Báo cáo khoa học: Novel target genes of the Wnt pathway and statistical insights into Wnt target promoter regulation potx

Novel target genes of the Wnt pathway and statistical
insights into Wnt target promoter regulation
Slava Ziegler
1
, Sonja Ro
¨
hrs
1
, Lara Tickenbrock
2
, Tarik Mo
¨
ro
¨
y
3
, Ludger Klein-Hitpass
3
,
Ingrid R. Vetter
1
and Oliver Mu
¨
ller
1
1 Max-Planck-Institut fu
¨
r molekulare Physiologie, Dortmund, Germany
2 Interdisziplina
¨
res Zentrum fu

¨
r Klinische Forschung, Universita
¨
tsklinikum Mu
¨
nster, Mu
¨
nster, Germany
3 Institut fu
¨
r Zellbiologie (Tumorforschung), Universita
¨
tsklinikum, Essen, Germany
The Wnt signalling pathway plays an important role in
embryonic development, cell differentiation and cell
proliferation [1]. The biological effects of the Wnt
pathway are mediated via the regulation of direct (pri-
mary) and indirect (secondary) target genes (for a list
of target genes see />pathways/targets.html) [2,3]. The transcription activa-
tion by TCF ⁄ Lef and b-catenin is a crucial step in the
direct target gene activation by the pathway. Thus the
activation of a Wnt target gene by TCF via a TCF ⁄ Lef
binding motif within the promoter region indicates the
direct regulation. Direct Wnt target genes are the cell
cycle kinase activator cyclin D1 and the transcription
factors MYC and PEA3 [4–6].
Keywords
microarray analysis; statistical analysis of
transcription factor motifs; transcription
factor binding site; Wnt pathway; Wnt

target gene
Correspondence
O. Mu
¨
ller, Max-Planck-Institut fu
¨
r
molekulare Physiologie, Otto-Hahn-Straße
11, 44227 Dortmund, Germany
Fax: +49 231 133 2199
Tel: +49 231 133 2158
E-mail: oliver.mueller@mpi-dortmund.
mpg.de
(Received 15 December 2004, revised 19
January 2005, accepted 24 January 2005)
doi:10.1111/j.1742-4658.2005.04581.x
The Wnt pathway controls biological processes via the regulation of target
gene expression. The expression of direct Wnt target genes, e.g. cyclin D1
and MYC, is activated by the transcription factor TCF, which binds to
specific sequence motifs in the promoter. Indirect target genes are regulated
via transcription regulators, which are targets of the Wnt pathway. As an
example, MYC regulates the MYC interacting zinc finger protein-1 (MIZ-
1), which is able to inhibit the expression of the indirect target p21WAF1.
We intended to identify new Wnt target genes and to get a deeper insight
into the regulatory mechanisms of Wnt target gene expression. For this we
analyzed the differential expression pattern of Wnt-1 activated cells by
microarray analysis. We identified 43 sequences including eight expressed
sequence tags (ESTs), which showed increased transcript levels, and 104
sequences including 19 ESTs with decreased RNA levels. Northern blot
and real-time quantitative PCR analysis of the differential expression levels

of 15 genes confirmed the differential expression trends of eight candidate
genes. When the Wnt pathway was regulated at the lower level of glycogen
synthase kinase-3b (GSK-3b) or adenomatous polyposis coli (APC), we
detected discrepant expression trends. We compared the number of binding
sites of transcription factors in the genomic regions of all candidate target
genes with the number of sites in control genes. We found that the genomic
regions of the down-regulated genes include an increased number of puta-
tive MIZ-1 binding sites. Our study introduces several new Wnt target
genes and provides indications that the specific gene expression pattern
depends on the type of the activation trigger or the level of interference
with the Wnt pathway. Furthermore, our data indicate that a high propor-
tion of Wnt target genes are regulated by indirect mechanisms.
Abbreviations
APC, adenomatous polyposis coli; EST, expressed sequence tag; GEO, gene expression omnibus; GSK-3b, glycogen synthase kinase-3b;
MIZ-1, MYC interacting zinc finger protein-1; qPCR, real-time quantitative PCR; RT-PCR, reverse transcription PCR; TSS, transcription start
site.
1600 FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS
While the regulatory mechanism of direct target gene
expression is well established, there are only few data
available about the regulation of indirect Wnt targets. A
prominent indirect negative target is the cell cycle kinase
inhibitor p21WAF1, which is induced in cells expressing
dominant negative TCF [7]. There are indications that
p21WAF1 expression is regulated by the relative levels
of the MYC interacting zinc finger protein-1 (MIZ-1)
and the direct Wnt target MYC [8]. In the absence of
MYC, MIZ-1 activates p21WAF1 expression, while in
the presence of high MYC levels, MIZ-1 together
with MYC functions as a repressor of p21WAF1 tran-
scription. In epithelial cells of the intestine, the MYC ⁄

MIZ-1 ⁄ p21WAF1 cascade acts as a switch between dif-
ferentiation and proliferation [7]. There are several other
known genes, which show a decreased expression level
in Wnt-activated cells. Examples are targets of the AP-1
family of transcription factors, which show decreased
expression in Wnt-activated cells [9].
Many microarray screenings have been performed
with the aim of identifying new target genes of the Wnt
pathway [9–12]. Until now, only a limited number of
candidate target genes have been confirmed and further
characterized by experimental methods. Recently, a new
statistical approach was introduced to analyse the distri-
bution of TCF sites in promoters of genes, which have
been identified as potential candidates of b-catenin ⁄ TCF
signalling by microarray screening [11]. We expanded
this strategy to gain general insights into the regulation
of promoters of potential target genes of the Wnt path-
way, which we identified by microarray screening.
Results
Evaluation of cell models
At the beginning we evaluated our cell models. First
we proved the presence of the Wnt-1 transcript in the
C57MG ⁄ Wnt-1 cells (Fig. 1A). A clear RT-PCR pro-
duct of the expected size indicated Wnt-1 expression in
C57MG ⁄ Wnt-1 cells of the same passage as the cells,
which we used for further analysis. Second, we showed
that the incubation of C57MG cells with lithium or
with Wnt-3a containing conditioned medium led to an
increase of the relative b-catenin level (Fig. 1B,C). We
conclude that the Wnt pathway in C57MG cells can

be activated by lithium and by conditioned medium
from L Wnt-3a cells.
Microarray analysis
We compared the gene expression patterns of
C57MG ⁄ Wnt-1 cells and C57MG ⁄ PLNCx cells by
DNA microarray analysis. As we used only one array
per condition, this analysis served only as a very first
screening for the collection of gene data with potentially
differential expression. The differential expression as
well as the function of the genes as Wnt pathway target
genes had to be evaluated in further experiments. The
raw data of the analysis with the list of all analysed
genes and the expression factors were deposited in the
GEO (Gene Expression Omnibus) database (http://
www.ncbi.nlm.nih.gov/projects/geo) under the GEO
accession numbers GSM32828 and GSM32829. When
using the described criteria for the definition of candi-
date target genes, we were able to identify 147 candidate
targets of the Wnt pathway: 43 up-regulated, among
them 35 known genes and eight ESTs. We found 104
down-regulated sequences representing 85 known genes
and 19 ESTs (supplementary tables S2, S3). Nineteen of
A
B
C
Fig. 1. Evaluation of cell models. (A) Agarose gel electrophoresis of
RT-PCR products using template RNA purified from C57MG ⁄ PLNCx
cells (P) or from C57MG ⁄ Wnt-1 cells (W). The PCR product of the
length of the Wnt-1 transcript is marked. The lengths of the exem-
plary marker fragments are indicated in base pairs (bp). M, Marker.

(B) Western blot to analyse the relative b-catenin levels in C57MG
cells after incubation with 20 m
M KCl (K
+
)or20mM LiCl (Li
+
). The
size of a marker fragment is shown on the left in kilodaltons (kD).
(C) Western-blot to analyse the relative b-catenin levels in C57MG
cells after incubation with the conditioned medium from non-trans-
fected L-M(TK-) cells (L-CM) and from Wnt-3a transfected L-M(TK-)
cells (L-Wnt-3a-CM). The size of a marker fragment is shown on
the left in kilodaltons (kDa).
S. Ziegler et al. Wnt target genes and the regulation of their promoters
FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS 1601
these candidate genes have already been described as
target genes in Wnt-activated murine cells or in APC
inactivated mouse model systems (supplementary tables
S2, S3). Of these, 12 genes were described with expres-
sion trends opposite to the trends that we found.
Evaluation of gene expression in C57MG/Wnt-1
cells
Several methods were used to evaluate microarray data
and to identify and to confirm target genes of the Wnt
pathway. All biological experiments were performed at
least twice. Representative results of the northern blot
and western blot experiments are shown. To evaluate the
microarray data the expression levels of 15 selected candi-
date genes were compared in C57MG ⁄ Wnt-1 cells and in
C57MG ⁄ PLNCx cells by northern blot: the up-regulated

candidates Lcn2, Fos, Gnb1, Fabp5, Slc7a2, Osf2, Sdc4,
Ccl2, Pedf and Fas and the down-regulated candidates
Kit1, Lgals8, Gbp3, Mrp ⁄ Plf and Ramp3. While the dif-
ferential expression of the genes Lcn2, Fos, Fabp5, Slc7a2,
Ccl2, Pedf, Fas, Mrp ⁄ Plf and Ramp3 could be confirmed
(Fig. 2), the genes Gnb1, Osf2, Sdc4, Kit1, Lgals8 and
Gbp3 showed similar expression levels in Wnt-1 trans-
fected cells compared to the levels in control cells (not
shown). Next we asked whether the differential expres-
sion trends depend on the type of the trigger and the
method of Wnt pathway activation. For this, we tested
the transcript levels after activation of the Wnt pathway
by lithium or by Wnt-3a. We are aware that lithium influ-
ences several pathways and inhibits other enzymes in
addition to GSK-3b (e.g. Na ⁄ K ATPase, adenylcyclase,
enzymes of the prostaglandins E1 synthesis and inositol-
1-phosphatase). Nevertheless lithium incubation is a
common way to activate the Wnt pathway [32–34]. The
candidates Fos, Slc7a2 and Ramp3 showed expression
trends in the presence of lithium, which correlated to the
corresponding trends in C57MG ⁄ Wnt-1 cells, whereas
Lcn2, Fabp5, Ccl2, Pedf, Fas and Mrp ⁄ Plf showed trends
contrary to the corresponding trends (Fig. 3). Next we
analysed the transcript levels after activation of the Wnt
pathway by Wnt-3a containing medium (L-Wnt-3a-CM)
[24]. We found that L-Wnt-3a-CM incubation leads to
an increased relative expression rates of the genes Fos,
Fabp5, Slc7a2, Ccl2, Pedf and Fas, and to decreased
rates of the genes Mrp ⁄ Plf and Ramp3 (Fig. 4). These
experimental results are consistent with the microarray

results and indicate that the activation level in the
Fig. 2. Northern blot analysis of expression
of candidate target genes in C57MG ⁄ PLNCx
(P) and C57MG ⁄ Wnt-1 (W) cells. The rele-
vant bands are indicated and marked by the
abbreviation codes of the corresponding
genes. The ethidium-bromide stained gels
with the 18S and 28S rRNA species are
shown for demonstrating equal loading.
Wnt target genes and the regulation of their promoters S. Ziegler et al.
1602 FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS
pathway and the type of the activation trigger are crucial
for the target gene regulation.
To see whether the differential gene expression can
also be found on the translation level, we analyzed the
relative amounts of the extracellular proteins Lcn2,
Ccl2, Pedf and Mrp ⁄ Plf in the culture medium and of
Ramp3 in the cellular lysates of Wnt-1 transfected and
of lithium stimulated C57MG cells (Fig. 5). The west-
ern blots confirmed the findings of the northern blot
analysis with one exception. The level of Mrp ⁄ Plf was
increased in Wnt transfected cells, whereas the RNA
level was decreased in these cells as found by micro-
array and by northern blot. We decided to analyze
more closely the Mrp ⁄ Plf protein levels in Wnt-activa-
ted cells. For this we tested the relative amount of
intracellular Mrp ⁄ Plf in lithium activated cells (Fig. 5I)
and detected a higher protein level as compared to
nonactivated cells; this correlates to the high RNA
level in these cells and to the high level of the extracel-

lular protein (Figs 3 and 5G,H).
There are 4 Mrp ⁄ Plf proteins, Plf1, Plf2, Mrp3 and
Mrp4, which are highly homologous on the sequence
level but differ in their patterns of glycosylation. The
corresponding mRNAs are transcribed from overlap-
ping genomic regions [27]. Using RT-PCR and restric-
tion enzyme digestion we found that the RNA, which
we purified from PLNCx or Wnt-1 transfected
C57MG cells or from cells treated with K
+
or Li
+
contains the transcripts of Plf1 and Mrp4, rather than
Fig. 3. Northern blot analysis of expression
of candidate target genes in C57MG cells
after treatment with 20 m
M LiCl. The
relevant bands are indicated. The ethidium-
bromide stained gels with the 18S and 28S
rRNA species are shown to demonstrate
equal loading.
Fig. 4. Analysis of transcript levels by real-time quantitative PCR
(qPCR). The relative expression rates of the indicated genes in
L-Wnt-3a-CM treated C57MG cells are shown (grey bars) in relation
to the expression rates in L-CM treated cells (white bars). Results
are the means of two independent analyses, which were per-
formed in triplicates.
S. Ziegler et al. Wnt target genes and the regulation of their promoters
FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS 1603
these of the two other isoforms (Fig. 6). These results

correlate to the protein bands of  38 kDa (Plf1) and
27 kDa (Mrp4) [27], which were detected in the west-
ern blot analysis (Fig. 5).
Analysis of promoter regulation by Wnt
signalling
We screened databases for the promoters of the 15
genes that we had further analyzed by northern blot
and found the promoter regions of Lcn2 [35], Fos [36],
Gnb1 (AB066210), Fabp5 [37], Sdc4 (D89572), Pedf
(AF017050), Kit1 [38] and Mrp ⁄ Plf [39] (Table 1). Pro-
moter sequences of the genes Lcn2, Fos, Fabp5, Pedf
and Kit1 were used in luciferase reporter genes to test
the regulatory activities of the promoters in cells with
activated Wnt pathway. When we compared the activit-
ies of some of these reporter constructs in RK13 and
SW480 cells and the TCF ⁄ Lef responsive topflash motif
[29] we confirmed our results from the RNA and pro-
tein expression analysis (Table 2) (Fig. 7). Lithium acti-
vation of the Wnt pathway regulated the Lcn2, Fos and
Pedf promoters as expected from the transcript levels
in the northern blot experiments. Pathway inactivation
by APC transfection in SW480 cells supported the
assumption that the trend of expression regulation
depends on the level within the Wnt pathway, on which
the trigger interferes with the pathway. The activation
or inactivation on the GSK-3b level by lithium or on a
further down-stream level by APC transfection had
similar effects on the promoters Lcn2 and Pedf. These
effects are opposite to the effects caused by activation
of the pathway on the Wnt level by Wnt-1 transfection.

Classification of analyzed genes
We screened the known promoter sequences for binding
sites of the transcription factors TCF, MYC, AP-1,
PEA3 and MIZ-1 (Table 1). Based on the results of this
analysis we tried to define the identified genes as direct
or indirect target genes. When analysing other path-
ways, this discrimination could be made experimentally.
The inhibition of protein translation (e.g. by cyclohexi-
mide) led to the selective repression of indirect targets.
When analysing the Wnt pathway, this approach is not
readily applicable because signal transduction depends
on the ongoing protein synthesis of b-catenin [10].
The Lcn2 promoter does not comprise any binding
site for the transcription factors TCF, MYC, AP-1 or
PEA3, which are assumed to activate direct or indirect
Wnt target genes suggesting that Lcn2 expression is
activated via motifs outside the reported promoter
region. Indeed, six TCF ⁄ Lef binding sites were found
at positions outside the reported Lcn2 promoter in the
genomic region from position )3000 to +100 bp:
)2644, )1875, )1809, )1377, )926, )827. The promot-
ers of the genes Gnb1, Fabp5, Sdc4 and Pedf comprise
binding sites for transcription activating, and the genes
Kit1 and Mrp ⁄ Plf include binding sites for expression
repressing factors (Table 1).
Screening of regulatory regions of differentially
expressed genes and statistical evaluation
We estimated the overall relevance of the microarray
analysis using statistical analysis [11]. First we screened
public databases to get the genomic sequence of the

ABCD
EFGH
LKI
Fig. 5. Western blot analysis of protein levels in culture medium or in lysates from C57MG ⁄ PLNCx (P), C57MG ⁄ Wnt-1 (W), or C57MG cells
treated with either 20 m
M KCl (K
+
) or with 20 mM LiCl (Li
+
). (A–H) The extracellular proteins Lcn2, Ccl2, Pedf and Mrp ⁄ Plf were analyzed in
the concentrated culture medium. I–L, Mrp ⁄ Plf was additionally and Ramp3 was exclusively analyzed in the cellular lysates. The expected
bands of the proteins are indicated. The sizes of exemplary marker proteins on the corresponding stained gels are shown in kilodaltons
(kDa) on the right sides of the gels.
Wnt target genes and the regulation of their promoters S. Ziegler et al.
1604 FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS
differentially expressed genes. Unknown genes (ESTs
in supplementary tables S2, S3) were not included.
We were able to identify the genomic regions from
)3000 to +100 bp relative to the transcription start
of 119 of the 120 candidate genes. All retrieved seq-
uences are available upon request. We looked for the
presence of transcription factor motifs in these regions
using the public signal scan service (http://bimas.
dcrt.nih.gov/molbio/signal) and in-house software.
Analyzing the genomic regions of the up-regulated
candidates, we included the motifs of four transcrip-
tion factors, which are important for the direct or indi-
rect activation of gene expression in Wnt-activated
cells. These are TCF, Myc and PEA3 [5,6] and AP-1
[31] (supplementary table S3, Figs 2 and 3). In the

analysis of the down-regulated candidates we included
the putative motifs of the transcription factor MIZ-1,
which is assumed to be involved in the regulation of
negative Wnt targets [7]. In this analysis we again
included the AP-1 promoter element because others
identified Fos as a gene that is down-regulated in
Wnt-activated cells [9].
Results of the analysis are summarized in
Tables 3–5. (For detailed results with all identified
positions of the screened transcription factors, see sup-
plementary tables S4 and S5.) All genes showing
decreased transcription levels include MIZ-1 sites.
Remarkably, the total number of MIZ-1c sites is signi-
ficantly increased in the region )1000 to )501 bp of
target genes with decreased expression, when compared
to the number of sites in the corresponding region in
control genes (Table 6). The number of the transcrip-
tion factors TCF ⁄ Lef, MYC, AP-1 and PEA3 showed
no significant accumulation in the genes with increased
or decreased expression (Tables 4 and 6).
Fig. 6. Analysis of the proliferin gene transcript. (A) Schematic pres-
entation of the four possible fragments obtained by RT-PCR of the
transcripts of Mrp ⁄ Plf isoforms [27]. The highly homologous frag-
ments differ in the numbers and positions of Fnu4H1 ⁄ BstX1
restriction sites. All four isoforms comprise BstX1 and Fnu4H1
sites at positions 34 and 598 bp. Plf1 and Mrp3 show BstX1 sites
at position 352, and Plf2 and Mrp3 show Fnu4H1 sites at posi-
tion 323. In the horizontal grey bars the lengths of the expected
restriction fragments are given in base pairs. (B) Agarose gel
electrophoresis of single digestions of RT-PCR products with the

indicated restriction enzymes. RNA purified from C57MG ⁄ PLNCx
cells was used as template. (C) Agarose gel electrophoresis of the
Fnu4H1 ⁄ BstX1 double digestions of RT-PCR products. RNA purified
from C57MG ⁄ PLNCx (P), from C57MG ⁄ Wnt-1 (W) cells, or from
C57MG cells treated either with KCl (K
+
)orLiCl(Li
+
) was used as
template. The sizes of exemplary marker fragments are indicated in
base pairs (bp). M, marker lane.
Table 1. Number of transcription factor binding sites in promoters.
The promoters of the up-regulated genes were analysed for binding
sites of TCF, MYC, AP-1 and PEA3. The promoters of the down-
regulated genes were analysed for binding sites of AP-1 and MIZ-1.
Gene
Promoter
length
Promoter
accession ID
Binding sites of promoters of
up-regulated genes
Number of transcription
factor binding sites
TCF MYC AP-1 PEA3
Lcn2 793 (bp) X81627 0 0 0 0
Fos 2 (kb) AF332140 4 1 2 3
Gnb1 1089 (bp) AB066210 1 3 1 4
Fabp5 2 (kb) AJ223066 5 3 3 0
Sdc4 690 (bp) D89572 1 3 0 0

Pedf 1.4 (kb) AH005932 1 1 0 2
Binding sites of promoters of
down-regulated genes
Number of transcription
factor binding sites
AP-1 MIZ-1a MIZ-1b MIZ-1c
Kit1 1482 bp U44724 1 2 0 8
Mrp ⁄ Plf 679 bp X05787 2 2 1 1
S. Ziegler et al. Wnt target genes and the regulation of their promoters
FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS 1605
Discussion
Over recent years, a vast number of candidate Wnt
target genes have been identified by microarray
analysis [9–12]. In the majority of these studies, one or
a few of the identified target genes were characterized.
In this study, we followed more general aims. First we
intended to identify and to confirm novel targets of
the Wnt pathway. We identified 147 candidate targets,
while screening the differential gene expression pattern
of Wnt-1 transfected murine cells by microarray analy-
sis. When judging these data principal constraints have
to be made. First, the limited number of arrays might
have led to the identification of candidate genes, which
are no true target genes. Second, the Wnt specific
expression pattern in Wnt-1 transfected cells might be
masked by unspecific side-effects of the stable transfec-
tion with a viral vector and the constitutively active
Wnt pathway. Both limitations can be smoothened by
the experimental evaluation of the microarray data
using alternative methods and experimental models.

We analyzed the differential expression levels of exem-
plary candidate genes by northern and western blots
and by qPCR. The differential expression trend of the
9 genes Lcn2, Fos, Fabp5, Slc7a2, Ccl2, Pedf, Fas,
Mrp ⁄ Plf and Ramp3 could be confirmed by northern
and western blot of Wnt-1 transfected cells. These
genes have been described as important for apoptosis,
cell proliferation and angiogenesis in several other
studies. The qPCR analysis confirmed the expression
trend of eight of these genes in Wnt-3a stimulated
cells. The analysis of the levels of selected proteins cor-
related with the northern blots with one exception.
The Mrp⁄ Plf protein level in Wnt transfected cells is
increased, though the RNA level is decreased in these
cells. We speculate that Mrp ⁄ Plf is regulated by post-
transcriptional mechanisms, which lead to the relative
accumulation in Wnt transfected cells or to the relative
protein degradation in control cells. After lithium acti-
vation, the genes Fos, Slc7a2 and Ramp3 showed the
same regulation trend as in Wnt-1 transfected cells.
The genes Lcn2, Fabp5, Ccl2, Pedf, Fas and Mrp ⁄ Plf
showed different regulation trends in lithium treated
cells and in Wnt-1 transfected cells. We presume that
the effects induced by lithium are due to its inhibitory
effects on GSK-3b. Though we can not exclude that
other lithium regulated enzymes have influences on
Wnt target gene expression. There are two possible
explanations for the discrepancies between the gene
expression pattern in lithium activated cells and the
pattern in Wnt-1 transfected cells. First, the type of

trigger and the activation level in pathway might play
a role in the regulation of gene expression. The acti-
vation at an upper level of the pathway by the Wnt
factor might have effects that are different to those
caused by activation at the GSK-3b level by lithium.
This difference might be caused by activation or
by inhibitory feedback loops, which are present at
specific levels of the pathway. Second, the detected
discrepancies might be due to different methods of
Table 2. Summary of experimental results. Increased levels or activation (+), decreased levels or inhibition (–) or no alteration (0) are
indicated.
Gene
Regulation of gene at the following levels using methods appropriate each:
Transcription Translation Promoter
Microarray Northern blot qPCR Western blot Reporter gene assay
C57MG ⁄ Wnt-1 C57MG ⁄ Wnt-1 C57MG + Li C57MG + Wnt-3a C57MG ⁄ Wnt-1 C57MG + Li RK13 + Li SW480 APC
Lcn2++– +––+
Fos++++ +0
Gnb1 + 0 0
Fabp5 + + – + 0 0
Slc7a2 + + + +
Osf2 + 0
Sdc4 + 0 0
Ccl2 + + – + + –
Pedf + + – + + – – +
Fas + + – +
Kit1 – 0 0 0 0 0 0
Lgals8 – 0 0
Gbp3 – 0 0
Mrp ⁄ Plf––+– ++

Ramp3 – – – – – –
Wnt target genes and the regulation of their promoters S. Ziegler et al.
1606 FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS
interference with the cellular metabolism. The effects
caused by short-term stimuli like lithium or by the
Wnt-3a protein might differ from the effects caused by
long-term and constitutive stimulation of the Wnt
pathway like it is caused by stable transfection of
Wnt-1. This difference might be due to: promoters,
activation loops or regulatory feedback loops that are
activated only after a certain time in cells with a con-
stitutively active Wnt pathway. Our results supported
the first possibility. The effects caused by the activa-
tion of the Wnt pathway by transfection with Wnt-1
are similar to the effects caused by the extracellular
Wnt-3a protein. The activities of the Lcn2, Fos and
Pedf promoters in lithium incubated cells correlate to
the detected expression trends of the corresponding
genes in lithium activated C57MG cells. In SW480
cells, the pathway is constitutively active due to the
lack of functional APC. When the Wnt pathway was
inhibited by transfection with APCwt the activities of
the Lcn2 and Pedf promoters increased. This result
correlates to the results that were obtained when the
pathway was activated by lithium; but it is opposite to
the result obtained with Wnt-1 transfected cells. We
conclude from these findings that the activating trigger
and the level of activation within the pathway are cru-
cial for the selection of a target gene and the trend of
the promoter regulation.

For our second aim we intended to draw general
conclusions about the significance of the microarray
data. As the number of genes that can be analyzed
experimentally by northern or western blot is limited,
we used a bioinformational approach [11]. We ana-
lyzed the genomic up-stream regions of all candidate
targets for the presence of binding motifs for transcrip-
tion factors, which play a role in the regulation of
Wnt target genes. Generally, the highest number of
relevant transcription factor binding motifs are located
in the region )500 bp to the transcription start site
(TSS) [40]. At the beginning, we screened only this
region for transcription factor binding sites and did
not find any significant accumulation of sites (Tables 3
and 4). To make sure that our analysis comprised
all relevant binding motifs we decided to screen the
genomic region )3000 to +100 bp relative to the TSS.
We did not find a significant accumulation of TCF ⁄ Lef
binding sites. Others have found an increased number
of TCF ⁄ Lef sites in the region )1000 to )501 bp and
in the 5¢ untranslated region of positive Wnt targets in
human tumours [11]. We ascribe this discrepancy to
the different experimental models and the different
pools of candidate target genes identified by microar-
ray analysis. Analyzing the genomic regions, we found
that the number of putative MIZ-1 binding sites is sig-
nificantly increased in the region )1000 to )501 bp of
down-regulated genes as compared to the same region
in control genes. This outcome is a strong indication
for the negative regulation of Wnt target genes via the

MYC ⁄ MIZ-1 cascade. We are still aware that
the MIZ-1 binding motifs, which we used in our
screen, are yet to be confirmed by experimental data.
In summary, we introduced and confirmed the novel
Wnt pathway target genes Lcn2, Fos, Fabp5, Slc7a2,
Ccl2, Pedf, Fas, Mrp ⁄ Plf and Ramp3. The genes Lcn2,
Fabp5, Ccl2, Pedf, Fas and Mrp ⁄ Plf show differential
expression trends, which depend on the trigger type
and on the level of interference with the Wnt pathway.
We conclude that the type of the trigger and the regu-
lation level within the Wnt pathway decide about the
gene expression pattern and thus about the kind of the
specific biological answer. In the second part of our
study we found that the number of MIZ-1 binding
sites is statistically high in the genomic regions neigh-
bouring the genes, which show decreased expression
levels in Wnt-activated cells.
Fig. 7. Exemplary results of the reporter gene assays. (A) The bars
represent the relative activities of the topflash motif or the Pedf
promoter in untreated (grey) or Li
+
treated RK13 cells (black). (B)
The bars represent the relative activities of the Topflash motif or
the Pedf promoter in pCMV transfected (black) or APCwt trans-
fected SW480 cells (grey). Average results from three experiments
are shown. Vertical lines indicate SD.
S. Ziegler et al. Wnt target genes and the regulation of their promoters
FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS 1607
Experimental procedures
Cell culture

Murine mammary C57MG cells can be transformed by the
retroviral infection with oncogenic Wnt-1 [13–15]. The result-
ing C57MG ⁄ Wnt-1 cells have been used in many studies to
characterize the Wnt pathway and to identify novel Wnt
target genes [16–20]. The culture of C57MG ⁄ Wnt-1 and of
C57MG ⁄ PLNCx cells, which are C57MG cells transfected
with an empty control vector, have been described previously
[17,21]. To evaluate the sustained Wnt-1 expression in
C57MG ⁄ Wnt-1 cells we tested the presence of the Wnt-1
transcript by reverse transcription RT-PCR. We used
the following primers for RT-PCR: 5¢-CATCGAGTC
CTGCACCTG-3¢;5¢-TGGGCGATTTCTCGAAGTAG-3¢.
The GSK-3b can be inhibited by lithium ions (Li
+
), which
are added to the medium of cultured cells [22,23]. To test the
effects of the Wnt pathway activation by Li
+
, we incubated
C57MG cells with 20 mm LiCl for 24 h. As a negative con-
trol we used untreated cells or cells treated with 20 mm KCl.
L-M(TK-) cells (L cells) and Wnt-3a transfected L-M(TK-)
cells (L Wnt-3a cells) from murine subcutaneous connective
tissue were purchased from ATCC. L Wnt-3a cells express
and secrete biologically active Wnt-3a [24]. For preparation
of conditioned media from L cells or from L Wnt-3a cells we
followed the protocol from ATCC (distributed by LGC Pro-
mochem, Wesel, Germany). C57MG, C57MG-pLNCx and
C57MG/Wnt-1 cells were kindly provided by J. Kitajewski
(Columbia University, NY, USA).

Analysis of differential gene expression
Total RNA was purified by standard methods from semi-
confluent C57MG ⁄ PLNCx and from C57MG ⁄ Wnt-1 cells.
Target preparation and hybridization to Affymetrix MG-
U74Av2 microarrays (Affymetrix, Santa Clara, CA, USA)
was performed as described [25]. On the MG-U74Av2 chip,
the major part of all known murine genes and 6000 murine
ESTs are represented. Gene chip array analysis and data
evaluation were performed according to protocols provided
by the microarray manufacturer. Northern blot analysis was
performed to validate the results of the microarray analysis.
Denaturing RNA electrophoresis, blotting and detection of
membrane immobilized RNA fragments by hybridization
with radioactively labeled probes were done by following
standard protocols and as described [26]. The relative
levels of proteins were measured by western blot of cell
lysates or of concentrated culture medium. Cell lysates were
prepared by sonification after re-suspension in NaCl ⁄ P
i
with
protease inhibitors (Roche, Mannheim, Germany) at 4 °C
followed by centrifugation at 16 000 g for 15 min. The
supernatants were calibrated to equal protein concentrations
by dilution with NaCl ⁄ P
i
and analyzed by SDS ⁄ PAGE
and western blot. Poly(vinylidene difluoride) (PVDF) mem-
branes (Amersham Biosciences Europe, Freiburg, Germany)
were probed with polyclonal rabbit antibodies against
Lcn2 antibody (kindly provided by S T. Chu, Institute of

Biological Chemistry, Academia Sinica, Taiwan), Pedf
(kindly provided by G. Maik-Rachline, The Weizmann
Institute of Science, Rehovot, Israel), Mrp ⁄ Plf (kindly
provided by M. Nilsen-Hamilton, Iowa State University,
Ames, IA, USA), Ccl2 (eBioscience, San Diego, CA,
USA), Ramp3 (Santa Cruz, CA, USA) or b-catenin
Table 3. Number of transcription factor binding sites in genes up-
regulated in Wnt-1 transfected cells and statistical evaluation. Num-
ber of transcription factor binding sites in the gene regions )3000
to +100 bp relative to the transcription start site (TSS). All known
genes identified as up-regulated by microarray analysis are inclu-
ded. TO, transcription orientation; NA not analysed.
Chromosome ⁄
genomic contig
TSS on
contig TO
Transcription factor
binding sites
TCF MYC AP-1 PEA3
Gene
Lcn2 2 ⁄ NT_039206 9905136 – 6 4 4
Fdps 3 ⁄ NT_078386 3020729 – 3 4 1 2
Myh9 15 ⁄ NT_081921 5432045 – 5 2 1
Fos 12 ⁄ NT_039551 44973723 + 4 2 2 4
Gnb1 4 ⁄ NT_039268 8769575 + 2 6 1 8
Fabp5 3 ⁄ NT_039226 7147260 + 8 9 4 2
Slc7a2 8 ⁄ NT_039460 890127 + 7 5 2 4
Osf2 3 ⁄ NT_039230 3529205 + 3 4 1 4
Sdc4 2 ⁄ NT_039210 26799702 – 5 9 1 3
Ccl2 11 ⁄ NT_039515 78607616 + 5 4 2 9

Pedf 11 ⁄ NT_039515 71993778 – 5 8 2
Mvk 5 ⁄ NT_078458 704837 + 3 6 1 4
Il6 5 ⁄ NT_039300 4719320 – 9 5 1 1
Kdelr2 5 ⁄ NT_039324 681130 + 2 12 3
Fas 19 ⁄ NT_082766 3757837 + 5 5 7
Actb 5 ⁄ NT_039324 72295 + 2 2 2
Ces1 8 ⁄ NT_078586 1628966 – 2 4 1 3
Sod1 16 ⁄ NT_039625 22848124 + 2 2 1 2
Col8a1 16 ⁄ NT_039624 55078952 – 6 2 7
Npm3 6 ⁄ NT_039350 15534227 – 3 1 4
Ccl7 11 ⁄ NT_039515 78617774 + 3 2 1 4
Il1rn 2 ⁄ NT_039206 1848408 + 7 6 1 3
Wisp2 2 ⁄ NT_039210 26177851 + 2 2 2 2
Reg2 6 ⁄ NT_039350 8560953 + 7 3 6
Cebpd 16 ⁄ NT_039624 12488939 + 2 4 5
Col1a1 11 ⁄ NT_039521 6337681 + 3 3 4
Cxcl5 5 ⁄ NT_039308 4924854 + 5 2 1 5
Mtap6 NA
Zfpm2 15 ⁄ NT_078780 2016644 – 5 10 3 2
Egr1 18 ⁄ NT_078847 3754230 + 1 5 1 2
Fdps 3 ⁄ NT_078386 3020829 – 2 4 1 2
Tyms 5 ⁄ NT_039300 4496307 + 6 9 5
Nfkbia 12 ⁄ NT_039551 14347872 – 3 1 6
Rfc4 16 ⁄ NT_039624 19760731 – 3 6 2
Homer3 8 ⁄ NT_081824 721551 + 1 5 2
Total 138 158 28 126
Wnt target genes and the regulation of their promoters S. Ziegler et al.
1608 FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS
(BD-Transduction, Heidelberg, Germany). The expression
of the Mrp ⁄ Plf genes in the used cell line was analyzed by

RT-PCR using the following primers: forward 5¢ -CTCTG
CAGAGATGCTCCCTTC-3¢, reverse 5¢-CATGATATTTC
AGAAGCAGAGCAC-3¢. These primers border a genomic
fragment of 715 bp, which is part of the genes Plf1 and
Plf2, and also a fragment of 721 bp, which itself is part of
the genes Mrp3 and Mrp4 [27] (Fig. 6). To discriminate
between the four different Mrp ⁄ Plf isoforms, the RT-PCR
products were digested with the enzymes BstX1 and ⁄ or
Fnu4H1 (New England Biolabs, Beverly, MA, USA) and
analyzed by agarose gel electrophoresis.
Evaluation of microarray data
Array images were processed to determine signals (Avg
Diff) and absolute calls (Present, Absent, Marginal) for
each probe set using the Affymetrix Microarray Suite 4.0
software (mas 4.0; empirical algorithm). Data on quality
control of the microarray data are listed in the
supplementary file (supplementary table S1). Scaling across
all probe sets of a given array to an average intensity of
1000 was performed to compensate for variations in the
amount and quality of the cRNA samples and other experi-
mental variables of nonbiological origin. Affymetrix probe
Table 4. Statistical analysis of transcription factor binding sites in the genes up-regulated in Wnt-activated cells. Analysed gene region is rel-
ative to the TSS; N
u
, number of motifs in up-regulated candidate target genes; T
u
, theoretically possible number of motifs in this region; Q
u
,
number of motifs in the control genes; N

c
, possible number; T
c
, relation of N
c
to T
c
;Q
c
, relation of Q
u
to Q
c
,Q;P, probability that the ana-
lysed regions of candidate genes and control genes comprise the same number of binding sites as calculated from Z-test.
Analysed gene region
(bp)
Candidate genes
(n ¼ 34)
N
u
Possible number
T
u
N
u
⁄ T
u
· 10000
Q

u
Control genes
(n ¼ 100)
N
c
Possible number
T
c
N
c ⁄
T
c
· 10000
Q
c
Q
u
⁄ Q
c
P (Z-test)
TCF ⁄ Lef
)3000: +100 138 105400 13.09 361 310000 11.65 1.12 0.5546
)3000 : 2501 24 17000 14.12 59 50000 11.8 1.2 0.3313
)2500 : 2001 19 17000 11.18 64 50000 12.8 0.87 0.4996
)2000 : 1501 25 17000 14.71 67 50000 13.4 1.1 0.3241
)1500 : 1001 28 17000 16.47 64 50000 12.8 1.29 0.1411
)1000 : 501 24 17000 14.12 54 50000 10.8 1.31 0.179
)500:TSS 18 17000 10.59 38 50000 7.6 1.39 0.0995
TSS: +100 0 3400 0 4 10000 4.0 0 –
MYC

)3000: +100 158 105400 14.99 420 310000 13.55 1.11 0.255
)3000 : 2501 29 17000 17.06 71 50000 14.2 1.20 0.4424
)2500 : 2001 25 17000 14.71 76 50000 15.2 0.97 0.1184
)2000 : 1501 30 17000 17.65 77 50000 15.4 1.15 0.0823
)1500 : 1001 30 17000 17.65 82 50000 16.4 1.08 0.3491
)1000 : 501 25 17000 14.71 71 50000 14.2 1.04 0.4048
)500:TSS 16 17000 9.41 39 50000 8 1.18 0.35
TSS: +100 3 3400 8.82 4 10000 4 2.21 0.5
AP)1
)3000: +100 28 105400 2.66 69 310000 2.23 1.19 0.2521
)3000 : 2501 1 17000 0.59 6 50000 1.2 0.49 0.1987
)2500 : 2001 5 17000 2.94 14 50000 2.8 1.05 0.4886
)2000 : 1501 9 17000 5.29 13 50000 2.6 2.04 0.0629
)1500 : 1001 6 17000 3.53 10 50000 2.0 1.77 0.1966
)1000 : 501 3 17000 1.77 13 50000 2.6 0.68 0.2247
)500:TSS 3 17000 1.77 10 50000 2.0 0.88 0.3959
TSS: +100 1 3400 2.94 3 10000 3.0 0.98 0.4787
PEA3
)3000: +100 126 105400 11.95 393 310000 12.68 0.94 0.2047
)3000 : 2501 26 17000 15.29 63 50000 12.6 1.21 0.2637
)2500 : 2001 18 17000 10.59 58 50000 11.6 0.91 0.2948
)2000 : 1501 17 17000 10.0 64 50000 12.8 0.78 0.1754
)1500 : 1001 30 17000 17.65 66 50000 13.2 1.34 0.125
)1000 : 501 22 17000 12.94 77 50000 15.4 0.84 0.2107
)500:TSS 11 17000 6.47 65 50000 13.0 0.5 0.0021
TSS: +100 2 3400 5.88 0 10000 0 – –
S. Ziegler et al. Wnt target genes and the regulation of their promoters
FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS 1609
Table 5. Number of MIZ-1 and AP-1 sites in the gene regions )3000 to +100 bp relative to the TSS of down-regulated genes. All known
candidate genes identified as down-regulated by microarray are listed. TO, transcription orientation.

Chromosome ⁄
genomic contig
TSS on
contig TO
Transcription factor binding sites
MIZ-1a MIZ-1b MIZ-1c AP-1
Gene
Cd14 18 ⁄ NT_078847 5579687 – 4 1 2 3
Fin15 6 ⁄ NT_039343 1853841 – 2 3 2
Areg 5 ⁄ NT_039308 5310834 – 1 1 6 1
Crabp2 3 ⁄ NT_078386 1746451 + 2 1 8 1
Hspc121 9 ⁄ NT_039474 11584627 – 1 1 7 2
Ank 15 ⁄ NT_039618 7952405 + 2 3
Sprr1b 3 ⁄ NT_039237 504491 – 2 7 1
Hmgcr 13 ⁄ NT_039590 46774651 – 5 3 1
Ctgf 10 ⁄ NT_039492 2139443 + 2 3 3
Cntn1 15 ⁄ NT_039621 8974893 + 4 2 5 1
Itgb7 15 ⁄ NT_039621 18581494 + 4 2 5 1
Kit1 10 ⁄ NT_039500 13631471 + 3 12 2
Aldh3a1 11 ⁄ NT_039515 57780011 + 3 6
Idh1 1 ⁄ NT_039170 143243304 – 4 2 6 2
Sps2 7 ⁄ NT_039435 9096451 + 2 5
Pkia 3 ⁄ NT_039226 4439759 + 1 7
Wsb1 11 ⁄ NT_039515 75825701 – 1 4 1 1
Nck2 1 ⁄ NT_080070 12640146 + 1 2 3
Spin 13 ⁄ NT_039585 160036 + 1 2 3 1
Mda5 2 ⁄ NT_039207 3772525 – 1 5 2
Iigp 18 ⁄ NT_082381 5230204 + 5 1 6 2
Nr4a1 15 ⁄ NT_039621 18309084 + 2 1 5 1
Usp22 11 ⁄ NT_039515 57755601 – 2 2 1

Nfe2l1 11 ⁄ NT_039521 8229366 – 5 1 3
Dusp6 10 ⁄ NT_039500 12864561 + 1 1 3
Gnpi 18 ⁄ NT_078847 2296344 + 5 4 6 1
Ptgs1 2 ⁄ NT_039206 13748585 + 2 2 5
Idb4 13 ⁄ NT_039580 8616441 + 3 6
Usp18 6 ⁄ NT_039353 35747881 + 5 2 1
Pcdh7 5 ⁄ NT_080434 1009320 + 2 1 2
Lgals8 13 ⁄ NT_039576 1377216 – 3 3 5 1
Adar 3 ⁄ NT_078386 3687256 + 5 6 1
Stc 14 ⁄ NT_039606 16046575 + 4 1 3 1
Cdkn1a 17 ⁄ NT_039649 5680506 + 2 4
C1qr1 2 ⁄ NT_039210 10582654 – 2 2 2 1
Lbp 2 ⁄ NT_039210 20675029 + 4 2 7
Plf 13 ⁄ NT_078738 139306 – 3 3 2 2
Clecsf8 6 ⁄ NT_039353 37983026 + 1 1 3
Csf2rb1 15 ⁄ NT_081921 5915126 + 3
H2-Q10 17 ⁄ NT_039662 1512800 + 3 2 2
Glp2 17 ⁄ NT_039655 6110216 + 2 3 6 1
Plaur 7 ⁄ NT_081126 98802 – 2 2 7 1
Tgfbr1 4 ⁄ NT_039260 12558491 + 3 2 1 1
Efnb2 8 ⁄ NT_039455 5197747 – 1 2 1 2
Pcx 19 ⁄ NT_039687 1358567 + 12 1 5 1
Tera 6 ⁄ NT_039361 2880461 – 2 3 2
Ddx46 13 ⁄ NT_039586 1291069 + 2 5
Ets1 9 ⁄ NT_039472 14158330 + 1 3 3
Cd44 2 ⁄ NT_039209 4903886 – 1 3 1
Csprs 1 ⁄ NT_039172 6738837 – 6 3 1 1
Thbd 2 ⁄ NT_039210 10547213 – 2 3
Dio2 12 ⁄ NT_039552 2471159 – 2 2 14
Cugbp1 2 ⁄ NT_078346 697630 + 1 4 1

Wnt target genes and the regulation of their promoters S. Ziegler et al.
1610 FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS
sets have been described [28]. Microarray annotation data
are based on the Affymetrix database from October 2003.
The software affymetrix data mining tool 2.0 was used
to compare the microarrays hybridized with RNA from
Wnt-activated and control cells.
Genes were defined as candidate target genes, when
they fulfilled the following differential criteria between Wnt-
activated cells and control cells: average difference change
(difference between number of signals of different intensities)
higher than 200 or lower than )200; fold change (difference
of signal intensity) higher than 2.5 or lower than )2.5; sort
score (statistically determined difference between average dif-
ference change and fold change) higher than 1 or lower than
)1; difference call (qualitative estimation of mRNA amount)
I (increased) or D (decreased); increase or decrease ratio
(number of increased or decreased probe pairs ⁄ number of
probe pairs used) of greater than 0.5. Using the murine
genome blast function on the NCBI website (http://
www.ncbi.nlm.nih.gov/genome/seq/MmBlast.html) we iden-
tified the contigs of 119 of the 120 identified candidate target
genes (Tables 3 and 4 and supplementary tables S2, S3).
Real-time quantitative PCR (qPCR)
C57MG cells were cultured for 24 h in conditioned media
from nontransfected L-M(TK-) cells or from Wnt-3a trans-
fected L-M(TK-) cells, which had been prepared according
to the protocol supplied by ATCC. Total RNA was puri-
fied using a commercial kit including digestion of DNA
(on-column DNase digestion) with RNase-free DNase

(Qiagen, Hilden, Germany). To produce template cDNA
the RNA was reverse transcribed using oligo-dT as a pri-
mer and Omniscript Reverse Transcriptase (Qiagen). For
qPCR the QuantiTectTM SYBR Green PCR kit (Qiagen)
was used. Fold induction was obtained by using the DDCt
method. All samples were first normalized to the level of
GAPDH in each sample. Relative and normalized units
were compared between the experimental sample (L-Wnt-
Table 5. (Continued).
Chromosome ⁄
genomic contig
TSS on
contig TO
Transcription factor binding sites
MIZ-1a MIZ-1b MIZ-1c AP-1
Tgoln2 6 ⁄ NT_039350 2723867 – 1 1 1
Abca1 4 ⁄ NT_039260 18430088 – 3 2 6 2
Irf7 7 ⁄ NT_081122 903526 + 2 1 1 1
Gbp3 3 ⁄ NT_039242 11502005 + 1 9 3
Csprs 1 ⁄ NT_039172 6738837 – 6 3 1 1
Zfp36l2 17 ⁄ NT_039658 10176825 – 1 2
Igfbp6 15 ⁄ NT_039621 18669019 – 5 3
Trfr 16 ⁄ NT_039624 29318743 + 1 5
Siat7f 2 ⁄ NT_039206 10124423 + 4
Prkr 17 ⁄ NT_039658 4824693 – 2 4
Adam12 7 ⁄ NT_039435 15858179 – 2 4 3 1
Ywhag 5 ⁄ NT_039314 5872776 – 1 6 1
Hsd3b7 7 ⁄ NT_039435 9670224 + 3 3 3 1
Abca1 4 ⁄ NT_039260 18430089 – 3 2 6 2
Vegfa 17 ⁄ NT_039655 5754842 – 4 2 4 1

Thbd 2 ⁄ NT_039210 10547213 – 2 3
Plf2 13 ⁄ NT_078738 139368 – 3 3 2 2
Ptgs1 2 ⁄ NT_039206 13748585 + 2 2 5
H2-D1 17 ⁄ NT_039662 1512790 + 4 2 2
Isgf3g 14 ⁄ NT_039606 1982529 + 2 1 2 2
mH-2D4(q) 17 ⁄ NT_082375 2291 + 4 4 2 1
Il1rl1 1 ⁄ NT_080070 9625618 + 4 4 5
Q8 ⁄ 9d 17 ⁄ NT_039662 1512821 + 4 2 2 2
Mrp ⁄ Plf 13 ⁄ NT_078738 139370 – 3 3 2 2
Bpgmut 6 ⁄ NT_039341 3756572 + 2 6 1
Agt 8 ⁄ NT_078575 13496446 – 3 5 3 1
Glp2 17 ⁄ NT_039655 6110216 + 2 3 6
Ptgs2 1 ⁄ NT_039184 2437324 + 1 1 6
Gsta4 9 ⁄ NT_039474 24912146 + 4 3
Ppicap 11 ⁄ NT_081857 862653 – 5 3 3 1
Mmp3 9 ⁄ NT_039471 4449959 + 4 1 7
Ramp3 11 ⁄ NT_039515 3556446 + 1 4 1
Total 205 154 339 65
S. Ziegler et al. Wnt target genes and the regulation of their promoters
FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS 1611
3a-CM treated) and its control (L-CM treated). The follow-
ing primers were used: GAPDH-fwd, 5¢-TGTGTCCGTCG
TGGATCTGA-3¢; GAPDH-rev, 5¢-CCTGCTTCACCACCT
TCTTGA-3¢; c-fos-fwd, 5¢-CCTGCCCCTTCTCAACGA-3¢;
c-fos-rev, 5¢-TCCACGTTGCTGATGCTCTT-3¢; Fabpe-fwd,
5¢-AGACGGTCTGCACCTTCCAA-3¢; Fabpe-rev, 5¢-TCTC
CTTCCCGTCCCATTG-3¢; CAT2-fwd, 5¢-TTCTCGCCCT
GTTCCTTGTC-3¢; CAT2-rev, 5¢-TTCTGTGGCTGCCTCC
AAA-3¢; JE-fwd, 5¢-CAGCACCTTTGAATGTGAAGTTG-3¢;
JE-rev, 5¢-TGAGGTGGTTGTGGAAAAGGTA-3¢; PED

F-fwd, 5¢-CCGGTCCACGAGGGAAAT-3¢; PEDF-rev,
5¢-GAAGTAAGCCACGCCAAGGA-3¢; Fas-fwd, 5¢-CCA
AGACACAGCTGAGCAGAAA-3¢; Fas-rev, 5¢-TGCATC
ACTCTTCCCATGAGAT-3¢, Proliferin-fwd: 5¢-CCATGT
GTGCAATGAGGAATG-3¢; Proliferin-rev, 5¢-TGCCGGC
TAATTCAAATGTG-3¢; Ramp3-fwd, 5¢-CTGGCCCAGA
GCTTCATCA-3¢; Ramp3-rev, 5¢-CCTGTCCACCGTGCA
GTTG-3¢.
Reporter gene assay
The reporter gene assays were performed as described with
small modifications [26,29]. RK13 or SW480 cells were trans-
Table 6. Statistical evaluation of binding site numbers of the transcription factors MIZ-1 and AP-1 factors. Analysed gene region is relative to
the TSS; N
d
, number of motifs in down-regulated candidate target genes; T
d
, the theoretically possible number of motifs in this region; Q
d
,
the number of motifs in the control genes; N
c
, possible number; T
c
, relation of N
c
to T
c
;Q
c
, relation of Q

d
to Q
c
,Q;P, probability that the
analysed regions of candidate genes and control genes comprise the same number of binding sites as calculated from Z-test.
Analysed
gene
region (bp)
Candidate
genes
(n ¼ 85) N
d
Possible
number T
d
N
d ⁄
T
d
· 10000
Q
d
Control genes
(n ¼ 100)
N
c
Possible
number
T
c

N
c
⁄ T
c
· 10000
Q
c
Q
d
⁄ Q
c
P (Z-test)
MIZ-1a
)3000: +100 205 263500 7.78 212 310000 6.84 1.14 0.1729
)3000 : 2501 32 42500 7.53 37 50000 7.4 1.02 0.4956
)2500 : 2001 26 42500 6.12 23 50000 4.6 1.33 0.1748
)2000 : 1501 28 42500 6.59 32 50000 6.4 1.03 0.4861
)1500 : 1001 30 42500 7.06 32 50000 6.4 1.0 0.3896
)1000 : 501 37 42500 8.71 39 50000 7.8 1.12 0.3409
)500:TSS 46 42500 10.82 44 50000 8.8 1.23 0.2002
TSS: +100 6 8500 7.06 5 10000 5.0 1.41 0.2866
MIZ-1b
)3000: +100 154 263500 5.84 191 310000 6.16 0.95 0.2934
)3000 : 2501 22 42500 5.18 21 50000 4.2 1.23 0.2717
)2500 : 2001 34 42500 8.0 36 50000 7.2 1.11 0.2877
)2000 : 1501 29 42500 6.82 38 50000 7.6 0.9 0.2737
)1500 : 1001 25 42500 5.88 33 50000 6.6 0.89 0.3156
)1000 : 501 22 42500 5.18 27 50000 5.4 0.96 0.4241
)500:TSS 20 42500 4.71 28 50000 5.6 0.84 0.2456
TSS: +100 2 8500 2.35 6 10000 6.0 0.39 0.1027

MIZ-1c
)3000: +100 339 263500 12.87 386 310000 12.45 1.03 0.413
)3000 : 2501 47 42500 11.06 62 50000 12.4 0.89 0.2657
)2500 : 2001 63 42500 14.82 98 50000 19.6 0.76 0.1633
)2000 : 1501 46 42500 10.82 51 50000 10.2 1.06 0.4149
)1500 : 1001 51 42500 12.0 56 50000 11.2 1.07 0.3841
)1000 : 501 66 42500 15.53 49 50000 9.8 1.59 0.0222
a
)500:TSS 59 42500 13.88 57 50000 11.4 1.22 0.1768
TSS: +100 7 8500 8.24 12 10000 12.0 0.69 0.2042
AP-1
)3000: +100 65 263500 2.47 69 310000 2.23 1.11 0.2649
)3000 : 2501 4 42500 0.94 6 50000 1.2 0.78 0.3374
)2500 : 2001 10 42500 2.35 14 50000 2.8 0.84 0.3291
)2000 : 1501 11 42500 2.58 13 50000 2.6 0.99 0.4651
)1500 : 1001 8 42500 1.88 10 50000 2.0 0.94 0.4213
)1000 : 501 13 42500 3.06 13 50000 2.6 1.18 0.3626
)500:TSS 14 42500 3.29 10 50000 2.0 1.65 0.1705
TSS: +100 5 8500 5.88 3 10000 3.0 1.96 0.1848
a
P<0.05.
Wnt target genes and the regulation of their promoters S. Ziegler et al.
1612 FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS
fected with the topflash-motif in pKS+ ⁄ Ltk80-luc, the empty
vector pKS+ ⁄ Ltk80-luc, and the b-galactosidase expression
vector pEQ176. The signal was normalized to the signal of the
cells transfected with the empty control vector pKS+ ⁄ Ltk80-
luc. SW480 cells were transfected with full length wild-type
APCwt in pCMV (kindly provided by K. Kinzler, Johns Hop-
kins University School of Medicine, Baltimore, MD, USA). In

SW480 cells, the Wnt pathway is activated by the loss of func-
tional APC [30]. The activated Wnt pathway in SW480 cells
can be inhibited by the transfection of APCwt. In reporter
gene assays of RK13 cells the Wnt pathway was activated by
the addition of 20 mm LiCl to the medium.
Analysis of regulatory gene regions
We screened the regions )3000 to +100 bp relative to the
transcription start for the presence of transcription factor
binding motifs using an online binding site screening pro-
gram ( and in-house
software. The genes showing increased expression were
screened for binding motifs of transcription factors, which
activate direct or might activate indirect Wnt targets.
Up-regulated candidates were screened for the following
motifs: 5¢-WWCAAWG-3¢ (TCF), 5¢-TCTCTTA-3¢ and the
E-box motif 5¢-CAYGTG-3¢ (MYC), 5¢-TGA(C)ATCA-3¢
and 5¢-TCAGTCAG-3¢ (AP-1), 5¢-AGGAAA-3¢ and 5¢-CA
GGATGT-3¢ (PEA3). The AP-1 promoter element was
included because our microarray data indicated that Fos is
up-regulated in Wnt-activated cells (supplementary table
S2). This finding is consistent with the results of others,
who have shown that both Jun and Fra-1 are target genes
of b-catenin ⁄ TCF signalling in colorectal tumour cells [31].
Contrary to these results, Fos gene expression and the levels
of Fos targets were found to be decreased in Wnt-activated
cells [9]. Therefore we included AP-1 sites also in the screen
for transcription factor binding sites of these candidates,
which we identified with a decreased expression level on the
microarray. As MIZ-1 has a high number of zinc-fingers,
the MIZ-1 protein might bind to several different DNA

motifs [M. Eilers, Institut fu
¨
r Molekulare Tumorforschung,
Marburg, Germany, personal communication]. Neverthe-
less, three putative MIZ-1 binding motifs have been
proposed [8]: 5¢-TCAGGG-3¢ (MIZ-1a), 5¢-TGAGGT-3¢
(MIZ-1b) and 5¢-TTCCTT-3¢ (MIZ-1c). The Wnt down-
regulated candidates were screened for these MIZ-1 binding
motifs and also for the two AP-1 binding motifs: 5¢-TGA
(C)ATCA-3¢ and 5¢-TCAGTCAG-3¢.
Statistical evaluation of transcription factor
binding sites
To get a general insight into the overall relevance of the
microarray data and of the binding site screens we followed a
statistical approach. Recently, the principle of this approach
was introduced [11]. The relative number of transcription
factor binding sites in genomic segments of the analyzed
candidates was compared to the number of sites in the
corresponding segments of 100 control genes. Control genes
showed expression level differences between Wnt-1 and
PLNCx transfected cells within the range from 0.99 and 1.01
in the microarray analysis. The list of control genes is avail-
able upon request. The statistical significance of the differ-
ence between the site number in candidate genes and in
control genes was calculated by a one-sided Z-test.
Acknowledgements
We are grateful to Ken Kinzler, Sin-Tak Chu, Galia
Maik-Rachline and Marit Nilsen-Hamilton, who pro-
vided reagents.
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Supplementary material
The following material is available from http://www.
blackwellpublishing.com/products/journals/suppmat/EJB/
EJB4581/EJB4581sm.htm
Appendix S1. Supplementary tables S1–S5.
FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS 1615
S. Ziegler et al. Wnt target genes and the regulation of their promoters