The homeobox transcription factor MSX2 partially mediatesthe effects of bone morphogenetic protein 4 (BMP4) onsomatic cell reprogrammingReceived for publication, May 9, 2018, and in revised form, August 2, 2018 Published, Papers in Press, August 10, 2018, DOI 10.1074/jbc.RA118.003913

Lilong Lin‡§¶�**1, Lining Liang‡§¶�**1, Xiao Yang‡§¶�**1, Hao Sun‡§¶�, Yuan Li‡§¶�, Duanqing Pei‡§¶�,and Hui Zheng‡§¶�**2

From the ‡Chinese Academy of Sciences Key Laboratory of Regenerative Biology, Joint School of Life Sciences, GuangzhouInstitutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China, Guangzhou Medical University,Guangzhou 511436, China, the §University of the Chinese Academy of Sciences, Beijing 100049, China, the ¶Guangdong ProvincialKey Laboratory of Stem Cell and Regenerative Medicine, Guangzhou 510530, China, the �Guangzhou Regenerative Medicine andHealth-Guangdong Laboratory (GRMH-GDL), Guangzhou 510530, China, and the **Institutes for Stem Cell and Regeneration,Chinese Academy of Sciences, Beijing 100101, China

Edited by Joel Gottesfeld

Bone morphogenetic proteins (BMPs) induce mesenchymal–epithelial transition (MET) and enhance the generation ofinduced pluripotent stem cells (iPSCs). However, BMPs are alsosignaling molecules critical for arresting reprogramming in thepre-iPSC state. In this study, using mouse embryonic fibro-blasts, we found that the time- and concentration-dependenteffects of BMPs on reprogramming are mediated by Mshhomeobox 2 (MSX2), a homeobox-containing transcription fac-tor. BMPs up-regulated Msx2 by activating SMAD1/5, andMSX2 then directly bound to the promoters and up-regulatedthe expression of the cadherin 1 (Cdh1, also known as E-cad-herin), GATA-binding protein 3 (Gata3), and Nanog genes.Cdh1 contributed to BMP4- and MSX2-induced MET andsubsequently promoted reprogramming. On the other hand,GATA3 promoted reprogramming, possibly by up-regulatingSpalt-like transcription factor 4 (SALL4) expression. As keytranscriptional factors in maintaining pluripotency, up-regula-tion of SALL4 and NANOG enhanced reprogramming. More-over, the ability of MSX2 to up-regulate Cdh1, Gata3, Nanog,and Sall4 was further potentiated in the presence of Krüppel-like factor 4 (KLF4). However, MSX2 did not mediate the effectsof BMP4 signaling on activation of the microRNAs miR-205 andmiR-200 or the inhibitory effects that arrested reprogramming

in the pre-iPSC state. In conclusion, MSX2 partially mediatesthe effects of BMP4 signaling during reprogramming, improv-ing our understanding of the role of BMP signaling in MET andof the connection between cell lineage specifiers such as MSX2and GATA3 and pluripotency.

The mechanisms underlying the generation of inducedpluripotent stem cells (iPSCs)3 have been studied for a longtime. At first, only pluripotency-associated factors were con-sidered as potential inducers or mediators of reprogram-ming (1–3). Later studies suggested that lineage specifiersare able to substitute for particular Yamanaka factors in pro-moting iPSC generation (4, 5). Thus, the balance of differentlineage-specifying forces, such as for the mesoendodermallineage and ectodermal lineage, may maintain pluripotency(5, 6).

MSX2, a homeobox-containing transcription factor (7, 8),controls the mesendoderm lineage commitment of humanPSCs by suppressing Sox2 expression and inducing Nodalexpression (9). In addition, Msx2 plays a crucial role in cranio-facial, limb, and ectodermal organogenesis, and knockout ofMsx2 results in developmental defects (10, 11). Thus, it is pos-sible that Msx2 also has beneficial roles during reprogrammingas a lineage specifier. In addition, as both a transcriptionalrepressor and activator (9, 14, 15), Msx2 is a downstream targetof bone morphogenetic protein (BMP) signaling and possiblymediates the effects of BMP signaling during reprogramming(16, 17).

BMPs play important roles in early embryonic patterning(18, 19) and modulate lineage commitment toward tropho-blast or mesoderm in embryonic stem (ES) cells (16, 20, 21).However, BMPs have complex and sometimes controversial

This work was supported by the National Natural Science Foundation ofChina (U1601228, 31671475, 31422032, and 31421004); the StrategicPriority Research Program of the Chinese Academy of Sciences(XDA16000000); the Key Research Program of Frontier Sciences, Chi-nese Academy of Sciences (QYZDB-SSW-SMC031); the Guangdong Nat-ural Science Foundation (2014A030308002); and the Science and Tech-nology Planning Project of Guangdong Province (2017B030314056).This work was also supported by the Guangzhou branch of the Super-computing Center of the Chinese Academy of Sciences. The authorsdeclare that they have no conflicts of interest with the contents of thisarticle.

This article contains Figs. S1–S6 and Tables S1 and S2.The results of the current RNA-seq experiments were deposited in the NCBI

Gene Expression Omnibus database under accession number GSE114120.1 These authors contributed equally to this work.2 To whom correspondence should be addressed: Guangzhou Institutes of

Biomedicine and Health, Chinese Academy of Sciences, 190 Kaiyuan Ave.,Science City, Guangzhou 510530, China. Tel.: 86-20-32015334; Fax: 86-20-32015231; E-mail: zheng_hui@gibh.ac.cn.

3 The abbreviations used are: iPSC, induced pluripotent stem cell; AP, alkalinephosphatase; BMP, bone morphogenetic protein; ES cell, embryonicstem cell; MEF, mouse embryonic fibroblast; miR, microRNA; MET,mesenchymal– epithelial transition; EMT, epithelial–mesenchymal transi-tion; shRNA, short hairpin RNA; TSS, transcription start site; GO, gene ontol-ogy; qPCR, quantitative PCR; RNA-seq, RNA sequencing; FBS, fetal bovineserum; ANOVA, analysis of variance.

croARTICLE

J. Biol. Chem. (2018) 293(38) 14905–14915 14905© 2018 Lin et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.

by guest on March 14, 2020

http://ww

w.jbc.org/

Dow

nloaded from

functions during reprogramming, from mouse embryonicfibroblasts (MEFs) to iPSCs (22). BMP-dependent inductionof miR-205 and miR-200a/b/c leads to mesenchymal–epithelial transition (MET), which has been suggested to bea critical step during the initial phase of reprogramming(23). In addition to MET, BMP-dependent signaling alsoenhances the expression of Nanog and Sall4, which arerequired for the establishment of pluripotency in MEFs(24, 25).

Colonies with ESC-like morphology but without activationof the endogenous locus encoding Oct4 were identified as pre-iPSCs during reprogramming (26, 27). BMPs have been identi-fied as critical signaling molecules in arresting reprogrammingin the pre-iPSC state (28). Thus, not all BMP-dependent signal-ing is beneficial for reprogramming. In addition, the BMP4 –MSX2 axis promotes mesodermal commitment by inducingepithelial–mesenchymal transition (EMT), which is not consis-tent with BMP’s functions during reprogramming. The abilityof Msx2 to induce both MET and EMT has been observed indifferent experimental systems (29, 30), possibly because of thedifferent partners with which MSX2 associates. Therefore, inthis study, by using reprogramming from MEFs to iPSCs as amajor experimental model, we elucidated the mechanismsunderlying modulation of the BMP4 –MSX2 axis during repro-gramming and how Msx2 mediates the beneficial or inhibitoryroles of BMPs.

Results

Msx2 mediates the beneficial effects of BMP signaling

Different concentrations of BMP4 and LDN-193189, aninhibitor of the BMP signaling pathway, were used at differentperiods during reprogramming from MEFs to iPSCs. A lowconcentration of BMP4 (2.5 ng/ml) promoted reprogrammingat the early stage (days 0 –5) but had little effect at the middle(days 5–10) or late (days 10 –15) stages (Fig. 1A). A high con-centration of BMP4 (10 ng/ml) inhibited reprogramming con-stantly (Fig. 1A). However, LDN-193189 impaired reprogram-ming at all tested concentrations and mainly at the early stageduring reprogramming (Fig. 1B), which suggested that a low tomedium level of activation of BMP signaling was required forreprogramming. In summary, the beneficial roles of BMP sig-naling could be observed only at relatively low concentrationsand at an early stage during reprogramming.

We then checked the expression of various factors in theBMP pathway (Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway 04350) based on a previously reportedmicroarray dataset (GSE21757) (22). Of the 36 genes ana-lyzed, only four genes were up-regulated to more than 2-foldor down-regulated to below 50% of basal levels (Fig. S1A).This phenomenon supports the hypothesis that low tomedium levels of activation of the BMP pathway are requiredfor reprogramming.

Day0-1

5

Day5-1

0

Day10

-150

100

200

300

GFP

+C

olon

ies

(%of

Con

trol

) Msx2sh-Msx2

***

***

***

Control

**

**

ControlMsx

2

Msx2-N

H

Msx2-H

C

Msx2-H

Msx2-M

U0

50100150200250300350

GFP

+C

olon

ies

***

***

*** **

*

ns

***

Day0-1

5

Day0-5

Day5-1

0

Day10

-150

50

100

150

200

GFP

+C

olon

ies

2.5 ng/ml BMP45.0 ng/ml BMP410 ng/ml BMP4

***

***

***

Control

***

*** ***

Day0-1

5

Day0-5

Day5-1

0

Day10

-150

15

30

45

6080

100120

GFP

+C

olon

ies

1.25�g/ml LDN2.55.0

***

******

Control

***

***

***

***

***

***

***

***

***

MEF

Repro.

0

5

10

15

20

25

30

Expr

essi

onofMsx2

(%of

Con

trol

)

2.5 ng/ml BMP45.0 ng/ml BMP410 ng/ml BMP4

***

***

***

Control

***

***

***

ControlMsx

1Msx

2Msx

30

100

200

300

400

GFP

+C

olon

ies

***

***

***

***

***

A B C

Msx2

D

E F G H1 142 201 267

N-terminal C-terminal

Homeobox

Msx2-NH

Msx2-HC

Msx2-H

Msx2-MUN-192-IR-194-K

Con

trol

sh-L

ucsh

-Msx

1sh

-Msx

2sh

-Msx

30

50

100

150

200

GFP

+C

olon

ies

***

***

***

***

2.5 ng/ml BMP4

***

�g/ml LDN�g/ml LDN

Figure 1. Msx2 mediated the beneficial functions of BMP4. A–C, different concentrations of BMP4 and LDN-193189 were used for different periodsduring reprogramming. The ability of BMP4 to up-regulate Msx2 were determined by qPCR on day 4 in both in MEFs and during reprogramming. D andE, Msx1/2/3 were overexpressed during reprogramming without BMP4 or suppressed during reprogramming with 2.5 ng/ml BMP4. GFP� colonies werecounted on day 15. F, the expression of Msx2 was modulated for different periods during reprogramming. GFP� colonies were counted on day 15. G andH, mutations of MSX2 with deletion of different domains were constructed. Their functions during reprogramming were determined. All experimentswere repeated at least five times. One-way ANOVA with Dunnett’s post hoc test was used. *, p � 0.05; **, p � 0.01; ***, p � 0.001; ns, not significant.

Msx2 mediates BMP signals

14906 J. Biol. Chem. (2018) 293(38) 14905–14915

by guest on March 14, 2020

http://ww

w.jbc.org/

Dow

nloaded from

We then determined the ability of BMP4 to up-regulatedownstream effectors such as Msx1/2/3 (16, 17). As indicatedin Fig. 1C and Fig. S1, B and C, BMP4 up-regulated Msx1/2/3 inconcentration-dependent manner and up-regulated Msx2more than Msx1 and Msx3. Retroviruses encoding Msx genesand shRNAs against them were delivered into MEFs. Msx2 hada higher ability to promote reprogramming than Msx1 andMsx3 (Fig. 1D). In addition, only shRNA against Msx2, but notagainst Msx1 or Msx3, significantly attenuated the ability of 2.5ng/ml BMP4 to promote reprogramming (Fig. 1E). The GFP�

colonies generated with Msx2 overexpression had typical char-acteristics of iPSCs, including suppressed expression of exoge-nous Yamanaka factors, high expression of endogenous pluri-potent factors, normal karyotypes, low DNA methylation ofNanog and Oct4 promoters, and the ability to generate chimeramice (Fig. S2).

When retroviruses encoding Msx2 and the correspondingshRNA were delivered into MEFs at different time points dur-ing reprogramming, larger modulations on reprogrammingwere observed when infections were performed at the begin-ning of reprogramming (Fig. 1F). Therefore, Msx2 also regu-lates reprogramming at the early stage, which is consistent withthe functions of BMP4.

Retroviruses encoding different truncations of MSX2 wereprepared and tested (Fig. 1G). Both the C terminus and homeo-box domain, a DNA binding domain, were required for MSX2to promote reprogramming (Fig. 1H) (15). In addition, mutat-ing the DNA binding site of MSX2 (N192A and T194K) totallydestroyed its ability to promote reprogramming, and thehomeobox domain functioned as a dominant-negative factorwhen used alone. Therefore, Msx2 mediates the beneficial rolesof BMP signaling during reprogramming by functioning as atranscriptional factor.

BMP4 up-regulates Msx2 via the conventional Smad1/5cascade

Phosphorylation of SMAD1/5/8 are conventional down-stream cascades of the BMP pathway. It was reasonable to sug-gest a direct pathway from BMP4 to Msx2 via phosphorylatedSMAD1/5/8. To confirm this, shRNAs against Smad1 andSmad5 were used. On one hand, either shRNA against Smad1or against Smad5 inhibited the ability of 2.5 ng/ml BMP4 topromote reprogramming, possibly because of the ability ofthese shRNAs to block BMP4-induced up-regulation of Msx2both in MEFs and during reprogramming (Fig. 2, A and B).

On the other hand, overexpression of Smad1 or Smad5 pro-moted the generation of iPSCs, which could be attenuated bysuppressing Msx2 (Fig. 2C). This phenomenon was consistentwith the ability of Smad1 and Smad5 to increase the expressionof Msx2 both in MEFs and during reprogramming (Fig. 2D). Aluciferase assay with the Msx2 promoter further supported tar-geting of SMAD1/5 to Msx2. Overexpression of Smad1 orSmad5 increased the activity of the WT Msx promoter but notthat of the mutated promoter (deletion in �773 to �768 rela-tive to the transcriptional start site (TSS)) (Fig. 2E). Therefore,Msx2 is a direct downstream target of BMP4 and SMAD1/5 notonly in MEFs but also during reprogramming.

Msx2 slightly promoted MET by targeting Cdh1

Retroviruses encoding Msx2 and the corresponding shRNAswere used during reprogramming, and gene expression profileswere analyzed on day 4 and day 8 (GSE114120) (Table S1).MEFs and R1 ES cells were used as controls.

We focused on genes whose expression in R1 ES cells wasmore than 2-fold or less than 50% of that in MEFs (Fig. 3A).Introducing Msx2 and the corresponding shRNA did notchange the overall progress of reprogramming but did inducesome expression changes (Fig. 3A).

Genes that were significantly regulated by Msx2 (expressionin the Msx2 group was more than 2-fold or less than 50% of thatin the control group) were subjected to gene ontology (GO)analysis. Genes up-regulated by Msx2 on day 4 were enrichedfor GO terms linked to the extracellular matrix, lung develop-ment, ion binding, and regulation of the biosynthetic process,whereas down-regulated genes were enriched for tube develop-ment and regulation of transcription (Fig. 3B). Of the 593up-regulated genes, 425 genes (71.7%) had higher or equalexpression in R1 ES cells compared with MEFs. Of the 579down-regulated genes, 419 genes (72.4%) had lower or equalexpression in R1 ES cells compared with MEFs (Fig. 3C). Thus,the changes in gene expression profiles were consistent with theability of Msx2 to promote reprogramming. Among the genes

Con

trol

sh-L

ucsh

-Sm

ad1

sh-S

mad

50

50

100

150

200

GFP

+C

olon

ies

***

* *

******

2.5 ng/mlBMP4

Control

Smad1

Smad1+

sh-M

sx2

Smad5

Smad5+

sh-M

sx2

0

100

200

300

400

500

GFP

+C

olon

ies

***

***

***

***

***

***

A

MEF

Repro.

0

5

10

15

20

Expr

essi

onofMsx2

(%of

Con

trol

)

Smad1Smad5

***

***

Control

***

***

MEF

Repro.

0

1

2

3

4

5

Rel

ativ

eEx

pres

sion

ofMsx2

(%of

DM

SO)

sh-Lucsh-Smad1

***

Control

***

sh-Smad5

***

******

****

******

2.5 ng/mlBMP4

B C

D E Luciferase-1600 +120

Msx2 promoter

-779 GCTGGGTCTGCTGG -766 Msx2-mu

Msx2 pro Msx2 pro-mu0

2

4

6

8

Luci

fera

seA

ctiv

ity(F

old

ofC

ontr

ol)

BMP4Smad1***

Control

***

Smad5

***

**

BMP4+sh-Smad1BMP4+sh-Smad5**

*

******

Figure 2. Msx2 was downstream of the BMP4 –SMAD1/5 axis. A and B,shRNA against luciferase, Smad1, or Smad5 was used in MEFs or during repro-gramming in the presence of 2. 5 ng/ml BMP4. Msx2 expression and repro-gramming efficiency were determined. C and D, a lentivirus encoding Smad1,or Smad5 was used in MEFs or during reprogramming with or without shRNAagainst Msx2. Msx2 expression and reprogramming efficiency were deter-mined. E, a promoter of Msx2 (�1.6 to �0.12 kb relative to the TSS) was usedto drive the expression of luciferase, which was determined when 2.5 ng/mlBMP4 was used or the expression of Smad1/5 was modulated. All experimentswere repeated at least five times. One-way ANOVA with Dunnett’s post hoctest was used. *, p � 0.05; **, p � 0.01; ***, p � 0.001.

Msx2 mediates BMP signals

J. Biol. Chem. (2018) 293(38) 14905–14915 14907

by guest on March 14, 2020

http://ww

w.jbc.org/

Dow

nloaded from

regulated by Msx2, we identified multiple genes, like Cdh1,Gata3, Nanog, and Sall4 (Fig. 3D), that are beneficial for repro-gramming (3, 23, 25, 31).

Cdh1, also known as E-cadherin, is an important marker ofepithelial cells and plays critical roles in MET during earlyreprogramming (23, 32). In addition, BMP signaling promotesreprogramming by facilitating MET at least partially (22). Thus,it was reasonable for us to suggest that Msx2 mediates the abil-ity of BMP signaling to facilitate MET.

Three epithelial markers and eight mesenchymal markerswere monitored after Msx2 overexpression with qPCR. Sevenmarkers were significantly regulated by Msx2, and all detectedmodulations on these markers suggested promoted MET (Fig. 3E).Another method that calculates the changes of epithelial and mes-enchymal characteristics of cells using the expression of selectedmarkers was also used (33). Based on current RNA-seq results, anincrease in the epithelial score and decrease in the mesenchymalscore were observed after Msx2 overexpression (Fig. 3F).

A

Cdh1Gata

3

Nanog

Sall4

0.0

0.5

1.03

6

9

Expr

essi

on(F

old

ofC

ontr

ol)

ControlMsx2sh-Msx2

***

***

***

***

***

***

***

***

-3.0

-1.5

0

1.5

3.0

Expr

essi

on (L

og v

s M

EF)

2

MEFContro

l

Msx2

sh-M

sx2

Control

Msx2

sh-M

sx2

R1 ESC

Day 4 Day 8

Extrac

ellular

matrix

Lung develo

pment

Ion binding

Regulat

ion of biosyntheti

c process

Tube develo

pment

Regulat

ion of Transc

riptio

n0

1

2

3

4

Enric

hmen

tSco

res

Up-regulation

Down-regulation

B

Up-

regu

late

d by

M

sx2

on d

ay4

Dow

n-re

gula

ted

by

Msx

2 on

day

4

C

258168

167

247160

172

Up

Down

No change

Down

Up

No change

R1 ESCs vs MEFs

R1 ESCs vs MEFs

D E

F G

MEF

Control

Msx2

sh-M

sx2-2

-1

0

1

2

Epith

elia

l/Mes

ench

ymal

Scor

esR

elat

ive

toM

EF

MesenchymalEpithelial

00

H I J

Krt8Cldn3

Ocln Cdh2 Fn1Snai

1Snai

2Zeb

1Zeb

2Twist

1

Twist2

0

50

100

150

200Ex

pres

sion

(%of

Con

trol

) *** Msx2

sh-Msx2

Control

***

***

******

*** ***

***

***

***

ControlMsx

2

sh-M

sx2

05

101520253035

Mig

ratio

n(�

m/c

ellp

erho

ur)

***

ns

ControlMsx

2

sh-M

sx2

0

20

40

60

80

100

120

Tran

swel

l(%

ofC

ontr

ol)

***

ns

ControlBMP4

BMP4+sh

-Msx

2

Smad1

Smad1+

sh-M

sx2

05

101520253035

Mig

ratio

n(�

m/c

ellp

erho

ur)

*** ***

*** ***

******

ControlBMP4

BMP4+sh

-Msx

2

Smad1

Smad1+

sh-M

sx2

0

20

40

60

80

100

120

Tran

swel

l(%

ofC

ontr

ol)

*** ***

*****

***

***

Msx2

Actin

Sall4

Nanog

Gata3

Cdh1

Control

Msx2

sh-M

sx2

100kD

55kD

35kD

100kD

130kD

25kD

35kD

40kD

Figure 3. Msx2 up-regulated genes with the ability to promote reprogramming. A–C, a lentivirus encoding Msx2 or shRNA against Msx2 was delivered toMEFs. The expression profiles of cells were assayed on day 4 and day 8 during reprogramming with RNA-seq. MEFs and R1 ES cells were used as controls. Geneshaving significantly higher (over 2-fold) or lower (below 50%) expression in R1 ES cells than in MEFs were analyzed (A). Genes that were significantly regulatedby Msx2 on day 4 were subjected to GO analysis (B) and categorized into three groups based on their expression in R1 ES cells and MEFs (C). D and E, a lentivirusencoding Msx2 or shRNA against Msx2 was delivered to MEFs. The expression of the indicated genes was determined with qPCR on day 4 of reprogramming.F, epithelial and mesenchymal scores relative to MEFs were calculated basing current RNA-seq. G–J, the ability of cells to migrate was determined with atranswell assay or with a live-cell tracing system that measured the migration distances of cells. Cells were modulated from different aspects of BMP4 –Smad1–Msx2. All experiments except RNA-seq were repeated at least five times. One-way ANOVA with Dunnett’s post hoc test was used. **, p � 0.01; ***, p � 0.001; ns,not significant.

Msx2 mediates BMP signals

14908 J. Biol. Chem. (2018) 293(38) 14905–14915

by guest on March 14, 2020

http://ww

w.jbc.org/

Dow

nloaded from

The migration ability of cells was determined with a tran-swell assay and live imaging system. Overexpression of Msx2decreased the migration ability of cells, and shRNA againstMsx2 attenuated the ability of BMP4 to modulate cell migration(Fig. 3, G–J). Therefore, Msx2 mediates the ability of BMP sig-naling to facilitate partial MET.

Because shRNA against Msx2 did not fully attenuate the abil-ities of BMP4 to promote reprogramming and to modulate cellmigration (Figs. 1E and 3, I and J), BMP signaling might facili-tate MET via additional pathways, such as the miR-205 andmiR-200 families, as suggested in a previous report (22). As indi-cated in Fig. S3, 2.5 ng/ml BMP4, but not Msx2, increased theexpression of miR-205 and miR-200a/b/c. In addition, shRNAagainst Msx2 did not affect the ability of BMP signaling to affectmiRNAs. Therefore, Msx2 partially mediates the ability of BMPsignaling to facilitate MET, possibility by up-regulating Cdh1.

Msx2 mediates the effect of BMP4 on Cdh1, Gata3, Nanog, andSall4

Overexpression of Msx2 up-regulates Cdh1, Gata3, Nanog,and Sall4 and subsequently promotes reprogramming. How-ever, whether BMP signaling promotes reprogramming simi-larly requires further investigation.

BMP4 at a 2.5 ng/ml concentration and a retrovirus encodingSmad1 up-regulated the expression of Cdh1, Gata3, and Nanogboth in MEFs and during reprogramming (Fig. 4, A–C). shRNAagainst Msx2 attenuated this up-regulation, which confirmedthat both BMP signaling and Msx2 promoted reprogrammingby activating Cdh1, Gata3, and Nanog.

However, significant up-regulation of Sall4 was only ob-served during reprogramming, but not in MEFs (Fig. 4D), pos-sibly because Gata3, but not Msx2, was the direct upstreamregulator of Sall4 (31). Actually, overexpression of Gata3 up-regulated Sall4 both in MEFs and during reprogramming (Fig.4E). shRNA against Gata3 attenuated the ability of Msx2 toup-regulate Sall4 during reprogramming (Fig. 4E). Thus, Msx2up-regulates Sall4 by up-regulating Gata3.

We then used the Cdh1, Gata3, Nanog, and Sall4 promoters(�1. 6 to �0.12 kb relative to the TSS) to drive luciferaseexpression. The potential binding sites of MSX2 were predictedand deleted on these promoters based on the information pro-vided by JASPAR 2018 (Fig. 4F) (12). A luciferase assay con-firmed direct targeting of MSX2 to Cdh1, Gata3, and Nanogpromoters but not Sall4 promoter (Fig. 4G). By using primerssurrounding the predicted binding sites (Table S2), ChIP–qPCR analysis further confirmed the targeting (Fig. 4H).

The targeting of GATA3 to the Sall4 promoter was con-firmed in a similar manner. Overexpression of Gata3 activatedthe WT Sall4 promoter. Mutating either the binding domain ofGATA3 or the binding motif on the Sall4 promoter diminishedthe increase in luciferase expression (Fig. 4I). ChIP–qPCR exper-iments further confirmed the binding of GATA3 to the Sall4 pro-moter (Fig. 4J). Therefore, both BMP4 and Msx2 up-regulateCdh1, Gata3, and Nanog and up-regulates Sall4 via Gata3.

In addition, overexpression of Msx1 during reprogrammingup-regulated Cdh1 and Nanog (Fig. S4). Overexpression ofMsx3 during reprogramming only up-regulated Cdh1 (Fig. S4).This up-regulation was less than that induced by Msx2 (Fig. 3D).

Therefore, although Msx1/2/3 are expressed in overlappingdomains and function similarly under certain circumstances,Msx2 is a more important mediator for BMP signaling duringreprogramming.

Klf4 potentiates the ability of Msx2 to activate downstreamfactors

When the abilities of Msx2 to up-regulate Cdh1, Gata3,Nanog, and Sall4 were compared, it was found that Msx2induced larger up-regulation during reprogramming than inMEFs (Fig. 5A). Thus, it was reasonable to suggest cooperationbetween MSX2 and an additional factor or factors in activatingdownstream factors.

Two groups of genes, genes that were up-regulated by Msx2on day 4 during reprogramming (day 4 up) and genes that wereup-regulated by Msx2 and also had higher expression in R1 EScells than in MEFs (day 4 up and ES cells up), were then ana-lyzed with Pscan (13). Of the four Yamanaka factors, only theKLF4 binding motif was enriched in the promoters of bothgroups of genes (Fig. 5B). Thus, Klf4 could help Msx2 to activatedownstream factors.

We further tested whether KLF4 and MSX2 cooperate toup-regulate genes like Cdh1, Gata3, Nanog, and Sall4. Msx2had a higher ability to up-regulate Cdh1, Gata3, Nanog, andSall4 in MEFs in the presence of Klf4 (Fig. 5, C and D). In addi-tion, Msx2 induced larger up-regulation of Cdh1, Gata3, Nanog,and Sall4 during reprogramming with Oct4, Klf4, c-Myc, and Sox2(OKMS) than during reprogramming with Oct4, c-Myc, and Sox2(OMS) (Fig. 5, E and F). Thus, Klf4 potentiated the ability of Msx2to activate downstream factors. Similar observations were notmade when potential cooperation between Msx2 and other threeYamanaka factors was tested (Fig. S5).

Members of the GATA family can induce somatic cell repro-gramming independent of Oct4 (31). Whether Msx2 canreplace Oct4 or other Yamanaka factors to reprogram somaticcells to iPSCs was determined. Msx2 could replace Oct4, Klf4,or Sox2 to reprogram somatic cells to iPSCs (Fig. S6). MoreiPSCs were generated when Oct4, Klf4, or Sox2 was replacedwith Gata3, possibly because overexpression of Msx2 only up-regulated Gata3 by about 6- to 8-fold (Fig. 4B). However, Msx2can replace Oct4 to induce reprograming.

Msx2 does not mediate the inhibitory roles of BMP duringreprogramming

BMPs have been identified as critical molecules to arrestreprogramming in the pre-iPSC state (28). This function of theBMP pathway explains the fact that 2.5 ng/ml BMP4 not onlyincreased the number of AP�GFP� colonies but also increasedthat of AP�GFP� colonies (pre-iPSCs colonies) (Fig. 6, A andB). In addition, high concentrations of BMP4 decreased thenumber of AP�GFP� colonies but increased that of AP�GFP�

colonies (Fig. 6, A and B). However, overexpression of Msx2 didnot increase the number of AP�GFP� colonies, and shRNAagainst Msx2 did not attenuate the ability of BMP4 to increasethe number of AP�GFP� colonies (Fig. 6, C and D).

Actually, overexpression of Msx2 facilitated the conversionfrom pre-iPSCs to iPSCs. Vitamin C-sensitive and -insensitivepre-iPSCs colonies established in a previous report were used

Msx2 mediates BMP signals

J. Biol. Chem. (2018) 293(38) 14905–14915 14909

by guest on March 14, 2020

http://ww

w.jbc.org/

Dow

nloaded from

(28). Vitamin C at 50 �g/ml concentration was used to inducethe conversion from pre-iPSCs to iPSCs. BMP4 attenuated andMsx2 facilitated this process (Fig. 6, E and F). Therefore, Msx2does not mediate the ability of BMP signaling to arrest cells inthe pre-iPSC state.

Discussion

In summary, Msx2 mediated the ability of BMP signaling(at low concentration and at an early stage) to up-regulateCdh1, Gatas-Sall4, and Nanog and to promote reprogram-

ming from MEFs to iPSCs (Fig. 7). The inability of Msx2 toup-regulate miR-205 and miR-200a/b/c and to arrest cells inthe pre-iPSC state suggested that Msx2 did not mediate theinhibitory effects of BMP signaling during reprograming(Figs. S3 and S6). Although we did not test all functions ofBMP signaling during reprogramming, it was still reasonableto conclude that Msx2 mediated the beneficial roles of BMPsignaling during reprogramming.

Msx2 is just one member of the Msh family, and the Mshfamily has two additional members, Msx1 and Msx3. MSX1/

MEF

Repro.

0

2

4

6

8

10

Expr

essi

onofGata 3

(Fol

dof

Con

trol

)

BMP4BMP4+sh-msx2

***

ns

Control

***

**

Smad1Smad1+sh-Msx2Msx2

***

******

***

***

***

***

***

******

MEF

Repro.

0

1

2

3

4

5

Expr

essi

onofNanog

(Fol

dof

Con

trol

)

BMP4BMP4+sh-msx2

***

ns

Control

***

**

Smad1Smad1+sh-Msx2Msx2

***

******

***

******

***

***

******

MEF

Repro.

0

1

2

3

4

5

Expr

essi

onofSall4

(Fol

dof

Con

trol

)

BMP4BMP4+sh-msx2

Control Smad1Smad1+sh-Msx2Msx2

***

***

***

***

***

******

GADPHCdh1

Gata3

Nanog

Sall4

0

1

2

3

4

5

%of

Inpu

t

@IgG@Msx2

GAPDHSall

40

1

2

3

4

5

%of

Inpu

t

@IgG@Gata3

MEF

Repro.

0

1

2

3

4

5

Expr

essi

onofCdh1

(Fol

dof

Con

trol

)

BMP4BMP4+sh-msx2

***

ns

Control

***

**

Smad1Smad1+sh-Msx2Msx2

*********

***

***

***

***

*********

A B C

D

Sall4 pro

Sall4 pro-m

u20

100

200

300

400

500

600

Luci

fera

seA

ctiv

ity(%

ofC

ontr

olin

wild

type

prom

oter

)

***

ControlGata3Gata3-MU

Cdh1 pro

Cdh1 pro-mu

Gata3 pro

Gata3 pro-m

u

Nanog pro

Nanog pro-m

u

Sall4 pro

Sall4 pro-m

u10

100

200

300

400

500

Luci

fera

seA

ctiv

ity(%

ofC

ontr

olin

wild

type

prom

oter

)

***

***

***

ControlMsx2Msx2-MU

Luciferase-1600 +120

Cdh1 pro

-653 AAAATTAGATTAGC -740

Luciferase-1600 +120

-404 TGGAAATTAGCTTT -391

Luciferase-1600 +120

-674 TACATTAATTTAAA -661

Luciferase-1600 +120

-926 AGCCAATTAAACTT -913

Luciferase-1600 +120

+12 TCATGATAAATCGC +25

Cdh1 pro-mu

Gata3 pro

Gata3 pro-mu

Nanog pro

Nanog pro-mu

Sall4 pro

Sall4 pro-mu1

Sall4 pro

Sall4 pro-mu2Fo

r Msx

2Fo

r Gat

a3

F

G HI J

MEF

Repro.

0

2

4

6

8

Rel

ativ

eEx

pres

sion

ofSall4

(%of

DM

SO)

Gata3Msx2+sh-Luc

Control

Msx2+sh-Gata3

***

***

***

***

***

E

***

***

***

***

***

***

Figure 4. Msx2 promoted reprogramming by up-regulating several pluripotency-related genes. A–D, 2.5 ng/ml BMP4 and a lentivirus encoding Msx2,sh-Msx2, or sh-Smad1 were used in MEFs or during reprogramming. The expression of Cdh1, Gata3, Nanog, and Sall4 was determined with qPCR on day 4. E, alentivirus encoding Msx2, Gata3, or sh-Gata3 was used in MEFs or during reprogramming. The expression of Sall4 was determined with qPCR on day 4. F,promoters (�1.6 to �0.12 kb relative to the TSS) of Cdh1, Gata3, Nanog, and Sall4 were constructed. The binding sites of MSX2 or GATA3 with the bestprediction were indicated and deleted. G and H, the responses of WT and mutated promoters of Cdh1, Gata3, Nanog, and Sall4 to WT and mutated MSX2 weredetermined (G). The binding between MSX2 and regions on the promoters indicated in F was assayed with ChIP– qPCR (H). I and J, the responses of WT andmutated promoters of Sall4 to WT and mutated GATA3 were determined (I). The binding between GATA3 and regions on the promoters indicated in F wasassayed with ChIP– qPCR (J). All experiments were repeated at least five times. Two-tailed Student’s t test (H–J) and one-way ANOVA with Dunnett’s post hoctest were used. **, p � 0.01; ***, p � 0.001; ns, not significant.

Msx2 mediates BMP signals

14910 J. Biol. Chem. (2018) 293(38) 14905–14915

by guest on March 14, 2020

http://ww

w.jbc.org/

Dow

nloaded from

2/3 have similar binding motifs and similar functions (7, 12).Thus, we speculated that these three proteins have a similarfunction during reprogramming. However, although BMP4did up-regulate Msx1 and Msx3 in a concentration-depen-dent manner, and Msx1 and Msx3 did promote reprogram-ming, their connection with BMP4 and reprogramming wasnot as tight as that of Msx2. For example, shRNAs againstMsx1 and Msx3 did not affect the function of BMP4. Thus,we suggested that Msx2 mediated the majority of the bene-ficial roles of BMP signaling. However, we did not excludethe possibility of Msx1 and Msx3 mediating the function of

BMP signaling, such as up-regulating Cdh1, Gata3, Nanog,and Sall4. In addition, Msx1 and Msx3 may mediate theother functions of BMP signaling, such as up-regulatingmiR-205 and miR-200a/b/c and arresting cells in the pre-iPSC state. Therefore, further investigation is required toanswer these questions.

In addition to core pluripotency regulators such as Oct4,Sox2, and Nanog, lineage specifiers such as GATA familymembers have also been suggested to substitute for particu-lar Yamanaka factors and to promote iPSCs generation (4, 5).Thus, the balance of different lineage-specifying forces, such

A B

Oct4

Oc t4::Sox2 Klf4

c-Myc

Sox20

1

2

3

Enric

hmen

t Sco

res

Day 4 UpDay 4 UpiPSCs Up

C D

E

B

F

Cdh1Gata

3

Nanog

Sall4

0

5

10

15

20

25

Expr

essi

on(F

old

ofC

ontr

ol)

ControlMsx2Klf4Msx2+Klf4

******

***

***

***

***

*** ***

***

***

***

Cdh1Gata

3

Nanog

Sall4

0

2

4

6

8

Expr

essi

on D

iffer

ence

(bet

wee

n in

dica

ted

grou

ps)

Msx2 vs ControlKlf4+Msx2 vs Klf4

***

***

***

***

Cdh1Gata

3

Nanog

Sall4

0

1

2

3

4

Expr

essi

onC

hang

e(in

MFE

svs

durin

gR

epro

.)

BMP4 ratioSmad1 ratioMsx2 ratio

Cdh1Gata

3

Nanog

Sall4

0

5

10

15

20

25

Expr

essi

on(F

old

ofC

ontr

ol)

ControlMsx2Klf4Msx2+Klf4

******

***

***

***

***

***

***

***

***

Cdh1Gata

3

Nanog

Sall4

0

2

4

6

8

Msx2 vs ControlKlf4+Msx2 vs Klf4

***

***

***

***

in M

EFs

durin

g re

pro.

with

OM

S

Expr

essi

on D

iffer

ence

(bet

wee

n in

dica

ted

grou

ps)

Figure 5. Klf4 potentiated the functions of Msx2. A, the ability of 2. 5 ng/ml BMP4, Smad1, and Msx2 to up-regulate Cdh1, Gata3, Nanog, and Sall4 duringreprogramming was normalized to that in MEFs based on the results summarized in Fig. 4, A–D. B, enrichment of the binding motifs of OCT4, KLF4, c-MYC, andSOX2 was determined with Pscan software. C and D, Klf4, Msx2, or both Klf4 and Msx2 were overexpressed in MEFs. The expression of Cdh1, Gata3, Nanog, andSall4 was determined on day 4 (C). The ability of Msx2 to up-regulate these genes was calculated by performing the comparisons indicated in D. E and F, Klf4,Msx2, or both Klf4 and Msx2 were overexpressed during reprogramming with only OMS. The expression of Cdh1, Gata3, Nanog, and Sall4 was determined onday 4 (C). The ability of Msx2 to up-regulate these genes was calculated by performing the comparisons indicated in D. All experiments were repeated at leastfive times. One-way ANOVA with Dunnett’s post hoc test was used. ***, p � 0.001.

Msx2 mediates BMP signals

J. Biol. Chem. (2018) 293(38) 14905–14915 14911

by guest on March 14, 2020

http://ww

w.jbc.org/

Dow

nloaded from

as those for the mesoendodermal and ectodermal lineages,may maintain pluripotency (5, 6). Both Msx2 and BMP sig-naling have been reported to modulate lineage commitmenttoward mesoderm in ES cells (9, 16, 20, 21). Therefore, it wasexpected to observe beneficial roles of Msx2 and BMP4 inpromoting reprogramming.

As an upstream regulator, BMP4 has more functions thanMsx2, which partially explains why Msx2 only mediates partof the beneficial roles of BMP signaling. Another explana-tion, based on the “seesaw” model (5), is that BMP signalingis a strong committer for the mesoderm lineage, whichresults in imbalance when used at high concentrations. As

a downstream effect of BMP signaling, Msx2 has a lowerability to induce mesoderm lineage commitment and imbal-ance between pluripotency factors and/or counteracting lin-eage specifiers, which leads to the observation that Msx2only mediates the beneficial roles of BMP signaling duringreprogramming.

Other homeobox transcription factors that also func-tion as lineage specifiers may also affect reprogramming.Their function in cell fate conversion may provide a newunderstanding of pluripotency and homeobox transcriptionfactors and will enrich the foundation of regenerativemedicine.

ControlBMP4

BMP4+sh

-Msx

2

Smad1Msx

20

200

400

600

GFP

+C

olon

ies

*** *** ***

ns

***

AP+ GFP

+

AP+ GFP

-0

50100150200300

600

900

Col

onie

s

***

***

***

***

***

0

20

40

60

80

100

120

%of

AP+ G

FP- C

olon

ies 2.5 ng/ml BMP4

5.0 ng/ml BMP410 ng/ml BMP4

ns

*** Control

***

AP+ GFP

+

AP+ GFP

-0

50100150200

300

500

700

Col

onie

s ***

***

***

***

***

***

ns

*** ns

0

20

40

60

80

100

AP+ G

FP- C

olon

ies

(%)

Msx2BMP4BMP4+sh-Msx2

Control

***

ControlBMP4

BMP4+sh

-Msx

2

Smad1Msx

20

50

100

150

GFP

+C

olon

ies

*** *** ***

ns

***

Vc-s

ensi

tive

pre-

iPSC

s

Vc-in

sens

itive

pre

-iPSC

s

A B

C D

E F

Figure 6. Msx2 did not mediate the functions of BMP4 to generate more pre-iPSCs. A and B, different concentrations of BMP4 were used duringreprogramming. AP�GFP� and AP�GFP� colonies were counted on day 15 (A). The percentages of AP�GFP� colonies (pre-iPSCs) were calculated (B). C and D,the expression of Msx2 was modulated during reprogramming with or without 2.5 ng/ml BMP4. AP�GFP� and AP�GFP� colonies were counted on day 15 (C).The percentages of AP�GFP� colonies (pre-iPSCs) were calculated (D). E and F, vitamin C–sensitive (E) and -insensitive (F) pre-iPSCs colonies were converted toiPSCs with 50 �g/ml vitamin C. The ability of BMP4, Smad1, and Msx2 to affect this conversion was determined. All experiments were repeated at least five times.One-way ANOVA with Dunnett’s post hoc test was used. ***, p � 0.001; ns, not significant.

Msx2 mediates BMP signals

14912 J. Biol. Chem. (2018) 293(38) 14905–14915

by guest on March 14, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Experimental procedures

Materials

The chemicals, antibodies, and primers used in this study arelisted in Table S2.

Animal studies

Experiments with mice were performed to obtain MEFs andto determine the pluripotency of the generated iPSCs. Ourstudy followed the Guidelines for the Care and Use of Labora-tory Animals of the National Institutes of Health. The protocolswere approved by the Committee on the Ethics of AnimalExperiments of the Guangzhou Institutes of Biomedicine andHealth. All efforts were made to minimize animal discomfort.

Generation of mouse iPSCs

MEFs were derived from 13.5-day-old mouse embryos car-rying the Oct4-GFP transgenic allele. MEFs, human embryonickidney 293T cells, and Plat-E cells were maintained in FBSmedium. FBS medium was supplemented with Dulbecco’smodified Eagle’s medium (Gibco), 10% FBS (Gibco), 1% nones-sential amino acids (Gibco), and 1% L-glutamine.

MEFs within two passages were seeded into a 6-well or12-well plate (2.5 � 104 or 1.5 � 104 cells/well). MEFs wereinfected with a retrovirus (produced with Plat-E cells andpMXs-based retroviral vectors as reported previously). MESmedium was used during reprogramming and was supple-mented with Dulbecco’s modified Eagle’s medium, 15% FBS, 1%Glutamax, 1% nonessential amino acids, 1% sodium pyruvate,0.1 mM 2-mercaptoentanol, leukemia inhibitory factor, and 50�g/ml vitamin C (Sigma).

After adding Polybrene to 4 �g/ml, the viral supernatantwas used for infection. iPSC colonies were counted or pickedaccording to their Oct4-GFP expression and ES cell–likemorphology.

Cell line characterization

Characterization of iPSCs was performed as reported previ-ously (32). Primary antibodies against NANOG and REX1 wereused together with the appropriate Alexa-conjugated second-ary antibodies. Immunofluorescence results were obtainedusing a Zeiss LSM 800 confocal microscope.

For karyotype analysis, demecolcine (50 �g/ml, Sigma) wasadded to the cells for 1 h. The cells were trypsinized, pelleted,resuspended in 0.075 M KCl, and incubated for 20 min at 37 °C.The cells were then fixed with acetic acid:methanol (1:3) for 10min at 37 °C. The cells were collected by centrifugation, resus-pended in fixative solution, dropped on a cold slide, and incu-bated at 75 °C for 3 h. The slides were treated with trypsin andcolorant, and metaphase chromosomes were analyzed using anOlympus BX51 microscope.

RNA-seq

RNA was extracted from cells using TRIzol reagent (Invitro-gen). Illumina mRNA-seq libraries were prepared for each RNAsample using the TruSeq RNA Sample Preparation Kit v2; themRNA-seq libraries were then sequenced on an IlluminaNextSeq 500 instrument with the NextSeq 500 Mid OutputKit v2.

qPCR

Total RNA was extracted from cells using TRIzol (Invitro-gen), and 5 �g of RNA was used to synthesize complementaryDNA with ReverTra Ace� (Toyobo) and oligo(dT) (Takara)according to the manufacturer’s instructions. The transcriptlevels of genes were determined using SYBR Premix Ex TaqII(Tli RNaseH Plus) (Takara) and a CFX-96 real-time system(Bio-Rad). The primers used are listed in Table S2.

ChIP– qPCR

Cells were cross-linked with 1% formaldehyde for 10 min atroom temperature. After quenching formaldehyde with 125mM glycine for 5 min, cells were washed twice with cold PBS.Cells were then lysed in ChIP buffer A (50 mM HEPES–KOH(pH 7.3), 140 mM NaCl, 1 mM EDTA (pH 8.0), 10% glycerol,0.5% NP-40, 0.25% Triton X-100, and protease inhibitor mix-ture) for 10 min at 4 °C. Samples were centrifuged at 1,400 � gfor 5 min at 4 °C. Pellets were resuspended in ChIP buffer B (1%SDS, 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, and proteaseinhibitor mixture) for 10 min at 4 °C and sheared by sonication.

Sheared chromatin was centrifuged (12,000 � g for 1 min at4 °C) to discard the pellets, and the supernatant was dilutedwith ChIP buffer (0.01% SDS, 1% Triton X-100,2 mM EDTA, 50mM Tris-HCl (pH 8.0), 150 mM NaCl, and protease inhibitormixture) to make the final SDS concentration lower than 0.1%.Antibodies were coupled to Dynabeads with protein A or G formore than 3 h at 4 °C in PBS supplemented with 0.01% Tween20, and beads were washed with PBS supplemented with 0.01%Tween 20.

Antibodies were incubated with sheared chromatin over-night at 4 °C. After immunoprecipitation, beads were washedwith low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM

EDTA, 20 mM Tris-HCl (pH 8.0), and 150 mM NaCl), high-

BMP4

BMPR

Smad1/5

Nanog

Gata3 Sall4

Cdh1 MET

Reprogramming

Msx2Klf4

Figure 7. Msx2 mediated the functions of BMP4 to up-regulate severalpluripotency-related genes. BMP4 induces up-regulation of Msx2 viaSMAD1/5. MSX2 cooperates with KLF4 to up-regulate Cdh1, Gata3-Sall4, andNanog. Cdh1 promotes reprogramming by facilitating MET, whereas Sall4 andNanog promote reprogramming by functioning as transcriptional factors thatare critical in maintaining pluripotency.

Msx2 mediates BMP signals

J. Biol. Chem. (2018) 293(38) 14905–14915 14913

by guest on March 14, 2020

http://ww

w.jbc.org/

Dow

nloaded from

salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20mM Tris-HCl (pH 8.0), and 500 mM NaCl), LiCl wash buffer(0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, and 10mM Tris-HCl (pH 8.1)), and TE buffer (10 mM Tris-HCl and 1mM EDTA (pH 8.0)). DNA was extracted and used for analysis.The sequences of all primers used in this study are given inTable S2.

Data deposition

The current RNA-seq were performed on days 4 and 8during reprogramming with modulations on the expressionof Msx2. The results were deposited in GEO under accessionnumber GSE114120. A previously reported microarray dataset(GSE21757) was also used (22).

Statistical analysis

All experiments were repeated at least five times (n � 5), withthe exception of RNA-seq and chimera experiments. The datawere analyzed and compared using a two-tailed t test or one-way ANOVA with Dunnett’s post hoc test in GraphPad Prism7.0. Error bars and n represent the standard deviation (standarderror if indicated) and the number of independent experiments,respectively. Asterisks represent significant differences (*, p �0.05; **, p � 0.01; ***, p � 0.001) from the indicated controlgroups.

For control samples, when BMP4 was dissolved in DMSOand was used to treat cells, we used the same amount of DMSOto treat control samples; when Msx2 or other genes were over-expressed, a retrovirus encoding FLAG was introduced to con-trol samples; when shRNA against Msx2 or other genes wasoverexpressed, a retrovirus encoding shRNA against luciferasewas introduced to control samples; and control samples wereboth treated with DMSO and infected with a retrovirus encod-ing FLAG and shRNA against luciferase when complex treat-ment was studied.

Author contributions—L. Lin, L. Liang, X. Y., H. S., and Y. L. formalanalysis; L. Lin, L. Liang, X. Y., H. S., Y. L., and H. Z. investigation;X. Y. writing-original draft; D. P. and H. Z. funding acquisition; D. P.and H. Z. project administration; H. Z. supervision; H. Z. writing-review and editing.

References1. Takahashi, K., and Yamanaka, S. (2006) Induction of pluripotent stem

cells from mouse embryonic and adult fibroblast cultures by defined fac-tors. Cell 126, 663– 676 CrossRef Medline

2. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda,K., and Yamanaka, S. (2007) Induction of pluripotent stem cells from adulthuman fibroblasts by defined factors. Cell 131, 861– 872 CrossRefMedline

3. Yu, J., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane,J. L., Tian, S., Nie, J., Jonsdottir, G. A., Ruotti, V., Stewart, R., Slukvin, I. I.,and Thomson, J. A. (2007) Induced pluripotent stem cell lines derivedfrom human somatic cells. Science 318, 1917–1920 CrossRef Medline

4. Montserrat, N., Nivet, E., Sancho-Martinez, I., Hishida, T., Kumar, S.,Miquel, L., Cortina, C., Hishida, Y., Xia, Y., Esteban, C. R., and IzpisuaBelmonte, J. C. (2013) Reprogramming of human fibroblasts to pluripo-tency with lineage specifiers. Cell Stem Cell 13, 341–350 CrossRefMedline

5. Shu, J., Wu, C., Wu, Y., Li, Z., Shao, S., Zhao, W., Tang, X., Yang, H., Shen,L., Zuo, X., Yang, W., Shi, Y., Chi, X., Zhang, H., Gao, G., et al. (2013)Induction of pluripotency in mouse somatic cells with lineage specifiers.Cell 153, 963–975 CrossRef Medline

6. Loh, K. M., and Lim, B. (2011) A precarious balance: pluripotency factorsas lineage specifiers. Cell Stem Cell 8, 363–369 CrossRef Medline

7. Davidson, D. (1995) The function and evolution of Msx genes: pointersand paradoxes. Trends Genet. 11, 405– 411 CrossRef Medline

8. Bell, J. R., Noveen, A., Liu, Y. H., Ma, L., Dobias, S., Kundu, R., Luo, W., Xia,Y., Lusis, A. J., Snead, M. L., and Maxson, R. (1993) Genomic structure,chromosomal location, and evolution of the mouse Hox 8 gene. Genomics16, 123–131 CrossRef Medline

9. Wu, Q., Zhang, L., Su, P., Lei, X., Liu, X., Wang, H., Lu, L., Bai, Y., Xiong, T.,Li, D., Zhu, Z., Duan, E., Jiang, E., Feng, S., Han, M., et al. (2015) MSX2mediates entry of human pluripotent stem cells into mesendoderm bysimultaneously suppressing SOX2 and activating NODAL signaling. CellRes. 25, 1314 –1332 CrossRef Medline

10. MacKenzie, A., Ferguson, M. W., and Sharpe, P. T. (1992) Expressionpatterns of the homeobox gene, Hox-8, in the mouse embryo suggest arole in specifying tooth initiation and shape. Development 115, 403– 420Medline

11. Satokata, I., Ma, L., Ohshima, H., Bei, M., Woo, I., Nishizawa, K., Maeda,T., Takano, Y., Uchiyama, M., Heaney, S., Peters, H., Tang, Z., Maxson, R.,and Maas, R. (2000) Msx2 deficiency in mice causes pleiotropic defects inbone growth and ectodermal organ formation. Nat. Genet. 24, 391–395CrossRef Medline

12. Khan, A., Fornes, O., Stigliani, A., Gheorghe, M., Castro-Mondragon, J. A.,van der Lee, R., Bessy, A., Chèneby, J., Kulkarni, S. R., Tan, G., Baranasic,D., Arenillas, D. J., Sandelin, A., Vandepoele, K., Lenhard, B., et al. (2018)JASPAR 2018: update of the open-access database of transcription factorbinding profiles and its web framework. Nucleic Acids Res. 46,D260 –D266 CrossRef Medline

13. Zambelli, F., Pesole, G., and Pavesi, G. (2009) Pscan: finding over-repre-sented transcription factor binding site motifs in sequences from co-reg-ulated or co-expressed genes. Nucleic Acids Res. 37, W247–W252CrossRef Medline

14. Li, Y., Liu, J., Hudson, M., Kim, S., and Hatch, N. E. (2010) FGF2 promotesMsx2 stimulated PC-1 expression via Frs2/MAPK signaling. J. Cell.Biochem. 111, 1346 –1358 CrossRef Medline

15. Newberry, E. P., Latifi, T., Battaile, J. T., and Towler, D. A. (1997) Struc-ture-function analysis of Msx2-mediated transcriptional suppression.Biochemistry 36, 10451–10462 CrossRef Medline

16. Xu, R. H., Chen, X., Li, D. S., Li, R., Addicks, G. C., Glennon, C., Zwaka, T. P.,and Thomson, J. A. (2002) BMP4 initiates human embryonic stem cell differ-entiation to trophoblast. Nat. Biotechnol. 20, 1261–1264 CrossRef Medline

17. Zhou, J., Su, P., Li, D., Tsang, S., Duan, E., and Wang, F. (2010) High-efficiency induction of neural conversion in human ESCs and human in-duced pluripotent stem cells with a single chemical inhibitor of transform-ing growth factor � superfamily receptors. Stem Cells 28, 1741–1750CrossRef Medline

18. Kishigami, S., and Mishina, Y. (2005) BMP signaling and early embryonicpatterning. Cytokine Growth Factor Rev. 16, 265–278 CrossRef Medline

19. Tam, P. P., and Loebel, D. A. (2007) Gene function in mouse embryogen-esis: get set for gastrulation. Nat. Rev. Genet. 8, 368 –381 CrossRef Medline

20. Richter, A., Valdimarsdottir, L., Hrafnkelsdottir, H. E., Runarsson, J. F.,Omarsdottir, A. R., Ward-van Oostwaard, D., Mummery, C., andValdimarsdottir, G. (2014) BMP4 promotes EMT and mesodermalcommitment in human embryonic stem cells via SLUG and MSX2.Stem Cells 32, 636 – 648 CrossRef Medline

21. Zhang, P., Li, J., Tan, Z., Wang, C., Liu, T., Chen, L., Yong, J., Jiang, W., Sun,X., Du, L., Ding, M., and Deng, H. (2008) Short-term BMP-4 treatmentinitiates mesoderm induction in human embryonic stem cells. Blood 111,1933–1941 CrossRef Medline

22. Samavarchi-Tehrani, P., Golipour, A., David, L., Sung, H. K., Beyer, T. A.,Datti, A., Woltjen, K., Nagy, A., and Wrana, J. L. (2010) Functional genom-ics reveals a BMP-driven mesenchymal-to-epithelial transition in the ini-tiation of somatic cell reprogramming. Cell Stem Cell 7, 64 –77 CrossRefMedline

Msx2 mediates BMP signals

14914 J. Biol. Chem. (2018) 293(38) 14905–14915

by guest on March 14, 2020

http://ww

w.jbc.org/

Dow

nloaded from

23. Li, R., Liang, J., Ni, S., Zhou, T., Qing, X., Li, H., He, W., Chen, J., Li, F.,Zhuang, Q., Qin, B., Xu, J., Li, W., Yang, J., Gan, Y., et al. (2010) A mesen-chymal-to-epithelial transition initiates and is required for the nuclearreprogramming of mouse fibroblasts. Cell Stem Cell 7, 51– 63 CrossRefMedline

24. Brambrink, T., Foreman, R., Welstead, G. G., Lengner, C. J., Wernig, M.,Suh, H., and Jaenisch, R. (2008) Sequential expression of pluripotencymarkers during direct reprogramming of mouse somatic cells. Cell StemCell 2, 151–159 CrossRef Medline

25. Tsubooka, N., Ichisaka, T., Okita, K., Takahashi, K., Nakagawa, M., andYamanaka, S. (2009) Roles of Sall4 in the generation of pluripotent stemcells from blastocysts and fibroblasts. Genes Cells 14, 683– 694 CrossRefMedline

26. Sridharan, R., Tchieu, J., Mason, M. J., Yachechko, R., Kuoy, E., Horvath,S., Zhou, Q., and Plath, K. (2009) Role of the murine reprogrammingfactors in the induction of pluripotency. Cell 136, 364 –377 CrossRefMedline

27. Mikkelsen, T. S., Hanna, J., Zhang, X., Ku, M., Wernig, M., Schorderet, P.,Bernstein, B. E., Jaenisch, R., Lander, E. S., and Meissner, A. (2008) Dis-secting direct reprogramming through integrative genomic analysis. Na-ture 454, 49 –55 CrossRef Medline

28. Chen, J., Liu, H., Liu, J., Qi, J., Wei, B., Yang, J., Liang, H., Chen, Y., Chen,J., Wu, Y., Guo, L., Zhu, J., Zhao, X., Peng, T., Zhang, Y., et al. (2013) H3K9

methylation is a barrier during somatic cell reprogramming into iPSCs.Nat. Genet. 45, 34 – 42 Medline

29. Cheng, S. L., Shao, J. S., Behrmann, A., Krchma, K., and Towler, D. A.(2013) Dkk1 and MSX2-Wnt7b signaling reciprocally regulate the endo-thelial-mesenchymal transition in aortic endothelial cells. Arterioscler.Thromb. Vasc. Biol. 33, 1679 –1689 CrossRef Medline

30. di Bari, M. G., Ginsburg, E., Plant, J., Strizzi, L., Salomon, D. S., and Von-derhaar, B. K. (2009) Msx2 induces epithelial-mesenchymal transition inmouse mammary epithelial cells through upregulation of Cripto-1. J. Cell.Physiol. 219, 659 – 666 CrossRef Medline

31. Shu, J., Zhang, K., Zhang, M., Yao, A., Shao, S., Du, F., Yang, C., Chen, W.,Wu, C., Yang, W., Sun, Y., and Deng, H. (2015) GATA family members asinducers for cellular reprogramming to pluripotency. Cell Res. 25,169 –180 CrossRef Medline

32. Liu, X., Sun, H., Qi, J., Wang, L., He, S., Liu, J., Feng, C., Chen, C., Li, W.,Guo, Y., Qin, D., Pan, G., Chen, J., Pei, D., and Zheng, H. (2013) Sequentialintroduction of reprogramming factors reveals a time-sensitive require-ment for individual factors and a sequential EMT-MET mechanism foroptimal reprogramming. Nat. Cell Biol. 15, 829 – 838 CrossRef Medline

33. Liang, L., Sun, H., Zhang, W., Zhang, M., Yang, X., Kuang, R., and Zheng,H. (2016) Meta-analysis of EMT datasets reveals different types of EMT.PLoS ONE 11, e0156839 CrossRef Medline

Msx2 mediates BMP signals

J. Biol. Chem. (2018) 293(38) 14905–14915 14915

by guest on March 14, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Lilong Lin, Lining Liang, Xiao Yang, Hao Sun, Yuan Li, Duanqing Pei and Hui Zhengmorphogenetic protein 4 (BMP4) on somatic cell reprogramming

The homeobox transcription factor MSX2 partially mediates the effects of bone

doi: 10.1074/jbc.RA118.003913 originally published online August 10, 20182018, 293:14905-14915.J. Biol. Chem. 

  10.1074/jbc.RA118.003913Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/293/38/14905.full.html#ref-list-1

This article cites 33 references, 4 of which can be accessed free at

by guest on March 14, 2020

http://ww

w.jbc.org/

Dow

nloaded from