counted and plotted (Figure S10). At
day 5 after injection with 25 ml of CTX and at day 7 after injection
of the larger volume, the numbers of cells with centrally located
nuclei decreased dramatically compared to earlier time points
(Figure S10). This suggests that under our experimental condi-
tions, wild-type mice largely recovered from wounding at day
5�7 in the case of 25 ml injections or day 7�11 in the case of
100 ml injections. Next, we examined the regeneration status of
FoxO3�/� mice injected with CTX at day 5, 7 and day 12, 16.
In sharp contrast to the wild-type, FoxO3�/�mice showed signif-
icantly higher percentages of cells containing center located
nuclei (Figures 7C and 7D), characteristic of incomplete muscle
cell regeneration. These injury and reparation experiments reveal
a distinct delay in skeletal muscle regeneration in FoxO3�/�mice
compared to wild-type animals and are consistent with our pro-
posal that FoxO3 plays an important role in vivo in skeletal mus-
cle differentiation, most likely via activating myod transcription.
Taken together, these observations suggest that FoxO3 null
mice more or less phenocopy MyoD null mice with respect to
muscle defects and support the notion that FoxO3 is an impor-
tant activator required for MyoD expression in vivo.
542 Developmental Cell 15, 534–546, October 14, 2008 ª2008 Else
DISCUSSION
Although FoxOs have previously been implicated functioning in
muscle differentiation (Bois and Grosveld, 2003; Hribal et al.,
2003; Kitamura et al., 2007; Machida et al., 2003; Sandri et al.,
2004), skeletal muscle-specific genes directly targeted by FoxOs
had not been identified. Our findings indicate that FoxO3 (but not
FoxO1 or FoxO4) binds a subset of FREs in the myod promoter to
work in concert with Pax3/7 in regulating cell type-specific tran-
scription activation in myoblasts. The contribution of FoxO3 in
directing myod transcription activation in vivo was further con-
firmed by the observed muscle regeneration defects in FoxO3
null mice.
Identification of FoxO3 as an important myod transcription ac-
tivator may provide a handle to explore the potential signaling
pathways governing muscle regeneration. Interestingly, FoxO1
was found to negatively regulate MyoD expression indirectly
through the Delta-Notch pathway (Holterman et al., 2007; Kita-
mura et al., 2007). The potential repression of MyoD by FoxO1
together with our results of direct activation of MyoD by FoxO3
suggests an intriguing mechanism to fine tune MyoD expression.
Muscle differentiation may therefore utilize selected FoxOs in
partnership with Pax3/7 to integrate inputs from multiple signal-
ing pathways.
The apparent Kd of FoxO3 and Pax3/7 binding individually to
the DNA elements in the myod promoter is on the order of
10�7M in vitro (P.H., unpublished data), which is rather modest
compared to a typical DNA binding protein, such as GAL4 (ap-
parent Kd, �10�11M) (Kamachi et al., 2000; Parthun and Jaehn-
ing, 1990). This is consistent with our finding that neither FoxO3
nor Pax3/7 alone binds promoter DNA efficiently to form a stable
DNA-activator complex capable of recruiting a functional PIC. It
appears that to efficiently assemble an active PIC via FoxO3 and
Pax3/7 at the myod promoter both protein-DNA and protein-
protein interactions mediated by this hitherto unknown activator
partnership must take place to trigger transcription activation
synergistically. This may therefore represent a useful and effi-
cient combinatorial mechanism to direct cell type-specific tran-
scription while utilizing two activators shared by many cell types.
This codependence is reminiscent of the mechanism utilized by
Sox2 and Pax6 to drive lens-specific transcription of d-crystallin
gene (Kondoh et al., 2004; Lang et al., 2007; Lefebvre et al.,
2007).
Although under normal conditions FoxO3 is predominantly de-
tected occupying the FRE at �1598 of the myod promoter, curi-
ously, we found that some FoxO4 can be detected at this site in
myoblasts when Pax3/7 is depleted. It is known that Pax3/7 is
not expressed in myotubes, but MyoD expression persists in
myotubes. It will be interesting to survey the identity of FoxOs
binding to the myod promoter in myotubes versus myoblasts
and explore additional mechanisms involved in cell type-specific
transcription activation during later stages of myogenesis. In-
triguingly, we now know there is a switching of the core transcrip-
tion machinery from the canonical holo-TFIID to a TRF3/TAF3
complex during myoblasts differentiation to myotubes (Deato
and Tjian, 2007). While FoxO3 and Pax3/7 appear to work in
concert with TFIID at the MyoD promoter in myoblasts, it will be
important to identify the key activator(s) that function together
with the TRF3/TAF3 complex in myotubes.
vier Inc.
Developmental Cell
Activators Direct MyoD Transcription
Figure 7. FoxO3 Null Mice Display Muscle
Regeneration Defects
(A) Immunofluorescent staining images of myosin
in differentiated satellite cells isolated from +/+,
+/�, and �/�mice. The average number of nuclei
per cell is indicated next to the images. Bar,
330 mm.
(B) qRT-PCR analysis of MyoD, Pax3, Pax7,
FoxO1, and FoxO4 mRNA levels in satellite
cells isolated from +/+, +/�, �/� mice. The error
bars represent values from standard deviation
calculations.
(C) Immunohistology staining images of laminin
and DAPI with frozen skeletal muscle sections
from +/+ and �/� mice injected with CTX or
PBS. CTX was injected intramuscularly in the right
hind leg. PBS was injected intramuscularly in the
left hind leg of the same mouse. Laminin staining
was indicated by red color. Green color was artifi-
cially applied to DAPI staining. Representative
staining images of 25 ml CTX injection after 5 days
are illustrated in panels (a) and (b). Representative
staining images of 100 ml CTX injection after
12 days are illustrated in panels (c) and (d). Repre-
sentative staining images of PBS injection are
illustrated in panels (e) and (f). Bar, 30 mm.
(D) Percentages of cells containing center located
nuclei in skeletal muscles sections from wild-type
or FoxO3 null mice. Days of recovery after injury
and volumes of injections are indicated below
each bar.
Developmental Cell 15, 534–546, October 14, 2008 ª2008 Elsevier Inc. 543
Developmental Cell
Activators Direct MyoD Transcription
EXPERIMENTAL PROCEDURES
Constructs and Antibodies
The mouse myod promoter was PCR from genomic DNA isolated from C2C12
cells. Plasmids containing FoxO1 and FoxO3 were kind gifts from Dr. D. Accili
(Columbia University, New York, NY). Plasmid containing FoxO4 was a kind
gift from Dr. O. Puig (University of California, Berkeley, Berkeley, CA). Pax3
and Pax7 genes were cloned from cDNA pool made from C2C12 cells. DNA
encoding shRNA was synthesized and cloned into pSM vector (Paddison
et al., 2004).
Rabbit polyclonal antibodies were affinity purified with antigen. Pax3 and
Pax7 monoclonal antibody (for immunofluorescent staining) were from The
Developmental Studies Hybridoma Bank. Anti-MyoD was from BD biosci-
ences. Anti-Myosin was from Upstate Biotechnology. Pax polyclonal anti-
bodies (for ChIP), a-GAPDH, a-c-met, and a-RNA polymerase II Ser5, a-lam-
inin was from Abcam. FITC-anti-CD34 is from Beckman Coulter. a-TBP was
from Biodesign International.
Cell Culture, Transfection, RNAi, Differentiation, and Muscle Injury
C2C12 cells (ATCC) were maintained in DMEM (Sigma) supplemented with
10% fetal bovine serum (FBS) (Sigma) at 37�C with 5% CO2. C2C12 cells
were transfected with lipofectamin 2000 (Invitrogen). Satellite cells were iso-
lated from mice legs as described (Carlson and Conboy, 2007). In brief,
mice younger than 1 month were sacrificed, and muscle tissues were dis-
sected out. The dissected muscle tissues were digested with collagenase
(Roche) and dispase (Roche), triturated followed by multiple sedimentations,
washes, and filtration. They were grown in F10 nutrition mix (Invitrogen) sup-
plemented with 15% FBS, 10 ng/ml bFGF (Invitrogen), 1000 units of LIF (Milli-
pore) at 37�C, 5% CO2. The cells were passed for less than 20 passages. Both
C2C12 cells and satellite cells were differentiated in DMEM containing 2%
horse serum (Invitrogen). C2C12 cells were grown to 100% confluence before
differentiation.
For skeletal muscle regeneration experiments, 25 ml of 42 mM or 100 ml of
10 mM CTX dissolved in PBS was injected intramuscularly to the right hind
leg of 6-week-old +/+ or �/� mice. As control, PBS was injected to the left
leg. The mice were sacrificed at 1, 2, 3, 4, 5, 6, 7, 11, 12, 14, and 16 days,
and frozen sections were made from skeletal muscles.
shRNA sequences against FoxOs were obtained from http://codex.cshl.
edu/scripts/main.pl. Control RNAi construct was obtained from Open Biosys-
tems. After transfection, the cells were selected and maintained in complete
DMEM medium containing 2 mg/ml puromycin. siRNA against Pax3 and
Pax7 were obtained from Dharmacon and transfected into C2C12 cells using
Oligofectamine (Invitrogen). Sequences were listed in Supplemental Experi-
mental Procedures.
DNA Microarrays
Microarray analysis was performed utilizing Affymetrix murine 430 2.0 chips.
The data were analyzed by GCOS software (Affymetrix). All chips have compa-
rable background values. The change p value is set at higher than 0.999. Other
values were listed in the Supplemental Experimental Procedures. Promoters of
the potential target genes were searched for FREs [(G/A/C)T(C/A)AA(T/C)A(A/
C)] at http://rna.berkeley.edu/�siwu/dnascanner.htm.
qRT-PCR
RNA was isolated from cells with RNeasy mini kit (QIAGEN) followed by
reverse transcription using M-LV RT (Ambion). cDNA was used as template
for qPCR with SYBR green master mix (Bio-Rad), followed by analysis with
DDCt method from at least four replicas. The error bars represent values
from standard deviation calculations (see Supplemental Experimental Proce-
dures for formula).
Immunofluorescent and Immunohistology Staining
Cells were fixed with 4% formaldehyde and permeablized in 0.1% Triton
X-100, then blocked with 0.5% goat serum and incubated with primary
antibody. Alexa 594- or Alexa 488-conjugated secondary antibodies (Molecu-
lar Probes) were used. The DNA contents of cells were stained with 300 nM
DAPI (Molecular Probes). Actin was stained with Alexa 488-conjugated
phalloidin (Molecular Probes). Frozen sections were made by cryostat
544 Developmental Cell 15, 534–546, October 14, 2008 ª2008 Else
(Brighter Instrument). After fixed in acetone for 10min at 4�C, the sections
were stained. Samples were visualized by 510 Meta Confocal Microscope
(Zeiss) or AxioImager M1 fluorescence microscope (Zeiss).
ChIP
Proliferating C2C12, or satellite cells, or C2C12 cells depleted FoxO3/Pax3/7
were crosslinked and processed as described in (Deato and Tjian, 2007)
except that wash buffer 2 contains 1 M LiCl.
EMSA
FoxO3 EMSA was performed as described in Puig and Tjian (2005). Pax3/7
EMSA was performed in the same buffer as FoxO3 in addition of 500 ng calf
thymus DNA (GE Healthcare) and 2 mM spermidine (Sigma) and ran on 5%
polyacrylamide gel at room temperature. Probe sequences were listed in
Supplemental Experimental Procedures.
In Vitro Transcription
Proliferating C2C12 nuclear extracts were used to transcribe from myod
promoter driven CAT in vitro. Transcription analysis was performed by primer
extension as described (Puig et al., 2003).
Chromatography
Proliferating C2C12 nuclear extracts were subject to 30%�40% ammonium
sulfate precipitation, followed by Poros-HQ 20 column (Applied Biosystems)
and eluted by linear KCl gradient in Buffer D (25 mM HEPES [pH 7.9],
0.2 mM EDTA, 2 mM MgCl2, 10% glycerol, 1 mM DTT, and KCl). The fractions
containing FoxO3 were pooled and applied to DNA affinity column. The DNA
affinity chromatography was performed as described (Kadonaga and Tjian,
1986). DNA bait was oligomers of the basic oligo unit containing three repeats
of FRE �940 followed by three FRE �1598 ranging from 50 to 70 mers.
ACCESSION NUMBERS
The complete DNA microarray data were deposited in the Gene Expression
Omnibus (GEO) at NCBI (http://www.ncbi.nlm.nih.gov/geo/) with series acces-
sion number GSE12582.
SUPPLEMENTAL DATA
The Supplemental Data include ten figures, one table, and Supplemental Ex-
perimental Procedures and can be found with this article online at http://
www.developmentalcell.com/cgi/content/full/15/4/534/DC1/.
ACKNOWLEDGMENTS
We thank M. Haggart for technical assistance, A. Fisher for assistance in large
scale cell culture, D. Schichnes for help on microscope usage, and D. King and
L. Kohlstaedt for help on peptide synthesis and mass spec. We thank I. Con-
boy and M. Carlson for help on satellite cell isolation, M. Marr for help on in vitro
transcription assays, and B. Gan for help on isolation of primary myoblasts
from knockout mice. We also thank M. Deato, K. Wright, Y. Fong, W. Liu, R.
Colemen, F. Herrera, B. Guglielmi, E. Olson, M.E. Buckingham, and M. Rud-
nicki for critical reading of the manuscript. We thank members of the Tjian lab-
oratory for valuable suggestions and discussions. J.-H.P. is Damon Runyon
Fellows supported by the Damon Runyon Cancer Research Foundation.
R.T. is an investigator of the Howard Hughes Medical Institute and Director
of the Li Ka-Shing Center for Biomedical and Health Sciences.
Received: January 25, 2008
Revised: July 21, 2008
Accepted: August 29, 2008
Published: October 13, 2008
REFERENCES
Accili, D., and Arden, K.C. (2004). FoxOs at the crossroads of cellular metab-
olism, differentiation and transformation. Cell 117, 421–426.
vier Inc.
Developmental Cell
Activators Direct MyoD Transcription
Anderson, M.J., Viars, C.S., Czekay, S., Cavenee, W.K., and Arden, K.C.
(1998). Cloning and characterization of three human forkhead genes that com-
prise an FKHR-like gene subfamily. Genomics 47, 187–199.
Arden, K.C. (2006). Multiple roles of FOXO transcription factors in mammalian
cells point to multiple roles in cancer. Exp. Gerontol. 41, 709–717.
Asakura, A., Lyons, G.E., and Tapscott, S.J. (1995). The regulation of MyoD
gene expression: conserved elements mediate expression in embryonic axial
muscle. Dev. Biol. 171, 386–398.
Bajard, L., Relaix, F., Lagha, M., Rocancourt, D., Daubas, P., and Buckingham,
M.E. (2006). A novel genetic hierarchy functions during hypaxial myogenesis:
Pax3 directly activates Myf5 in muscle progenitor cells in the limb. Genes Dev.
20, 2450–2464.
Berkes, C.A., and Tapscott, S.J. (2005). MyoD and the transcriptional control
of myogenesis. Semin. Cell Dev. Biol. 16, 585–595.
Biggs, W.H., 3rd, Cavenee, W.K., and Arden, K.C. (2001). Identification and
characterization of members of the FKHR (FOX O) subclass of winged-helix
transcription factors in the mouse. Mamm. Genome 12, 416–425.
Bober, E., Franz, T., Arnold, H.H., Gruss, P., and Tremblay, P. (1994). Pax-3 is
required for the development of limb muscles: a possible role for the migration
of dermomyotomal muscle progenitor cells. Development 120, 603–612.
Bois, P.R., and Grosveld, G.C. (2003). FKHR (FOXO1a) is required for myotube
fusion of primary mouse myoblasts. EMBO J. 22, 1147–1157.
Buckingham, M., and Relaix, F. (2007). The role of Pax genes in the develop-
ment of tissues and organs: Pax3 and Pax7 regulate muscle progenitor cell
functions. Annu. Rev. Cell Dev. Biol. 23, 645–673.
Buckingham, M., Bajard, L., Daubas, P., Esner, M., Lagha, M., Relaix, F., and
Rocancourt, D. (2006). Myogenic progenitor cells in the mouse embryo are
marked by the expression of Pax3/7 genes that regulate their survival and
myogenic potential. Anat. Embryol. (Berl.) 211 (Suppl 1), 51–56.
Carlson, M.E., and Conboy, I.M. (2007). Loss of stem cell regenerative capac-
ity within aged niches. Aging Cell 6, 371–382.
Carter, M.E., and Brunet, A. (2007). FOXO transcription factors. Curr. Biol. 17,
R113–R114.
Castrillon, D.H., Miao, L., Kollipara, R., Horner, J.W., and DePinho, R.A. (2003).
Suppression of ovarian follicle activation in mice by the transcription factor
Foxo3a. Science 301, 215–218.
Charge, S.B., and Rudnicki, M.A. (2004). Cellular and molecular regulation of
muscle regeneration. Physiol. Rev. 84, 209–238.
Chen, J.C., Ramachandran, R., and Goldhamer, D.J. (2002). Essential and
redundant functions of the MyoD distal regulatory region revealed by targeted
mutagenesis. Dev. Biol. 245, 213–223.
Choi, J., Costa, M.L., Mermelstein, C.S., Chagas, C., Holtzer, S., and Holtzer,
H. (1990). MyoD converts primary dermal fibroblasts, chondroblasts, smooth
muscle, and retinal pigmented epithelial cells into striated mononucleated
myoblasts and multinucleated myotubes. Proc. Natl. Acad. Sci. USA 87,
7988–7992.
Collins, C.A. (2006). Satellite cell self-renewal. Curr. Opin. Pharmacol. 6, 301–
306.
Conboy, I.M., Conboy, M.J., Wagers, A.J., Girma, E.R., Weissman, I.L., and
Rando, T.A. (2005). Rejuvenation of aged progenitor cells by exposure to
a young systemic environment. Nature 433, 760–764.
Cornelison, D.D., Olwin, B.B., Rudnicki, M.A., and Wold, B.J. (2000).
MyoD(�/�) satellite cells in single-fiber culture are differentiation defective
and MRF4 deficient. Dev. Biol. 224, 122–137.
Deato, M.D., and Tjian, R. (2007). Switching of the core transcription machin-
ery during myogenesis. Genes Dev. 21, 2137–2149.
Furuyama, T., Nakazawa, T., Nakano, I., and Mori, N. (2000). Identification of
the differential distribution patterns of mRNAs and consensus binding
sequences for mouse DAF-16 homologues. Biochem. J. 349, 629–634.
Gauthier-Rouviere, C., Vandromme, M., Tuil, D., Lautredou, N., Morris, M.,
Soulez, M., Kahn, A., Fernandez, A., and Lamb, N. (1996). Expression and
activity of serum response factor is required for expression of the muscle-
Develop
determining factor MyoD in both dividing and differentiating mouse C2C12
myoblasts. Mol. Biol. Cell 7, 719–729.
Goldhamer, D.J., Brunk, B.P., Faerman, A., King, A., Shani, M., and Emerson,
C.P., Jr. (1995). Embryonic activation of the myoD gene is regulated by a highly
conserved distal control element. Development 121, 637–649.
Gomis, R.R., Alarcon, C., He, W., Wang, Q., Seoane, J., Lash, A., and Mas-
sague, J. (2006). A FoxO-Smad synexpression group in human keratinocytes.
Proc. Natl. Acad. Sci. USA 103, 12747–12752.
Goulding, M., Lumsden, A., and Paquette, A.J. (1994). Regulation of Pax-3
expression in the dermomyotome and its role in muscle development. Devel-
opment 120, 957–971.
Holterman, C.E., Le Grand, F., Kuang, S., Seale, P., and Rudnicki, M.A. (2007).
Megf10 regulates the progression of the satellite cell myogenic program.
J. Cell Biol. 179, 911–922.
Hosaka, T., Biggs, W.H., 3rd, Tieu, D., Boyer, A.D., Varki, N.M., Cavenee, W.K.,
and Arden, K.C. (2004). Disruption of forkhead transcription factor (FOXO)
family members in mice reveals their functional diversification. Proc. Natl.
Acad. Sci. USA 101, 2975–2980.
Hribal, M.L., Nakae, J., Kitamura, T., Shutter, J.R., and Accili, D. (2003).
Regulation of insulin-like growth factor-dependent myoblast differentiation
by Foxo forkhead transcription factors. J. Cell Biol. 162, 535–541.
Huang, H., and Tindall, D.J. (2007). Dynamic FoxO transcription factors. J. Cell
Sci. 120, 2479–2487.
Jacobs, F.M., van der Heide, L.P., Wijchers, P.J., Burbach, J.P., Hoekman,
M.F., and Smidt, M.P. (2003). FoxO6, a novel member of the FoxO class of
transcription factors with distinct shuttling dynamics. J. Biol. Chem. 278,
35959–35967.
Jonsson, H., Allen, P., and Peng, S.L. (2005). Inflammatory arthritis requires
Foxo3a to prevent Fas ligand-induced neutrophil apoptosis. Nat. Med. 11,
666–671.
Kadonaga, J.T., and Tjian, R. (1986). Affinity purification of sequence-specific
DNA binding proteins. Proc. Natl. Acad. Sci. USA 83, 5889–5893.
Kamachi, Y., Uchikawa, M., and Kondoh, H. (2000). Pairing SOX off: with part-
ners in the regulation of embryonic development. Trends Genet. 16, 182–187.
Kassar-Duchossoy, L., Giacone, E., Gayraud-Morel, B., Jory, A., Gomes, D.,
and Tajbakhsh, S. (2005). Pax3/Pax7 mark a novel population of primitive myo-
genic cells during development. Genes Dev. 19, 1426–1431.
Khan, J., Bittner, M.L., Saal, L.H., Teichmann, U., Azorsa, D.O., Gooden, G.C.,
Pavan, W.J., Trent, J.M., and Meltzer, P.S. (1999). cDNA microarrays detect
activation of a myogenic transcription program by the PAX3-FKHR fusion
oncogene. Proc. Natl. Acad. Sci. USA 96, 13264–13269.
Kitamura, T., Kitamura, Y.I., Funahashi, Y., Shawber, C.J., Castrillon, D.H.,
Kollipara, R., DePinho, R.A., Kitajewski, J., and Accili, D. (2007). A Foxo/Notch
pathway controls myogenic differentiation and fiber type specification. J. Clin.
Invest. 117, 2477–2485.
Kondoh, H., Uchikawa, M., and Kamachi, Y. (2004). Interplay of Pax6 and
SOX2 in lens development as a paradigm of genetic switch mechanisms for
cell differentiation. Int. J. Dev. Biol. 48, 819–827.
Kucharczuk, K.L., Love, C.M., Dougherty, N.M., and Goldhamer, D.J. (1999).
Fine-scale transgenic mapping of the MyoD core enhancer: MyoD is regulated
by distinct but overlapping mechanisms in myotomal and non-myotomal mus-
cle lineages. Development 126, 1957–1965.
L’Honore, A., Lamb, N.J., Vandromme, M., Turowski, P., Carnac, G., and Fer-
nandez, A. (2003). MyoD distal regulatory region contains an SRF binding
CArG element required for MyoD expression in skeletal myoblasts and during
muscle regeneration. Mol. Biol. Cell 14, 2151–2162.
Lam, E.W., Francis, R.E., and Petkovic, M. (2006). FOXO transcription factors:
key regulators of cell fate. Biochem. Soc. Trans. 34, 722–726.
Lang, D., Powell, S.K., Plummer, R.S., Young, K.P., and Ruggeri, B.A. (2007).
PAX genes: roles in development, pathophysiology, and cancer. Biochem.
Pharmacol. 73, 1–14.
Lefebvre, V., Dumitriu, B., Penzo-Mendez, A., Han, Y., and Pallavi, B. (2007).
Control of cell fate and differentiation by Sry-related high-mobility-group box
(Sox) transcription factors. Int. J. Biochem. Cell Biol. 39, 2195–2214.
mental Cell 15, 534–546, October 14, 2008 ª2008 Elsevier Inc. 545
Developmental Cell
Activators Direct MyoD Transcription
Li, H., Liang, J., Castrillon, D.H., DePinho, R.A., Olson, E.N., and Liu, Z.P.
(2007). FoxO4 regulates tumor necrosis factor alpha-directed smooth muscle
cell migration by activating matrix metalloproteinase 9 gene transcription. Mol.
Cell. Biol. 27, 2676–2686.
Lin, L., Hron, J.D., and Peng, S.L. (2004). Regulation of NF-kappaB, Th activa-
tion, and autoinflammation by the forkhead transcription factor Foxo3a. Immu-
nity 21, 203–213.
Liu, Z.P., Wang, Z., Yanagisawa, H., and Olson, E.N. (2005). Phenotypic mod-
ulation of smooth muscle cells through interaction of Foxo4 and myocardin.
Dev. Cell 9, 261–270.
Machida, S., Spangenburg, E.E., and Booth, F.W. (2003). Forkhead transcrip-
tion factor FoxO1 transduces insulin-like growth factor’s signal to p27Kip1 in
primary skeletal muscle satellite cells. J. Cell. Physiol. 196, 523–531.
Mammucari, C., Milan, G., Romanello, V., Masiero, E., Rudolf, R., Del Piccolo,
P., Burden, S.J., Di Lisi, R., Sandri, C., Zhao, J., et al. (2007). FoxO3 controls
autophagy in skeletal muscle in vivo. Cell Metab. 6, 458–471.
Maroto, M., Reshef, R., Munsterberg, A.E., Koester, S., Goulding, M., and
Lassar, A.B. (1997). Ectopic Pax-3 activates MyoD and Myf-5 expression in
embryonic mesoderm and neural tissue. Cell 89, 139–148.
Marr, M.T., 2nd, D’Alessio, J.A., Puig, O., and Tjian, R. (2007). IRES-mediated
functional coupling of transcription and translation amplifies insulin receptor
feedback. Genes Dev. 21, 175–183.
McKinnell, I.W., Ishibashi, J., Le Grand, F., Punch, V.G., Addicks, G.C., Green-
blatt, J.F., Dilworth, F.J., and Rudnicki, M.A. (2008). Pax7 activates myogenic
genes by recruitment of a histone methyltransferase complex. Nat. Cell Biol.
10, 77–84. Published online December 9, 2007. 10.1038/ncb1671.
Mercado, G.E., and Barr, F.G. (2007). Fusions involving PAX and FOX genes in
the molecular pathogenesis of alveolar rhabdomyosarcoma: recent advances.
Curr. Mol. Med. 7, 47–61.
Morosetti, R., Mirabella, M., Gliubizzi, C., Broccolini, A., De Angelis, L., Taglia-
fico, E., Sampaolesi, M., Gidaro, T., Papacci, M., Roncaglia, E., et al. (2006).
MyoD expression restores defective myogenic differentiation of human meso-
angioblasts from inclusion-body myositis muscle. Proc. Natl. Acad. Sci. USA
103, 16995–17000.
Paddison, P.J., Cleary, M., Silva, J.M., Chang, K., Sheth, N., Sachidanandam,
R., and Hannon, G.J. (2004). Cloning of short hairpin RNAs for gene knock-
down in mammalian cells. Nat. Methods 1, 163–167.
Paik, J.H., Kollipara, R., Chu, G., Ji, H., Xiao, Y., Ding, Z., Miao, L., Tothova, Z.,
Horner, J.W., Carrasco, D.R., et al. (2007). FoxOs are lineage-restricted redun-
dant tumor suppressors and regulate endothelial cell homeostasis. Cell 128,
309–323.
Parthun, M.R., and Jaehning, J.A. (1990). Purification and characterization of
the yeast transcriptional activator GAL4. J. Biol. Chem. 265, 209–213.
Puig, O., and Tjian, R. (2005). Transcriptional feedback control of insulin recep-
tor by dFOXO/FOXO1. Genes Dev. 19, 2435–2446.
Puig, O., Marr, M.T., Ruhf, M.L., and Tjian, R. (2003). Control of cell number by
Drosophila FOXO: downstream and feedback regulation of the insulin receptor
pathway. Genes Dev. 17, 2006–2020.
Relaix, F., Rocancourt, D., Mansouri, A., and Buckingham, M. (2005). A Pax3/
Pax7-dependent population of skeletal muscle progenitor cells. Nature 435,
948–953.
546 Developmental Cell 15, 534–546, October 14, 2008 ª2008 Else
Roth, J.F., Shikama, N., Henzen, C., Desbaillets, I., Lutz, W., Marino, S.,
Wittwer, J., Schorle, H., Gassmann, M., and Eckner, R. (2003). Differential
role of p300 and CBP acetyltransferase during myogenesis: p300 acts
upstream of MyoD and Myf5. EMBO J. 22, 5186–5196.
Rudnicki, M.A., Schnegelsberg, P.N., Stead, R.H., Braun, T., Arnold, H.H., and
Jaenisch, R. (1993). MyoD or Myf-5 is required for the formation of skeletal
muscle. Cell 75, 1351–1359.
Sabourin, L.A., Girgis-Gabardo, A., Seale, P., Asakura, A., and Rudnicki, M.A.
(1999). Reduced differentiation potential of primary MyoD�/� myogenic cells
derived from adult skeletal muscle. J. Cell Biol. 144, 631–643.
Sandri, M., Sandri, C., Gilbert, A., Skurk, C., Calabria, E., Picard, A., Walsh, K.,
Schiaffino, S., Lecker, S.H., and Goldberg, A.L. (2004). Foxo transcription fac-
tors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal
muscle atrophy. Cell 117, 399–412.
Seale, P., Sabourin, L.A., Girgis-Gabardo, A., Mansouri, A., Gruss, P., and
Rudnicki, M.A. (2000). Pax7 is required for the specification of myogenic
satellite cells. Cell 102, 777–786.
Seoane, J., Le, H.V., Shen, L., Anderson, S.A., and Massague, J. (2004). Inte-
gration of Smad and forkhead pathways in the control of neuroepithelial and
glioblastoma cell proliferation. Cell 117, 211–223.
Tapscott, S.J. (2005). The circuitry of a master switch: Myod and the regulation
of skeletal muscle gene transcription. Development 132, 2685–2695.
Tothova, Z., Kollipara, R., Huntly, B.J., Lee, B.H., Castrillon, D.H., Cullen, D.E.,
McDowell, E.P., Lazo-Kallanian, S., Williams, I.R., Sears, C., et al. (2007).
FoxOs are critical mediators of hematopoietic stem cell resistance to physio-
logic oxidative stress. Cell 128, 325–339.
van der Heide, L.P., Jacobs, F.M., Burbach, J.P., Hoekman, M.F., and Smidt,
M.P. (2005). FoxO6 transcriptional activity is regulated by Thr26 and Ser184,
independent of nucleo-cytoplasmic shuttling. Biochem. J. 391, 623–629.
Wagers, A.J., and Conboy, I.M. (2005). Cellular and molecular signatures of
muscle regeneration: current concepts and controversies in adult myogenesis.
Cell 122, 659–667.
Weintraub, H., Tapscott, S.J., Davis, R.L., Thayer, M.J., Adam, M.A., Lassar,
A.B., and Miller, A.D. (1989). Activation of muscle-specific genes in pigment,
nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD. Proc.
Natl. Acad. Sci. USA 86, 5434–5438.
Weintraub, H., Dwarki, V.J., Verma, I., Davis, R., Hollenberg, S., Snider, L.,
Lassar, A., and Tapscott, S.J. (1991). Muscle-specific transcriptional activa-
tion by MyoD. Genes Dev. 5, 1377–1386.
Wilson, E.M., and Rotwein, P. (2006). Control of MyoD function during initiation
of muscle differentiation by an autocrine signaling pathway activated by
insulin-like growth factor-II. J. Biol. Chem. 281, 29962–29971.
Xuan, Z., and Zhang, M.Q. (2005). From worm to human: bioinformatics
approaches to identify FOXO target genes. Mech. Ageing Dev. 126, 209–215.
Yablonka-Reuveni, Z., Rudnicki, M.A., Rivera, A.J., Primig, M., Anderson, J.E.,
and Natanson, P. (1999). The transition from proliferation to differentiation is
delayed in satellite cells from mice lacking MyoD. Dev. Biol. 210, 440–455.
Yan, Z., Choi, S., Liu, X., Zhang, M., Schageman, J.J., Lee, S.Y., Hart, R., Lin,
L., Thurmond, F.A., and Williams, R.S. (2003). Highly coordinated gene regula-
tion in mouse skeletal muscle regeneration. J. Biol. Chem. 278, 8826–8836.
vier Inc.
top related