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Foxk1 promotes cell proliferation and repressesmyogenic differentiation by regulating Foxo4 and Mef2

Xiaozhong Shi1, Alicia M. Wallis1, Robert D. Gerard2, Kevin A. Voelker3, Robert W. Grange3,Ronald A. DePinho4, Mary G. Garry1 and Daniel J. Garry1,*1Lillehei Heart Institute, University of Minnesota-Twin Cities, Minneapolis, MN 55455, USA2Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA3Department of Human Nutrition, Foods and Exercise, Virginia, USA Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA4Belfer Institute for Applied Cancer Science, Departments of Medical Oncology, Medicine and Genetics, Dana-Farber Cancer Institute, HarvardMedical School, Boston, MA 02115, USA

*Author for correspondence (garry@umn.edu)

Accepted 9 August 2012Journal of Cell Science 125, 5329–5337� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.105239

SummaryIn response to severe injury, adult skeletal muscle exhibits a remarkable regenerative capacity due to a resident muscle stem/progenitorcell population. While a number of factors are expressed in the muscle progenitor cell (MPC) population, the molecular networks that

govern this cell population remain an area of active investigation. In this study, utilizing knockdown techniques and overexpression ofFoxk1 in the myogenic lineage, we observed dysregulation of Foxo and Mef2 downstream targets. Utilizing an array of technologies, weestablish that Foxk1 represses the transcriptional activity of Foxo4 and Mef2 and physically interacts with Foxo4 and Mef2, thus

promoting MPC proliferation and antagonizing the myogenic lineage differentiation program, respectively. Correspondingly,knockdown of Foxk1 in C2C12 myoblasts results in cell cycle arrest, and Foxk1 overexpression in C2C12CAR myoblasts retards muscledifferentiation. Collectively, we have established that Foxk1 promotes MPC proliferation by repressing Foxo4 transcriptional activity

and inhibits myogenic differentiation by repressing Mef2 activity. These studies enhance our understanding of the transcriptionalnetworks that regulate the MPC population and muscle regeneration.

Key words: Foxk1, Foxo4, Mef2, Cell proliferation, Cell differentiation

IntroductionAdult skeletal muscle is a dynamic and highly regenerative tissue

due to a resident myogenic progenitor cell (MPC) population

(Mauro, 1961). In response to a severe injury that involves more

than 90% of the muscle, the MPC population is capable of

completely restoring the cellular architecture within a three-week

period. Recent studies using genetic mouse models and

transcriptome analysis have identified molecular markers for

the MPC population that include Foxk1, CD29, C-met, integrin

alpha7, m-cadherin, Pax3, Pax7 and Syndecan3/4 (Biressi and

Rando, 2010; Shi and Garry, 2006). In addition, the C-met/Hgf,

Igf, Tgfb/Myostatin/Smad3/4, Notch/Numb signaling pathways

have also been shown to be essential for the MPC population

(Buckingham and Vincent, 2009; Ten Broek et al., 2010).

Despite these recent insights, the molecular networks that govern

the MPC population remain an area of active investigation.

Forkhead/winged helix transcription factors are known to exert

important regulatory functions in developmental processes

including the determination of cell fate, cell cycle kinetics, cell

differentiation and tissue morphogenesis (Hannenhalli and

Kaestner, 2009; Myatt and Lam, 2007; Wijchers et al., 2006;

Yang et al., 2009). We have previously established that Foxk1 is

restricted to the MSC/MPC population in adult skeletal muscle

(Garry et al., 1997). Foxk1 deficient mice have severely impaired

skeletal muscle regeneration, decreased number of muscle

progenitor cells, impaired progenitor cell activation, increased

expression of the cyclin dependent kinase inhibitor, p21, and

perturbed cell cycle kinetics of the muscle progenitor cell population

(Garry et al., 2000; Hawke et al., 2003a). Transgenic, molecular

biological and biochemical studies have demonstrated that Sox15 is

a potent transcriptional activator of Foxk1 in the myogenic

progenitor cell population, although Foxk1’s downstream

transcriptional program in this lineage has yet to be defined

(Meeson et al., 2007). Our recent studies have demonstrated that

Foxk1 recruits Sin3/Sds3 repression complex and functions to

activate the myogenic progenitor although the mechanisms are

incompletely defined (Shi and Garry, 2012; Shi et al., 2012).

Foxo proteins have been shown to have a broad functional role

in the regulation of catabolic pathways, cell cycle kinetics, cell

fate, aging and life span (Burgering, 2008; Ho et al., 2008;

Partridge and Bruning, 2008). Recent studies have demonstrated

that Foxo1 transgenic overexpression in skeletal muscle results in

decreased body size, decreased muscle mass and increased

atrogin 1 (ubiquitin ligase) expression (Kamei et al., 2004). In

addition, molecular biological and biochemical studies have

demonstrated that Foxo proteins directly interact with the Tgf-

beta downstream effectors, Smad3/4 and transcriptionally co-

activate the cyclin dependent kinase inhibitor, p21CIP and

maintain the hematopoietic stem cell population in a quiescent

state (Seoane et al., 2004; Tothova et al., 2007).

In the present study, we have utilized an array of techniques to

uncover the functional role of Foxk1 in the MPC population. We

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have knocked down Foxk1 using siRNA techniques andoverexpressed Foxk1 using a transgenic technique in the

myogenic lineage. Our analysis of Foxk1 knockdown andoverexpression revealed dysregulation of Foxo and Mef2downstream target genes, respectively. We demonstrate that

Foxk1 directly interacts with Foxo4 and represses Foxo4transcriptional activity, and that the repression of Foxo4 results

in decreased p21 expression and increased cellular proliferationof the MPC population. We further demonstrate that Foxk1 bindsto and represses Mef2c thereby restraining myogenic

differentiation. Collectively, our current data concerning Foxk1provide direct evidence for a specific role for members of thisForkhead gene family in the regulation of progenitor/stem cell

function.

ResultsFoxk1 is required for the cell cycle progression

Our previous studies have defined the expression of Foxk1 inmyogenic progenitor cells. Further, we have reported that loss of

Foxk1 resulted in the perturbation of skeletal muscle regenerationdue to impaired cell cycle regulation of the myogenic progenitor

cell population. To further define the underlying mechanisms forFoxk1, we knocked down Foxk1 in C2C12 cells using siRNAoligonucleotides. From the four candidates, we identified two

siRNA olgionucleotides, which efficiently knocked down Foxk1in C2C12 cells (Fig. 1A). Using these reagents, we analyzed theeffect of Foxk1 knockdown on cell cycle kinetics. As shown in

Fig. 1B, the knockdown of Foxk1 resulted in cell cycle arrestusing FACS analysis, which was further quantified in Fig. 1C.The gene expression studies revealed the upregulation of Foxo

target genes (Fig. 1D). In addition, we observed decreasedcellular proliferation with Foxk1 siRNA treatment (Fig. 1E).

Taken together, these data support the notion that Foxk1 has animportant functional role in the proliferation of the myogenicprogenitor cell population.

Foxk1 represses transcription through a DNA-bindingindependent mechanism

The interaction between the winged helix domain (WHD) ofFoxk1/Foxk2 and the consensus motif has been characterizedusing NMR spectroscopy and crystallography techniques

(Chuang et al., 2002; Liu et al., 2002; Tsai et al., 2006). Usingthese techniques, a number of conserved amino acid residueswithin the WHD have been shown to contact with the DNA (Liu

et al., 2002; Tsai et al., 2006). To further investigate thetranscriptional repression of Foxk1, we constructed two Foxk1

WHD (winged helix domain) mutants: K333A and R340A asthese conserved amino acids were important in DNA-binding(Liu et al., 2002; Tsai et al., 2006) (Fig. 2A). These mutations did

not affect the protein stability in vitro (Fig. 2B). We observedthat the DNA binding ability is attenuated in the K333A mutantand abolished in the R340A mutant (Fig. 2C). Transcriptional

assays revealed that both mutants did not affect the Foxk1repression activity in two distinct promoter-reporter constructs

(Fig. 2D,E). Collectively, these studies suggested that Foxk1represses transcription through a DNA-binding independentmechanism.

Foxk1 represses and interacts with Foxo4

Our above studies support the hypothesis that Foxk1 regulates

gene expression via Foxo proteins. To test our hypothesis, weused conventional transcriptional assays to evaluate the role ofFoxk1 on Foxo transcriptional activity. We transfected a

multimerized Foxo binding element (86FBE) fused to theluciferase reporter to evaluate Foxo transcriptional activity inthe presence and absence of Foxo factors and Foxk1 in C2C12

myoblasts. As expected, we observed that Foxo4 was a potenttranscriptional activator of gene expression (Fig. 3A) (Shi et al.,

2010). In a dose-dependent manner, Foxk1 repressed Foxo4transcriptional activity (Fig. 3A; supplementary material Fig.S1A). Knockdown of Foxk1 enhanced the transcriptional activity

Fig. 1. Foxk1 promotes cellular

proliferation. (A) Selection of Foxk1

siRNA oligonucleotides using qPCR

analysis. All four siRNA oligonucleotides

knocked down the endogenous Foxk1

mRNA. The No.1 and No. 2 siRNA

oligonucleotides were selected for further

analysis as they knocked down Foxk1 gene

expression with higher efficiency. Ctrl,

control. (B) Knockdown of Foxk1 using

siRNA results in G0/G1 cell cycle arrest.

The FACS profile is a representative sample

using siRNA No. 2. (C) The quantification

of the cell cycle phases from data presented

in panel B (*P50.02, n54). (D) The gene

expression profile was evaluated using

qPCR following siRNA No. 2 treatment.

The Foxo target genes are upregulated,

including Gadd45a, p21, p27 and p57. Note

that the expression of Foxo1, Foxo3a and

Foxo4 were not affected. (E) Growth curve

of C2C12 cells with Foxk1 siRNA No. 2

treatment. Ctrl, control.

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(supplementary material Fig. S1B). In addition, we utilized the

Gal4 reporter system, where Foxo4 was fused to the Gal4 DNA-

binding domain and Gal4 UAS-luc was the reporter (Sadowski

et al., 1992). We observed that Foxk1 repressed Gal4-Foxo4

activity in a dose-dependent fashion (supplementary material Fig.

S1C).

To uncover the regulatory mechanism of Foxo4 activity by

Foxk1, we first examined the protein interaction between Foxk1

and Foxo4 using a co-immunoprecipitation assay. As shown in

Fig. 3B, Foxk1 can be co-immunoprecipitated by Foxo4, and the

reverse is also true. Further studies revealed the interaction of

endogenous Foxk1 and Foxo4 (Fig. 3C). Using GST pulldown

assays, we observed that a construct which harbors the Forkhead

Associated (FHA) and WHD of Foxk1 interacts with Foxo4(Fig. 3D–F). Similarly, we demonstrated that Foxk1 directlyinteracted with the winged helix (DNA binding) domain of Foxo4

(Fig. 3G–I). To complement these binding studies, we examinedthe Foxk1 deletional constructs for their ability to repress Foxo4transcriptional activity. Using transcriptional assays, thetruncated Foxk1 construct, which harbors the FHA and DNA-

binding domains (81–406), which is capable of binding Foxo4, issufficient to repress Foxo4 activity (Fig. 3J; supplementarymaterial Fig. S1D). These functional studies are reinforced by

biochemical studies revealing that Foxk1 most avidly interactswith Foxo4, and to a lesser extent with Foxo1 and Foxo3a, inGST pulldown assays (Fig. 3K). Using transcriptional assays,

Foxk1 also represses the activity of Foxo1 and Foxo3a in a dose-dependent fashion (supplementary material Fig. S1E–F). Wepropose that Foxk1 governs gene expression via a DNA binding

independent mechanism, which is context dependent, therebymodulating the quiescent/proliferative state of the MPCpopulation. Collectively, these data support the hypothesis thatFoxk1 directly binds to the DNA-binding domain of Foxo4 and

represses Foxo4 transcriptional activity, thereby decreasing itsdownstream target genes including p21.

Perturbed skeletal muscle regeneration in Foxo4 null mice

Our previous studies have demonstrated that the skeletal muscleregeneration is delayed in p21 knockout mice (Hawke et al.,2003b). As the cell cycle inhibitor genes are downstream targets

of Foxo factors, we examined the muscle regeneration capacity inFoxo4 null mice. To examine the regenerative capacity of theFoxo4 mutant skeletal muscle, cardiotoxin was injected into the

gastrocnemius (GAS) muscles. In the wild-type skeletal muscle,the cellular architecture was restored within 2 weeks followingcardiotoxin injury. In contrast, the Foxo4 mutant skeletal musclehad perturbed regeneration that was evident with smaller

myofibers 2 weeks following cardiotoxin injury (Fig. 4A,B).To label proliferating cells two weeks following CTX injury, theinjured mice of wild type and Foxo4 null were pulsed with

BrdU for a 48-hour period (24 hours62) and the respectivegastrocnemius muscles were processed for BrdU and Myodimmunostaining (Fig. 4C). We observed increased numbers of

BrdU and Myod labeled myoblasts using morphological andquantitative assays (Fig. 4C,D). To analyze the gene expressionprofile, we isolated RNA from the primary myoblasts from

Foxo4 wild-type (WT) and null neonatal mice. Our datademonstrated that Foxo4 target genes (Gadd45a, p21, p27 andp57) were downregulated in Foxo4 null myoblasts using qRT-PCR analysis (Fig. 4E).

Overexpression of Foxk1 perturbs skeletal muscledifferentiation

Our previous studies demonstrated that Foxk1 expression is

downregulated during cell differentiation (Shi et al., 2010). Toexamine the functional role of Foxk1 in cell differentiation, weutilized the 4.8 kb MCK promoter to overexpress Foxk1 using

transgenic techniques as shown in Fig. 5A (Sternberg et al.,1988). Here, western blot analysis revealed abundant HA–Foxk1overexpression in the fast twitch extensor digitorum longus

(EDL), the slow twitch soleus (SOL) and the mixed fiber muscles[tibialis anterior (TA) and gastrocnemius (GAS)] of thetransgenic mice, and absence of HA–Foxk1 expression in the

Fig. 2. Transcriptional repression by Foxk1 is independent of its DNA-

binding capacity. (A) Schematic illustration of the mutation in the Foxk1

DNA-binding domain (WHD). (B) In vitro translated product of Foxk1 (wild-

type, WT) and its DNA-binding domain (WHD) mutants (K333A and

R340A). (C) The DNA-binding ability of Foxk1 is impaired in the Foxk1

mutant K333A, and abolished in the Foxk1 mutant R340A using an EMSA.

(D) The transcriptional repression by the Foxk1 mutants (K333A and R340A)

is similar to the wild-type control using the Fox-binding element reporter.

(E) A reporter construct which harbors a multimerized (86) fragment from

the p21 promoter is utilized in the transcriptional assays as described for panel

(D). Ctrl, control.

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wild-type controls (Fig. 5B). Initial transcriptome analysis

revealed the dysregulation of Mef2 target genes (data not

shown) (Black and Olson, 1998). Using qRT-PCR assays, we

have further verified the dysregulation of Mef2 downstream

target genes in Foxk1 TG muscle (Fig. 5C). In addition, we did

not observe any changes in Mef2 mRNA (Fig. 5B) or the

Mef2 protein in the Foxk1 overexpression transgenic mice

(supplementary material Fig. S2).

Evaluation of the transgenic skeletal muscle revealed

essentially normal skeletal muscle cellular architecture at 2

months of age (Fig. 5C). While we observed no evidence of

tissue degeneration, there were increased number of nuclei

associated with the transgenic muscle and occasional evidence of

centronucleated myofibers (Fig. 5D). Furthermore, we isolated

the primary myoblasts from the neonatal mice and examined their

capacity for differentiation. As shown in Fig. 5E,F, the cellular

differentiation (i.e. formation of multinucleated myotubes) is

reduced in Foxk1 TG myoblasts.

To further explore the functional role of Foxk1, we engineered

an adenoviral vector that overexpresses Foxk1. We infected

C2C12CAR myoblasts with experimental (Ad-Foxk1) and

control (Ad-GFP) viruses and exposed both samples to

differentiation media for 48 hours. We observed a relative

absence of multinucleated myotubes with Foxk1 overexpression

(Fig. 5G,H). In contrast, the sample infected with the GFP

expressing vector or the mock control had many multinucleated

myotubes (performed in triplicate and in three separate

experiments). To further support these morphological findings,

we harvested the respective samples and undertook western blot

analysis for Foxk1 and myogenic differentiation markers. As

shown in Fig. 5I, Ad-Foxk1 overexpression was associated with

decreased expression of myoglobin and MHC, which further

supports the hypothesis that Foxk1 retards muscle differentiation.

Foxk1 inhibits and binds to Mef2

Our above data implicated a dual role for Foxk1 in muscle

regeneration through the promotion of MPC expansion (i.e.

cellular proliferation) and restraining of myogenic differentiation

(through the repression of Mef2 activity). To test this hypothesis,

we utilized transcriptional assays and cotransfected a

multimerized Mef2-binding motif fused to the luciferase

reporter in the absence and presence of increasing amounts of

Fig. 3. Foxk1 represses and interacts with

Foxo4. (A) Transcriptional assays (using the

86FBE-luc) reveals that Foxk1 in a dose-

dependent fashion represses Foxo4 transcriptional

activity. (B) Using co-immunoprecipitation (IP)

assays, the protein interaction between tagged

Myc-Foxk1 and Flag-Foxo4 was confirmed in the

overexpression studies. WB, western blot.

(C) The endogenous Foxk1 and Foxo4 could also

form a complex in C2C12 myoblasts using co-IP

assays. (D) Schematic summary of the Foxk1

deletion constructs (FHA, forkhead domain

associated domain; WHD, winged helix domain).

(E) Coomassie blue staining of the purified GST-

Foxk1 deletions and the GST-control proteins.

(F) The GST pulldown assay reveals that the

construct containing the FHA and WHD (81–406)

interacts with Foxo4. (G) Schematic summarizing

of the Foxo4 deletional constructs. (H) Coomassie

blue staining of the purified GST-Foxo4 deletions

and the GST control protein. (I) GST pulldown

assay reveals that full length Foxo4 (1–505) and

the WHD (97–215) interact with Foxk1.

(J) Transcriptional assays further verify that the

Foxk1 truncated protein that contains the FHA

and WHD (81–406) fully represses Foxo4

transcriptional activity. (K) GST pulldown assays

reveal that Foxk1 could bind to all the members of

the Foxo1, Fox3a and Foxo4 family with various

affinities, and most avidly to Foxo4. Ctrl, control.

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Foxk1. We performed the studies with Mef2c as a representative

member of the Mef2 family. We observed that Foxk1, in a dose

dependent fashion, repressed Mef2c transcriptional activity

(Fig. 6A; supplementary material Fig. S3A). We further

analyzed the Foxk1 mediated repression in the Gal4-UAS

system. We observed that Foxk1 repressed Gal4-Mef2c activity

in a dose-dependent fashion (supplementary material Fig. S3B).

Using co-immunoprecipitation assays, we further determined that

Foxk1 interacted with Mef2c using an overexpression strategy or

endogenous protein in C2C12 myoblasts (Fig. 6B,C).

To map the interacting domains between Foxk1 and Mef2c, we

utilized GST pulldown assays. These assays revealed that a

construct that harbors both the FHA and WHD of Foxk1 directly

interacts with the MADS domain of Mef2c (Fig. 6D–H). Further,

we used these deletional mutants and transcriptional assays to

verify that the same Foxk1 deletional construct that interacted

with Mef2c also repressed Mef2c transcriptional activity (Fig. 6I;

supplementary material Fig. S3C). As the Mef2 MADS domain is

involved in DNA-binding and protein–protein interactions, we

hypothesized that Foxk1 prevents the interaction between Mef2c

and its DNA binding motif in the target genes or alternatively

Foxk1 represses the formation of the Mef2c transactivation

complex and inhibits the activation of the myogenic

differentiation program. To discriminate between these

possibilities, we utilized electrophoretic mobility shift assays

(EMSA) to examine the effect of Foxk1 on the Mef2c–DNA

interaction. As shown in supplementary material Fig. S3D, the

addition of Foxk1 reduced the formation of the high molecular

weight Mef2c complex without affecting the low molecular

complex. These studies support the notion that Foxk1 perturbs

the Mef2 transcriptional complex.

DiscussionMuscle progenitor cells reside in adult skeletal muscle and

promote tissue regeneration in response to an injury or disease.

While muscle progenitor cells have a tremendous proliferative

capacity, the molecular regulation of this cell population is

incompletely defined (Kuang and Rudnicki, 2008). In the

present study, we made three discoveries, which significantly

enhance our understanding regarding the molecular mechanisms

that govern the MPC population proliferation. Our first

discovery demonstrates that Foxk1 represses transcription

through a DNA-binding independent mechanism. This Foxk1

mechanism is via the interaction with Foxo4 resulting in the

repression of Foxo4 activity, thereby promoting MPC

proliferation. This protein–protein interaction confirmed that

the FHA and winged helix domains of Foxk1 interacted with the

winged helix domain of Foxo4 thereby repressing its

transactivation of its downstream target genes including the

cyclin dependent kinase inhibitor p21.

To date there are more than 300 members that belong to the

forkhead/winged helix transcription factor family based on

relative homology of a 110 amino acid DNA-binding domain

(also referred to as winged helix domain) since the discovery of

the original member Fkh in Drosophila (Clark et al., 1993;

Shimeld et al., 2010; Weigel and Jackle, 1990; Weigel et al.,

1989). Many of the forkhead/winged helix factors bind directly to

cognate binding motifs of genes and transactivate or repress gene

expression (Wijchers et al., 2006). Typically, these Fox factor

DNA binding mechanisms are mediated by interacting cofactors

that result in altered transcriptional responses (i.e. transcriptional

synergy through the interaction of Foxo factors and Smads)

(Gomis et al., 2006; van der Vos and Coffer, 2008). Some Fox

Fig. 4. Foxo4 null skeletal muscle has

increased cellular proliferation. (A) The

gastrocnemius muscles of Foxo4 wild-type

and null mice were injured with cardiotoxin

(CTX). The gastrocnemius muscles were

harvested 1 week (CTX-1w) or 2 weeks

(CTX-2w) following the injury. The

uninjured muscle was utilized as the control

(Uninjured). Note that the muscle

regeneration is perturbed in Foxo4 null

skeletal muscle compared with the wild-type

(WT) controls (scale bar, 50 mm).

(B) Quantification of the myofiber cross-

sectional area (CSA) in the regenerating

muscle in panel A (*P50.04; **P50.03;

n53). (C) There are increased numbers of

BrdU- and Myod-positive cells (indicated by

arrowheads) in the Foxo4 null skeletal muscle

compared with the WT control 2 weeks

following cardiotoxin injury. DAPI staining

indicates the nuclear compartment.

(D) Quantification of the BrdU+ and Myod+

cells in panel C (*P50.01; n53). (E) qRT-

PCR analysis reveals decreased expression

(Exp) of cell cycle inhibitors (i.e. p21

expression) in the Foxo4 null myoblasts

versus the WT controls (n53).

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family members modulate gene expression through protein–

protein interactions and have DNA-binding independent

functions (Foxe1, Foxg1, Foxp3, or Foxo-mediated protein

degradation) (Bettelli et al., 2005; Hanashima et al., 2002;

Perrone et al., 2000; Zhao et al., 2007). In the present study, we

propose that Foxk1 governs gene expression via a DNA-binding

independent mechanism, which is context dependent, thereby

modulating the quiescent/proliferative state of the MPC

population. Our data define one pathway whereby the MPC

population re-enters the cell cycle resulting in an increased

number of myogenic progenitors. This expansion of the

myogenic progenitors is a critical regenerative response in the

repair of damaged muscle.

Our second discovery revealed that the Foxo4 knockout

mouse had increased cellular proliferation following cardiotoxin

injury and decreased expression of cell cycle inhibitors

including p21CIP. Previous gene disruption studies have

verified that Foxo1 null embryos are lethal by E10.5 due to

vascular perturbations (Furuyama et al., 2004; Hosaka et al.,

2004) and mice lacking Foxo3a have perturbed ovarian

follicular development and are infertile (Castrillon et al.,

2003; Hosaka et al., 2004). However, initial analysis of the

Foxo4 null mouse revealed no overt phenotype (Hosaka et al.,

2004). Due to the redundancy and overlapping expression of the

Foxo factors, recent efforts were undertaken to conditionally

delete Foxo1, Foxo3a and Foxo4 (Paik et al., 2007; Tothova

et al., 2007). These studies revealed that lineage-specific loss of

the Foxo factors resulted in decreased number and impaired cell

cycle kinetics of the hematopoietic stem cell pool (Tothova et al.,

2007). These hematopoietic stem cell studies are conceptually

aligned with our findings in the myogenic lineage where we

demonstrate a prominent role for Foxo4 as a key cell cycle

regulator in the MPC population.

Transgenic technologies have been useful in uncovering the

physiological role of proteins in a temporal and spatial context.

Such a transgenic strategy was used to overexpress Foxo

members in the muscle lineage (Kamei et al., 2004; Skurk

et al., 2005). Enforced Foxo1 expression in the skeletal muscle

lineage resulted in smaller body size, reduced skeletal muscle

mass, perturbed fiber type diversity (i.e. a shift towards increased

number of oxidative slow twitch myofibers with myogenic Foxo1

overexpression) and an altered gene expression program that

enabled definition of Foxo transcriptome in this tissue (Kamei

et al., 2004). In contrast to the phenotype of the Foxo1

overexpression in skeletal muscle, our data demonstrate a

distinct phenotype for transgenic Foxk1 overexpression in the

skeletal muscle lineage that results in normal body size,

preserved cellular function but altered gene expression that

includes decreased expression of Foxo and Mef2 downstream

target genes (data not shown). This genetic strategy uncovered an

important functional mechanism for Foxk1 and its interacting

proteins in the myogenic stem/progenitor cell population.

Fig. 5. Overexpression of Foxk1 represses muscle differentiation. (A) Schematic representation of the transgenic construct using the muscle creatine kinase

(MCK) promoter to direct the HA–Foxk1 fusion protein to the myogenic lineage. (B) Western blot analysis of adult skeletal muscle isolated from wild-type (WT)

and transgenic (TG) mice. The anti-HA serum identifies the Foxk1 fusion protein in the extensor digitorum longus (EDL), gastrocnemius (GAS), soleus (SOL) and

tibialis anterior (TA) muscles. Anti-tubulin serum was used as the loading control. (C) qPCR analysis was utilized to examine the relative gene expression (Rel

Exp) of Mef2 downstream targets. (D) Representative histological analysis of skeletal muscle isolated from the 2-month-old transgenic male mice reveals normal

cellular architecture (n53) with occasional centronucleated myofibers (indicated by arrowheads; scale bar, 50 mm). (E) Differentiation of myogenic progenitor

cells is delayed in Foxk1 transgenic myogenic progenitor cells compared with the wild-type control (scale bar, 100 mm; myotubes are marked with arrowheads).

(F) Quantification of the cell fusion index in panel E (*P50.05, n53). (G) Using adenoviral vectors to overexpress Foxk1 (Ad-Foxk1) or GFP as a control (Ad-

GFP), we observed that overexpression of Foxk1 repressed muscle differentiation and the formation of multinucleated myotubes (scale bar, 100 mm).

(H) Quantification of the fusion index in panel G (*P50.01, n53). (I) Western blot analysis of the samples in panel G reveals that overexpression of Foxk1 results

in decreased expression of the myogenic differentiation program (decreased expression of MHC and myoglobin; tubulin was used as a loading control).

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Previous studies in vertebrates support a role for Mef2c in

skeletal myogenesis (Naya and Olson, 1999; Potthoff and Olson,

2007). Recent studies undertaken by Hughes and colleagues

demonstrated that simultaneous morpholino knockdown of

zebrafish Mef2c and Mef2d resulted in a loss of thick filament

proteins and the disruption of the sarcomeric structure (Hinits

and Hughes, 2007). In addition, it has been conclusively

demonstrated that Mef2c is an essential upstream

transcriptional activator of troponins in skeletal muscle and

myofiber identity (Bassel-Duby and Olson, 2006; Blais et al.,

2005). Conditional transgenic technologies have revealed a

broader role for Mef2c in cellular maintenance in various

lineages. The conditional deletion of Mef2c in skeletal muscle

lineages using a MCK-cre transgenic line resulted in a severe

decrease of type I fibers (Potthoff et al., 2007). Potthoff and

colleagues clearly demonstrated that HDAC-mediated inhibition

of Mef2c was the essential regulatory step in the conversion of

oxidative fibers to slow-twitch, non-oxidative, fast-twitch fibers.

The authors further demonstrated that the conditional loss of

Mef2c in the skeletal muscle lineage, results in decreased

expression of structural Mef2c target genes, including troponins I

and T and myomesin2. Our third discovery demonstrated that

Foxk1 interacted with the MADS domain of Mef2c and

precluded its activation of the myogenic differentiation

molecular program. Moreover, the overexpression of Foxk1resulted in a delay in myogenic differentiation. These resultssuggest that Foxk1 has a dual function within the MPC

population that includes the retardation of the differentiationprocess. In this fashion, the MPC population can expand to forma pool of MPCs that will respond to local cues and participate in

the regenerative process.

Collectively, these studies support a model whereby Foxk1directly interacts with Foxo4 and represses Foxo4 transcriptionalactivity (supplementary material Fig. S4). The repression of Foxo4

results in decreased p21 expression and increased cellularproliferation of the MPC population. We further demonstrate thatFoxk1 interacts with Mef2 and inhibits Mef2 transcriptional activity

thereby restraining myoblast terminal differentiation. Our currentdata concerning Foxk1 provide direct evidence for a specific dualrole for members of this extended gene family in the regulation of

progenitor/stem cell function and skeletal muscle regeneration. Inthis fashion, the MPC population can expand to form a pool ofMPCs that will respond to local cues and participate in theregenerative process (supplementary material Fig. S4).

Materials and MethodsDNA and RNA manipulation

Mef2c expression plasmids and the Mef2 reporter were kindly provided by Dr EricOlson (Molkentin et al., 1996; Naya et al., 1999). All of other plasmids were

Fig. 6. Foxk1 binds to Mef2 and inhibits Mef2 activity. (A) Transcriptional assays reveal that Foxk1, in a dose-dependent fashion, represses Mef2c

transcriptional activity. (B) Co-immunoprecipitation (IP) assays reveal the protein interaction between Myc–Mef2c and HA–Foxk1. WB, western blot. (C) The

protein interaction of endogenous Foxk1 and Mef2 is confirmed in C2C12 cells. (D) Schematic summary of the Foxk1 deletional constructs. (E) Coomassie blue

staining of the purified GST-Foxk1 deletional proteins. (F) The GST pulldown assay reveals that the construct containing the FHA and WHD (81–406) interacts

with Mef2c. (G) Schematic summary of the Mef2c deletional constructs. The activation domains are designated as TAD I and TAD II. (H) In vitro translated

Mef2c constructs (upper panel) and the GST pulldown assay (lower panel) reveal that the MADS domain of Mef2c interacts with Foxk1. (I) Transcriptional assays

further verify that the Foxk1 truncated protein that contains the FHA and WHD (81–406) represses Mef2c transcriptional activity.

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constructed by PCR and verified by DNA sequencing. An electrophoreticmobility-shift assay (EMSA) was done according to the protocol outlined in ourprevious studies. RNA extraction, cDNA synthesis, microarray, and qPCR wereperformed as previously described (Gallardo et al., 2003; Hawke et al., 2003b).

Western blot, co-immunoprecipitation, in vitro translation and GSTpulldown

Western blot and co-immunoprecipitation were performed as described in thestandard protocols with the following antibodies: anti-HA (Santa Cruz), anti-Myc(Santa Cruz), anti-Flag (Sigma), anti-Fox4 (Cell Signaling and Santa Cruz), anti-Mef2 (Santa Cruz), anti-tubulin (Sigma), anti-myoglobin (Dako), MF20(Hyridoma Bank), and anti-Foxk1 sera as preciously described (Shi et al., 2010).In vitro protein expression was performed with TNT Quick systems (Promega) asoutlined in the standard manual. GST pulldown assays utilized E. coli BL21expressing GST fusion proteins, which were extracted with B-PER bacterialProtein Extraction Reagent (Pierce Biochemicals) and then purified withglutathione-Sepharose CL-4B (GE Healthcare). GST fusion proteins bound toSepharose beads were incubated with 35S-labeled protein product and the BL21cell extract. The beads pulldown complex was washed (four times) andresuspended in sample loading buffer, analyzed using a 4–20% polyacrylamidegel and imaged as previously described (Shi et al., 2010).

Tissue culture, transcriptional assays and primary myoblast preparation

C2C12 myoblasts were cultured in 35 mm dishes containing DMEMsupplemented with 10% fetal bovine serum and penicillin/streptomycin.Approximately, 1.06105 of cells were transfected with 4 ml of lipofectamine(Invitrogen) and assayed for both luciferase and b-galactosidase activity.Luciferase assays were performed using the Promega Luciferase Assay Systemfollowing the manufacturer’s instructions. All fold changes in luciferase activitywere normalized to b-galactosidase activity, and to the vector alone as previouslydescribed (Alexander et al., 2010). All transfection experiments were performed intriplicate and replicated three times. Preparation of the primary myoblasts fromFoxk1 transgenic, Foxo4 null, or wild type neonates was performed as previouslydescribed (Shi et al., 2010). Myogenic differentiation was promoted by exposing todifferentiation medium (DMEM supplemented with 2% heat inactivated horseserum, antibiotics, insulin and transferrin) as previously described and wasevaluated immunohistochemically by anti-MHC serum (clone MF20). The fusionindex was defined as the ratio of the number of the nuclei in myotubes versus thetotal number of nuclei.

Adenoviral infection

The mouse Foxk1 cDNA was inserted under the control of the cytomegalovirus(CMV) promoter/enhancer upstream of an ires-GFP fragment to produce abicistronic pAC shuttle plasmid (Ivanciu et al., 2007). To construct a negativecontrol, no cDNA was inserted into this vector. Recombinant adenovirusesoverexpressing Foxk1 and GFP (Ad-Foxk1) or GFP alone (Ad-GFP) wereconstructed using cre-loxP recombination in vitro (Aoki et al., 1999). A single cellclone of C2C12CAR myoblasts stably transformed with a human coxsackieadenovirus receptor (CAR) expression plasmid (kindly provided by Dr SusanStevenson of Novartis) was infected with a multiplicity of 300 viruses per cell.

siRNA and cell cycle analysis

C2C12 cells were transfected with the Foxk1 siRNA oligonucleotides(Dharmacon) or with RISC (RNA-induced silencing complex)-free nontargetingduplexes as control, as previously described (Shi et al., 2010). The treated cellswere fixed with cold enthanol for FACS analysis or lysed with Tripure for RNAextraction. The cell cycle profiles were analyzed on a FACScan and processedwith Cell Quest software (Shi et al., 2010). For the cell growth analysis, C2C12cells were seeded 26104 cells/well into the 6-well plate 24 hours before siRNAtreatment, and harvested 72 hours later for quantification.

Animal care, cardiotoxin-induced muscle regeneration, BrdU pulse andhistology

All mice used were maintained, crossed, genotyped, injected and sacrificed inaccordance with an approved Institutional Animal Care and Use Committeeprotocol at the University of Minnesota. Cardiotoxin (CTX, Calbiochem) inducedmuscle injury/regeneration model in adult mouse is an established, reliable modelto study muscle regeneration (Goetsch et al., 2003). 100 ml CTX (10 mM) weredelivered using an intramuscular injection into the gastrocnemius (mixed fiber typemuscle group) of the adult 2-month-old male mice and the mice were sacrificed atdefined time periods: control (uninjured), one weeks and two weeks (n53 at eachtime period). BrdU labeling reagent was injected into mice (Invitrogen) via theintraperitoneal route at 48 hours and 24 hours prior to sacrifice. Mice wereanesthetized, perfusion fixed with 4% paraformaldehyde. The selected skeletalmuscle groups were harvested and fixed in 4% paraformaldehyde, paraffin-embedded, sectioned and stained with hematoxylin and eosin (H&E) to assessskeletal muscle fiber architecture. The muscle cross-sectional area (CSA) of the

regenerating gastrocnemius muscles was quantified using AxioVision 4.8. Forimmunostaining, the sections were incubated with a rat monoclonal anti-BrdUserum (AbD Serotec) and a polyclonal rabbit anti-Myod serum (Santa Cruz). Theprimary antisera were detected with species specific AlexaFluor 647 andAlexaFluor 594 fluorophore conjugated antisera (Jackson ImmunoResearch) andcoverslipped with Vectashield mounting medium with DAPI and imaged using the

Zeiss Axio Imager M1 microscope equipped with the AxioCam HRc camera andAxioVision 4.8 software as previously described (Meeson et al., 2007).

Generation of transgenic mice

The transgene construct (HA–Foxk1) was subcloned into the 4.8 kb MCKpromoter cassette, which harbors the MCK upstream 4.8 kb to +1 base pairfragment (Sternberg et al., 1988). Transgenic mice were generated by themicroinjection of the linearized constructs into fertilized F2 eggs (B6SJLF1;Jackson Labs), which were reimplanted into pseudopregnant F1 foster ICR females(Harlan) as previously described (Shi and Garry, 2010).

Statistics

Student’s t-tests were performed to identify significant difference (P,0.05) in dataobtained from control and experimental samples. Data are presented as mean 6

standard error of mean (SEM).

AcknowledgementsWe are grateful to Eric Olson (UT Southwestern Medical Center) forgenerously providing the Mef2c expression plasmids and Mef2creporter plasmids. We further acknowledge the support of JenniferL. Springsteen and Kathy M. Bowlin for the assistance with theimmunohistochemical analyses.

FundingFunding support was obtained from the National Institutes of Health(National Institute of Arthritis and Musculoskeletal and Skin) [grantnumbers 5R01AR047850 and 5R01AR055906 to D.J.G.]. R.A.D. issupported by the Robert A. and Renee E. Belfer Institute for AppliedCancer Science. Deposited in PMC for release after 12 months.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.105239/-/DC1

ReferencesAlexander, M. S., Shi, X., Voelker, K. A., Grange, R. W., Garcia, J. A., Hammer,

R. E. and Garry, D. J. (2010). Foxj3 transcriptionally activates Mef2c and regulates

adult skeletal muscle fiber type identity. Dev. Biol. 337, 396-404.

Aoki, K., Barker, C., Danthinne, X., Imperiale, M. J. and Nabel, G. J. (1999).

Efficient generation of recombinant adenoviral vectors by Cre-lox recombination in

vitro. Mol. Med. 5, 224-231.

Bassel-Duby, R. and Olson, E. N. (2006). Signaling pathways in skeletal muscle

remodeling. Annu. Rev. Biochem. 75, 19-37.

Bettelli, E., Dastrange, M. and Oukka, M. (2005). Foxp3 interacts with nuclear factor

of activated T cells and NF-kappa B to repress cytokine gene expression and effector

functions of T helper cells. Proc. Natl. Acad. Sci. USA 102, 5138-5143.

Biressi, S. and Rando, T. A. (2010). Heterogeneity in the muscle satellite cell

population. Semin. Cell Dev. Biol. 21, 845-854.

Black, B. L. and Olson, E. N. (1998). Transcriptional control of muscle development by

myocyte enhancer factor-2 (MEF2) proteins. Annu. Rev. Cell Dev. Biol. 14, 167-196.

Blais, A., Tsikitis, M., Acosta-Alvear, D., Sharan, R., Kluger, Y. and Dynlacht, B. D.

(2005). An initial blueprint for myogenic differentiation. Genes Dev. 19, 553-569.

Buckingham, M. and Vincent, S. D. (2009). Distinct and dynamic myogenic

populations in the vertebrate embryo. Curr. Opin. Genet. Dev. 19, 444-453.

Burgering, B. M. (2008). A brief introduction to FOXOlogy. Oncogene 27, 2258-2262.

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.

Chuang, W. J., Yeh, I. J., Hsieh, Y. H., Liu, P. P., Chen, S. W. and Jeng, W. Y.

(2002). 1H, 15N and 13C resonance assignments for the DNA-binding domain of

myocyte nuclear factor (Foxk1). J. Biomol. NMR 24, 75-76.

Clark, K. L., Halay, E. D., Lai, E. and Burley, S. K. (1993). Co-crystal structure of the

HNF-3/fork head DNA-recognition motif resembles histone H5. Nature 364, 412-420.

Furuyama, T., Kitayama, K., Shimoda, Y., Ogawa, M., Sone, K., Yoshida-Araki, K.,

Hisatsune, H., Nishikawa, S., Nakayama, K., Nakayama, K. et al. (2004).

Abnormal angiogenesis in Foxo1 (Fkhr)-deficient mice. J. Biol. Chem. 279, 34741-

34749.

Gallardo, T. D., Hammer, R. E. and Garry, D. J. (2003). RNA amplification and

transcriptional profiling for analysis of stem cell populations. Genesis 37, 57-63.

Journal of Cell Science 125 (22)5336

Journ

alof

Cell

Scie

nce

Garry, D. J., Yang, Q., Bassel-Duby, R. and Williams, R. S. (1997). Persistentexpression of MNF identifies myogenic stem cells in postnatal muscles. Dev. Biol.

188, 280-294.Garry, D. J., Meeson, A., Elterman, J., Zhao, Y., Yang, P., Bassel-Duby, R. and

Williams, R. S. (2000). Myogenic stem cell function is impaired in mice lacking theforkhead/winged helix protein MNF. Proc. Natl. Acad. Sci. USA 97, 5416-5421.

Goetsch, S. C., Hawke, T. J., Gallardo, T. D., Richardson, J. A. and Garry, D. J.

(2003). Transcriptional profiling and regulation of the extracellular matrix duringmuscle regeneration. Physiol. Genomics 14, 261-271.

Gomis, R. R., Alarcon, C., He, W., Wang, Q., Seoane, J., Lash, A. and Massague, J.

(2006). A FoxO-Smad synexpression group in human keratinocytes. Proc. Natl. Acad.

Sci. USA 103, 12747-12752.Hanashima, C., Shen, L., Li, S. C. and Lai, E. (2002). Brain factor-1 controls the

proliferation and differentiation of neocortical progenitor cells through independentmechanisms. J. Neurosci. 22, 6526-6536.

Hannenhalli, S. and Kaestner, K. H. (2009). The evolution of Fox genes and their rolein development and disease. Nat. Rev. Genet. 10, 233-240.

Hawke, T. J., Jiang, N. and Garry, D. J. (2003a). Absence of p21CIP rescuesmyogenic progenitor cell proliferative and regenerative capacity in Foxk1 null mice.J. Biol. Chem. 278, 4015-4020.

Hawke, T. J., Meeson, A. P., Jiang, N., Graham, S., Hutcheson, K., DiMaio, J. M.

and Garry, D. J. (2003b). p21 is essential for normal myogenic progenitor cellfunction in regenerating skeletal muscle. Am. J. Physiol. Cell Physiol. 285, C1019-C1027.

Hinits, Y. and Hughes, S. M. (2007). Mef2s are required for thick filament formation innascent muscle fibres. Development 134, 2511-2519.

Ho, K. K., Myatt, S. S. and Lam, E. W. (2008). Many forks in the path: cycling withFoxO. Oncogene 27, 2300-2311.

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) familymembers in mice reveals their functional diversification. Proc. Natl. Acad. Sci. USA

101, 2975-2980.Ivanciu, L., Gerard, R. D., Tang, H., Lupu, F. and Lupu, C. (2007). Adenovirus-

mediated expression of tissue factor pathway inhibitor-2 inhibits endothelial cellmigration and angiogenesis. Arterioscler. Thromb. Vasc. Biol. 27, 310-316.

Kamei, Y., Miura, S., Suzuki, M., Kai, Y., Mizukami, J., Taniguchi, T., Mochida,

K., Hata, T., Matsuda, J., Aburatani, H. et al. (2004). Skeletal muscle FOXO1(FKHR) transgenic mice have less skeletal muscle mass, down-regulated Type I (slowtwitch/red muscle) fiber genes, and impaired glycemic control. J. Biol. Chem. 279,41114-41123.

Kuang, S. and Rudnicki, M. A. (2008). The emerging biology of satellite cells and theirtherapeutic potential. Trends Mol. Med. 14, 82-91.

Liu, P. P., Chen, Y. C., Li, C., Hsieh, Y. H., Chen, S. W., Chen, S. H., Jeng, W. Y.

and Chuang, W. J. (2002). Solution structure of the DNA-binding domain ofinterleukin enhancer binding factor 1 (FOXK1a). Proteins 49, 543-553.

Mauro, A. (1961). Satellite cell of skeletal muscle fibers. J. Biophys. Biochem. Cytol. 9,493-495.

Meeson, A. P., Shi, X., Alexander, M. S., Williams, R. S., Allen, R. E., Jiang, N.,

Adham, I. M., Goetsch, S. C., Hammer, R. E. and Garry, D. J. (2007). Sox15 andFhl3 transcriptionally coactivate Foxk1 and regulate myogenic progenitor cells.EMBO J. 26, 1902-1912.

Molkentin, J. D., Black, B. L., Martin, J. F. and Olson, E. N. (1996). Mutationalanalysis of the DNA binding, dimerization, and transcriptional activation domains ofMEF2C. Mol. Cell. Biol. 16, 2627-2636.

Myatt, S. S. and Lam, E. W. (2007). The emerging roles of forkhead box (Fox) proteinsin cancer. Nat. Rev. Cancer 7, 847-859.

Naya, F. J. and Olson, E. (1999). MEF2: a transcriptional target for signaling pathwayscontrolling skeletal muscle growth and differentiation. Curr. Opin. Cell Biol. 11, 683-688.

Naya, F. J., Wu, C., Richardson, J. A., Overbeek, P. and Olson, E. N. (1999).Transcriptional activity of MEF2 during mouse embryogenesis monitored with aMEF2-dependent transgene. Development 126, 2045-2052.

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 redundanttumor suppressors and regulate endothelial cell homeostasis. Cell 128, 309-323.

Partridge, L. and Bruning, J. C. (2008). Forkhead transcription factors and ageing.

Oncogene 27, 2351-2363.

Perrone, L., Pasca di Magliano, M., Zannini, M. and Di Lauro, R. (2000). The

thyroid transcription factor 2 (TTF-2) is a promoter-specific DNA-binding

independent transcriptional repressor. Biochem. Biophys. Res. Commun. 275, 203-

208.

Potthoff, M. J. and Olson, E. N. (2007). MEF2: a central regulator of diverse

developmental programs. Development 134, 4131-4140.

Potthoff, M. J., Arnold, M. A., McAnally, J., Richardson, J. A., Bassel-Duby, R. and

Olson, E. N. (2007). Regulation of skeletal muscle sarcomere integrity and postnatal

muscle function by Mef2c. Mol. Cell. Biol. 27, 8143-8151.

Sadowski, I., Bell, B., Broad, P. and Hollis, M. (1992). GAL4 fusion vectors for

expression in yeast or mammalian cells. Gene 118, 137-141.

Seoane, J., Le, H. V., Shen, L., Anderson, S. A. and Massague, J. (2004). Integration

of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell

proliferation. Cell 117, 211-223.

Shi, X. and Garry, D. J. (2006). Muscle stem cells in development, regeneration, and

disease. Genes Dev. 20, 1692-1708.

Shi, X. and Garry, D. J. (2010). Myogenic regulatory factors transactivate the Tceal7

gene and modulate muscle differentiation. Biochem. J. 428, 213-221.

Shi, X. and Garry, D. J. (2012). Sin3 interacts with Foxk1 and regulates myogenic

progenitors. Mol. Cell. Biochem. 366, 251-258.

Shi, X., Bowlin, K. M. and Garry, D. J. (2010). Fhl2 interacts with Foxk1 and

corepresses Foxo4 activity in myogenic progenitors. Stem Cells 28, 462-469.

Shi, X., Seldin, D. C. and Garry, D. J. (2012). Foxk1 recruits the Sds3 complex and

represses gene expression in myogenic progenitors. Biochem. J. 446, 349-357.

Shimeld, S. M., Degnan, B. and Luke, G. N. (2010). Evolutionary genomics of the Fox

genes: origin of gene families and the ancestry of gene clusters. Genomics 95, 256-

260.

Skurk, C., Izumiya, Y., Maatz, H., Razeghi, P., Shiojima, I., Sandri, M., Sato, K.,

Zeng, L., Schiekofer, S., Pimentel, D. et al. (2005). The FOXO3a transcription

factor regulates cardiac myocyte size downstream of AKT signaling. J. Biol. Chem.

280, 20814-20823.

Sternberg, E. A., Spizz, G., Perry, W. M., Vizard, D., Weil, T. and Olson, E. N.

(1988). Identification of upstream and intragenic regulatory elements that confer cell-

type-restricted and differentiation-specific expression on the muscle creatine kinase

gene. Mol. Cell. Biol. 8, 2896-2909.

Ten Broek, R. W., Grefte, S. and Von den Hoff, J. W. (2010). Regulatory factors and

cell populations involved in skeletal muscle regeneration. J. Cell. Physiol. 224, 7-16.

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 physiologic oxidative

stress. Cell 128, 325-339.

Tsai, K. L., Huang, C. Y., Chang, C. H., Sun, Y. J., Chuang, W. J. and Hsiao, C. D.

(2006). Crystal structure of the human FOXK1a-DNA complex and its implications

on the diverse binding specificity of winged helix/forkhead proteins. J. Biol. Chem.

281, 17400-17409.

van der Vos, K. E. and Coffer, P. J. (2008). FOXO-binding partners: it takes two to

tango. Oncogene 27, 2289-2299.

Weigel, D. and Jackle, H. (1990). The fork head domain: a novel DNA binding motif of

eukaryotic transcription factors? Cell 63, 455-456.

Weigel, D., Jurgens, G., Kuttner, F., Seifert, E. and Jackle, H. (1989). The homeotic

gene fork head encodes a nuclear protein and is expressed in the terminal regions of

the Drosophila embryo. Cell 57, 645-658.

Wijchers, P. J., Burbach, J. P. and Smidt, M. P. (2006). In control of biology: of mice,

men and Foxes. Biochem. J. 397, 233-246.

Yang, X. F., Fang, P., Meng, S., Jan, M., Xiong, X., Yin, Y. and Wang, H. (2009).

The FOX transcription factors regulate vascular pathology, diabetes and Tregs. Front.

Biosci. (Schol Ed.) 1, 420-436.

Zhao, J., Brault, J. J., Schild, A., Cao, P., Sandri, M., Schiaffino, S., Lecker, S. H.

and Goldberg, A. L. (2007). FoxO3 coordinately activates protein degradation by the

autophagic/lysosomal and proteasomal pathways in atrophying muscle cells. Cell

Metab. 6, 472-483.

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