pone.0034327 1..10

Synergistic Effect of SRY and Its Direct Target, WDR5, on

Sox9 Expression

Zhen Xu

, Xinxing Gao

, Yinghong He

1,3.

, Junyi Ju

, Miaomiao Zhang

, Ronghua Liu

, Yupeng Wu

Chunyan Ma

, Chi Ma

, Zhaoyu Lin

, Xingxu Huang

, Quan Zhao

1 The State Key Laboratory of Pharmaceutical Biotechnology, Molecular Immunology and Cancer Research Center, School of Life Sciences, Nanjing University, Nanjing,

China, 2 Model Animal Research Center, Nanjing University, Nanjing, China, 3 School of Basic Medicine, Dali University, Yunnan, China

Abstract

SRY is a sex-determining gene that encodes a transcription factor, which triggers male development in most mammals. The

molecular mechanism of SRY action in testis determination is, however, poorly understood. In this study, we demonstrate

that WDR5, which encodes a WD-40 repeat protein, is a direct target of SRY. EMSA experiments and ChIP assays showed that

SRY could bind to the WDR5 gene promoter directly. Overexpression of SRY in LNCaP cells significantly increased WDR5

expression concurrent with histone H3K4 methylation on the WDR5 promoter. To specifically address whether SRY

contributes to WDR5 regulation, we introduced a 4-hydroxy-tamoxifen-inducible SRY allele into LNCaP cells. Conditional SRY

expression triggered enrichment of SRY on the WDR5 promoter resulting in induction of WDR5 transcription. We found that

WDR5 was self regulating through a positive feedback loop. WDR5 and SRY interacted and were colocalized in cells. In

addition, the interaction of WDR5 with SRY resulted in activation of Sox9 while repressing the expression of b-catenin. These

results suggest that, in conjunction with SRY, WDR5 plays an important role in sex determination.

Citation: Xu Z, Gao X, He Y, Ju J, Zhang M, et al. (2012) Synergistic Effect of SRY and Its Direct Target, WDR5, on Sox9 Expression. PLoS ONE 7(4): e34327.

doi:10.1371/journal.pone.0034327

Editor: Andy T. Y. Lau, Shantou University Medical College, China

Received July 27, 2011; Accepted February 25, 2012; Published April 16, 2012

Copyright: ß 2012 Xu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted

use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by National Natural Science Foundation of China (NSFC#31071118, NSFC#31170716, NSFC#81121062, SBK201140017(QZ),

NSFC#81060248(YH), the Fundamental Research Funds for the Central Universities, and the Doctoral Program of Higher Education of China

RFDP#20090091110033. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have decl ared that no competing interests exist.

* E-mail: [email protected]

. These authors contributed equally to this work.

Introduction

WDR5, also named BIG-3, was first identified in a murine

prechondroblastic cell line by differential display PCR following

induction with BMP-2. Analysis of liver, spleen, kidney, and other

tissues showed that the highest level of WDR5 expression was in

testis [1]. WDR5 is a member of the WD-40 repeat protein

family, which exhibits a seven-bladed propeller-like structure with

a narrow channel running through the center [2,3].

In the last several years,studies have focused on the role of

WDR5 associated with MLL and SET1 complexes, which trigger

methylation of histone H3K4. WDR5 is the core component of

the MLL/SET1 complex, and it is indispensable for assembly and

effective methyltransferase activity of the complex [2]. It was

shown that WDR5 interacts with histone H3 regardless of the

methylation status of the Lysine 4 residue [2]. WDR5 regulates

osteoblast differentiation and vertebrate development [4,5]. In

addition, WDR5 has been shown to be a regulator of embryonic

stem cell self-renewal, and its expression correlates with the

undifferentiated state [6]. However, it is not known how WDR5

itself is regulated.

SRY i s a testis-determining gene located on the Y-chromo-

some, which triggers male development in most mammalian

embryos. Mutations in SRY are associa ted with hu man X Y

gonadal dysgenesis [7]. The open reading frame ( ORF) of human

SRY contains only a single exon and encodes a 204-amino- acid

protein, which is composed of three regions: a central 79 amino

acids HMG domain, C-terminal domain, and N-terminal

domain. The HMG domain, which is highly conserved between

species, binds sites in the minor groove of DNA and introduces

local conformational changes that influence transcrip tion of genes

downstream [8–10]. SRY, a member of th e Sox (SRY-related

HMG box) gene family, interacts with CaM (calmodulin) and

importin b and facilitates translocation of proteins from

cytoplasm to nucleus [11,12]. Unlike mouse, human SRY lacks

a C terminal transcription activation domain necessary for male

sex determination, suggesting that human SRY may function

through interaction with additional transcriptional co-activators.

In vit ro analysis of recombinant SRY protein suggests that it

recognizes a dege nerate motif (A/T)AACAA(A/T), making it

difficult to id entify its in vivo targets [13]. To date, few regulatory

target genes of SRY have been identified; these include Sox9,

Cbln4, TCF21, and NTF3 [14–17]. The molecular mechanism

of SRY action in testis determination is poorly understood

[18,19].

In this study, we show that WDR5 is a direct target of SRY.

The interaction of WDR5 and SRY activates Sox9 expression. As

Sox9 is the master regulator of sex determination [18,19], we

hypothesize that WDR5 interacts with SRY to promote testis

development.

PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e34327

Results

Identification of potential SRY binding sites in the

proximal WDR5 promoter

The human WDR5 gene has two transcript variants, which

encode the same protein but differ in the 59UTR (Fig. S1A).

Alignment of the proximal promoter of the WDR5 gene (between

22000 bp and +1 bp) in different vertebrates reveals that the

proximal promoter of Variant 2 (the shorter variant) is more

conserved (Fig. S1B). Thus, in this study, we focused on the

Variant 2 promoter to investigate transcriptional regulatory

mechanisms of WDR5.

To predict potential transcription factors, we used the TF-

SEARCH program (http://www.cbrc.jp/research/db/

TFSEARCH.html) to search a 2.0-kb fragment of genomic

DNA that contains part of the first exon of WDR5 and 59UTR.

The result revealed that within the proximal 200 bp promoter

region several transcription factors were likely to bind, including

CdxA, GATA-1, and SRY (Fig. 1A). Notably, there were two

predicted SRY binding sites in this region (sequence: TTTGTTT),

which were exactly complementary to the SRY binding consensus

sequence (A/T)AACAA(A/T). These sites were further checked

using ConSite (http://mordor.cgb.ki.se/cgibin/CONSITE/

consite/) software (Fig. 1B). Alignment of the region (2101 bp

to +1 bp) between human, rhesus, and mouse showed that the

binding site of SRY at –11 bp was more conserved than the site at

294bp. We hypothesized that these two sites might be SRY

binding sites.

To isolate the 59-flanking region of the WDR5 gene containing

the two SRY binding sites, a 249-bp fragment of genomic DNA

(2134 to +115 of the WDR5 gene) was amplified and subcloned

into a pGL3-basic vector. In order to test whether SRY could

function on this reporter construct, the reporter construct was

cotransfected with either an SRY expression plasmid (pCXN-2-

SRY-3HA) or an empty plasmid (pCXN-2) into human prostate

adenocarcinoma LNCaP cells, which have very low expression

levels of endogenous SRY, and relative luciferase activity was

analyzed. Compared to empty vector, luciferase activity was

increased 3.6-fold when cells were transfected with SRY

expression plasmid (Fig. 1C). To verify that the SRY binding

elements functioned for activation of human WDR5 expression,

we constructed three reporter plasmids in which point-mutations

were introduced into one or both SRY binding sites, and tested

luciferase activity. Luciferase activity from mutants in which either

SRY binding element was mutated was significantly reduced

compare to the wild type. Moreover, luciferase activity from a

mutant in which both SRY binding sites were mutated was further

reduced (Fig. 1D). These data indicated that these two SRY

binding sites contributed to the transcriptional activity of human

WDR5.

SRY binds to the promoter of WDR5

To determine if SRY bound the WDR5 promoter directly,

electrophoretic mobility shift assays were performed. A wild type

labeled probe bound the nuclear extract from transfected LNCaP

cells overexpressing HA-tagged SRY (Fig. 2A, lane 2). This

binding was ablated by competition with unlabeled wild type

probe, but not by competing mutant probe (Fig. 2A, lanes 3, 4). A

supershift band was observed when anti-HA antibody was added

in nuclear extract incubated with wild type labeled probe, but not

with control IgG (Fig. 2A, lanes 5, 6). This experiment indicated

that SRY bound the WDR5 promoter in vitro.

Because of a lack of a high quality ChIP-grade antibody against

human SRY, we performed ChIP assays on LNCaP cells

overexpressing HA-tagged SRY, as above, to confirm that SRY

associated with the endogenous WDR5 promoter. The results

demonstrated that precipitation of HA-SRY brought down the

WDR5 promoter, but did not bring down a control region

upstream of the WDR5 promoter, or a negative control promoter,

MyoD (Fig. 2B). Taken together, results of both EMSA and ChIP

assays indicated that SRY binds WDR5 promoter.

SRY activates WDR5 expression

In order to assess the effect of SRY on endogenous protein, both

WDR5 mRNA and protein expression levels were analyzed in

LNCaP cells stably overexpressing HA tagged SRY. Consistent

with reporter gene assays, overexpression of SRY induced

endogenous WDR5 expression as shown in Western blot assay

with anti-WDR5 antibodies (Fig. 3A). To confirm this result, we

performed real-time PCR using specific primer for WDR5 cDNA.

We found that the WDR5 mRNA level was three-fold higher in

SRY overexpressing cells than control (Fig. 3B). In order to

examine changes in epigenetic histone modification marks, we

performed ChIP analyses of the WDR5 gene promoter with

antibodies to H3K4me2, H3K4me3, and H3K27me3. Consistent

with the expression data, histone H3K4me2 and H3K4me3 on the

WDR5 promoter were enriched whereas H3K27me3 remained

unchanged. We observed that WDR5 binding to its own promoter

was significantly increased as well (Fig. 3C).

To further confirm that SRY activates WDR5 expression

specifically, we established an inducible system using 4-OHT

induction, and performed a time course assay. The ERtm domain

(amino acids 281–599 of murine estrogen receptor with a Glycine

to Arginine substitution at amino acid 525) was fused to the full

length HA tagged SRY, separated by a glycine-rich linker

(Fig. 3D). The modified ERtm domain is deficient in binding

endogenous estrogen, but remains responsive to activation by the

synthetic estrogen derivative 4-OHT or its precursor tamoxifen.

Proteins fused to ERtm are retained outside the nucleus in a

complex with heat shock proteins (Hsp), such as Hsp90. Upon

binding to 4-OHT, the fusion protein is released and shuttled into

the nucleus where it acts as a transcription factor [20]. The

assembled cDNA was inserted into a murine stem cell virus

(MSCV)-based retroviral vector MSCV-IRES-GFP, yielding

MSCV-SRY-HA-ERtm-IRES-GFP. Using FACS sorting, we

generated a stable LNCaP cell line overexpressing SRY-HA-

ERtm. Expression of SRY-HA-ERtm was determined by

immunoblotting with a monoclonal antibody against HA

(Fig. 3D). LNCaP cells stably overexpressing SRY were harvested

at different times after 4-OHT treatment. RNA from these cells

was isolated and analyzed by quantitative real time PCR, which

revealed that WDR5 mRNA levels were significantly increased

after 24 hours induction (Fig. 3E). Consistent with this, anti-HA

antibody ChIP experiments indicated that HA-tagged SRY was

increasingly enriched on the WDR5 promoter following 4-OHT

treatment (Fig. 3F). This result suggested that the accumulated

SRY on the WDR5 promoter could be directly activating WDR5

expression.

WDR5 regulates itself through a feedback loop

Since WDR5 binds its own promoter (Fig. 3C), we investigated

whether WDR5 regulated its own expression. To test this, we

generated a stable LNCaP cell line which overexpressed WDR5

protein exogenously. Endogenous mRNA can be distinguished

from total mRNA by real time-RT-PCR using two different pairs

of primers (Fig. 4A). Q-RT-PCR analysis revealed that the

endogenous WDR5 mRNA level was increased 2-fold compare to

the control, while the total WDR5 mRNA level was increased

WDR5 Is a Direct Target of SRY

PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e34327

about 10-fold compare to the control (Fig. 4B, 4C). These results

indicated that WDR5 could activate itself through positive

feedback.

SRY cooperates with WDR5 to induce Sox9 expression

and repress b-catenin expression

Next, we examined whether WDR5 could interact with SRY in

vivo. To test this, we performed immunofluorescent staining

experiments. The results showed that WDR5 co-localized with

Figure 1. Identification of potential SRY binding sites in WDR5 proximal promoter. (A) Schematic representation of WDR5 proximal

promoter with two potential SRY binding sites and GATA1 and CdxA binding sites. (B) Alignment of WDR5 proximal promoters between human,

rhesus, and mouse. Regions in frame represent the SRY binding sites. (C) Relative luciferase activity assay from cells containing either vector or

overexpressing SRY. Graphs show mean 6 SD, n = 3 (left panel). Western blot analysis of HA-tagged SRY and loading control, Hsp70 (right panel). (D)

Relative luciferase activity assay from cells containing either wild type promoter or mutant promoters with either vector or HA tagged SRY. Graphs

show mean 6 SD, n = 3. *P,0.05, **P,0.01 compared to wild type (top panel); Student’s t-test.

doi:10.1371/journal.pone.0034327.g001

WDR5 Is a Direct Target of SRY

PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e34327

SRY in the nucleus of LNCaP cells (Fig. 5A). In addition, a ChIP-

reChIP (anti-HA followed by anti-WDR5) experiment demon-

strated that SRY and WDR5 interacted on the Sox9 promoter,

but not a control region upstream of the promoter (Fig. 5B). The

interaction between WDR5 and SRY was verified by co-

immunoprecipitation experiments in which cellular extracts from

LNCaP cells overexpressing SRY-3HA were co-immunoprecipi-

tated with anti-HA antibody and blotted with anti-WDR5

antibody (Fig. 5C). SRY was expected to recruit more MLL

complex components rather than to disrupt the complex (Fig. S2).

SRY is the critical gene that initiates male sex determination in

most mammals. The best direct target for SRY is Sox9. In order to

determine whether SRY or WDR5 can regulate Sox9 expression,

we established LNCaP cell lines which expressed either SRY,

WDR5, or both together with the nuclear orphan receptor,

steroidogenic factor (SF1). Real time PCR results indicated that

SRY and WDR5 together activated the expression of Sox9

significantly more than either alone (Fig. 5D). Consistent with their

function in triggering male sex determination, SRY and WDR5

significantly reduced the expression levels of b-catenin, which is

important for development of ovaries (Fig. 5E). Expression of

WDR5, HA tagged SRY, and HA tagged SF1 was confirmed by

Western blot (Fig. 5F). To further probe the direct role of WDR5

alone on Sox9 expression, we generated two WDR5 knockdown

(WDR5i-1 and WDR5i-2) LNCaP cell lines using specific short

hairpin RNAs, and a scrambled control line. In the knockdown

lines, WDR5 levels were reduced to about 20% of the scrambled

cells (Fig. 5G, 5H), and Q-RT-PCR demonstrated a 20-45%

decrease in Sox9 expression in these cells compared to control cells

(Fig. 5I). In aggregate, these results suggest that WDR5 together

with SRY could play an important role in sex determination.

Localization of the WDR5 protein and expression of the

WDR5 gene in the murine embryonic testis

To study the expression pattern of WDR5 in mouse embryonic

testis, timed-pregnant mice were set up. Genital ridges from

different days of these mice were collected and sectioned for

immunofluorescent analysis with specific antibodies. At 11.5 dpc

(days post-coitum), WDR5 was localized to germ cells and co-

localized with Sox9 in somatic cells (Fig. 6A). At 12.5 dpc, WDR5

immunopositive cells were found predominantly in germ cells and

occasionally in Sertoli cells (Fig. 6B). At 13.5 dpc, WDR5 was

detected in germ cells, Sertoli cells, and in the interstitium

(Fig. 6C). Of note, WDR5 was mostly localized in the cytoplasm

rather than in the nucleus. It has previously been reported that

WDR5 is more abundant in the cytoplasm than in the nucleus of

human embryonic kidney 293 cells, and that it can be translocated

from the nucleus to the cytoplasm during viral infection [21].

However, the movement of WDR5 between nucleus and

cytoplasm in developing testes is currently unknown. The

differential localization of WDR5 at different stages of develop-

ment suggested different roles and functions for WDR5 in testis

differentiation and sex determination. In order to confirm whether

SRY binds the WDR5 promoter in vivo, ChIP analysis was

performed from E13 rat gonads. The results demonstrated

significant enrichment of SRY on the WDR5 promoter, similar

to SRY binding on the Tcf21 promoter, a positive control

(Fig. 6D). Moreover, Q-RT-PCR revealed that WDR5 and Sox9

displayed a similar expression profile in rat gonads from E13 to

E15 (Fig. 6E). In aggregate, these results indicted that WDR5 and

SRY are likely to have direct regulatory effects on Sox9

expression.

Discussion

Although WDR5 is a core subunit of MLL/SET1 complexes, it

also functions as a subunit of other complexes. WDR5 has been

shown to be important in bone morphogenesis, vertebrate

development, and embryonic stem cell renewal [4–6]. However,

a function of WDR5 in sex determination has not been previously

reported. In this study, we demonstrated that WDR5 is a direct

target of SRY. In addition, WDR5 can regulate itself through a

positive feedback loop. Furthermore, WDR5 can synergize with

SRY to activate Sox9 while repressing the expression of b-catenin.

All these results suggest that WDR5 may have an important role in

sex determination.

Since the SRY gene was discovered in 1990, an answer to the

question as to how SRY promotes testis differentiation has

remained elusive [7,22,23]. Although some pathways regulating

sexual differentiation have been elucidated, the details of SRY

function were poorly understood. Currently, the dominant theory

is that SRY plays a critical role in early gonad development in

either direction (male or female) by pushing the balance to favor

the male development pathway [18,19]. In this process, Sox9,

which is regulated by SRY, controls Sertoli cell formation and,

consequently, testis differentiation. In our experiment, we showed

Figure 2. SRY binds to WDR5 promoter. (A) EMSA analysis of HA

tagged SRY with wild type and mutant probes. Arrows indicate SRY-

probe complex and supershift. (B) ChIP analysis of HA tagged SRY on

WDR5 promoter,WDR5 promoter proximal upstream region, or MyoD

promoter. Mouse IgG serves as a negative control. Graphs show mean

6 SD, n = 3.

doi:10.1371/journal.pone.0034327.g002

WDR5 Is a Direct Target of SRY

PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e34327

that SRY can directly regulate WDR5 expression. The expressed

WDR5 subsequently acts together with SRY to promote Sox9

expression.

The auto feedback loop involved in the regulation of WDR5 is

interesting. During testis differentiation, many feedback loops have

been observed. In mouse, SRY is expressed for only a short

developmental period (dpc10.5–12.5) [24,25], but Sox9 expression

needs to be maintained for testis formation. Sox9 has been shown

to contribute to its own expression [14]. In addition, a target gene

of Sox9, Fgf9, also displays a positive feedback loop in which its

expression helps to activate Sox9 [26]. In contrast to Fgf9, WDR5

seems to act upstream of Sox9. This may be a common scenario of

how sex differentiation is achieved.

Most studies of mammalian sexual determination have been

carried out using mouse models. In mice, at dpc10, genital ridges

are formed without morphological differences between male and

female [18]. At this time in male differentiation, SRY begins to be

expressed and triggers expression of other genes involved in the

differentiation of Sertoli cells. Female-specific gene expression

leading to differentiation of granulose cells must be repressed [19].

b-catenin, a signature gene in ovary development, needs to be

repressed [27]. This is consistent with our results that SRY and

WDR5 together not only increase Sox9 expression, but also

repress b-catenin expression.

The question arises, how can SRY and WDR5 play opposite

roles on different genes? There is little doubt that different

transcription factors or epigenetic modifiers and co-factors

recruited by SRY and WDR5 must help to determine gene

activity. In fact, histone H3K4 methylation, which is usually

associated with WDR5, has been shown to be a dual factor.

Although H3K4 methylation is largely associated with transcrip-

tion initiation and elongation [2,28], some evidence indicated that

this mark could also be involved in gene repression. In yeast,

H3K4me2/3 induced by Set1 can directly contribute to repressive

machinery on PHO5 and PHO84 genes [29,30]. H3K4me3 can

also be recognized by ING2, a component of the Sin3-HDAC

complex, to repress the Cyclin D gene in mammalian cells [31].

However, we could not exclude the possibilities that WDR5 and

SRY recruit different co-factors to act on different genes.

Figure 3. SRY activates WDR5 expression. (A) Western blot analysis of HA tagged SRY, Hsp70, and WDR5 from LNCaP cells containing either

control vector (Ctrl) or HA-tagged SRY. Relative quantitation of WDR5 protein with ImageJ software (NIH, USA) is shown on the bottom. (B)

Quantitative real-time analysis of WDR5 levels as in (A). Graphs show mean 6 SD, n = 3. (C) ChIP analysis on WDR5 promoter with indicated antibodies

as in (A). Graphs show mean 6 SD, n = 3. (D) Schematic representation of HA-SRY-ERtm construct. Numbers show the respective amino acid positions

of the individual constituents (upper panel). Western blot analysis of SRY-HA-ERtm expression with anti-HA antibody. A triangle indicates non-specific

bands (lower panel). (E) Quantitative real time analysis of WDR5 levels from cells with SRY-3HA-ERtm after induction by 4-OHT at indicated time

points. Graphs show mean 6 SD, n = 3. (F) ChIP analysis on WDR5 promoter with either IgG or anti-HA antibody as in (E). Graphs show mean 6 SD,

n = 3. *P,0.05, **P,0.01, #P.0.05 compared to control; Student’s t-test.

doi:10.1371/journal.pone.0034327.g003

WDR5 Is a Direct Target of SRY

PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e34327

In summery, we have identified WDR5 as a novel direct target

of SRY. More interestingly, WDR5 can further cooperate with

SRY to regulate Sox9 and b-catenin expression. It would be

interesting to determine the molecular mechanism by which SRY

plays a dual role at the early stage of mammalian sex

determination. The finding that WDR5 cooperates with SRY will

help probe their roles in testis differentiation.

Materials and Methods

Antibodies and reagents

Anti-HA (12CA5) antibody was purchased from Roche. Anti-

WDR5, anti-H3K4me2, and anti-H3K27me3 antibodies were

purchased from Abcam. Anti-H3K4me3 antibody was purchased

from Millipore. Anti-Hsp70 and anti-SRY (mouse) antibodies

were purchased from Santa Cruz. The pGL3-Basic luciferase

vector was purchased from Promega. DMEM, RPMI 1640, and

fetal bovine serum were obtained from Life Technologies.

Cell lines

LNCaP cells (gifted by Dr. Jiemin Wong, East China Normal

University, China) were seeded in a 100-mm dish and transfected

with 10 mg pCXN-2-SRY-3HA plasmid when the confluency

reached 80%; 36 hours after transfection, cells were supplied with

fresh media containing 600

mg/ml G418. Resistant clones were

selected within 15–20 days and expanded. The G418 concentra-

tion was maintained at 400

mg/ml in the cell culture medium.

The WDR5 coding regions and SRY-3HA-ERtm sequence

were cloned into the retroviral vector plasmid 3HA-MSCV-IRES-

GFP at unique XhoI or EcoRI sites and amphotropic viral

supernatant was obtained as described previously [32].

The siRNA target sequences for WDR5 were inserted into the

XhoI/HpaI sites in the pLL3.7 lentiviral vector according to the

manufacturer’s recommendations (American Type Culture Col-

lection, USA). The oligonucleotides are:

WDR5i-1 sequences: GTGGAAGAGTGACTGCTAA;

WDR5i-2 sequences: GAATGAGAAATACTGCATA

Retroviral supernatant was filtered and added to LNCaP cells

every 24 hours for 3 days. Lentivirus production in 293T cells and

infection of LNCaP cells were performed as described previously

[33]. Cells expressing GFP were sorted by sterile flow cytometry

and expanded.

Site-directed mutagenesis and luciferase reporter assay

Genomic DNA was extracted from 293T cells as described

previously [34]. A fragment (2134 to +115) containing the

proximal promoter of WDR5 was amplified by PCR using the

following primers: sense, 59-GCGGTACCAGGACTTAGGG-

GAATTAATAG -39, which contained a KpnI restriction site

and antisense, 59-CGCAGATCTGTCTCGGGCTTCTTCTC-

39, which contained a BglII restriction site. The PCR product was

cloned into the pGL-3 vector. Mutations were obtained using a

site-directed mutagenesis kit (SBS technologies, Shanghai). All

mutated insert fragments were confirmed by sequencing.

For the luciferase reporter assay, 3610

LNCaP cells were

plated in 12 well plates 24 hours prior to transfection. Triplicate

wells were transiently transfected with the indicated plasmids using

lipofectamine 2000 (Life technologies), and 36 hours after

transfection, relative luciferase activity was measured using the

Luciferase Reporter Assay System (Promega). Beta-galactosidase

assays were performed as normalization controls according to the

Cold Spring Harbor Protocol [35]. b-galactosidase enzyme

activity was measured using a Universal Microplate Spectropho-

tometer.

Co-immunoprecipitation, immunofluorescence, and

histology

For co-immunoprecipitation studies, nuclear extracts were

extracted from LNCaP cells and incubated with HA or WDR5

antibodies for 2 hours at 4uC. A 50% slurry of protein G

Sepharose was added and incubated overnight at 4uC. The

mixture was then centrifuged and the pellet was washed 4 times in

50 mM Tris-HCl, pH 7.9, containing 150 mM NaCl prior to

being resuspended in SDS loading buffer. Samples were separated

by SDS-PAGE, immunoblotted, and probed with the relevant

antibodies. Western blots were detected by using an ECL kit

according to the instructions of the manufacturer (Thermo

Scientific).

For immunofluorescence, LNCaP cells overexpressing SRY-

3HA were mounted on polylysine slides and fixed in 4%

paraformaldehyde for 30 min. After being permeabilized with

0.1% (v/v) Triton X-100, cells were blocked in PBS containing

10% goat serum for 30 min at room temperature. The cells were

then incubated with the polyclonal anti-rabbit WDR5 and anti-

mouse HA primary antibodies at 4uC overnight. After washing

cells four times with PBS, secondary antibodies, goat TexasRed

anti-rabbit IgG and FITC anti-mouse IgG (Vector Laboratories)

were applied in PBS for 1 h at room temperature. The slides were

washed and counterstained with 4,6-diamidino-2-phenylindole

(DAPI) for 3 min before imaging with a Nikon Eclipse 80i

microscope (Nikon).

For mouse histology and immunofluorescence analysis, genital

ridges from timed-pregnant mice were collected at E11.5, E12.5,

and E13.5. Sex was determined for Sry on genomic DNA from

embryo tails by standard PCR. Animal studies were approved by

the Animal Care and Use Committee of the Model Animal

Research Center, the host for the National Resource Center for

Figure 4. WDR5 regulates itself through positive feedback

loop. (A) Schematic representation of primers to detect total (F1 and

R1) and endogenous (F2 and R2) WDR5 mRNA. (B) Total WDR5 mRNA

levels from cells containing either control vector (Ctrl) or overexpressing

WDR5. Graphs show mean6SD, n = 3. (C) Endogenous WDR5 mRNA

levels as in (B). Graphs show mean 6 SD, n = 3. *P,0.05 compared to

control; Student’s t-test.

doi:10.1371/journal.pone.0034327.g004

WDR5 Is a Direct Target of SRY

PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e34327

Mutant Mice in China, Nanjing University. Genital ridges were

fixed in 4% paraformaldehyde and dehydrated through 30%

sucrose solution two hours later. Fixed tissues were embedded in

Jung Tissue Freezing Medium, and serially sectioned. Sections

were washed three times in PBS,transferred to blocking solution

containing 5% donkey serum in 0.1% Tween/PBS for 1 hour,

and incubated with primary antibody at 4uC overnight. After

washing with PBS, the secondary antibody was added and sections

were incubated for 2 hours at room temperature. Slides were

mounted and analyzed with a BX51 Olympus fluorescence

microscope connected to a DP 20 digital camera (Olympus

Corporation, Japan). Combinations of the first and secondary

antibody were as follows: rabbit anti-Sox9 (1:50; Santa Cruz, sc-

20095) and goat anti-WDR5 (1:50; R&D, AF5810); FITC donkey

Figure 5. SRY cooperates with WDR5 to induce Sox9 expression. (A) Immunofluorescence analysis of HA-tagged SRY and WDR5 in LNCaP

cells. (B) ChIP-reChIP (anti-HA antibody ChIP followed by anti-WDR5 antibody ChIP) analysis of HA-tagged SRY and WDR5 on Sox9 promoter, or Sox9

promoter proximal upstream region. Graphs show mean 6 SD, n = 3. (C) Coimmunoprecipitation of WDR5 and HA-tagged SRY from LNCaP cells. (D)

Quantitative real time PCR analysis of Sox9 levels from LNCaP cells containing SF1, SF1+WDR5, SF1+SRY, or SF1+SRY+WDR5. Graphs show mean 6

SD, n = 3. *P,0.05, **P,0.01 compared to SF1 control; Student’s t-test. (E) Quantitative real time PCR analysis of b-catenin levels as in (D). Graphs

show mean 6 SD, n = 3. *P,0.05, **P,0.01 compared to SF1 control; Student’s t-test. (F) Western blot analysis of cellular lysate from cells as in (D).

(G) Western blot analyses of cellular extracts from WDR5i-1 and WDR5i-2 or scrambled control (Ctrl) LNCaP cells with indicated antibodies. (H) WDR5

gene expression analysis by Q-RT-PCR of RNA from WDR5i-1, WDR5i-2 and scrambled control (Ctrl) LNCaP cells. Graphs show mean 6 SD, n = 3.

*P,0.05 compared to the scrambled control, Student’s t-test. (I) Sox9 gene expression analysis by Q-RT-PCR of RNA from WDR5i-1, WDR5i-2 and

scrambled control (Ctrl) LNCaP cells. Graphs show mean6SD, n = 3. **P,0.01 compared to the scrambled control, Student’s t-test.

doi:10.1371/journal.pone.0034327.g005

WDR5 Is a Direct Target of SRY

PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e34327

anti-goat IgG (1:200; Abcam, ab6881) and Alexa Fluor 594

donkey anti-rabbit IgG (1:200; Invitrogen, A21207).

RNA isolation and Real time-PCR

RNA was isolated from cells with Trizol reagent (Life

Technologies) according to the manufacturer’s protocol. cDNA

was synthesized with the SuperScript first-strand synthesis system

(Life Technologies). Q-RT-PCR primers are provided as follow-

ing. Real-time quantitative RT-PCR was performed using the

FastStart Universal SYBR Green Master (Roche) in a Rotorgene

6000 (Corbett Research) in a final volume of 20

ml. Cycling

conditions were 94uC for 15 s, 60uC for 30 s and 72uC for 30 s.

Each reaction was done in triplicate.

The primers for human hypoxanthine guanine phosphoribosyl-

transferase (HPRT) were:

forward 59-ATGGACAGGACTGAACGTCT,

reverse 59-CTTGCGACCTTGACCATCTT.

The primers for human WDR5 were:

forward 59-CACAAGCTGGGAATATCCGATG,

reverse 59-GGGGATTGAAGTTGCAGCAAAA.

The primers for human WDR5 (UTR) were:

forward 59-CGAGAGACTGTCGGGAAGTTG,

Figure 6. Immunolocalization of WDR5 protein and gene expression in embryonic testis. (A) Immunofluorescent staining of WDR5 (in

green) and Sox9 (in red) protein in mouse E11.5 testis. Soc, Somatic cells; GC, Germ cells. Scale bar, 20

mM. (B) Immunofluorescent staining of WDR5

(in green) and Sox9 (in red) protein in mouse E12.5 testis. SC, Sertoli cells; GC, Germ cells. Scale bar, 20

mM. (C) Immunofluorescent staining of WDR5

(in green) and Sox9 (in red) protein in mouse E13.5 testis. IC, interstitial cells. Scale bar, 20

mM. Data shown are representative of three independent

experiments. (D) ChIP analysis of SRY on rat WDR5 promoter and Tcf21 promoter. Graphs show mean 6SD, n = 3. **P,0.01 compared to control;

Student’s t-test. (E) Quantitative real time PCR analysis of WDR5, Sox9, and SRY levels relative to b-actin in male rat gonads at indicated embryonic

stages.

doi:10.1371/journal.pone.0034327.g006

WDR5 Is a Direct Target of SRY

PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e34327

reverse 59- TCCCTAGACAGTGTTAGAAT.

The primers for human Sox9 were:

forward 59-TACGACTGGACGCTGGTG,

reverse 59-TCTCCAGAGCTTGCCCAGCGT.

The primers for human b-catenin were:

forward 59-GAAACGGCTTTCAGTTGAGC,

reverse 59-CTGGCCATATCCACCAGAGT

The primers for rat WDR5 were:

forward 59-CGTGAGTTCCGGAAAGTGTCTGAAG,

reverse 59-GAAATGAACGGCTGAGACTGGAT

The primers for rat Sox9 were:

forward 59-TGAAGATGACCGACGAGCAGGAGAAG,

reverse 59-CTTCCTCGCTCTCCTTCTTCAG

The primers for rat SRY were:

forward 59-CATCGAAGGGTTAAAGTGCCA,

reverse 59-ATAGTGTGTAGGTTGTTGTCC

The primers for rat b-Actin were:

forward 59-GTCGACAACGGCTCCGGCA,

reverse 59-AGGTCTCAAACATGATCTGGGT

Electrophoretic mobility shift assays (EMSA)

To assess the DNA binding activity of SRY in vitro, EMSA was

performed. Nuclear extracts were prepared from LNCaP cells

overexpressing SRY-3HA as described previously [36]. EMSA

was performed by using a LightShift EMSA optimization and

control kit (Pierce, Rockford, USA). The double-stranded

oligonucleotides correspond to the sequence 215 to +9(59-

biotin-ATAGTTTGTTTCTTGGCTCCCTGT-39) of the

WDR5 promoter region. For the binding reaction, 20 fmol

biotin-labeled, double-stranded oligonucleotides were incubated

with nuclear extract (2–5 mg) in 16 binding buffer, 1 mg poly-

dI:dC, 20 min at room temperature. For competition studies,

unlabeled wild-type or mutant double-stranded oligonucleotides

(50-fold molar excess) were pre-incubated with nuclear extract

before addition of labeled oligonucleotides. For supershift assays,

extracts were preincubated with 2

mg mouse IgG or anti-HA

antibody for 15 min at room temperature before addition of the

probe. Reaction products were separated in 6.5% native

polyacrylamide gels in 0.56 TBE buffer and visualized using the

LightShift EMSA kit (Pierce).

Chromatin Immunoprecipitation (ChIP) Assays

ChIP assays were performed as described previously [33,37].

For re-ChIP experiments, immunoprecipitates from the single

ChIP were eluted by incubation for 30 min, 37uCin25ml10mM

dithiothreitol. The supernatant was removed, diluted at least

70 times using ChIP dilution buffer (1% Triton X-100, 2 mM

EDTA, 150 mM NaCl, 20 mM Tris-HCl [pH 8.1]) and then

subjected to another round of immunoprecipitation. PCR

amplification or realtime PCR was performed using the purified

DNA from either the single ChIP or the re-ChIP. For the in vivo

ChIP, carrier ChIP (cChIP) analysis was adopted form Bhandari

et al. [16]. Ten 13dpc rat gonads were used for the assay.

The ChIP primers for human WDR5 promoter were:

forward 59-CTGCTGCATTCTTACAGACTTCTGG,

reverse 59-TGACTACCATATTGAGCCCTGTAGC.

The ChIP primers for human WDR5 promoter proximal region

were:

forward 59-CCAGACCCACCAAGCCACTCAGT,

reverse 59-GGAACGTAACCGCTCAAAATGGCT

The ChIP primers for human Sox9 promoter were:

forward 59-ACCCTACCGTCCGCCCTTTG,

reverse 59-CCGCCTCACCTTAGAGCCAC.

The ChIP primers for human Sox9 promoter proximal region

were:

forward 59-CATCTATTCGATCAGTCAACAG,

reverse 59-CGCTGGGCTTGGAGAGTGTTTAT.

The ChIP primers for rat WDR5 promoter were:

forward 59-GTCAGCCAGGCAGTTGAGAGTAC,

reverse 59-AGCAGCCATCAGTCTCCCTCCAAT

The ChIP primers for rat Tcf21 promoter were:

forward 59-TCTCCACACTGGTGATTAACAAA,

reverse 59-TAATCCAGGCTCAGCTGAGA

Supporting Information

Figure S1 Diagram and alignment of WDR5 genes. (A)

Schematic representation of human WDR5 variants. Filled

rectangles indicate exons, empty rectangles indicate UTR. (B)

2000 bp upstream of the two WDR5 variants from different

vertebrates were aligned using the UCSC Genome Browser.

(TIF)

Figure S2 Co-immunoprecipitation and Western blot

analysis of MLL complex and SRY. WDR5 antibody

immunoprecipitates from vector-containing control (Ctrl) and

SRY-3HA-overexpressing (SRY-OE) cells were blotted with

indicated antibodies on the right.

(TIF)

Author Contributions

Conceived and designed the experiments: ZX XG YH XH QZ. Performed

the experiments: ZX XG YH JJ MZ RL YW Chunyan Ma Chi Ma ZL.

Analyzed the data: QZ XG YH XH. Wrote the paper: ZX XG YH XH

QZ.

References

1. Gori F, Divieti P, Demay MB (2001) Cloning and characterization of a novel

WD-40 repeat protein that dramatically accelerates osteoblastic differentiation.

J Biol Chem 276: 46515–46522.

2. Trievel RC, Shilatifard A (2009) WDR5, a complexed protein. Nat Struct Mol

Biol 16: 678–680.

3. Han Z, Guo L, Wang H, Shen Y, Deng XW, et al. (2006) Structural basis for the

specific recognition of methylated histone H3 lysine 4 by the WD-40 protein

WDR5. Mol Cell 22: 137–144.

4. Gori F, Friedman LG, Demay MB (2006) Wdr5, a WD-40 protein, regulates

osteoblast differentiation during embryonic bone development. Dev Biol 295:

498–506.

5. Wysocka J, Swigut T, Milne TA, Dou Y, Zhang X, et al. (2005) WDR5

associates with histone H3 methylated at K4 and is essential for H3 K4

methylation and vertebrate development. Cell 121: 859–872.

6. Ang YS, Tsai SY, Lee DF, Monk J, Su J, et al. (2011) Wdr5 mediates self-

renewal and reprogramming via the embryonic stem cell core transcriptional

network. Cell 145: 183–197.

7. Berta P, Hawkins JR, Sinclair AH, Taylor A, Griffiths BL, et al. (1990) Genetic

evidence equating SRY and the testis-determining factor. Nature 348: 448–450.

8. Ferrari S, Harley VR, Pontiggia A, Goodfellow PN, Lovell-Badge R, et al.

(1992) SRY, like HMG1, recognizes sharp angles in DNA. EMBO J 11:

4497–4506.

9. Connor F, Cary PD, Read CM, Preston NS, Driscoll PC, et al. (1994) DNA

binding and bending properties of the post-meiotically expressed Sry-relat ed

protein Sox-5. Nucleic Acids Res 22: 3339–3346.

10. Pontiggia A, Rimini R, Harley VR, Goodfellow PN, Lovell-Badge R, et al.

(1994) Sex-reversing mutations affect the architecture of SRY-DNA complexes.

EMBO J 13: 6115–6124.

11. Dubin RA, Ostrer H (1994) Sry is a transcriptional activator. Mol Endocrinol 8:

1182–1192.

12. Bowles J, Cooper L, Berkman J, Koopman P (1999) Sry requires a CAG repeat

domain for male sex determination in Mus musculus. Nat Genet 22: 405–408.

13. Harley VR, Lovell-Badge R, Goodfellow PN (1994) Definition of a consensu s

DNA binding site for SRY. Nucleic Acids Res 22: 1500–1501.

14. Sekido R, Lovell-Badge R (2008) Sex determination involves synergistic action

of SRY and SF1 on a specific Sox9 enhancer. Nature 453: 930–934.

WDR5 Is a Direct Target of SRY

PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e34327

15. Bradford ST, Hiramatsu R, Maddugoda MP, Bernard P, Chaboissier MC, et al.

(2009) The cerebellin 4 precursor gene is a direct target of SRY and SOX9 in

mice. Biol Reprod 80: 1178–1188.

16. Bhandari RK, Sadler-Riggleman I, Clement TM, Skinner MK (2011) Basic

Helix-Loop-Helix Transcription Factor TCF21 Is a Downstream Target of the

Male Sex Determining Gene SRY. PLoS One 6: e19935.

17. Clement TM, Bhandari RK, Sadler-Riggleman I, Skinner MK (2011) SRY

Directly Regulates the Neurotrophin 3 Promoter During Male Sex Determi-

nation and Testis Development in Rats. Biol Reprod.

18. Sekido R, Lovell-Badge R (2009) Sex determination and SRY: down to a wink

and a nudge? Trends Genet 25: 19–29.

19. Kashimada K, Koopman P (2010) Sry: the master switch in mammalian sex

determination. Development 137: 3921–3930.

20. Littlewood TD, Hancock DC, Danielian PS, Parker MG, Evan GI (1995) A

modified oestrogen receptor ligand-binding domain as an improved switch for

the regulation of heterologous proteins. Nucleic Acids Res 23: 1686–1690.

21. Wang YY, Liu LJ, Zhong B, Liu TT, Li Y, et al. (2010) WDR5 is essential for

assembly of the VISA-associated signaling complex and virus-triggered IRF3

and NF-kappaB activation. Proc Natl Acad Sci U S A 107: 815–820.

22. Sinclair AH, Berta P, Palmer MS, Hawkins JR, Griffiths BL, et al. (1990) A gene

from the human sex-determining region encodes a protein with homology to a

conserved DNA-binding motif. Natu re 346: 240–244.

23. Gubbay J, Collignon J, Koopman P, Capel B, Economou A, et al. (1990) A gene

mapping to the sex-determining region of the mouse Y chromosome is a

member of a novel family of embryonically expressed genes. Nature 346:

245–250.

24. Wilhelm D, Martinson F, Bradford S, Wilson MJ, Combes AN, et al. (2005)

Sertoli cell differentiation is induced both cell-autonomously and through

prostaglandin signaling during mammalian sex determination. Dev Biol 287:

111–124.

25. Taketo T, Lee CH, Zhang J, Li Y, Lee CY, et al. (2005) Expression of SRY

proteins in both normal and sex-reversed XY fetal mouse gonads. Dev Dyn 233:

612–622.

26. Kim Y, Kobayashi A, Sekido R, DiNapoli L, Brennan J, et al. (2006) Fgf9 and

Wnt4 act as antagonistic signals to regulate mammalian sex determination.

PLoS Biol 4: e187.

27. Maatouk DM, DiNapoli L, Alvers A, Parker KL, Taketo MM, et al. (2008)

Stabilization of beta-catenin in XY gonads causes male-to-female sex-reversal.

Hum Mol Genet 17: 2949–2955.

28. Dou Y, Milne TA, Tackett AJ, Smith ER, Fukuda A, et al. (2005) Physical

association and coordinate function of the H3 K4 methyltransferase MLL1 and

the H4 K16 acetyltransferase MOF. Cell 121: 873–885.

29. Carvin CD, Kladde MP (2004) Effectors of lysine 4 methylation of histone H3 in

Saccharomyces cerevisiae are negative regulators of PHO5 and GAL1-10. J Biol

Chem 279: 33057–33062.

30. Pinskaya M, Gourvennec S, Morillon A (2009) H3 lysine 4 di- and tri-

methylation deposited by cryptic transcription attenuates promoter activation.

EMBO J 28: 1697–1707.

31. Shi X, Hong T, Walter KL, Ewalt M, Michishita E, et al. (2006) ING2 PHD

domain links histone H3 lysine 4 methylation to active gene repression. Nature

442: 96–99.

32. Zhao Q, Zhou W, Rank G, Sutton R, Wang X, et al. (2006) Repression of

human gamma-globin gene expression by a short isoform of the NF-E4 protein

is associated with loss of NF-E2 and RNA polymerase II recruitment to the

promoter. Blood 107: 2138–2145.

33. Zhao Q, Rank G, Tan YT, Li H, Moritz RL, et al. (2009) PRMT5-mediated

methylation of histone H4R3 recruits DNMT3A, coupling histone and DNA

methylation in gene silencing. Nat Struc t Mol Biol 16: 304–311.

34. Yao YL, Dupont BR, Ghosh S, Fang Y, Leach RJ, et al. (1998) Cloning,

chromosomal localization and promoter analysis of the human transcription

factor YY1. Nucleic Acids Res 26: 3776–3783.

35. Smale ST (2010) Beta-galactosidase assay. Cold Spring Harb Protoc 2010: pdb

prot5423.

36. Zelivianski S, Glowacki R, Lin MF (2004) Transcriptional activation of the

human prostatic acid phosphatase gene by NF-kappaB via a novel hexanucleo-

tide-binding site. Nucleic Acids Res 32: 3566–3580.

37. Rank G, Cerr uti L, Simpson RJ, Moritz RL, Jane SM, et al. (2010)

Identification of a PRMT5-dependent repressor complex linked to silencing of

human fetal globin gene expression. Blood 116: 1585–1592.

WDR5 Is a Direct Target of SRY

PLoS ONE | www.plosone.org 10 April 2012 | Volume 7 | Issue 4 | e34327