Novel target genes of the Wnt pathway and statistical insights into Wnt target promoter regulation Slava Ziegler1, Sonja Rohrs1, Lara Tickenbrock2, Tarik Moroy3, Ludger Klein-Hitpass3, ă ă ă Ingrid R Vetter1 and Oliver Muller1 ă Max-Planck-Institut fur molekulare Physiologie, Dortmund, Germany ă Interdisziplinares Zentrum fur Klinische Forschung, Universitatsklinikum Munster, Munster, Germany ă ă ă ă ă Institut fur Zellbiologie (Tumorforschung), Universitatsklinikum, Essen, Germany ă ă Keywords microarray analysis; statistical analysis of transcription factor motifs; transcription factor binding site; Wnt pathway; Wnt target gene Correspondence O Muller, Max-Planck-Institut fur ă ă molekulare Physiologie, Otto-Hahn-Straòe 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 (MIZ1), 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 putative 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 proportion of Wnt target genes are regulated by indirect mechanisms 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 (primary) and indirect (secondary) target genes (for a list of target genes see http://www.stanford.edu/rnusse/ 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] 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 S Ziegler et al 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 transcription In epithelial cells of the intestine, the MYC ⁄ MIZ-1 ⁄ p21WAF1 cascade acts as a switch between differentiation 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 distribution 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 pathway, which we identified by microarray screening Results Wnt target genes and the regulation of their promoters A B C Fig 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 exemplary 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 mM KCl (K+) or 20 mM 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-transfected 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) 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 product 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 FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS 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 candidate 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 1601 Wnt target genes and the regulation of their promoters 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 expression 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 candidate 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 differential 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- S Ziegler et al fected cells compared to the levels in control cells (not shown) Next we asked whether the differential expression 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 influences several pathways and inhibits other enzymes in addition to GSK-3b (e.g Na ⁄ K ATPase, adenylcyclase, enzymes of the prostaglandins E1 synthesis and inositol1-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 Northern blot analysis of expression of candidate target genes in C57MG ⁄ PLNCx (P) and C57MG ⁄ Wnt-1 (W) cells The relevant 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 1602 FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS S Ziegler et al Wnt target genes and the regulation of their promoters Fig Northern blot analysis of expression of candidate target genes in C57MG cells after treatment with 20 mM LiCl The relevant bands are indicated The ethidiumbromide stained gels with the 18S and 28S rRNA species are shown to demonstrate equal loading 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 Fig 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 performed in triplicates FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS 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 western 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 microarray and by northern blot We decided to analyze more closely the Mrp ⁄ Plf protein levels in Wnt-activated 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 extracellular protein (Figs and 5G,H) There are 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 overlapping genomic regions [27] Using RT-PCR and restriction 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 1603 Wnt target genes and the regulation of their promoters S Ziegler et al A B C D E F G H I K L Fig 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 mM 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 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 western 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) Promoter 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 activities 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 protein expression analysis (Table 2) (Fig 7) Lithium activation 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 1604 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 pathways, this discrimination could be made experimentally The inhibition of protein translation (e.g by cycloheximide) 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 promoters 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 FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS S Ziegler et al Wnt target genes and the regulation of their promoters Table 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 downregulated genes were analysed for binding sites of AP-1 and MIZ-1 Binding sites of promoters of up-regulated genes Number of transcription factor binding sites Gene Promoter length Promoter accession ID TCF MYC AP-1 PEA3 Lcn2 Fos Gnb1 Fabp5 Sdc4 Pedf 793 (bp) (kb) 1089 (bp) (kb) 690 (bp) 1.4 (kb) X81627 AF332140 AB066210 AJ223066 D89572 AH005932 1 3 3 0 0 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 Mrp ⁄ Plf 679 bp Fig Analysis of the proliferin gene transcript (A) Schematic presentation of the four possible fragments obtained by RT-PCR of the transcripts of Mrp ⁄ Plf isoforms [27] The highly homologous fragments 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 position 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+) or LiCl (Li+) was used as template The sizes of exemplary marker fragments are indicated in base pairs (bp) M, marker lane 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 sequences 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 FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS U44724 X05787 2 Analyzing the genomic regions of the up-regulated candidates, we included the motifs of four transcription factors, which are important for the direct or indirect activation of gene expression in Wnt-activated cells These are TCF, Myc and PEA3 [5,6] and AP-1 [31] (supplementary table S3, Figs 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 supplementary tables S4 and S5.) All genes showing decreased transcription levels include MIZ-1 sites Remarkably, the total number of MIZ-1c sites is significantly 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 transcription factors TCF ⁄ Lef, MYC, AP-1 and PEA3 showed no significant accumulation in the genes with increased or decreased expression (Tables and 6) 1605 Wnt target genes and the regulation of their promoters S Ziegler et al Table Summary of experimental results Increased levels or activation (+), decreased levels or inhibition (–) or no alteration (0) are indicated Regulation of gene at the following levels using methods appropriate each: Transcription Translation qPCR Microarray Northern blot Gene C57MG ⁄ Wnt-1 C57MG ⁄ Wnt-1 C57MG + Li Lcn2 Fos Gnb1 Fabp5 Slc7a2 Osf2 Sdc4 Ccl2 Pedf Fas Kit1 Lgals8 Gbp3 Mrp ⁄ Plf Ramp3 + + + + + + + + + + – – – – – + + + + 0 + + + 0 – – – + – + – – – 0 + – Promoter Western blot Reporter gene assay C57MG + Wnt-3a C57MG ⁄ Wnt-1 C57MG + Li RK13 + Li SW480 APC + – – + + 0 + + + + + + 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 analysis 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 transfection 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 exemplary candidate genes by northern and western blots and by qPCR The differential expression trend of the 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 1606 – – – + – – + + 0 + – + – trend of eight of these genes in Wnt-3a stimulated cells The analysis of the levels of selected proteins correlated 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 posttranscriptional mechanisms, which lead to the relative accumulation in Wnt transfected cells or to the relative protein degradation in control cells After lithium activation, 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 activation 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 FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS S Ziegler et al Fig 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 transfected SW480 cells (grey) Average results from three experiments are shown Vertical lines indicate SD 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 constitutively active Wnt pathway Our results supported the first possibility The effects caused by the activation 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 FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS Wnt target genes and the regulation of their promoters 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 crucial 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 analyzed the genomic up-stream regions of all candidate targets for the presence of binding motifs for transcription 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 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 microarray analysis Analyzing the genomic regions, we found that the number of putative MIZ-1 binding sites is significantly 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 regulation 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 neighbouring the genes, which show decreased expression levels in Wnt-activated cells 1607 Wnt target genes and the regulation of their promoters S Ziegler et al Table Number of transcription factor binding sites in genes upregulated in Wnt-1 transfected cells and statistical evaluation Number 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 included TO, transcription orientation; NA not analysed Transcription factor binding sites Chromosome ⁄ TSS on genomic contig contig Gene Lcn2 Fdps Myh9 Fos Gnb1 Fabp5 Slc7a2 Osf2 Sdc4 Ccl2 Pedf Mvk Il6 Kdelr2 Fas Actb Ces1 Sod1 Col8a1 Npm3 Ccl7 Il1rn Wisp2 Reg2 Cebpd Col1a1 Cxcl5 Mtap6 Zfpm2 Egr1 Fdps Tyms Nfkbia Rfc4 Homer3 ⁄ NT_039206 ⁄ NT_078386 15 ⁄ NT_081921 12 ⁄ NT_039551 ⁄ NT_039268 ⁄ NT_039226 ⁄ NT_039460 ⁄ NT_039230 ⁄ NT_039210 11 ⁄ NT_039515 11 ⁄ NT_039515 ⁄ NT_078458 ⁄ NT_039300 ⁄ NT_039324 19 ⁄ NT_082766 ⁄ NT_039324 ⁄ NT_078586 16 ⁄ NT_039625 16 ⁄ NT_039624 ⁄ NT_039350 11 ⁄ NT_039515 ⁄ NT_039206 ⁄ NT_039210 ⁄ NT_039350 16 ⁄ NT_039624 11 ⁄ NT_039521 ⁄ NT_039308 NA 15 ⁄ NT_078780 18 ⁄ NT_078847 ⁄ NT_078386 ⁄ NT_039300 12 ⁄ NT_039551 16 ⁄ NT_039624 ⁄ NT_081824 TO TCF MYC AP-1 PEA3 9905136 3020729 5432045 44973723 8769575 7147260 890127 3529205 26799702 78607616 71993778 704837 4719320 681130 3757837 72295 1628966 22848124 55078952 15534227 78617774 1848408 26177851 8560953 12488939 6337681 4924854 – – – + + + + + – + – + – + + + – + – – + + + + + + + 5 2 3 7 4 2 9 12 2 2016644 3754230 3020829 4496307 14347872 19760731 721551 – + – + – – + 3 10 1 2 2 138 158 28 126 Total 1 1 1 1 4 4 7 4 5 Experimental procedures Cell culture Murine mammary C57MG cells can be transformed by the retroviral infection with oncogenic Wnt-1 [13–15] The resulting 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 1608 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 control 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 Promochem, 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 semiconfluent C57MG ⁄ PLNCx and from C57MG ⁄ Wnt-1 cells Target preparation and hybridization to Affymetrix MGU74Av2 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 ⁄ Pi with protease inhibitors (Roche, Mannheim, Germany) at °C followed by centrifugation at 16 000 g for 15 The supernatants were calibrated to equal protein concentrations by dilution with NaCl ⁄ Pi and analyzed by SDS ⁄ PAGE and western blot Poly(vinylidene difluoride) (PVDF) membranes (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 FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS S Ziegler et al Wnt target genes and the regulation of their promoters Table Statistical analysis of transcription factor binding sites in the genes up-regulated in Wnt-activated cells Analysed gene region is relative to the TSS; Nu, number of motifs in up-regulated candidate target genes; Tu, theoretically possible number of motifs in this region; Qu, number of motifs in the control genes; Nc, possible number; Tc, relation of Nc to Tc; Qc, relation of Qu to Qc, 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 34) Nu Possible number Tu Nu ⁄ Tu · 10000 Qu Control genes (n ¼ 100) Nc Possible number Tc Nc ⁄ Tc · 10000 Qc Qu ⁄ Qc P (Z-test) TCF ⁄ Lef )3000: +100 )3000 : 2501 )2500 : 2001 )2000 : 1501 )1500 : 1001 )1000 : 501 )500:TSS TSS: +100 138 24 19 25 28 24 18 105400 17000 17000 17000 17000 17000 17000 3400 13.09 14.12 11.18 14.71 16.47 14.12 10.59 361 59 64 67 64 54 38 310000 50000 50000 50000 50000 50000 50000 10000 11.65 11.8 12.8 13.4 12.8 10.8 7.6 4.0 1.12 1.2 0.87 1.1 1.29 1.31 1.39 0.5546 0.3313 0.4996 0.3241 0.1411 0.179 0.0995 – MYC )3000: +100 )3000 : 2501 )2500 : 2001 )2000 : 1501 )1500 : 1001 )1000 : 501 )500:TSS TSS: +100 158 29 25 30 30 25 16 105400 17000 17000 17000 17000 17000 17000 3400 14.99 17.06 14.71 17.65 17.65 14.71 9.41 8.82 420 71 76 77 82 71 39 310000 50000 50000 50000 50000 50000 50000 10000 13.55 14.2 15.2 15.4 16.4 14.2 1.11 1.20 0.97 1.15 1.08 1.04 1.18 2.21 0.255 0.4424 0.1184 0.0823 0.3491 0.4048 0.35 0.5 AP)1 )3000: +100 )3000 : 2501 )2500 : 2001 )2000 : 1501 )1500 : 1001 )1000 : 501 )500:TSS TSS: +100 28 3 105400 17000 17000 17000 17000 17000 17000 3400 2.66 0.59 2.94 5.29 3.53 1.77 1.77 2.94 69 14 13 10 13 10 310000 50000 50000 50000 50000 50000 50000 10000 2.23 1.2 2.8 2.6 2.0 2.6 2.0 3.0 1.19 0.49 1.05 2.04 1.77 0.68 0.88 0.98 0.2521 0.1987 0.4886 0.0629 0.1966 0.2247 0.3959 0.4787 PEA3 )3000: +100 )3000 : 2501 )2500 : 2001 )2000 : 1501 )1500 : 1001 )1000 : 501 )500:TSS TSS: +100 126 26 18 17 30 22 11 105400 17000 17000 17000 17000 17000 17000 3400 11.95 15.29 10.59 10.0 17.65 12.94 6.47 5.88 393 63 58 64 66 77 65 310000 50000 50000 50000 50000 50000 50000 10000 12.68 12.6 11.6 12.8 13.2 15.4 13.0 0.94 1.21 0.91 0.78 1.34 0.84 0.5 – 0.2047 0.2637 0.2948 0.1754 0.125 0.2107 0.0021 – (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 FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS 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 experimental variables of nonbiological origin Affymetrix probe 1609 Wnt target genes and the regulation of their promoters S Ziegler et al Table 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 Transcription factor binding sites Chromosome ⁄ genomic contig Gene Cd14 Fin15 Areg Crabp2 Hspc121 Ank Sprr1b Hmgcr Ctgf Cntn1 Itgb7 Kit1 Aldh3a1 Idh1 Sps2 Pkia Wsb1 Nck2 Spin Mda5 Iigp Nr4a1 Usp22 Nfe2l1 Dusp6 Gnpi Ptgs1 Idb4 Usp18 Pcdh7 Lgals8 Adar Stc Cdkn1a C1qr1 Lbp Plf Clecsf8 Csf2rb1 H2-Q10 Glp2 Plaur Tgfbr1 Efnb2 Pcx Tera Ddx46 Ets1 Cd44 Csprs Thbd Dio2 Cugbp1 1610 TSS on contig TO MIZ-1a 18 ⁄ NT_078847 ⁄ NT_039343 ⁄ NT_039308 ⁄ NT_078386 ⁄ NT_039474 15 ⁄ NT_039618 ⁄ NT_039237 13 ⁄ NT_039590 10 ⁄ NT_039492 15 ⁄ NT_039621 15 ⁄ NT_039621 10 ⁄ NT_039500 11 ⁄ NT_039515 ⁄ NT_039170 ⁄ NT_039435 ⁄ NT_039226 11 ⁄ NT_039515 ⁄ NT_080070 13 ⁄ NT_039585 ⁄ NT_039207 18 ⁄ NT_082381 15 ⁄ NT_039621 11 ⁄ NT_039515 11 ⁄ NT_039521 10 ⁄ NT_039500 18 ⁄ NT_078847 ⁄ NT_039206 13 ⁄ NT_039580 ⁄ NT_039353 ⁄ NT_080434 13 ⁄ NT_039576 ⁄ NT_078386 14 ⁄ NT_039606 17 ⁄ NT_039649 ⁄ NT_039210 ⁄ NT_039210 13 ⁄ NT_078738 ⁄ NT_039353 15 ⁄ NT_081921 17 ⁄ NT_039662 17 ⁄ NT_039655 ⁄ NT_081126 ⁄ NT_039260 ⁄ NT_039455 19 ⁄ NT_039687 ⁄ NT_039361 13 ⁄ NT_039586 ⁄ NT_039472 ⁄ NT_039209 ⁄ NT_039172 ⁄ NT_039210 12 ⁄ NT_039552 ⁄ NT_078346 5579687 1853841 5310834 1746451 11584627 7952405 504491 46774651 2139443 8974893 18581494 13631471 57780011 143243304 9096451 4439759 75825701 12640146 160036 3772525 5230204 18309084 57755601 8229366 12864561 2296344 13748585 8616441 35747881 1009320 1377216 3687256 16046575 5680506 10582654 20675029 139306 37983026 5915126 1512800 6110216 98802 12558491 5197747 1358567 2880461 1291069 14158330 4903886 6738837 10547213 2471159 697630 – – – + – + – – + + + + + – + + – + + – + + – – + + + + + + – + + + – + – + + + + – + – + – + + – – – – + 2 2 4 3 1 1 2 5 5 2 3 2 12 1 2 MIZ-1b 1 2 2 2 1 1 2 2 3 2 3 MIZ-1c 2 7 3 5 12 6 3 3 6 7 1 5 3 14 AP-1 1 1 1 2 1 2 1 1 1 1 1 1 FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS S Ziegler et al Wnt target genes and the regulation of their promoters Table (Continued) Transcription factor binding sites Chromosome ⁄ genomic contig Tgoln2 Abca1 Irf7 Gbp3 Csprs Zfp36l2 Igfbp6 Trfr Siat7f Prkr Adam12 Ywhag Hsd3b7 Abca1 Vegfa Thbd Plf2 Ptgs1 H2-D1 Isgf3g mH-2D4(q) Il1rl1 Q8 ⁄ 9d Mrp ⁄ Plf Bpgmut Agt Glp2 Ptgs2 Gsta4 Ppicap Mmp3 Ramp3 TSS on contig TO ⁄ NT_039350 ⁄ NT_039260 ⁄ NT_081122 ⁄ NT_039242 ⁄ NT_039172 17 ⁄ NT_039658 15 ⁄ NT_039621 16 ⁄ NT_039624 ⁄ NT_039206 17 ⁄ NT_039658 ⁄ NT_039435 ⁄ NT_039314 ⁄ NT_039435 ⁄ NT_039260 17 ⁄ NT_039655 ⁄ NT_039210 13 ⁄ NT_078738 ⁄ NT_039206 17 ⁄ NT_039662 14 ⁄ NT_039606 17 ⁄ NT_082375 ⁄ NT_080070 17 ⁄ NT_039662 13 ⁄ NT_078738 ⁄ NT_039341 ⁄ NT_078575 17 ⁄ NT_039655 ⁄ NT_039184 ⁄ NT_039474 11 ⁄ NT_081857 ⁄ NT_039471 11 ⁄ NT_039515 2723867 18430088 903526 11502005 6738837 10176825 18669019 29318743 10124423 4824693 15858179 5872776 9670224 18430089 5754842 10547213 139368 13748585 1512790 1982529 2291 9625618 1512821 139370 3756572 13496446 6110216 2437324 24912146 862653 4449959 3556446 – – + + – – – + + – – – + – – – – + + + + + + – + – + + + – + + Total 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 Wntactivated 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 difference change and fold change) higher than 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- FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS MIZ-1a MIZ-1b MIZ-1c AP-1 1 5 4 4 6 2 2 6 3 205 154 339 3 4 4 3 2 2 4 1 2 2 1 65 tified the contigs of 119 of the 120 identified candidate target genes (Tables and 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 transfected L-M(TK-) cells, which had been prepared according to the protocol supplied by ATCC Total RNA was purified 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 primer 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- 1611 Wnt target genes and the regulation of their promoters S Ziegler et al Table Statistical evaluation of binding site numbers of the transcription factors MIZ-1 and AP-1 factors Analysed gene region is relative to the TSS; Nd, number of motifs in down-regulated candidate target genes; Td, the theoretically possible number of motifs in this region; Qd, the number of motifs in the control genes; Nc, possible number; Tc, relation of Nc to Tc; Qc, relation of Qd to Qc, 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) Nd Possible number Td N d ⁄ Td · 10000 Qd Control genes (n ¼ 100) Nc Possible number Tc MIZ-1a )3000: +100 )3000 : 2501 )2500 : 2001 )2000 : 1501 )1500 : 1001 )1000 : 501 )500:TSS TSS: +100 205 32 26 28 30 37 46 263500 42500 42500 42500 42500 42500 42500 8500 7.78 7.53 6.12 6.59 7.06 8.71 10.82 7.06 212 37 23 32 32 39 44 310000 50000 50000 50000 50000 50000 50000 10000 MIZ-1b )3000: +100 )3000 : 2501 )2500 : 2001 )2000 : 1501 )1500 : 1001 )1000 : 501 )500:TSS TSS: +100 154 22 34 29 25 22 20 263500 42500 42500 42500 42500 42500 42500 8500 5.84 5.18 8.0 6.82 5.88 5.18 4.71 2.35 191 21 36 38 33 27 28 MIZ-1c )3000: +100 )3000 : 2501 )2500 : 2001 )2000 : 1501 )1500 : 1001 )1000 : 501 )500:TSS TSS: +100 339 47 63 46 51 66 59 263500 42500 42500 42500 42500 42500 42500 8500 12.87 11.06 14.82 10.82 12.0 15.53 13.88 8.24 AP-1 )3000: +100 )3000 : 2501 )2500 : 2001 )2000 : 1501 )1500 : 1001 )1000 : 501 )500:TSS TSS: +100 65 10 11 13 14 263500 42500 42500 42500 42500 42500 42500 8500 2.47 0.94 2.35 2.58 1.88 3.06 3.29 5.88 a Nc ⁄ Tc · 10000 Qc Qd ⁄ Qc P (Z-test) 6.84 7.4 4.6 6.4 6.4 7.8 8.8 5.0 1.14 1.02 1.33 1.03 1.0 1.12 1.23 1.41 0.1729 0.4956 0.1748 0.4861 0.3896 0.3409 0.2002 0.2866 310000 50000 50000 50000 50000 50000 50000 10000 6.16 4.2 7.2 7.6 6.6 5.4 5.6 6.0 0.95 1.23 1.11 0.9 0.89 0.96 0.84 0.39 0.2934 0.2717 0.2877 0.2737 0.3156 0.4241 0.2456 0.1027 386 62 98 51 56 49 57 12 310000 50000 50000 50000 50000 50000 50000 10000 12.45 12.4 19.6 10.2 11.2 9.8 11.4 12.0 1.03 0.89 0.76 1.06 1.07 1.59 1.22 0.69 0.413 0.2657 0.1633 0.4149 0.3841 0.0222a 0.1768 0.2042 69 14 13 10 13 10 310000 50000 50000 50000 50000 50000 50000 10000 2.23 1.2 2.8 2.6 2.0 2.6 2.0 3.0 1.11 0.78 0.84 0.99 0.94 1.18 1.65 1.96 0.2649 0.3374 0.3291 0.4651 0.4213 0.3626 0.1705 0.1848 P < 0.05 3a-CM treated) and its control (L-CM treated) The following 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 1612 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- FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS S Ziegler et al 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+ ⁄ Ltk80luc SW480 cells were transfected with full length wild-type APCwt in pCMV (kindly provided by K Kinzler, Johns Hopkins University School of Medicine, Baltimore, MD, USA) In SW480 cells, the Wnt pathway is activated by the loss of functional 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 program (http://bimas.dcrt.nih.gov/molbio/signal) 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 fur Molekulare Tumorforschung, ă Marburg, Germany, personal communication] Nevertheless, 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 downregulated 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 FEBS Journal 272 (2005) 1600–1615 ª 2005 FEBS Wnt target genes and the regulation 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gain general insights into the regulation of promoters of potential target genes of the Wnt pathway, which we identified by microarray screening Results Wnt target genes. .. 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 regulation level within the Wnt pathway decide about the gene... FEBS S Ziegler et al Wnt target genes and the regulation of their promoters Table Number of transcription factor binding sites in promoters The promoters of the up-regulated genes were analysed