Landscape of the SOX2 protein–protein interactome

RESEARCH ARTICLE

Landscape of the SOX2 protein–protein interactome

Xuefeng Fang1,2,3,4, Jae-Geun Yoon1, Lisha Li2, Yihsuan S. Tsai3, Shu Zheng4, Leroy Hood5,David R. Goodlett3, Gregory Foltz1 and Biaoyang Lin1

1 Swedish Neuroscience Institute, Swedish Medical Center, Seattle, WA, USA2 Zhejiang-California International NanoSystems Institute, Zhejiang University, Hangzhou, Zhejiang, P. R. China3 Department of Medicinal Chemistry, University of Washington, Seattle, WA, USA4 Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education),

The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, P. R. China5 The Institute for Systems Biology, Seattle, WA, USA

Received: July 7, 2010

Revised: November 19, 2010

Accepted: December 5, 2010

SOX2 is a key gene implicated in maintaining the stemness of embryonic and adult stem

cells that appears to re-activate in several human cancers including glioblastoma multiforme.

Using immunoprecipitation (IP)/MS/MS, we identified 144 proteins that are putative SOX2

interacting proteins. Of note, SOX2 was found to interact with several heterogeneous nuclear

ribonucleoprotein family proteins, including HNRNPA2B1, HNRNPA3, HNRNPC,

HNRNPK, HNRNPL, HNRNPM, HNRNPR, HNRNPU, as well as other ribonucleoproteins,

DNA repair proteins and helicases. Gene ontology (GO) analysis revealed that the SOX2

interactome was enriched for GO terms GO:0030529 ribonucleoprotein complex and

GO:0004386 helicase activity. These findings indicate that SOX2 associates with the hetero-

geneous nuclear ribonucleoprotein complex, suggesting a possible role for SOX2 in post-

transcriptional regulation in addition to its function as a transcription factor.

Keywords:

Biomedicine / Glioblastoma multiforme / Heterogeneous nuclear ribonucleoprotein /

Mass spectrometry / SOX2

1 Introduction

SOX (SRY-like HMG box) gene family represents a family of

transcriptional factors characterized by the presence of a

homologous sequence called the HMG (high mobility group)

box in their genes. SOX2, one of the key members of the SOX

family gene, is highly expressed in embryonic stem cells

(ESCs) [1]. Recently, Takahashi et al. showed that SOX2 is a

key transcription factor, in conjunction with KLK4, Oct4 and c-

Myc, whose overexpression can induce pluripotency in both

mouse and human somatic cells [2, 3]. SOX2 is one of the four

factors (OCT4, SOX2, NANOG, and LIN28) that Yu et al. used

to reprogram human somatic cells to pluripotent stem cells

that exhibit the essential characteristics of ESCs [4]. SOX2 is

one of the two factors (SOX2 and OCT4) that were sufficient

to generate induced pluripotent stem cells from human cord

blood cells [5]. These suggest that SOX2 is the key gene in

conferring stemness of cells. The stemness program can also

play an important role in cancer because self-renewal is a

hallmark of for both stem cells and cancer cells.

SOX2 are overexpressed in malignant glioma while

displaying minimal expression in normal tissues [6, 7].

We previously completed massively parallel signatureAbbreviations: ESC, embryonic stem cell; GBM, glioblastoma

multiforme; GO, gene ontology; HMG, high mobility group;

HNRNPU, heterogeneous nuclear ribonucleoprotein U; IP,

immunoprecipitation; mRNP, messenger ribonucleoprotein;

RBM14, RNA-binding motif protein 14; SOX2, SRY (sex-deter-

mining region Y)-box 2

�Additional corresponding author: Dr. Gregory Foltz

E-mail: [email protected]

Colour Online: See the article online to view Figs. 2 and 3 in colour

Correspondence: Dr. Biaoyang Lin, Swedish Neuroscience

Institute, Swedish Medical Center, 550 17th Ave., James Tower,

Suite 570, Seattle, WA 98122, USA

E-mail: [email protected]

Fax: 11-206-320-3166

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

Proteomics 2011, 11, 921–934 921DOI 10.1002/pmic.201000419

sequencing (MPSS) and identified SOX2 as significantly

overexpressed in glioblastoma multiforme (GBM) tissues

compared to normal brain tissues [8]. We identified two

MPSS tags that correspond to different polyadenylated

isoform, and both are up-regulated (unpublished result, data

not shown). More recently, Gangemi et al. showed that

silencing of the SOX2 in freshly derived glioblastoma tumor

initiating cells (TICs) stopped proliferation and the resulting

cells lost tumorigenicity in immunodeficient mice [9]. More

recently, Ikushima et al. showed that inhibition of TGF-bsignaling drastically deprived tumorigenicity of glioma-

initiating cells (GICs) by promoting their differentiation,

and that these effects were attenuated in GICs transduced

with SOX2 or SOX4 [10]. Their data again demonstrated that

SOX2 is a key gene in maintaining the stemness of glioma

stem cells. Therefore, SOX2 may be a gene that is predo-

minantly expressed in embryonic and adult stems cells

including neural progenitor cells and re-activates in cancers

including brain tumors. It is also a key gene involved in

tumorigenesis of gliomas and in the maintenance of glioma

stem cells.

Despite its importance, what other proteins interact

with SOX2 in glioma cells to achieve its functions

was not known. We therefore analyzed the SOX2 inter-

actome by immunoprecipitation (IP) coupled with MS

analysis.

2 Materials and methods

2.1 Immunoprecipitation (IP)

The LN229 cells were grown in DMEM with 10% FBS.

When the cells’ growth reached 80% confluence 4 days after

sub-culturing, the cells were washed three times with ice-

cold PBS. The cells were lysated in ice-cold lysis buffer

(20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10% glycerol, 1%

NP-40, and complete protease inhibitor from Roche Applied

Sciences). We used the Immunoprecipitation Kit – Dyna-

beadss Protein A for the IP. Fifty microliters of protein-A

Dynabeads (Invitrogen) and 5mg SOX2 antibody (ab59776,

Abcam) or heterogeneous nuclear ribonucleoprotein U

(HNRNPU) antibody [3G6] (ab10297, Abcam) were,

respectively, incubated to generate Protein-A–Dynabea-

d–antibody complex. In brief, the Protein-A–Dynabead and

antibody were incubated in pH 5.0 citrate phosphate buffer

at room temperature for 60 min with tilting/rotation.

Protein-A–Dynabead–antibody complex was washed twice in

cell lysis buffer and then resuspended in 100mL cell lysis

buffer. The supernatants of 1 mg cell lysate were incubated

with the Protein A–Dynabeads–Ab complex at 41C over-

night. The beads were washed three times with PBS

containing 0.05% Tween-20 at 41C. After washing, the

protein complex was eluted with 0.1 M citrate (pH 2.3) and

analyzed by Western blot with the SOX2 or the HNRNPU

antibody.

2.2 Mass spectrometry and data analysis

In-solution and in-gel digestion was carried out as described

previously [11]. Detailed MS analysis and its downstream

data processing are shown in Supporting Information 1.

High-Throughput GoMiner [12] was used to find statis-

tically overrepresented gene ontology (GO) terms using all

evidence levels and categories. One-sided Fisher’s exact

p-value corrected for multiple comparisons was used to

calculate the FDR (false discovery rate).

2.3 Western blot analysis

Primary antibodies used were: rabbit polyclonal Ku70 anti-

body (ab10878, Abcam), mouse monoclonal Ku80 antibody

[S10B1] (ab2173, Abcam), rabbit polyclonal TLS/FUS

(fusion gene in myxoid liposarcoma 2) antibody (ab70381,

Abcam), and mouse monoclonal HNRNPU antibody [3G6]

(ab10297, Abcam). Standard immunoblot protocol was used.

2.4 Taqman assays

The SOX2 taqman assay (Hs01053049_s1) and the

normalization control Human GUSB (b-glucuronidase)

(Life Technologies) were used. Standard protocol according

to the manufacturer’s instruction was used.

3 Results

3.1 IP-MS analysis of the SOX2 interactome

We screened by real-time PCR the SOX2 expression in eight

commonly used glioblastoma cell lines T98, U118, U87,

H683, M059J, M059K, LN229, and LN18. We found that

LN229 expressed the highest amount of SOX2 (Fig. 1). We

also compared the expression levels (using the Ct values of

the real-time quantitative PCR data) of LN229 and 19 GBM

tissue specimens (Supporting Information Fig. 1) and

showed that LN229 has comparable expression levels of

SOX2 with GBM tissues. We, therefore, decided to use

LN229 for our SOX2-IP experiments.

We first tested the SOX2 antibody for its specificity

using cell lysates from glioblastoma cell line LN229. We

showed that SOX2 antibody (ab59776, Abcam) detected

two bands, one at the right predicted size of 34 kDa and

another probably representing the post-translationally

modified form of around 36 kDa (Fig. 2B). For the IgG

control, we used the normal rabbit IgG (sc-2027, Santa Cruz

Biotechnology), which was widely used as a negative IP

control [13, 14]. We then performed two replicate IP

experiments for both SOX2 and IgG controls using LN229

glioblastoma cell line obtained from the American Type

Culture Collection.

922 X. Fang et al. Proteomics 2011, 11, 921–934


A Coomassie-stained protein gel revealed that there are

14 additional bands specific to SOX2-IP, which were not

found in the IgG-IP (Fig. 2A). Three major non-specific

bands were evident in the control IgG-IP, and additional

background was observable after adjusting the contrast of

the image for Supporting Information Fig. 2 (data not

shown). Western blot analysis confirmed that the SOX2-IP

product contained the SOX2 protein (Fig. 2B) with a specific

band at 34 kDa, whereas the IgG-IP did not contain the

SOX2 product, suggesting that the SOX2-IP is specific for

SOX2.

We then performed MS analysis. We carried out in-

solution trypsin digestion of the SOX2-IP and the IgG-IP

products, as well as in-gel digestion of individual bands

identified (Fig. 2A). After MS analysis, we identified a total

of 11 peptides that corresponded to the SOX2 protein

(Supporting Information Table 1), suggesting that our

SOX2-IP product contains the SOX2 protein, which is

consistent with our Western blot analysis data shown above

(Fig. 2B). For the SOX2-IP replicate 1, we obtained 321

entries (70 single hits) with Protein prophet p40.9 (with

estimated error rate of 0.8%). For the SOX2-IP replicate 2,

we obtained 294 entries (57 single hits) with Protein prophet

p40.9 (with estimated error rate of 0.6%). To remove as

much as potential contamination and/or non-specific bind-

ing of proteins from the IgG portion of the antibody, we

removed those proteins that were identified in the IgG-IP

with Protein Prophet p40.5 (with error rate of 6.7%). In

addition, we removed proteins with single peptide hits from

the SOX2-IP list and then obtained a list for the intersection

of two SOX2-IP analyses. In the end, we identified a total of

144 proteins that are putative SOX2 interacting proteins

(Table 1) in GBM cells. Supporting Information Table 2 lists

the number of peptides, the sequence and charge state of

each peptide, and other additional information for the

proteins identified.

We also analyzed the MS data separately for each indi-

vidual band. The Supporting Information Table 3 lists the

plausible identifications of the most abundant proteins from

each band: HNRNPU could be found in band 5, HNRMPR

and HNRNPL and FUS (HNRNP-P2) in band 8; SOX2,

HNRNPC, and HNRNP2B1 in band 9.

To confirm that our approach and analysis pipeline were

able to identify SOX2-interacting proteins, we randomly

picked several proteins for which antibodies were available

and good for Western blot analysis. The proteins are

HNRNPU, Ku70 (XRCC6), and TLS (FUS, hnRNP-P2).

Figure 2C–F shows that only the SOX2-IP product

contained these proteins, whereas the IgG-IP product did

not, confirming that our SOX2-IP was able to pull down

SOX2-interacting proteins specifically.

3.2 GO analysis of the SOX2 interactome

We found that the SOX2 interactome was enriched for GO

terms: GO:0030529 ribonucleoprotein complex, GO:0030530

heterogeneous nuclear ribonucleoprotein complex, GO:0031981

nuclear lumen, GO:0005730 nucleolus (Table 2), suggesting

that SOX2 is a protein involved in forming heterogeneous

nuclear ribonucleoprotein complex at the nucleolus. To further

confirm our finding that SOX2 is an HNRNP complex protein,

we performed reciprocal IP for HNRNPU using HNRNPU

mouse monoclonal antibody (ab10297, Abcam). We confirmed

that SOX2 could be detected in the HNRNPU-IP product

(Fig. 2G).

Other interesting enriched GO terms included the GO

terms related to helicase activity, which include GO:0004386

helicase activity, GO:0003724 RNA helicase activity, and

GO:0003678 DNA helicase activity (Table 2).

4 Discussion

The SOX2 interactome contains many proteins that belong

to the heterogeneous nuclear ribonucleoprotein family

Figure 1. Real-time quantitative PCR of SOX2

expression in eight glioblastoma cell lines

T98, U118, U87, H683, M059J, M059K, LN229,

and LN18. Relative fold of expression

normalized to GUSB was shown as bar chart.

Standard deviations of three replicate assays

were indicated as error bars.

Proteomics 2011, 11, 921–934 923


including HNRNPA2B1, HNRNPA3, HNRNPK, HNRNPL,

heterogeneous nuclear ribonucleoprotein M (HNRNPM),

HNRNPR, HNRNPU, FUS (which is heterogeneous nuclear

ribonucleoprotein P2), other ribonucleoproteins, DNA

repair proteins, and helicases (Table 1). HNRNPK is a

protein that is found at higher density at transcribed gene

loci compared with silent gene loci [15]. Mikula et al. used

the IP-MS/MS approach to identify proteins that interact

with hnrnpk in rat hepatoma cells and they identified 89

unique proteins from the hnrnpk protein complex (the

union of Supporting Information Tables 1–3 of their paper)

[16]. They identified several hnRNP proteins including

hnrnpl, hnrnpg, hnrnp2b1, and several dead box containing

proteins ddx1, ddx5, ddx17. Using the homologene table

from NCBI (www.ncbi.nlm.nih.gov/homologene), we were

able to find 74 human orthologs for hnrnpk protein

complexes. We then compared these 74 human orthologs

with our SOX2 interactome list, and we identified 16

proteins (about 22%) that are in common, which included

three HNRNP proteins, three DDX proteins, and two heat

shock proteins (Table 1). The overlap could be bigger as

several proteins have similar functions but those were given

different protein names and we did not consider them to be

exact orthologs based on the homologene table from NCBI.

Previous protein–protein interaction analysis revealed that

HNRNPK interacts with multiple proteins involved in

multiple steps in the gene expression including chromatin

remodeling, transcription, RNA processing, and translation

[16].

Many other ribonucleoproteins (Table 1) were identified

to be SOX2 binding partners, including PTBP1, YBX1,

RNA-binding motif protein 14 (RBM14), STAU1, and

KHDRBS1. PTBP1 is a multi-functional RNA-binding

protein that is aberrantly overexpressed in glioma. Knock-

down of PTBP1 in glioma cells slowed cell proliferation,

inhibited cell migration, and increased adhesion of cells to

fibronectin and vitronectin [17]. YBX1 is highly expressed

in primary GBM but not in normal brain tissues [18].

Figure 2. (A) A Coomassie-stained protein gel

of IP products for SOX2 and IgG control

revealed that there are about 14 additional

distinct bands specific to SOX2-IP, which

were not found in the IgG-IP. Positions

marked as 15–18 correspond to blank areas

between distinct bands. (B) Western blot

analysis of the cell lysate (left), IgG-IP

(middle), and SOX2-IP (right) with the SOX2

antibody. (C–F) Western blot confirmation of

HNRNPU, XRCC6, TLS, and XRCC5 proteins.

IP-products using IgG antibody and SOX2

antibody were analyzed by Western blot

using the corresponding antibodies.



Tab

le1.

SO

X2

inte

ract

ing

pro

tein

sid

en

tifi

ed

inre

pli

cate

SO

X2-I

Pb

yM

S

IPI

nu

mb

er

Nu

mb

er

of

un

iqu

ep

ep

tid

eh

its

Desc

rip

tio

n%

Seq

uen

ceco

vera

ge

iden

tifi

ed

fro

mM

S/M

Sd

ata

Gen

en

am

eId

en

tifi

ed

as

hn

RN

PK

inte

ract

ing

inm

ou

se

Iden

tifi

ed

inm

RN

Pco

mp

lexes

inra

t

Pu

tati

ve

dir

ect

or

ind

irect

?

Hete

rog

en

eo

us

nu

clear

rib

on

ucle

op

rote

ins

IPI0

0216592

8H

ete

rog

en

eo

us

nu

clear

rib

on

ucl

eo

pro

tein

sC

28

HN

RN

PC

Yes

IPI0

0396378

7H

ete

rog

en

eo

us

nu

clear

rib

on

ucl

eo

pro

tein

sA

2/B

121.2

HN

RN

PA

2B

1IP

I00419373

5H

ete

rog

en

eo

us

nu

clear

rib

on

ucl

eo

pro

tein

A3

19.5

HN

RN

PA

3IP

I00216049

9H

ete

rog

en

eo

us

nu

clear

rib

on

ucl

eo

pro

tein

K20.1

HN

RN

PK

Yes

IPI0

0027834

7H

ete

rog

en

eo

us

nu

clear

rib

on

ucl

eo

pro

tein

L21.5

HN

RN

PL

Yes

Yes

Ind

irect

IPI0

0171903

29

Hete

rog

en

eo

us

nu

clear

rib

on

ucl

eo

pro

tein

M29.4

HN

RN

PM

IPI0

0012074

8H

ete

rog

en

eo

us

nu

clear

rib

on

ucl

eo

pro

tein

R24.2

HN

RN

PR

Ind

irect

IPI0

0479217

13

Hete

rog

en

eo

us

nu

clear

rib

on

ucl

eo

pro

tein

U17.2

HN

RN

PU

Yes

Yes

IPI0

0221354

11

Fu

sio

ng

en

ein

myxo

idli

po

sarc

om

a2

14

TLS

/FU

S(H

nR

NP

-P2)

Rib

on

ucle

op

rote

ins

an

do

ther

RN

Ab

ind

ing

pro

tein

s

IPI0

0444262

9N

ucl

eo

lin

16.9

NC

LY

es

Ind

irect

IPI0

0041325

3N

ucl

eo

lar

pro

tein

fam

ily

A,

mem

ber

2(H

/AC

Asm

all

nu

cleo

lar

RN

Ps)

25.5

NO

LA

2

IPI0

0006379

6N

OP

5/N

OP

58

nu

cleo

lar

pro

tein

515.3

NO

P5/N

OP

58

IPI0

0220740

5N

ucl

eo

ph

osm

in(n

ucl

eo

lar

ph

osp

ho

pro

tein

B23,

nu

matr

in)

23

NP

M1

Ind

irect

IPI0

0215914

2A

DP

-rib

osy

lati

on

fact

or

319.3

AR

F3

IPI0

0215918

4A

DP

-rib

osy

lati

on

fact

or

425.6

AR

F4

IPI0

0002349

24

Nu

clear

frag

ile

Xm

en

tal

reta

rdati

on

pro

tein

inte

ract

ing

pro

tein

230.1

NU

FIP

2Y

es

IPI0

0179964

4P

oly

pyri

mid

ine

tract

bin

din

gp

rote

in1

12.2

PT

BP

1In

dir

ect

IPI0

0013174

15

RN

A-b

ind

ing

mo

tif

pro

tein

14

24.1

RB

M14

Ind

irect

IPI0

0008575

3K

Hd

om

ain

con

tain

ing

,R

NA

bin

din

g,

sig

nal

tran

sdu

ctio

nass

oci

ate

d1

6.9

KH

DR

BS

1In

dir

ect

IPI0

0000001

4S

tau

fen

,R

NA

-bin

din

gp

rote

in,h

om

olo

g1

(Dro

sop

hil

a)

8.5

ST

AU

1S

TA

U2

Ind

irect

IPI0

0031812

3Y

bo

xb

ind

ing

pro

tein

120.5

YB

X1

Yes

DN

Are

pair

pro

tein

san

dh

eli

cases

IPI0

0002564

4X

-ray

rep

air

com

ple

men

tin

gd

efe

ctiv

ere

pair

inC

hin

ese

ham

ster

cells

15.7

XR

CC

1In

dir

ect

IPI0

0220834

42

X-r

ay

rep

air

com

ple

men

tin

gd

efe

ctiv

ere

pair

inC

hin

ese

ham

ster

cells

5(d

ou

ble

-str

an

d-b

reak

rejo

inin

g;

Ku

au

toan

tig

en

,80

kDa)

39.5

XR

CC

5In

dir

ect

IPI0

0644712

35

X-r

ay

rep

air

com

ple

men

tin

gd

efe

ctiv

ere

pair

inC

hin

ese

ham

ster

cells

6(K

uau

toan

tig

en

,70

kDa)

48.8

XR

CC

6In

dir

ect

IPI0

0009841

12

Ew

ing

sarc

om

ab

reakp

oin

tre

gio

n1

15

EW

SR

1IP

I00293655

30

DE

AD

(Asp

-Glu

-Ala

-Asp

)b

ox

po

lyp

ep

tid

e1

42.6

DD

X1

Yes

IPI0

0215637

17

DE

AD

(Asp

-Glu

-Ala

-Asp

)b

ox

po

lyp

ep

tid

e3,

X-l

inke

d31.6

DD

X3X

IPI0

0007208

7D

EA

D(A

sp-G

lu-A

la-A

sp)

bo

xp

oly

pep

tid

e41

14.3

DD

X41

Proteomics 2011, 11, 921–934 925


Tab

le1.

Co

nti

nu

ed

IPI

nu

mb

er

Nu

mb

er

of

un

iqu

ep

ep

tid

eh

its

Desc

rip

tio

n%

Seq

uen

ceco

vera

ge

iden

tifi

ed

fro

mM

S/M

Sd

ata

Gen

en

am

eId

en

tifi

ed

as

hn

RN

PK

inte

ract

ing

inm

ou

se

Iden

tifi

ed

inm

RN

Pco

mp

lexes

inra

t

Pu

tati

ve

dir

ect

or

ind

irect

?

IPI0

0017617

12

DE

AD

(Asp

-Glu

-Ala

-Asp

)b

ox

po

lyp

ep

tid

e5

34.2

DD

X5

Yes

Yes

IPI0

0023785

8D

EA

D(A

sp-G

lu-A

la-A

sp)

bo

xp

oly

pep

tid

e17

23.3

DD

X17

Yes

Ind

irect

IPI0

0411733

3D

EA

H(A

sp-G

lu-A

la-H

is)

bo

xp

oly

pep

tid

e30

4.2

DH

X30

Yes

IPI0

0396435

8D

EA

H(A

sp-G

lu-A

la-H

is)

bo

xp

oly

pep

tid

e15

11.9

DH

X15

IPI0

0844578

30

DE

AH

(Asp

-Glu

-Ala

-His

)b

ox

po

lyp

ep

tid

e9

24.1

DH

X9

IPI0

0375358

7R

ep

lica

tio

nfa

cto

rC

(act

ivato

r1)

1,

145

kDa

8.1

RFC

1IP

I00029744

5S

ing

le-s

tran

ded

DN

Ab

ind

ing

pro

tein

122.3

SS

BP

1

Rib

oso

mal

pro

tein

s

IPI0

0376798

3R

ibo

som

al

pro

tein

L11

13

RP

L11

Yes

IPI0

0024933

6R

ibo

som

al

pro

tein

L12

54.5

RP

L12

IPI0

0026202

9R

ibo

som

al

pro

tein

L18a

38.1

RP

L18A

IPI0

0306332

5R

ibo

som

al

pro

tein

L24

36.4

RP

L24

IPI0

0007144

3R

ibo

som

al

pro

tein

L26

15.5

RP

L26

IPI0

0219155

7R

ibo

som

al

pro

tein

L27

36

RP

L27

IPI0

0398135

3R

ibo

som

al

pro

tein

L27a

22.3

RP

L27A

IPI0

0550021

5R

ibo

som

al

pro

tein

L3

13

RP

L3

IPI0

0219156

5R

ibo

som

al

pro

tein

L30

40.9

RP

L30

Yes

Ind

irect

IPI0

0219160

2R

ibo

som

al

pro

tein

L34

12.8

RP

L34

IPI0

0029731

3R

ibo

som

al

pro

tein

L35a

17

RP

L35A

IPI0

0003918

2R

ibo

som

al

pro

tein

L4

6.8

RP

L4

Yes

Ind

irect

IPI0

0025091

9R

ibo

som

al

pro

tein

S11

34.2

RP

S11

Ind

irect

IPI0

0013917

2R

ibo

som

al

pro

tein

S12

12.9

RP

S12

IPI0

0221089

9R

ibo

som

al

pro

tein

S13

42.4

RP

S13

Ind

irect

IPI0

0026271

4R

ibo

som

al

pro

tein

S14

29.1

RP

S14

Yes

IPI0

0221091

5R

ibo

som

al

pro

tein

S15a

29.2

RP

S15A

Ind

irect

IPI0

0221092

10

Rib

oso

mal

pro

tein

S16

48.6

RP

S16

IPI0

0221093

7R

ibo

som

al

pro

tein

S17

48.1

RP

S17

IPI0

0215780

6R

ibo

som

al

pro

tein

S19

28.3

RP

S19

Yes

IPI0

0012493

5R

ibo

som

al

pro

tein

S20

22.7

RP

S20

Yes

IPI0

0011253

14

Rib

oso

mal

pro

tein

S3

51.9

RP

S3

IPI0

0419880

7R

ibo

som

al

pro

tein

S3A

20.8

RP

S3A

Yes

Ind

irect

IPI0

0217030

15

Rib

oso

mal

pro

tein

S4,

X-l

inke

d42.2

RP

S4X

IPI0

0021840

6R

ibo

som

al

pro

tein

S6

18.1

RP

S6

IPI0

0012772

2R

ibo

som

al

pro

tein

L8

8.5

RP

L8

Ind

irect

IPI0

0013296

5R

ibo

som

al

pro

tein

S18

23.7

RP

S18

Tra

nsp

ort

er

an

dG

TP

-bin

din

gp

rote

ins

IPI0

0007188

7S

olu

teca

rrie

rfa

mil

y25

(mit

och

on

dri

alca

rrie

r;ad

en

ine

nu

cleo

tid

etr

an

slo

cato

r),

mem

ber

524.5

SLC

25A

5

IPI0

0012442

18

GT

Pase

act

ivati

ng

pro

tein

(SH

3d

om

ain

)b

ind

ing

pro

tein

139.3

G3B

P1



Tab

le1.

Co

nti

nu

ed

IPI

nu

mb

er

Nu

mb

er

of

un

iqu

ep

ep

tid

eh

its

Desc

rip

tio

n%

Seq

uen

ceco

vera

ge

iden

tifi

ed

fro

mM

S/M

Sd

ata

Gen

en

am

eId

en

tifi

ed

as

hn

RN

PK

inte

ract

ing

inm

ou

se

Iden

tifi

ed

inm

RN

Pco

mp

lexes

inra

t

Pu

tati

ve

dir

ect

or

ind

irect

?

IPI0

0009057

13

GT

Pase

act

ivati

ng

pro

tein

(SH

3d

om

ain

)b

ind

ing

pro

tein

224.5

G3B

P2

IPI0

0465121

3G

uan

ine

nu

cleo

tid

eb

ind

ing

pro

tein

(Gp

rote

in),a

inh

ibit

ing

act

ivit

yp

oly

pep

tid

e2

14.6

GN

AI2

Oth

er

TFs

IPI0

0019996

5S

AFB

-lik

e,

tran

scri

pti

on

mo

du

lato

r8.3

SLT

MIP

I00009703

16

SR

Y(s

ex

dete

rmin

ing

reg

ion

Y)-

bo

x2

25.2

SO

X2

IPI0

0006079

6B

CL2-a

sso

ciate

dtr

an

scri

pti

on

fact

or

110.6

BC

LA

F1

Str

uctu

rep

rs

IPI0

0005087

25

Tro

po

mo

du

lin

3(u

biq

uit

ou

s)52.3

TM

OD

3IP

I00216230

8T

hym

op

oie

tin

22.9

TM

PO

IPI0

0554811

4A

RP

C4

act

in-r

ela

ted

pro

tein

2/3

com

ple

xsu

bu

nit

422.6

TT

LL3;A

RP

C4

IPI0

0376344

39

Myo

sin

IB39.9

MY

O1B

IPI0

0414980

39

Myo

sin

IB41

MY

O1B

IPI0

0743335

16

Myo

sin

IC36.8

MY

O1C

IPI0

0010418

15

Myo

sin

IC36.4

MY

O1C

IPI0

0329672

29

Myo

sin

IE28.9

MY

O1E

IPI0

0302592

52

Fil

am

inA

,a

(act

in-b

ind

ing

pro

tein

280)

26.6

FLN

AIP

I00005162

5cD

NA

FLJ51245,

hig

hly

sim

ilar

toact

in-r

ela

ted

pro

tein

2/3

com

ple

xsu

bu

nit

322.2

–

IPI0

0003269

16

AC

TB

L2b-

act

in-l

ike

pro

tein

229.5

AC

TB

L2

IPI0

0013808

9A

ctin

in,a

411.2

AC

TN

4IP

I00028091

16

AR

P3

act

in-r

ela

ted

pro

tein

3h

om

olo

g(y

east

)40.9

AC

TR

3In

dir

ect

IPI0

0333068

2A

ctin

-rela

ted

pro

tein

2/3

com

ple

x,

sub

un

it1A

,41

kDa

5.9

AR

PC

1A

IPI0

0005161

5A

ctin

-rela

ted

pro

tein

2/3

com

ple

x,

sub

un

it2,

34

kDa

15.7

AR

PC

2In

dir

ect

IPI0

0550234

4A

ctin

-rela

ted

pro

tein

2/3

com

ple

x,

sub

un

it5,

16

kDa

34.4

AR

PC

5In

dir

ect

IPI0

0414554

4A

ctin

-rela

ted

pro

tein

2/3

com

ple

x,

sub

un

it5-l

ike

34

AR

PC

5L

IPI0

0012011

4C

ofi

lin

1(n

on

-mu

scle

)23.5

CFL1

IPI0

0291136

6C

oll

ag

en

,ty

pe

VI,a

18

CO

L6A

1IP

I00022200

28

Co

llag

en

,ty

pe

VI,a

310.8

CO

L6A

3IP

I00013933

26

Desm

op

laki

n8.4

DS

P

Un

kn

ow

nfu

ncti

on

s

IPI0

0012750

414

kDa

pro

tein

24.2

–IP

I00013415

319

kDa

pro

tein

20.1

–IP

I00174442

13

Hyp

oth

eti

cal

pro

tein

LO

C25940

25.3

FA

M98A

Oth

ers

IPI0

0182938

5S

-Ad

en

osy

lho

mo

cyst

ein

eh

yd

rola

se-l

ike

19.6

AH

CY

L1

IPI0

0418169

5A

nn

exin

A2

16.3

AN

XA

2Y

es

IPI0

0456359

25

Ata

xin

2-l

ike

23.3

AT

XN

2L

Proteomics 2011, 11, 921–934 927


Tab

le1.

Co

nti

nu

ed

IPI

nu

mb

er

Nu

mb

er

of

un

iqu

ep

ep

tid

eh

its

Desc

rip

tio

n%

Seq

uen

ceco

vera

ge

iden

tifi

ed

fro

mM

S/M

Sd

ata

Gen

en

am

eId

en

tifi

ed

as

hn

RN

PK

inte

ract

ing

inm

ou

se

Iden

tifi

ed

inm

RN

Pco

mp

lexes

inra

t

Pu

tati

ve

dir

ect

or

ind

irect

?

IPI0

0783872

7C

ell

-cycl

e-a

sso

ciate

dp

rote

in1

12.5

CA

PR

IN1

IPI0

0005969

9C

ap

pin

gp

rote

in(a

ctin

fila

men

t)m

usc

leZ

-lin

e,a

135.5

CA

PZ

A1

IPI0

0218782

13

Cap

pin

gp

rote

in(a

ctin

fila

men

t)m

usc

leZ

-lin

e,b

33.1

CA

PZ

BIP

I00290770

3C

hap

ero

nin

con

tain

ing

TC

P1,

sub

un

it3

(gam

ma)

6.5

CC

T3

Ind

irect

IPI0

0011302

2C

D59

mo

lecu

le,

com

ple

men

tre

gu

lato

ryp

rote

in9.4

CD

59

IPI0

0289773

3C

CA

AT

/en

han

cer

bin

din

gp

rote

in(C

/EB

P),b

8.1

CE

BP

BIP

I00295857

5C

oato

mer

pro

tein

com

ple

x,

sub

un

ita

4.8

CO

PA

IPI0

0029601

5C

ort

act

in15.1

CT

TN

IPI0

0013933

26

Desm

op

laki

n8.4

DS

PIP

I00186290

6E

uka

ryo

tic

tran

slati

on

elo

ng

ati

on

fact

or

28.3

EE

F2

IPI0

0060181

14

EF-h

an

dd

om

ain

fam

ily,

mem

ber

D2

39.6

EFH

D2

IPI0

0025491

3E

uka

ryo

tic

tran

slati

on

init

iati

on

fact

or

4A

,is

ofo

rm1

8.9

EIF

4A

1IP

I00039626

7Fam

ily

wit

hse

qu

en

cesi

mil

ari

ty120A

7.3

FA

M120A

IPI0

0031820

3P

hen

yla

lan

yl-

tRN

Asy

nth

eta

se,a

sub

un

it6.7

FA

RS

AIP

I00031023

31

Fli

gh

tless

Ih

om

olo

g(D

roso

ph

ila)

24.9

FLII

IPI0

0059366

7H

2A

his

ton

efa

mily,

mem

ber

Y26.6

H2A

FY

IPI0

0382470

4H

eat

sho

ckp

rote

in90

kDaa

(cyto

soli

c),

class

Am

em

ber

119.1

HS

P90A

A1

IPI0

0414676

9H

eat

sho

ckp

rote

in90

kDaa

(cyto

soli

c),

class

Bm

em

ber

124.9

HS

P90A

B1

Ind

irect

IPI0

0007765

9H

eat

sho

ck70

kDa

pro

tein

9(m

ort

alin

)18.4

HS

PA

9Y

es

IPI0

0784154

5H

eat

sho

ck60

kDa

pro

tein

1(c

hap

ero

nin

)13.3

HS

PD

1Y

es

IPI0

0179713

5In

sulin

-lik

eg

row

thfa

cto

r2

mR

NA

bin

din

gp

rote

in2

16.7

IGF2B

P2

IPI0

0005198

10

Inte

rleu

kin

en

han

cer

bin

din

gfa

cto

r2,

45

kDa

23.8

ILF2

IPI0

0298788

8In

terl

eu

kin

en

han

cer

bin

din

gfa

cto

r3,

90

kDa

11.7

ILF3

Yes

Ind

irect

IPI0

0291579

4K

inesi

nfa

mil

ym

em

ber

23

5.6

KIF

23

Ind

irect

IPI0

0294186

5Lact

am

ase

,b

16.3

LA

CT

BIP

I00219219

3Lect

in,

gala

cto

sid

e-b

ind

ing

,so

lub

le,

1(g

ale

ctin

1)

31.9

LG

ALS

1IP

I00008918

13

LIM

do

main

an

dact

inb

ind

ing

123.4

LIM

A1

IPI0

0016373

2LO

C100131294

sim

ilar

toR

AB

13

pro

tein

15.8

LO

C100131294

IPI0

0005160

4LO

C653888

sim

ilar

top

41-A

rc10.5

LO

C653888

IPI0

0296830

12

Leu

cin

ezi

pp

er

pro

tein

113.7

LU

ZP

1IP

I00008868

11

Mic

rotu

bu

le-a

sso

ciate

dp

rote

in1B

7.3

MA

P1B

IPI0

0017297

11

Matr

in3

19

MA

TR

3IP

I00335168

14

Myo

sin

,li

gh

tch

ain

6B

,alk

ali

,sm

oo

thm

usc

lean

dn

on

-m

usc

le60.7

MY

L6B

IPI0

0300127

5N

-ace

tylt

ran

sfera

se10

5.5

NA

T10

IPI0

0009456

12

50 -

Nu

cleo

tid

ase

,ect

o(C

D73)

29.8

NT

5E

IPI0

0012726

25

Po

ly(A

)b

ind

ing

pro

tein

,cy

top

lasm

ic4

(in

du

cib

lefo

rm)

35

PA

BP

C4

Yes

IPI0

0449049

40

Po

ly(A

DP

-rib

ose

)p

oly

mera

sefa

mil

y,

mem

ber

132.1

PA

RP

1



Inhibition of YBX1 in GBM cells reduced tumor cell inva-

sion and growth in monolayer as well as in soft agar, and

delayed tumor onset in mice [19]. In addition, inhibition of

YBX1 enhanced temozolomide sensitivity in a manner that

was independent of MGMT [19].

RBM14 (also named CoAA, coactivator activator) is a

nuclear receptor coactivator protein involved in transcrip-

tional coactivation and RNA splicing [20]. It also plays a role

in regulating stem/progenitor cell differentiation [21]. Kang

et al. recently showed that RBM14 (CoAA) is a potential

tumor suppressor in renal carcinoma and it represses the

proto-oncogene c-myc by recruiting HDAC3 protein and

decreasing both the acetylation of histone H3 and the

presence of RNA polymerase II on the c-myc promoter [22].

STAU1 is an RNA-binding protein that is required for the

survival and migration of primordial germ cells in zebrafish

and is required for ESC differentiation in mouse [23, 24].

KHDRBS1 (also named Sam68, Src-associated in mitosis of

68 kDa) is a known RNA-binding protein that involves in

modulating splicing of genes [25]. In prostate cancer, Sam68

expression supports proliferation and survival to cytotoxic

agents [26].

Another interesting family of proteins we identified as

SOX2 binding include nine members of the DEAD (Asp-

Glu-Ala-Asp) box containing genes (Table 1), which are

human RNA helicases participating in transcription,

splicing, and translation by modulating the structure of

RNA [27]. In addition, XRCC6 (Ku autoantigen, 70 kDa) and

XRCC5 (Ku autoantigen, 80 kDa) are single-stranded DNA-

dependent ATP-dependent helicases.

SOX2, together with Oct4, Klf4, c-Myc, Nanog, and

Lin28, are ESC markers [28] and different combination of

these genes were shown to be able to generate induced

pluripotent stem (iPS) cells [4, 29–31]. Physical interactions

among these members were shown previously. Wei et al.

showed that Klf4 could interact directly with Oct4 and Sox2

in iPS cells and mouse ESCs [32]. SOX2 and OCT4 interact

with each other in mouse ESCs [32]. However, we did not

detect OCT4 in our IP-MS analysis of SOX2. This could be

due to different cellular context of mouse ES and human

glioma cells, or due to the fact that Oct4 and Sox2 have low

affinity for each other in solution [33, 34]. Interestingly, in a

recent proteomics studies of the Sox2-IP in mouse ESCs, the

Oct4 protein was not identified either [35].

Transcriptional regulation and post-transcriptional regu-

lation may also exist among these six ESC markers. For

example, Sox2 and Oct4 bind to the promoter of Nanog [36].

Lin28 encodes a cytoplasmic mRNA-binding protein in

messenger ribonucleoprotein (mRNP) complexes [37].

Interestingly, many SOX2-binding proteins that we identi-

fied also belong to mRNP complexes. Angenstein et al.

conducted a proteomic analysis of mRNP complexes in the

rat cerebral cortex and they identified 30 proteins in the

mRNP complexes [38]. Comparing their list with our SOX2

binding protein list, we identified 10 proteins that are in

common including HNRNPC, HNRNPL, HNRNPU,Tab

le1.

Co

nti

nu

ed

IPI

nu

mb

er

Nu

mb

er

of

un

iqu

ep

ep

tid

eh

its

Desc

rip

tio

n%

Seq

uen

ceco

vera

ge

iden

tifi

ed

fro

mM

S/M

Sd

ata

Gen

en

am

eId

en

tifi

ed

as

hn

RN

PK

inte

ract

ing

inm

ou

se

Iden

tifi

ed

inm

RN

Pco

mp

lexes

inra

t

Pu

tati

ve

dir

ect

or

ind

irect

?

IPI0

0183002

11

Pro

tein

ph

osp

hata

se1,

reg

ula

tory

(in

hib

ito

r)su

bu

nit

12A

11.8

PP

P1R

12A

IPI0

0045550

5P

rote

inp

ho

sph

ata

se1,

reg

ula

tory

(in

hib

ito

r)su

bu

nit

9B

5.9

PP

P1R

9B

IPI0

0000874

8P

ero

xir

ed

oxin

144.2

PR

DX

1IP

I00292953

41

Reti

no

icaci

din

du

ced

14

38.2

RA

I14

IPI0

0297211

4S

WI/

SN

F-r

ela

ted

,m

atr

ix-a

sso

ciate

d,

act

in-d

ep

en

den

tre

gu

lato

ro

fch

rom

ati

n,

sub

fam

ily

a,

mem

ber

54.5

SM

AR

CA

5

IPI0

0178072

13

SP

EC

C1-l

ike

13.5

SP

EC

C1L

IPI0

0005154

11

Str

uct

ure

-sp

eci

fic

reco

gn

itio

np

rote

in1

15.2

SS

RP

1In

dir

ect

IPI0

0026970

10

Su

pp

ress

or

of

Ty

16

ho

mo

log

(S.

cere

vis

iae)

12.3

SU

PT

16H

IPI0

0020194

10

TA

F15

RN

Ap

oly

mera

seII,

TA

TA

bo

xb

ind

ing

pro

tein

(TB

P)-

ass

oci

ate

dfa

cto

r,68

kDa

20.5

TA

F15

Yes

IPI0

0104050

6T

hyro

idh

orm

on

ere

cep

tor

ass

oci

ate

dp

rote

in3

7.4

TH

RA

P3

Proteomics 2011, 11, 921–934 929


NUFIP2, YBX1, DDX5, DHX30, and ILF3 (Table 1). The

hypergeometric probability of the overlap (10 overlap genes

in two lists of 30 and 144 genes) with 17 512 human-rat

homologs at NCBI’s database is highly significant

(po2.690e-14). This suggests that SOX2 might also be a

member of mRNP complexes. Lin28 binds to IGF2 mRNA

and regulated its translation efficiency [38]. Although we

showed that SOX2-binding proteins or SOX2 might belong

to mRNP, whether SOX2 binds directly to any specific

species of mRNA remains to be determined.

The limitation of the IP/MS/MS approach is that it

cannot distinguish between direct and indirect interacting

partners of a protein as both can be immunoprecipitated

and therefore identified. The proteins that we identified

by SOX2-IP/MS analysis may include both direct SOX2

binding proteins and indirect SOX2-binding proteins. We

were not able to differentiate these two possibilities and

further experimentation is necessary to distinguish

them. We found that many proteins that we identified

(Table 1) show existing protein–protein interactions. For

example, hnRNP A2/B1 and TLS/FUS are interacting

partners in a mass spectrometry analysis [39]. XRCC5 and

XRCC6 form heterodimers [40]. KHDRBS1 (Sam68) was

found to be associated with heterogeneous nuclear ribonu-

cleoproteins hnRNP A1, A2/B1, G and L [41], and with

hnRNP K [42]. To systematically identifying all existing

protein–protein interactions among the SOX2-binding

proteins that identified, we searched the human HPRD

(www.hprd.org) using the Cytoscape (www.cytoscape.org)

MiMI plugin [43]. A protein–protein interacting map

showing SOX2 interactome as well as existing protein–-

protein interaction is shown in Fig. 3A. The protein–protein

interaction that was confirmed by us or in the literature is

shown in Fig. 3B.

Table 2. Interesting GO terms that are enriched in SOX2 interactome

GO category Totalgenes

Changedgenes

Enrichmentfolds

p-Value(log 10)

FDR

GO:0030529_ribonucleoprotein_complex 406 36 10.59 �26.59 0.0000GO:0030530_heterogeneous_nuclear_ribonucleoprotein_complex 10 5 59.69 �8.03 0.0000GO:0031981_nuclear_lumen 608 19 3.73 �6.17 0.0000GO:0005730_nucleolus 159 10 7.51 �6.06 0.0000GO:0003723_RNA_binding 645 55 10.05 �41.12 0.0000GO:0003735_structural_constituent_of_ribosome 159 25 18.53 �24.34 0.0000GO:0003676_nucleic_acid_binding 3070 70 2.69 �17.57 0.0000GO:0005198_structural_molecule_activity 656 33 5.93 �16.45 0.0000GO:0005515_protein_binding 6544 94 1.69 �12.27 0.0000GO:0008026_ATP-dependent_helicase_activity 89 11 14.57 �9.67 0.0000GO:0003779_actin_binding 287 17 6.98 �9.52 0.0000GO:0000166_nucleotide_binding 1889 43 2.68 �9.47 0.0000GO:0004386_helicase_activity 131 12 10.80 �8.97 0.0000GO:0003724_RNA_helicase_activity 28 7 29.47 �8.57 0.0000GO:0008092_cytoskeletal_protein_binding 403 18 5.26 �8.10 0.0000GO:0005488_binding 11 156 116 1.23 �7.73 0.0000GO:0042623_ATPase_activity__coupled 251 13 6.11 �6.69 0.0000GO:0016887_ATPase_activity 304 14 5.43 �6.54 0.0000GO:0017111_nucleoside-triphosphatase_activity 535 18 3.97 �6.24 0.0007GO:0016462_pyrophosphatase_activity 562 18 3.78 �5.94 0.0006GO:0016818_hydrolase_activity__acting_on_acid_anhydrides__in_phosphorus-

containing_anhydrides564 18 3.76 �5.91 0.0006

GO:0016817_hydrolase_activity__acting_on_acid_anhydrides 567 18 3.74 �5.88 0.0006GO:0003725_double-stranded_RNA_binding 31 5 19.01 �5.24 0.0005GO:0005524_ATP_binding 1240 26 2.47 �4.92 0.0005GO:0032553_ribonucleotide_binding 1577 30 2.24 �4.82 0.0005GO:0032555_purine_ribonucleotide_binding 1577 30 2.24 �4.82 0.0005GO:0032559_adenyl_ribonucleotide_binding 1259 26 2.43 �4.81 0.0004GO:0019843_rRNA_binding 19 4 24.82 �4.76 0.0004GO:0051082_unfolded_protein_binding 102 7 8.09 �4.60 0.0004GO:0008186_RNA-dependent_ATPase_activity 21 4 22.45 �4.58 0.0004GO:0017076_purine_nucleotide_binding 1648 30 2.15 �4.46 0.0004GO:0030554_adenyl_nucleotide_binding 1330 26 2.30 �4.39 0.0004GO:0004003_ATP-dependent_DNA_helicase_activity 19 3 18.61 �3.28 0.0048GO:0004004_ATP-dependent_RNA_helicase_activity 20 3 17.68 �3.21 0.0063GO:0003697_single-stranded_DNA_binding 47 4 10.03 �3.18 0.0066GO:0008094_DNA-dependent_ATPase_activity 47 4 10.03 �3.18 0.0066GO:0003678_DNA_helicase_activity 34 3 10.40 �2.53 0.0444



Recently, Mallanna et al. conducted a proteomic analysis

of SOX2-associated proteins during early stages of mouse

ESC differentiation [35], and they identified 60 nuclear

proteins that associate with Sox2 during early ESC differ-

entiation with high confidence scores. Additionally, they

identified 194 less confident but probable binding partners

of Sox2 in mouse ESC cells. Fifty-eight of the 60 high

confident mouse Sox2-associating proteins have human

homologs. There are 11 proteins that are common between

the 58 Sox2-associated proteins in mouse ESC and

the SOX2-binding proteins in glioblastoma cells. These

proteins are listed in Table 3, which include H2A histone

Figure 3. (A) A cytoscape map of the SOX2 interactome. Nodes indicate proteins and the edges indicate protein–protein interactions.

(B) The protein–protein interaction that was confirmed by us or in the literature. Nodes indicate proteins and the edges (marked by pp)

indicate protein–protein interactions.

Proteomics 2011, 11, 921–934 931


family, member Y (H2AFY), single-stranded DNA binding

protein 1 (SSBP1), RBM14, XRCC5 (double-strand-break

rejoining; Ku autoantigen, 80 kDa), XRCC6 (Ku autoanti-

gen, 70 kDa), and HNRNPM. Additionally, the mouse

heterogeneous nuclear ribonucleoprotein U-like 2

(Hnrnpul2) was identified in the mouse Sox2-IP, whereas in

human the HNRNPU was identified. The hypergeometric

probability of the overlap (12 genes) with 19 570 human–

mouse homologs at NCBI’s database is highly significant

(po1.056e–14).

The difference is not surprising as the SOX2-associated

proteins are from different cell types and different species.

Furthermore, the experimental approaches are different.

While we used natural SOX2 expression cancer cell line and

used SOX2 antibody to isolate SOX2 binding proteins,

Mallanna et al. overexpressed the SOX2 protein as a Flag-

Sox2 fusion protein in an inducible system and used anti-

Flag M2 affinity beads to isolate SOX2-binding proteins.

Finally, it is possible that some of the proteins are non-

specific binding proteins that we failed to filter out even

when we used stringent filtering criteria. Additional

confirmation studies are necessary when one wants to focus

on studying a specific interaction between SOX2 and a

protein.

4 Concluding remarks

A critical discovery in our analysis is that SOX2 belongs to the

hnRNP or mRNP complexes, which are key components of

transcription and post-transcription regulations. This suggests

a potential new role for SOX2 as not only a transcription factor

but also a factor involved in post-transcriptional regulation.

Such examples of dual functions of transcriptional factors have

been documented previously. Cassiday and Maher surveyed

and identified many examples of transcription factors that

bind to both DNAs and RNAs [44], including the prototypic

Xenopus TFIIIA protein, the Caenorhabditis elegans TRA-1, the

Drosophila bicoid, and the mammalian P53, WT-1, STAT1, and

TLS/FUS. The mammalian p53 binds to cognate site in

promoters target genes involved in growth arrest or apoptosis

[45]. Yoshida et al. showed that p53 interacts with RNA via its

C-terminal domain, which regulate its DNA-binding activity

and its oligomerization [46]. Recently, Grinberg et al. showed

that p52 could bind to double-stranded RNA and destroy them

[47]. The Ku protein, consists of a heterodimeric complex of 70

(Ku70) and 80 kDa (Ku80) subunits, is also both a DNA- and

RNA-binding protein in yeast [48]. It binds to double-stranded

DNA to repair double-stranded DNA breaks and it also binds

to telomerase RNA to promote telomere addition [48]. We also

identified Ku70 (XRCC6) and Ku80 (XRCC5) as SOX2-binding

proteins. TLS/FUS is another protein that possesses both

DNA- and RNA-binding activities [49, 50] and we also identi-

fied it as a SOX2-binding protein.

Tung et al. recently showed that SOX2 has RNA-binding

activity and the binding needs the HMG domains using an in

vitro pull down assay in transitional cell carcinoma (TCC).

They further showed that ectopic expression of SOX2 modu-

lates alternative splicing of genes [51]. They postulated that

SOX2 is an RNA splicer. Our data support this speculation.

Further experiments were necessary to establish the functional

consequences and detailed mechanisms of these interactions,

and SOX2’s exact roles in post-transcriptional regulation.

The authors have declared no conflict of interest

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H2AFY H2A histone family, member Y ��

HNRNPM Heterogeneous nuclear ribonucleoprotein M ��

ILF3 Interleukin enhancer binding factor 3, 90 kDa ��

PARP1 Poly (ADP-ribose) polymerase family,member 1

��

RBM14 RNA-binding motif protein 14 ��

RPS11 Ribosomal protein S11 ��

SSBP1 Single-stranded DNA-binding protein 1 ��

SSRP1 Structure-specific recognition protein 1 ��

SUPT16H Suppressor of Ty 16 homolog (S. cerevisiae) ��

XRCC5 X-ray repair complementing defective repairin Chinese hamster cells 5 (double-strand-break rejoining; Ku autoantigen, 80 kDa)

��

XRCC6 X-ray repair complementing defective repairin Chinese hamster cells 6 (Kuautoantigen, 70 kDa)

��

AHCYL1 S-adenosylhomocysteine hydrolase-like 1 �

CCT3 Chaperonin-containing TCP1, subunit 3(gamma)

�

KIF23 Kinesin family member 23 �

SMARCA5 SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin,subfamily a, member 5

�

TMPO Thymopoietin �

�� Identified in mouse with high-frequency and enrichmentvalues.

� Identified in mouse with low-frequency and enrichmentvalues.



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Landscape of the SOX2 protein–protein interactome