Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

SIP1/ZEB2 induces EMT by repressing genes of different epithelial cell–cell junctions

SIP1/ZEB2 induces EMT by repressing genes of different epithelial cell–cell junctions Published online November 24, 2005 6566–6578 Nucleic Acids Research, 2005, Vol. 33, No. 20 doi:10.1093/nar/gki965 SIP1/ZEB2 induces EMT by repressing genes of different epithelial cell–cell junctions Cindy Vandewalle, Joke Comijn, Bram De Craene, Petra Vermassen, Erik Bruyneel , 2 3 4 Henriette Andersen , Eugene Tulchinsky , Frans Van Roy and Geert Berx* Unit of Molecular and Cellular Oncology, Department for Molecular Biomedical Research, VIB-Ghent University, Belgium, Laboratory of Experimental Cancerology, Department of Radiotherapy and Nuclear Medicine, Ghent University, Belgium, Department of Molecular Cancer Biology, Danish Cancer Society, Denmark, 3 4 Department of Cancer Studies and Molecular Medicine, University of Leicester, UK and Molecular Cell Biology Unit, Department for Molecular Biomedical Research, VIB-Ghent University, Belgium Received August 12, 2005; Revised and Accepted November 2, 2005 INTRODUCTION ABSTRACT Smad Interacting Protein 1 (SIP1; also known as ZEB2, for SIP1/ZEB2 is a member of the dEF-1 family of two- zinc finger E-box-binding protein 2 and ZFHX1B) belongs to handed zinc finger nuclear factors. The expression the dEF-1 or ZEB protein family. These proteins are charac- of these transcription factors is associated with terized by a homeodomain flanked by two separated, highly epithelial mesenchymal transitions (EMT) during conserved zinc finger clusters: an N-terminal and a C-terminal development. SIP1 is also expressed in some breast one, which contain four and three zinc fingers, respectively cancer cell lines and was detected in intestinal (1). Each zinc finger cluster can bind independently to CACCT(G) sequences present in promoter regions of genes gastric carcinomas, where its expression is inversely involved in differentiation and development, such as the correlated with that of E-cadherin. Here, we show Xenopus Xbra2 promoter, the human a4-integrin promoter that expression of SIP1 in human epithelial cells and the E-cadherin promoter (2). The integrity of the two results in a clear morphological change from an zinc finger clusters of SIP1 is necessary for its binding as a epithelial to a mesenchymal phenotype. Induction monomer to the target promoter sequences (2). SIP1 acts as a of this epithelial dedifferentiation was accompanied transcriptional repressor and contains consensus binding sites by repression of several cell junctional proteins, with for the corepressor CtBP (3,4). Gene repression by SIP1 has concomitant repression of their mRNA levels. been reported to occur both dependent on and independent of a Besides E-cadherin, other genes coding for crucial CtBP corepressor complex (4,5). Recently it was reported that proteins of tight junctions, desmosomes and gap the SIP1 protein can be sumoylated, which attenuates gene junctions were found to be transcriptionally regulated repression by disruption of CtBP recruitment (6). We reported previously that binding of the E-cadherin pro- by the transcriptional repressor SIP1. Moreover, moter by SIP1 downregulates E-cadherin expression (7). In study of the promoter regions of selected genes by epithelial MDCK cells, this suppression of E-cadherin expres- luciferase reporter assays and chromatin immuno- sion was accompanied by loss of aggregation and acquisition precipitation shows that repression is directly medi- of invasive properties. An inverse correlation between SIP1 ated by SIP1. These data indicate that, during epithelial and E-cadherin expression levels was observed in several epi- dedifferentiation, SIP1 represses in a coordinated thelial tumor cell lines, such as MDA-MB-435S1 and MDA- manner the transcription of genes coding for junc- MB-231; high levels of SIP1 mRNA are observed in these tional proteins contributing to the dedifferentiated cells while E-cadherin transcripts are not detectable. Vice state; this repression occurs by a general mechanism versa, a transformed breast cancer cell line, MCF7/AZ, still mediated by Smad Interacting Protein 1 (SIP1)- expresses E-cadherin but lacks SIP1 expression (7). In the binding sites. intestinal type of gastric carcinomas, the downregulation of *To whom correspondence should be addressed at Department for Molecular Biomedical Research, VIB-Ghent University, Technologiepark 927, 9052 Ghent (Zwijnaarde), Belgium. Tel: +32.0 9.3313740; Fax: +32.0 9.3313609; Email: [email protected] The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors The Author 2005. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact [email protected] Nucleic Acids Research, 2005, Vol. 33, No. 20 6567 E-cadherin expression was again shown to be inversely doxycycline (Dox). The cells were cultured in RPMI with 10% correlated with SIP1 mRNA expression levels (8). SIP1 was fetal calf serum (FCS), 100 U/ml penicillin and 100 mg/ml also identified in a large-scale screen for cancer related genes, streptomycin. The cDNAs for wild-type SIP1 and for mutant which demonstrates its putative role in oncogenic transforma- SIP1 (mutated in both zinc finger clusters) (1) were cloned in tion (9). In addition, SIP1 expression is involved in neurogenesis the pcDNA4/TO vector (Invitrogen). To this end, the wild- of Xenopus laevis (10,11). SIP1 deletions as well as nonsense type and mutant SIP1 fragments (1), both of which contain an and frameshift mutations were demonstrated to play a role in N-terminal myc-tag, were cut from the pCS3 vectors, using Hirschsprung disease, a syndrome characterized by mental XbaI and ClaI, and blunted. Fragments were cloned into the retardation and multiple congenital anomalies (12–15). EcoRV digested pcDNA4/TO vector. Both constructs were In the adherens junction, E-cadherin complexes contain stably transfected in DLD1Tr21 cells by electroporation several catenins, through which E-cadherin is linked to the using 30 mg of the SIP1 expression plasmid. Transfectants actin cytoskeleton. Intercellular interactions between the were selected in 500 mg/ml Zeocin (Invitrogen) and 10 mg/ml E-cadherin proteins on adjacent cells result in strong cell– blasticidin (Invitrogen) for 2 weeks. Clones were isolated cell adhesion and explicit epithelial cell polarity. Abnormal- using cloning cylinders and designated DLD1Tr21/WTSIP1 ities in epithelial cells are at the root of the majority of human and DLD1Tr21/mutSIP1. SIP1 expression was induced using cancers. In these cells, E-cadherin fulfills the role of a major Dox (2 mg/ml, Sigma). As the transfected vectors encode cell–cell adhesion molecule and potently suppresses invasion. myc-tagged SIP1 fusion proteins, resistant colonies were Epithelial mesenchymal transition (EMT) occurs in patholo- tested for induction of SIP1 expression by immunofluorescent gical situations, such as wound healing, fibrosis and the staining with the anti-myc antibody 9E10 (22). A squamous acquisition of an invasive phenotype in epithelial tumors epidermoid carcinoma cell line A431 with Tet-on Dox- (16). This EMT allows cells to dissociate from epithelial tissue regulated SIP1 expression was constructed (J. Mejlvang et al., and become more motile. Furthermore, EMT also participates manuscript submitted). These cells, designated A431/WTSIP1 in mesoderm and neural crest formation during normal devel- were cultured in DMEM with 10% FCS, 100 mg/ml penicillin opment. The putative role of SIP1 in EMT processes was and 100 mg/ml streptomycin. suggested by the phenotype of the Zfhx1b-knock-out mouse, which displays delamination arrest of cranial neural crest cells SDS–PAGE and immunoblotting resulting in the loss of migratory behavior of these cells (17). Cells were rinsed with phosphate-buffered saline (PBS) and This delamination is normally mediated by the triggering of proteins were extracted with 1· Laemmli lysis buffer. Total EMT (16), indicating that SIP1 is a key player in EMT pro- protein lysates of cultured cells were loaded on an 8% one- cesses during development. The adherens junctions are not the dimensional SDS–PAGE gel and the separated proteins were only cell–cell junctions nullified during EMT processes. For transferred on to Immobilon-P membranes (Millipore Corp.). tight junctions, which are adjacent to the adherens junctions, it After blocking with 5% non-fat dry milk in Tris buffered saline was recently shown that the transcriptional repressor Snail (TBS, pH 7.4) containing 0.01% Tween-20, the membranes directly represses claudin and occludin expression, and were incubated with primary antibody. After several washing induces EMT concomitantly with the disappearance of tight steps in TBS, the membranes were incubated with secondary junctions (18,19). Disappearance of the desmosome, another horseradish peroxidase (HRP)-conjugated antibodies (dilution cell junction complex, was also reported in several EMT 1:3000; Amersham Pharmacia Biotech). Proteins were detec- events (19,20). ted using the enhanced chemiluminescence (ECL) detection It is unlikely that E-cadherin is the only SIP1 target gene system (Amersham Pharmacia Biotech). that is involved in the triggering of SIP1-induced conversion of epithelial cells to a more fibroblast-like morphology. Using Antibodies and immunofluorescence human epithelial cell lines with conditional SIP1 expression, we examined the molecular mechanism involved in the SIP1- Fixation and immunofluorescence were performed following induced EMT-like process. This study revealed that SIP1 standard procedures (23). Primary antibodies used for both expression downregulates several cell junctional genes. immunofluorescence and immunoblotting were mouse mono- Furthermore, we illustrate that this downregulation is caused clonal antibody HECD1, raised against human E-cadherin by SIP1-induced repression of promoter activity. Our results (1:75; Takara), polyclonal anti-aE-catenin antibody (1:500; demonstrate that by binding promoter regions containing Sigma), polyclonal anti-b-catenin antibody (1:1000; Sigma), SIP1 recognition sites, SIP1 performs its role in EMT-like mouse anti-myc antibody 9E10 [1:500; (22)], rabbit anti-myc processes by altering in a coordinated fashion the functionality antibody (1:100; Upstate), mouse anti-p120ctn antibody of adherens junctions, tight junctions, desmosomes and gap pp120 (1:200; Transduction) and mouse anti-N-cadherin anti- junctions. body (1:1000; Transduction). Mouse anti-plakophilin 2 anti- body (1:10; Progen), mouse anti-desmoplakin I + II antibody (1:10; Boehringer Mannheim) and mouse anti-claudin 4 anti- body (1:300; Zymed) were used for immunofluorescence only. MATERIALS AND METHODS A specific monoclonal antibody for SIP1 was generated Cell culture and transfection by immunizing C57/BL6 mice with a fusion protein composed The colon cancer cell line DLD1 was provided with the of glutathione S-transferase (GST) coupled to mouse SIP1 T-REX system (Invitrogen) by Van de Wetering et al. (21) amino acids 26–129, according to a described protocol (24). to yield DLD1Tr21 Tet-on cells. This Tet-on system activates After characterization of the antibody, supernatant from transcription of the gene of interest in the presence of hybridoma 7F7 was purified on a protein-G Sepharose column 6568 Nucleic Acids Research, 2005, Vol. 33, No. 20 0 0 0 (Amersham Pharmacia Biotech, Rainham, UK). Secondary were: 5 -CCCCCAGCAGGTGTG-3 and 5 -AAGGGG- antibodies were Alexa-488 and Alexa-594 coupled anti- GAAACTGATAGGAT-3 . Primers for a distal region of mouse, anti-rat or anti-rabbit Ig (1:500; Molecular Probes). the E-cadherin promoter (4834 to 4779) were: 5 - 0 0 TGCCAGGTGACAGGGTCTCT-3 and 5 -AGAGGCCTT- GCCCTTCAGAT-3 ; primers for a distal region of the plako- Real-time quantitative RT–PCR (Q-RT–PCR) philin 2 promoter (6039 to 5974) were: 5 -GGCAGCTG- 0 0 0 Primers and probes used for amplification were designed TGGTCATCCAT-3 and 5 -GGGCATGCAGAAGCACAGTAC-3 using Primer Express 1.0 software (Perkin-Elmer Applied primers for a distal region of the tight junction protein 3 pro- Biosystems). cDNA synthesis and PCR amplification were moter (4416 to 4337) were: 5 -CCGTGAAACATGTCC- 0 0 0 described previously (25). The average threshold cycle of CAGATT-3 and 5 -ACCTCACAGCCCACCTCATC-3 and triplicate reactions was used for all subsequent calculations primers for a distal region of the connexin 26 promoter (4294 0 0 using the delta Ct method. Sequences of primers for mouse to 4232) were: 5 -AAAAGCTACTGCCGTCCATCA-3 and 0 0 0 SIP1 cDNA amplification were: 5 -AGGCATATGGTGACG- 5 -ACAAGGGCAATAGAGCGATGA-3 . 0 0 0 CACAA-3 and 5 -CTTGAACTTGCGGTTACCTGC-3 . The Taqman probe sequence was: 5 -FAM-CAGATCAGCAC- Collagen invasion and fast aggregation assay CAAATGCTAACCCAAGG-TAMRA-3 (Eurogentec). For human E-cadherin, the primers were: 5 -GTCACTGACAC- For the collagen invasion assay, cells were seeded on gelified 0 0 CAACGATAATCCT-3 and 5 -TTTCAGTGTGGTGATTA- Collagen S (type I, 0.22%) solution (Seromed, Biochrom KG, CGACGTTA-3 . The E-cadherin Taqman probe sequence Berlin, Germany) and the invasion assay was performed as was: 5 -FAM-TTCAATCCCACCACGTACAAGGGTCAG- described (26). For a fast cell aggregation assay, single cell TAMRA-3 . For human N-cadherin, the primers were: suspensions were prepared according to an E-cadherin protein 0 0 0 5 -AGCCTGACACTGTGGAGCCT-3 and 5 -TCAGCGTG- saving procedure (27). Cells were incubated in an isotonic 2+ GATGGGTCTTTC-3 and the Taqman probe sequence was: buffer containing 1.25 mM Ca under continuous shaking. 5 -FAM-ATGCCATCAAGCCTGTGGGAATCCG-TAMRA- E-cadherin was functionally blocked using DECMA-1 0 0 3 . For human claudin 4, the primers were: 5 -GGCCGG- (Sigma; 1:500). Particle diameters were measured in a Coulter 0 0 CCTTATGGTGATA-3 and 5 -GCCACCAGCGGATTGTA- particle size counter LS200 (Coulter, Electronics Ltd) at the GA-3 ; for human tight junction protein 3 (ZO-3), the primers start (N0) and after 30 min of incubation (N30); these results 0 0 0 were: 5 -CGTCGCCTCTACGCACAAG-3 and 5 -TGAAG- were plotted against the percentage of volume distribution AGGTGGCTGCTGTGTT-3 ; for human P-cadherin (CDH3), (expressed as percent of the total cell volume). 0 0 the primers were: 5 -ATGACGTGGCACCAACCAT-3 and 0 0 5 -GTTAGCCGCCTTCAGGTTCTC-3 ; for human plako- Isolation of promoter fragments and reporter assays philin 2 (PKP2), the primers were: 5 -CGGAAATCTTCACC- 0 0 0 GAACCA-3 and 5 -AACGGCCTCCAACAAAATCAT-3 ; The human P-cadherin, claudin 4 and connexin 26 promoter for human desmoplakin (DSP), the primers were: 5 -CAG- sequences were identified by screening the public human 0 0 TGGTGTCAGCGATGATGT-3 and 5 -TGACGCTGGATA- genomic DNA database (http://genome.ucsc.edu) with the TGGTGGAA-3 ; for human connexin 26 (GJB2), the primers respective cDNA fragments. These promoter sequences 0 0 0 were: 5 -CTGGCTCACCGTCCTCTTCA-3 and 5 -GCAGC- were amplified by PCR from genomic DNA isolated from CACAACGAGGATCA-3 and for human connexin 31 MDA-MB-435S1 cells. Primers used for P-cadherin were: 0 0 0 0 (GJB3), the primers were: 5 -TCTGGCATGGCTTCAATA- 5 -ACGGGAGGTGGAGAAAGAG-3 and 5 -AGAGAGAG- 0 0 0 0 0 TGC-3 and 5 -GGCAATGTAGCAGTCCACGAT-3 . For GGGTGAAGCAG-3 ; for claudin 4: 5 -GGGGTACCTTCT- 0 0 human TBP (TATA-box binding protein), the primers were GGGGGACCTGTTCA-3 and 5 -CCCAAGCTTCTTAACG- 0 0 0 0 0 5 -CGGCTGTTTAACTTCGCTTC-3 and 5 -CACACGCC- TTCGCAGAGTG-3 and for connexin 26: 5 -GGGGTACC- 0 0 0 AAGAAACAGTGA-3 and the Taqman probe sequence GGGCGCCAATTTTTCAAG-3 and 5 -CCCAAGCTTGGC- 0 0 was: 5 -FAM-CATAGTGATCTTTGCAGTGACCCAG- CGCAACACCTGTCTC-3 . Amplified fragments were CAGC-TAMRA-3 ; for human UBC (Ubiquitin C), the cloned in the pGL3basic vector (Promega, Madison, WI), in 0 0 0 primers were: 5 -ATTTGGGTCGCGGTTCTTG-3 and 5 - which the multicloning site was exchanged for the promoter TGCCTTGACATTCTCGATGGT-3 and for human GAPD fragments. The 1554 bp connexin 26 promoter, containing (Glyceraldehyde-3-phosphate dehydrogenase), the primers 5 SIP1-binding sites, was shortened to a 1294 bp fragment 0 0 0 were: 5 -TGCACCACCAACTGCTTAGC-3 and 5 -GGCAT- by removing the most distal SIP1-binding element using KpnI GGACTGTGGTCATGAG-3 . Primers for PCR analysis for and NruI. Transient transfection with the luciferase reporter chromatin immunoprecipitation (ChIP) of a proximal frag- constructs and cotransfection with the pCS3SIP1FS expres- ment of the human E-cadherin promoter (86 to +60) sion vector in MCF7/AZ cells was performed using FuGENE 0 0 0 were: 5 -GGCCGGCAGGTGAAC-3 and 5 -GGGCTGGAG- 6 reagent (Roche). Approximately 200 000 cells were seeded 0 2 TCTGAACTGAC-3 ; primer sequences for the human plako- per 10 cm well. After 24 h, 500 ng of each plasmid DNA philin 2 proximal promoter (529 to 391) were: 5 -GCGA- was transfected. Luciferase activity was measured with a 0 0 CAAAGCCTGACTAACCA-3 and 5 -GGATGGATTTCC- Galacto-Star kit (Tropix) 48 h after transfection. Transfection GCTCGAT-3 ; primer sequences for the human tight junction was normalized by measuring b-galactosidase (Galacto-Star protein 3 proximal promoter (784 to 563) were: 5 -CTG- kit; Tropix), encoded by the co-transfected pUT651 plasmid 0 0 CAACTCAGGCGCTGTTC-3 and 5 -CCTGAGTAGCTGG- (Eurogentec). Mutagenesis of the SIP1-binding sites in the GCTCCTGAG-3 and primer sequences for the human human connexin 26 promoter segment was performed connexin 26 proximal promoter (1088 to 1017) with the QuickChange Multi Site-Directed Mutagenesis Kit Nucleic Acids Research, 2005, Vol. 33, No. 20 6569 Figure 1. The stable transfectants DLD1Tr21/WTSIP1 and A431/WTSIP1 expressing SIP1. (A) Phase contrast images demonstrate the morphological changes that follow induction of SIP1 in vitro. The non-induced DLD1Tr21/WTSIP1 and A431/WTSIP1 cell lines display a general epithelial phenotype. WTSIP1 induces a clear morphological change in these cells whereas mutant SIP1 does not. (B) Immunofluorescence analysis with a monoclonal antibody directed against the myc-tag confirmed the absence of SIP1 protein from the non-induced cell lines and its presence in the induced cell lines. (Stratagene) using 4 primers, each mutated in the SIP1-binding E-cadherin repressing transcription factors Snail or Slug, as sequence: Mut 1: 5 -AAGTGGGTGCCCGAGATGGGG- they showed no detectable mRNA expression in the 0 0 CGGGGGTTG-3 ; Mut 2: 5 -CCAGAAAGCCCCCAGCA- DLD1Tr21 cell line conditionally expressing SIP1 (data not 0 0 GATGTGCAGTGCAGAGC-3 ; Mut 3: 5 -CCTCACCCCG- shown). The status of the E-cadherin–catenin complex was 0 0 AAAGGAGTCATCTCCTTGCAGTTCC-3 ; Mut 4: 5 -CC- studied in detail by immunofluorescence and western blot ACGGCGGGAGACAGATGTTGCGGCCAAGC-3 . analysis (Figure 2). Expression of SIP1 resulted in the loss of membranous E-cadherin and aE-catenin expression and in ChIP assays their internalization. Western blot analysis revealed that pro- tein levels of E-cadherin and aE-catenin were significantly DLD1Tr21/WTSIP1 and A431/WTSIP1 cells were grown for decreased only after 4 days of SIP1 expression. However, Q- 24 h up to 80% confluency in the absence or presence of Dox. RT–PCR experiments showed that extensive repression of Cells were then crosslinked with 1% formaldehyde and pro- endogenous E-cadherin mRNA was already evident after 12 cessed using the ChIP-IT kit from Active Motif as described h of SIP1 protein expression (Figure 3). On the other hand, the (28). Purified immunoprecipitated DNA was used for real-time aE-catenin mRNA levels remained unchanged after SIP1 quantitative PCR. expression (data not shown). b-catenin protein was relocal- ized, but unlike aE-catenin, it was not strongly downregu- lated. It should be noted that APC is mutated in the RESULTS DLD1Tr21 cell line, and so b-catenin cannot be degraded Exogenous SIP1 expression induces morphological by the ubiquitin-proteasome machinery in the DLD1Tr21 changes in human epithelial cells derivatives. b-catenin was translocated to the cytoplasm and We reported previously that SIP1 induces downregulation of putatively also to the nucleus, even though nuclear b-catenin endogenous E-cadherin in the MDCK cell line (7). One way to staining was barely detected (Figure 2A). The SIP1-induced further elucidate the functional role of SIP1 in dedifferenti- changes in p120ctn protein levels are also remarkable. ation and invasion of epithelial cells is to analyze SIP1- Immunofluorescence analysis indicated that expression of mediated differential gene expression. The human p120ctn decreased at the cell contacts (Figure 2A). Due to DLD1Tr21 cell line is an E-cadherin positive colon cancer alternative splicing of internal exons and multiple translation cell line expressing high levels of the tetracycline repressor initiation sites, several p120ctn isoforms can be expressed (TetR) protein. Using stable transfection with expression vec- from a single gene (29,30). For the induced SIP1- tors under control of the Tet responsive promoter for myc- expressing DLD1Tr21/WTSIP1 cell line, western blot analysis revealed an upregulation of a protein of 120 kDa, represent- tagged wild-type and mutant SIP1, we created the inducible model cell systems DLD1Tr21/WTSIP1 and DLD1Tr21/ ing p120ctn isoform 1 (Figure 2B). On the other hand, isoform mutSIP1, respectively. Furthermore, a similar Tet-on indu- 3 was clearly downregulated upon SIP1 expression. This cible human epidermoid cancer cell line, A431/WTSIP1 inverse regulation of p120ctn isoforms by SIP1 was also detec- was constructed (J. Mejlvang et al., manuscript submitted). ted in A431/WTSIP1 (data not shown). Addition of Dox to the cell cultures resulted in a dose- dependent expression of SIP1. Nuclear expression of SIP1 Wild-type SIP1 induces loss of aggregation and invasion was detectable by immunofluorescence after 4 h of Dox treat- but mutant SIP1 does not ment. The cells expressing SIP1 underwent a dramatic morphological conversion, from an epithelial cell state to a The adhesion function of the E-cadherin–catenin complex is fibroblast-like phenotype, which was most apparent after lost in the induced SIP1-expressing cells. In a fast aggregation 4 days of SIP1 expression (Figure 1). This conversion was assay, induced DLD1Tr21/WTSIP1 cells showed loss of not due to changes in the expression status of the other cell–cell aggregation, whereas expression of the mutant 6570 Nucleic Acids Research, 2005, Vol. 33, No. 20 Figure 2. Behavior of the different proteins of the cadherin–catenin complex upon SIP1 induction. (A) Immunofluorescence microscopy of non-induced and induced DLD1Tr21/WTSIP1 cells using antibodies specific for adherens junction components. E-cadherin as well as aE-catenin, p120ctn and b-catenin became nearly undetectable at cell–cell contacts in the SIP1-induced cells. (B) Western blot analysis of the non-induced and induced DLD1Tr21/WTSIP1 cell line. E-cadherin and aE-catenin were downregulated at the protein level in the SIP1-expressing cells. b-catenin protein levels were unaltered in the SIP1-induced compared to non-induced cells. Protein expression of p120ctn isoform 1 was upregulated and that of isoform 3 downregulated after SIP1 induction (*: addition of Dox every 2 days, **: washing away Dox from the cell culture medium). Nucleic Acids Research, 2005, Vol. 33, No. 20 6571 Figure 3. Downregulation of E-cadherin mRNA due to induction of SIP1 expression in DLD1Tr21/WTSIP1 cells. Q-RT–PCR for mRNA expression of SIP1 and E-cadherin. E-cadherin downregulation is already clear after 12 h of SIP1 induction. Maximum inhibition was observed after 24 h of induction. TBP amplification was used to normalize the values. Figure 4. Wild-type SIP1 induces loss of cell aggregation and invasion whereas mutant SIP1 does not. (A) Fast aggregation assay. No cell aggregates were detected in liquid cell suspensions at time 0 (N0). After 30 min, cell–cell aggregation was detected for the DLD1Tr21 cell line expressing mutant SIP1, but no aggregates were detected when the cell line expressed wild-type SIP1. (B) Invasion into type I collagen was induced by induction (+Dox) of expression of wild-type SIP1 but it was not induced by mutant SIP1. SIP1 had no effect on the aggregation capacity compared to gene expression analysis using cDNA microarrays was per- the non-induced cells (Figure 4A). SIP1 expression in formed (will be reported elsewhere) 12, 24 and 48 h after SIP1 DLD1Tr21/WTSIP1 results in the induction of an invasive induction. Besides E-cadherin, a distinct but large set of genes phenotype (Figure 4B). Invasion into collagen type I was encoding proteins localized in the different epithelial cell junc- induced as efficiently by SIP1 as by the E-cadherin- tions showed modified expression. The differential expression blocking antibody DECMA-1 (data not shown). This is in of transcripts encoding tight junction, adherens junction, des- line with the demonstrated invasive behavior of MDCK mosome and gap junction proteins was confirmed by Q-RT– cells upon SIP1 expression (7). In contrast, the induction of PCR in the DLD1Tr21/WTSIP1 versus DLD1Tr21/mutSIP1 SIP1 protein, mutated in the zinc finger domains, in cell line (Figure 5). SIP1-mediated downregulation was shown DLD1Tr21/mutSIP1 cells had no influence on the in vitro for transcripts encoding E-cadherin, P-cadherin, claudin 4, invasive behavior of these cells (Figure 4B). These data indic- tight junction protein 3 (ZO-3), plakophilin 2, desmoplakin, ate that functional DNA-binding of the SIP1 protein is needed connexin 26 (GJB2) and connexin 31 (GJB3). None of the to convert the cells to a more mesenchymal phenotype. genes that were downregulated by wild-type SIP1 were repressed by mutant SIP1. To further verify the authenticity Wild-type SIP1 downregulates expression of tight of the observations made on the transcript level, we performed junction, adherens junction, desmosome and gap immunofluorescence analysis for proteins encoded by junction proteins at the mRNA level SIP1-repressed genes from both the non-induced and SIP1- In order to gain better insight into the functional impact of induced DLD1Tr21/WTSIP1 and A431/WTSIP1 cell lines. SIP1 expression in epithelial cells, a comparative differential The desmosomal proteins desmoplakin and plakophilin 2 6572 Nucleic Acids Research, 2005, Vol. 33, No. 20 Figure 5. SIP1 downregulates the mRNA expression levels of constituents of tight, adherens, desmosomal and gap junctions in epithelial cells. Total RNA was isolated from DLD1Tr21/WTSIP1 and DLD1Tr21/mutSIP1 cell lines after induction with Dox (at 12 h, 24 h and 48 h) or without Dox. Q-RT–PCR analysis for the genes indicated was compared with the microarray results. It is clear that all these transcripts are downregulated by WTSIP1, but the expression of none of them was affected in the cell line expressing mutant SIP1. Amplification of TBP, UBC and GAPD was used to normalize the values. were clearly downregulated (Figure 6). The tight junction SIP1 regulated genes in the SIP1-induced cell line were protein claudin 4 was also repressed at the protein level by repressed only by the wild-type SIP1, not by the mutated induction of SIP1. SIP1 protein. This indicates that the SIP1-induced morpholo- gical changes observed are caused by direct functional pro- moter regulation of target genes. Until now, E-cadherin is the SIP1 induces cadherin switching in A431/WTSIP1 cells only known target gene of SIP1 which is known to be involved in EMT and invasion processes (7). Therefore, we investigated In contrast to E-cadherin, N-cadherin is believed to promote whether the promoters of several putative SIP1 target genes cell migration and tumor progression, and has been shown are directly regulated by SIP1. Initially, the promoters of previously to be upregulated in invasive cancer cell lines P-cadherin, claudin 4 and connexin 26 (GJB2) were screened (31,32). Q-RT–PCR revealed upregulation of N-cadherin for the presence of SIP1-binding sites [CACCT(G)], and suit- mRNA in A431/WTSIP1 cells 48 h after induction of SIP1, able fragments were cloned in the pGL3basic vector upstream while E-cadherin expression was strongly downregulated of the luciferase reporter gene. In the P-cadherin promoter (Figure 7A). This cadherin switching was confirmed at the (531 bp) 1 AGGTG and 3 CACCTG sequences are present. protein level (Figure 7B). One CACCT and two AGGTG sequences were identified in the claudin 4 promoter (635 bp) (Figure 8A). Finally, the Effect of SIP1 expression on promoter activities isolated connexin 26 promoter fragment that was cloned After induction of SIP1, morphological changes were (1294 bp) contains 1 CACCT, 1 AGGTG and 2 CAGGTG observed only in the DLD1Tr21/WTSIP1 cell line, not in sequences. To elucidate whether SIP1-binding affects the the DLD1Tr21/mutSIP1, in which SIP1 is mutated in both transcriptional activity of these cloned promoters, we transi- zinc finger clusters and is therefore unable to bind promoter ently co-transfected the reporter plasmids together with a SIP1 sequences (2,7). Indeed Q-RT–PCR analysis revealed that the expression vector in the E-cadherin positive epithelial cell line Nucleic Acids Research, 2005, Vol. 33, No. 20 6573 Figure 6. SIP1 downregulates different proteins of the tight junctions and desmosomes. Immunofluorescence microscopy of non-induced and induced DLD1Tr21/ WTSIP1 and A431/WTSIP1 cells using specific antibodies for tight junctional and desmosomal components. SIP1 expression decreases expression of desmoplakin and PKP2 at contact regions. Claudin 4 expression at the tight junctions was affected by SIP1 expression. DAPI staining was done in DLD1Tr21/WTSIP1 and PI (propidium iodide) staining in A431/WTSIP1 to visualize nuclei of the same cell fields. MCF7/AZ. SIP1 expression caused significant decreases in the repressive activity was diminished (derepressed) only when promoter activities of P-cadherin, claudin 4 and connexin 26 all 4 SIP1-binding sites were mutated (Figure 8C). Mutating 1, (Figure 8C). To address the specificity of SIP1 action, the 4 2 or 3 SIP1 recognition sequences did not have a large impact SIP1-binding sites present in the human connexin 26 promoter on the repressed promoter activity. These data show that the were mutated, either separately or in different combinations integrity of a single SIP1-binding element in the connexin 26 (Figure 8B). When these mutant connexin 26 promoter promoter is sufficient for recruitment of SIP1 to the promoter constructs were co-transfected with SIP1 cDNA, the SIP1 and significant repressive activity. These findings are in line 6574 Nucleic Acids Research, 2005, Vol. 33, No. 20 Figure 7. Cadherin switching in A431/WTSIP1 upon SIP1 induction. (A) Q-RT–PCR for E-cadherin and N-cadherin mRNAs in A431/WTSIP1. E-cadherin was clearly downregulated after 48 h of SIP1 induction, while N-cadherin mRNA was upregulated. TBP and GAPD amplification was used to normalize the values. (B) Western blot analysis showed a similar inverse correlation between E-cadherin and N-cadherin protein levels after induction of SIP1. with the results obtained previously with the E-cadherin pro- DISCUSSION moter in which both SIP1-binding sites had to be mutated in order for SIP1 to lose its repressive activity EMT occurs frequently during normal development in pro- (Figure 8C) (7). cesses such as mesoderm and neural crest cell formation. During tumor progression, EMT is also crucial for loss of cell polarity of epithelial cells, thus facilitating migratory SIP1 associates at the chromatin level with promoters and invasive behavior. The involvement of the transcription containing SIP1-binding sites factor SIP1/ZEB2 during EMT in developmental processes We wanted to investigate whether SIP1 associates directly, at was indicated by the phenotype of the SIP1 knock-out the chromatin level, with these new target genes via their SIP1- mouse (17). Loss of SIP1 expression was correlated with binding sites. Therefore, we performed chromatin immuno- loss of the migratory capacities of neural crest cells. Retroviral precipitation (ChIP) assays in both the DLD1Tr21/WTSIP1 insertion mutagenesis suggested that SIP1 could contribute to and the A431/WTSIP1 inducible cell systems. Cells were oncogenic transformation (9). Furthermore, the upregulation grown to 80% confluency in the absence or presence of of ZEB-family members during EMT was recently demon- Dox. After 24 h the cells were crosslinked with 1% formal- strated (33). To study the role of SIP1 in more detail in EMT- dehyde and harvested. A SIP1-specific mouse monoclonal like processes, we generated human cell lines with conditional antibody was used to pull down any chromatin fragment phys- SIP1 expression. In these Tet-on cell systems, adding Dox to ically bound by SIP1. Background was determined using an the cell culture medium resulted in nuclear expression of SIP1. irrelevant IgG antibody. Quantitative PCR performed with A drastic morphological change was induced in these epithe- primers specific for SIP1-binding site containing promoter lial cells as a consequence of exogenous SIP1 expression. As fragments of E-cadherin (CDH1), plakophilin 2 (PKP2), the transmembrane cell adhesion protein E-cadherin is a direct tight junction protein 3 (ZO-3) and connexin 26 (CX26) target of SIP1, we analyzed the expression of the different revealed an enrichment of these sequences after induction components of the cadherin–catenin complex. Downregula- of SIP1 (Figure 9), whereas distal promoter sequences for tion of protein and mRNA levels was only detected for these genes showed no significant enrichment. This indicates E-cadherin. The complexing aE-catenin was altered only at that SIP1 can directly downregulate expression of epithelial the protein expression level, probably as a consequence of loss cell junctional genes in a direct manner by physically inter- of E-cadherin expression (34). Different p120ctn isoforms acting with the promoter regions containing SIP1-binding were inversely regulated during the SIP1-induced EMT-like sites. process. The upregulation of p120ctn isoform 1 (120 kDa) Nucleic Acids Research, 2005, Vol. 33, No. 20 6575 Figure 8. SIP1 transcriptionally downregulates genes of several intercellular junctional complexes. (A) Schematic overview of the cloned promoter regions of E-cadherin, P-cadherin, claudin 4 and connexin 26. The putative SIP1-binding sites are indicated. (B) Mutations generated in the SIP1-binding sites of the human E-cadherin promoter (7) and the human connexin 26 promoter. E2-boxes are shaded gray. Mut 4 carries the mutation CAGGTG ! CAGATG; Mut 2 + 4 carries 2 identical CAGGTG! CAGATG mutations; Mut 1 + 2 + 4 carries an additional AGGTG! AGATG mutation; Mut 1 + 2 + 3 + 4 carries the 3 mutations described above and has a CACCT ! CATCT mutation in SIP1-binding site 3. (C) Promoter activity assays on extracts of transfected MCF7/AZ cells. Cells were co- transfected with a SIP1 expression vector and luciferase promoter constructs for E-cadherin, P-cadherin, claudin 4 or connexin 26. Co-expression of SIP1 with the promoter constructs resulted in downregulation of promoter activities. Mutation of all 4 SIP1-binding elements in the connexin 26 promoter (see B) relieved the repressive activity of SIP1. Mutation of less than 4 SIP1-binding sequences preserved the repressive effect of SIP1. Luciferase values are normalized with b-galactosidase activities. and the downregulation of isoform 3 (100 kDa) indicate The E-cadherin promoter was previously identified as a putative specific roles for different isoforms in epithelial direct target of SIP1. SIP1 binds to the E2-boxes (CACCTG) and mesenchymal states. A similar shift in p120ctn isoform present in the E-cadherin promoter, resulting in downregula- expression was seen in FosER cells, in which EMT is induced tion of the promotor’s activity (7). Global gene expression as a consequence of FosER activation by estradiol addition analysis using the in vitro SIP1-induced cell models revealed (35). This shift is in line with the previously observed pre- that SIP1 expression results in downregulation of major con- dominant expression of 100 kDa and 120 kDa isoforms in stituents of different cell junctional complexes, such as tight epithelial cells and in highly motile fibroblastoid cells, respect- junctions, adherens junctions, desmosomes and gap junctions. ively (36). The functional difference between these isoforms Interestingly we found by Q-RT–PCR analyis that SIP1 down- remains to be elucidated. The other E-cadherin-binding Arma- regulates several cell junction genes on the transcript level. dillo protein b-catenin showed no decrease in mRNA and The fact that expression of a SIP1 mutant, with one missense protein expression levels, but b-catenin was no longer mutation in each of the zinc finger clusters, has no effect on expressed at the cell–cell contacts. SIP1 expression resulted mRNA expression levels of these regulated genes, suggests in the redistribution of b-catenin to the cytoplasm and possibly that downregulation by SIP1 is mediated mainly via promoter also to the nucleus. regulation. Both zinc finger clusters are indeed needed for 6576 Nucleic Acids Research, 2005, Vol. 33, No. 20 Figure 9. SIP1 associates with the promoter regions of cell junction genes at the chromatin level. (A) Promoter regions of junctional genes repressed by SIP1. The 0 0 promoter region, 5 untranslated region (5 -UTR), open reading frame (ORF) and intron were defined using the sequence information derived from the Database for Transcriptional Start Sites (http://dbtss.hgc.jp) and the public human genomic DNA database (http://genome.ucsc.edu) using the Refseq Ids as mentioned under the gene symbol. CACCT(G) and (C)AGGTG boxes were mapped. Amplicons analyzed in the chromatin immunoprecipitation are shown as black bars. (B) DLD1Tr21/ WTSIP1 and A431/WTSIP1 cells were either not induced or induced with Dox for 24 h. In vivo binding of SIP1 to proximal promoter sequences in DLDTr21/ WTSIP1 and A431/WTSIP1 cells, as determined by ChIP analysis. Enrichment of bound sequences was quantified by quantitative real-time PCR and is depicted as the fold increase of association of SIP1 detected with a SIP1-specific antibody in the Dox induced cells versus non-induced cells. Background was determined using an irrelevant IgG antibody and is also depicted as the fold increase in the induced versus non-induced cells. Irrelevant sequences (Irr. seq.) are amplified distal promoter sequences, 4 to 7 kb upstream of the transcription start site of E-cadherin, plakophilin 2, tight junction protein 3 and connexin 26. SIP1-dependent promoter repression via E-box-binding (7). in mesenchymal cells expressing EMT inducers such as Snail Cloning of the promoter regions of the regulated genes con- cannot restore the epithelial phenotype (19,40,41). Moreover, nexin 26 (in the gap junctions), P-cadherin (at the adherens downregulation of the tight junction components, occludins junction) and claudin 4 (in the tight junctions) revealed the and claudins, by Snail was linked to repression of their pro- presence of several SIP1-binding sequences in each of them. moter activity (18,19). Hence, we have to conclude that Mutation of these elements in the cloned connexin 26 pro- E-cadherin and other junctional genes are simultaneously moter showed the importance of the integrity of these downregulated as part of the SIP1 driven reprogramming dur- sequences in the SIP1-dependent suppressive activity. Further- ing EMT. It remains enigmatic though why these different more, we could demonstrate physical interaction at the junctional genes are repressed in a coordinated fashion. We chromatin level between SIP1 and the promoter regions of do know that some of these genes can be regarded as NACos, E-cadherin, plakophilin 2, connexin 26 and ZO-3, all of proteins that can localize both to the nucleus and adhesion which contain SIP1-binding sites. complexes (42). Such proteins have the intriguing potential to The change in expression and distribution of those SIP1 coordinate the regulation of cell adhesion and transcription. target genes during EMT could be explained as a secondary The function of several NACos proteins belonging to the consequence of repression of E-cadherin. A crucial role for Armadillo family such as PKP2, b-catenin and p120ctn E-cadherin in epithelial cell polarity has been well docu- seem to be affected in epithelial cells with SIP1 expression. mented (37–39). However, exogenous E-cadherin expression The desmosomal PKP2 has been reported to be present also in Nucleic Acids Research, 2005, Vol. 33, No. 20 6577 the nucleus at all times. This protein seems to be part of 3. Postigo,A.A. and Dean,D.C. (2000) Differential expression and function of members of the zfh-1 family of zinc finger/homeodomain repressors. particles containing RNA polymerases (43). Moreover the Proc. Natl Acad. Sci. USA, 97, 6391–6396. transcription factor Snail is also able to repress PKP2 expres- 4. Van Grunsven,L.A., Michiels,C., Van De Putte,T., Nelles,L., sion very potently (19). This strong downregulation of PKP2 Wuytens,G., Verschueren,K. and Huylebroeck,D. (2003) Interaction suggests that inhibiting the potential role of PKP2 in adhesion between Smad-interacting protein-1 and the corepressor C-terminal binding protein is dispensable for transcriptional repression of E-cadherin. and/or transcriptional regulation could be essential in the J. Biol. Chem., 278, 26135–26145. process of EMT. It is at present not clear if the SIP1-induced 5. Shi,Y., Sawada,J.I., Sui,G., Affar,E.B., Whetstine,J.R., Lan,F., isoform switching has functional consequences for a particular Ogawa,H., Luke,M.P., Nakatani,Y. and Shi,Y. (2003) Coordinated nuclear role of p120ctn (23). P120ctn can interact with Kaiso histone modifications mediated by a CtBP co-repressor complex. and has as such the potential to influence beta-catenin/ Nature, 422, 735–738. 6. Long,J., Zuo,D. and Park,M. (2005) Pc2-mediated sumoylation of Smad- TCF signaling (44). On the other hand, downregulation of interacting protein 1 attenuates transcriptional repression of E-cadherin. E-cadherin could result in loss of b-catenin sequestration to J. Biol. Chem., 280, 35477–35489. sites of cell–cell adhesion, enabling b-catenin/TCF mediated 7. Comijn,J., Berx,G., Vermassen,P., Verschueren,K., van Grunsven,L., transcription. However SIP1 did not induce clear nuclear Bruyneel,E., Mareel,M., Huylebroeck,D. and van Roy,F. (2001) The two-handed E-box-binding zinc finger protein SIP1 downregulates b-catenin localization nor enhanced WNT signaling (data E-cadherin and induces invasion. Mol. Cell, 7, 1267–1278. not shown). 8. Rosivatz,E., Becker,I., Specht,K., Fricke,E., Luber,B., Busch,R., Enhanced SIP1 expression has so far been reported in a Hofler,H. and Becker,K.F. (2002) Differential expression of the distinct set of cancers comprising gastric, hepatocellular, ovar- epithelial-mesenchymal transition regulators snail, SIP1, and twist ian and breast carcinomas (8,45,46). Here, the described in gastric cancer. Am. J. Pathol., 161, 1881–1891. 9. Mikkers,H., Allen,J., Knipscheer,P., Romeyn,L., Hart,A., Vink,E. and candidate SIP1 target genes have been documented to show Berns,A. (2002) High-throughput retroviral tagging to identify abberant expression in a variety of human cancer types components of specific signaling pathways in cancer. Nature Genet., (47–50). This suggests that repression of these genes could 32, 153–159. be due to enhanced SIP1 expression, although this has to be 10. Eisaki,A., Kurosda,H., Fukui,A. and Asashima,M. (2000) XSIP1,a member of two-handed zinc finger proteins, induced anterior neural examined in detail in the near future. markers in Xenopus Laevis animal cap. Biochem. Biophys. Res. Commun., Taken together, the present results identify the SIP1 protein 271, 151–157. as an important mediator of epithelial dedifferentiation 11. van Grunsven,L.A., Papin,C., Avalosse,B., Opdecamp,K., through direct downregulation of a distinct set of constituents Huylebroeck,D., Smith,J.C. and Bellefroid,E.J. (2000) XSIP1,a Xenopus of adherens junctions, tight junctions, desmosomes and gap zinc finger/homeodomain encoding gene highly expressed during early neural development. Mech. Dev., 94, 189–193. junctions, which are key determinants of the epithelial pheno- 12. Amiel,J., Espinosa-Parrilla,Y., Steffann,J., Gosset,P., Pelet,A., Prieur,M., type, including epithelial cell polarity. Boute,O., Choiset,A., Lacombe,D., Philip,N. et al. (2001) Large-scale deletions and SMADIP1 truncating mutations in syndromic Hirschsprung disease with involvement of midline structures. Am. J. Hum. Genet., 69, 1370–1377. ACKNOWLEDGEMENTS 13. Cacheux,V., Dastot-Le Moal,F., Kaariainen,H., Bondurand,N., We thank Dr Hans Clevers for providing us with the DLDTr21 Rintala,R., Boissier,B., Wilson,M., Mowat,D. and Goossens,M. (2001) Loss-of-function mutations in SIP1 Smad interacting protein 1 result in a cell line. We are grateful to B. Gilbert for expert assistance. J.C. syndromic Hirschsprung disease. Hum. Mol. Genet., 10, 1503–1510. was supported by the ‘Vlaams Instituut voor de Bevordering 14. Wakamatsu,N., Yamada,Y., Yamada,K., Ono,T., Nomura,N., van het Wetenschappelijk-Technologisch Onderzoek in de Taniguchi,H., Kitoh,H., Mutoh,N., Yamanaka,T., Mushiake,K. et al. industrie’ en the ‘Stichting Emmanuel Van der Schueren’. (2001) Mutations in SIP1, encoding Smad interacting protein-1, cause a form of Hirschsprung disease. Nature Genet., 27, 369–370. C.V. is a research assistant with the Fund for Scientific 15. Yamada,K., Yamada,Y., Nomura,N., Miura,K., Wakako,R., Research, Flanders (FWO). G.B. is a postdoctoral fellow Hayakawa,C., Matsumoto,A., Kumagai,T., Yoshimura,I., Miyazaki,S. with the FWO. The research was supported by the et al. (2001) Nonsense and frameshift mutations in ZFHX1B, encoding Association for International Cancer Research (AICR-UK), Smad-interacting protein 1, cause a complex developmental disorder with the Geconcerteerde Onderzoeksacties of Ghent University, a great variety of clinical features. Am. J. Hum. Genet., 69, 1178–1185. 16. Hay,E.D. (1995) An overview of epithelio-mesenchymal transformation. FWO-Flanders, Fortis Insurances (Belgium), and Acta Anat., 154, 8–20. Interuniversity Attraction Poles Programme (IUAP, Belgian 17. Van de Putte,T., Maruhashi,M., Francis,A., Nelles,L., Kondoh,H., Science Policy). Funding to pay the Open Access publication Huylebroeck,D. and Higashi,Y. (2003) Mice lacking Zfhx1b, the gene that charges for this article was provided by IUAP. codes for Smad-interacting protein-1, reveal a role for multiple neural crest cell defects in the etiology of Hirschsprung disease-mental Conflict of interest statement. None declared. retardation syndrome. Am. J. Hum. Genet., 72, 465–470. 18. Ikenouchi,J., Matsuda,M., Furuse,M. and Tsukita,S. (2003) Regulation of tight junctions during the epithelium-mesenchyme transition: direct repression of the gene expression of claudins/occludin by Snail. J. Cell REFERENCES Sci., 116, 1959–1967. 1. Verschueren,K., Remacle,J.E., Collart,C., Kraft,H., Baker,B.S., 19. De Craene,B., Gilbert,B., Stove,C., Bruyneel,E., Van Roy,F. and Berx,G. Tylzanowski,P., Nelles,L., Wuytens,G., Su,M.T., Bodmer,R., Smith,J.C. (2005) The transcription factor Snail induces tumor cell invasion through and Huylebroeck,D. (1999) SIP1, a novel zinc finger/homeodomain modulation of the epithelial cell differentiation program. Cancer Res., repressor, interacts with Smad proteins and binds to 5 -CACCT sequences 65, 6237–6244. in candidate target genes. J. Biol. Chem., 274, 20489–20498. 20. Savagner,P., Yamada,K.M. and Thiery,J.P. (1997) The zinc-finger 2. Remacle,J.E., Kraft,H., Lerchner,W., Wuytens,G., Collart,C., protein slug causes desmosome dissociation, an initial and necessary step Verschueren,K., Smith,J.C. and Huylebroeck,D. (1999) New mode of for growth factor-induced epithelial-mesenchymal transition. J. Cell DNA binding of multi-zinc finger transcription factors: dEF1 family Biol., 137, 1403–1419. members bind with two hands to two target sites. EMBO J., 18, 21. van de Wetering,M., Sancho,E., Verweij,C., deLau,W., Oving,I., 5073–5084. Hurlstone,A., van der Horn,K., Batlle,E., Coudreuse,D., Haramis,A.P. 6578 Nucleic Acids Research, 2005, Vol. 33, No. 20 et al. (2002) The beta-catenin/TCF-4 complex imposes a crypt progenitor involves nuclear translocation of beta-catenin and upregulation of phenotype on colorectal cancer cells. Cell, 111, 241–250. beta-catenin/lymphoid enhancer binding factor-1 transcriptional activity. 22. Evan,G.I., Lewis,G.K., Ramsay,G. and Bishop,J.M. (1985) Isolation of J. Cell Biol., 148, 173–188. ctn monoclonal antibodies specific for human c-myc proto-oncogene 36. Reynolds,A.B. and Daniel,J.M. (1997) p120 : A Src-substrate turned product. Mol. Cell Biol., 5, 3610–3616. catenin. In Cowin,P. and Klymkowsky,M.W. (eds), Cytoskeletal- 23. van Hengel,J., Vanhoenacker,P., Staes,K. and van Roy,F. (1999) Nuclear Membrane Interactions and Signal Transduction. Landes Company and localization of the p120ctn Armadillo-like catenin is counteracted by a Chapman & Hall, Austin, TX, pp. 31–48. nuclear export signal and by E-cadherin expression. Proc. Natl Acad. 37. Takeichi,M. (1991) Cadherin cell adhesion receptors as a morphogenetic Sci. USA, 96, 7980–7985. regulator. Science, 251, 1451–1455. 24. Scha ¨fer,S., Stumpp,S. and Franke,W.W. (1996) Immunological 38. Thiery,J.P. (2002) Epithelial-mesenchymal transitions in tumour identification and characterization of the desmosomal cadherin Dsg2 progression. Nature Rev. Cancer, 2, 442–454. in coupled and uncoupled epithelial cells and in human tissues. 39. Rodriguez-boulan,E. and Nelson,W.J. (1989) Morphogenesis of the Differentiation, 60, 99–108. polarized epithelial cell phenotype. Science, 245, 718–725. 25. Bieche,I., Laurendeau,I., Tozlu,S., Olivi,M., Vidaud,D., Lidereau,R. and 40. Ohkubo,T. and Ozawa,M. (2004) The transcription factor Snail Vidaud,M. (1999) Quantitation of MYC gene expression in sporadic downregulates the tight junction components independently of breast tumors with real-time reverse transcription-PCR assay. E-cadherin downregulation. J. Cell Sci., 117, 1675–1685. Cancer Res., 59, 2759–2765. 41. Yang,J., Mani,S.A., Donaher,J.L., Ramaswamy,S., Itzykson,R.A., 26. Vleminckx,K., Vakaet,L.Jr, Mareel,M., Fiers,W. and van Roy,F. (1991) Come,C., Savagner,P., Gitelman,I., Richardson,A. and Weinberg,R.A. Genetic manipulation of E-cadherin expression by epithelial tumor (2004) Twist, a master regulator of morphogenesis, plays an essential cells reveals an invasion suppressor role. Cell, 66, 107–119. role in tumor metastasis. Cell, 117, 927–939. 27. Bracke,M.E., Vyncke,B.M., Bruyneel,E.A., Vermeulen,S.J., 42. Balda,M.S. and Matter,K. (2003) Epithelial cell adhesion and the De Bruyne,G.K., Van Larebeke,N.A., Vleminckx,K., Van Roy,F.M. and regulation of gene expression. Trends Cell Biol., 13, 310–318. Mareel,M.M. (1993) Insulin-like growth factor I activates the invasion 43. Mertens,C., Hofmann,I., Wang,Z., Teichmann,M., Sepehri Chong,S., suppressor function of E-cadherin in MCF-7 human mammary Schnolzer,M. and Franke,W.W. (2001) Nuclear particles containing RNA carcinoma cells in vitro. Br. J. Cancer, 68, 282–289. polymerase III complexes associated with the junctional plaque protein 28. Vanpoucke,G., Goossens,S., De Craene,B., Gilbert,B., van Roy,F. and plakophilin 2. Proc. Natl Acad. Sci. USA, 98, 7795–7800. Berx,G. (2004) GATA-4 and MEF2C transcription factors control the 44. Park,J.I., Kim,S.W., Lyons,J.P., Ji,H., Nguyen,T.T., Cho,K., tissue-specific expression of the alphaT-catenin gene CTNNA3. Barton,M.C., Deroo,T., Vleminckx,K., Moon,R.T. and McCrea,P.D. Nucleic Acids Res., 32, 4155–4165. (2005) Kaiso/p120-catenin and TCF/beta-catenin complexes 29. Keirsebilck,A., Bonne ´,S., Staes,K., van Hengel,J., Nollet,F., Reynolds,A. coordinately regulate canonical Wnt gene targets. Dev. Cell, 8, and van Roy,F. (1998) Molecular cloning of the human p120ctn catenin 843–854. gene (CTNND1): expression of multiple alternatively spliced isoforms. 45. Elloul,S., Elstrand,M.B., Nesland,J.M., Trope,C.G., Kvalheim,G., Genomics, 50, 129–146. Goldberg,I., Reich,R. and Davidson,B. (2005) Snail, Slug, and Smad- 30. Mo,Y.Y. and Reynolds,A.B. (1996) Identification of murine p120(cas) interacting protein 1 as novel parameters of disease aggressiveness in isoforms and heterogeneous expression of p120(cas) isoforms in human metastatic ovarian and breast carcinoma. Cancer, 103, 1631–1643. tumor cell lines. Cancer Res., 56, 2633–2640. 46. Miyoshi,A., Kitajima,Y., Sumi,K., Sato,K., Hagiwara,A., Koga,Y. and 31. Hazan,R.B., Kang,L., Whooley,B.P. and Borgen,P.I. (1997) N-cadherin Miyazaki,K. (2004) Snail and SIP1 increase cancer invasion by promotes adhesion between invasive breast cancer cells and the stroma. upregulating MMP family in hepatocellular carcinoma cells. Br. J. Cell Adhes. Commun., 4, 399–411. Cancer, 90, 1265–1273. 32. Tran,N.L., Nagle,R.B., Cress,A.E. and Heimark,R.L. (1999) N-cadherin 47. Reynolds,A.B. and Carnahan,R.H. (2004) Regulation of cadherin stability expression in human prostate carcinoma cell lines. An epithelial- and turnover by p120ctn: implications in disease and cancer. Semin. mesenchymal transformation mediating adhesion with stromal cells. Cell Dev. Biol., 15, 657–663. Am. J. Pathol., 155, 787–798. 48. Aasen,T., Hodgins,M.B., Edward,M. and Graham,S.V. (2003) The 33. Jechlinger,M., Grunert,S., Tamir,I.H., Janda,E., Ludemann,S., relationship between connexins, gap junctions, tissue architecture and Waerner,T., Seither,P., Weith,A., Beug,H. and Kraut,N. (2003) tumour invasion, as studied in a novel in vitro model of HPV-16- Expression profiling of epithelial plasticity in tumor progression. associated cervical cancer progression. Oncogene, 22, 7969–7980. Oncogene, 22, 7155–7169. 49. Mertens,C., Kuhm,C., Moll,R., Schwetlick,I. and Franke,W.W. (1999) 34. Nagafuchi,A., Takeichi,M. and Tsukita,S. (1991) The 102 kd cadherin- Desmosomal plakophilin 2 as a differentiation marker in normal associated protein: similarity to vinculin and posttranscriptional and malignant tissues. Differentiation, 64, 277–290. regulation of expression. Cell, 65, 849–857. 50. Kanczuga-Koda,L., Sulkowski,S., Koda,M. and Sulkowska,M. (2005) 35. Eger,A., Stockinger,A., Schaffhauser,B., Beug,H. and Foisner,R. (2000) Alterations in connexin26 expression during colorectal carcinogenesis. Epithelial mesenchymal transition by c-Fos estrogen receptor activation Oncology, 68, 217–222. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

SIP1/ZEB2 induces EMT by repressing genes of different epithelial cell–cell junctions

Download PDF
13 pages

Loading next page...
 
/lp/oxford-university-press/sip1-zeb2-induces-emt-by-repressing-genes-of-different-epithelial-cell-GnWWAcHkY4

References (52)

Publisher
Oxford University Press
Copyright
© The Author 2005. Published by Oxford University Press. All rights reserved
 The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact [email protected]
ISSN
0305-1048
eISSN
1362-4962
DOI
10.1093/nar/gki965
pmid
16314317
Publisher site
See Article on Publisher Site

Abstract

Published online November 24, 2005 6566–6578 Nucleic Acids Research, 2005, Vol. 33, No. 20 doi:10.1093/nar/gki965 SIP1/ZEB2 induces EMT by repressing genes of different epithelial cell–cell junctions Cindy Vandewalle, Joke Comijn, Bram De Craene, Petra Vermassen, Erik Bruyneel , 2 3 4 Henriette Andersen , Eugene Tulchinsky , Frans Van Roy and Geert Berx* Unit of Molecular and Cellular Oncology, Department for Molecular Biomedical Research, VIB-Ghent University, Belgium, Laboratory of Experimental Cancerology, Department of Radiotherapy and Nuclear Medicine, Ghent University, Belgium, Department of Molecular Cancer Biology, Danish Cancer Society, Denmark, 3 4 Department of Cancer Studies and Molecular Medicine, University of Leicester, UK and Molecular Cell Biology Unit, Department for Molecular Biomedical Research, VIB-Ghent University, Belgium Received August 12, 2005; Revised and Accepted November 2, 2005 INTRODUCTION ABSTRACT Smad Interacting Protein 1 (SIP1; also known as ZEB2, for SIP1/ZEB2 is a member of the dEF-1 family of two- zinc finger E-box-binding protein 2 and ZFHX1B) belongs to handed zinc finger nuclear factors. The expression the dEF-1 or ZEB protein family. These proteins are charac- of these transcription factors is associated with terized by a homeodomain flanked by two separated, highly epithelial mesenchymal transitions (EMT) during conserved zinc finger clusters: an N-terminal and a C-terminal development. SIP1 is also expressed in some breast one, which contain four and three zinc fingers, respectively cancer cell lines and was detected in intestinal (1). Each zinc finger cluster can bind independently to CACCT(G) sequences present in promoter regions of genes gastric carcinomas, where its expression is inversely involved in differentiation and development, such as the correlated with that of E-cadherin. Here, we show Xenopus Xbra2 promoter, the human a4-integrin promoter that expression of SIP1 in human epithelial cells and the E-cadherin promoter (2). The integrity of the two results in a clear morphological change from an zinc finger clusters of SIP1 is necessary for its binding as a epithelial to a mesenchymal phenotype. Induction monomer to the target promoter sequences (2). SIP1 acts as a of this epithelial dedifferentiation was accompanied transcriptional repressor and contains consensus binding sites by repression of several cell junctional proteins, with for the corepressor CtBP (3,4). Gene repression by SIP1 has concomitant repression of their mRNA levels. been reported to occur both dependent on and independent of a Besides E-cadherin, other genes coding for crucial CtBP corepressor complex (4,5). Recently it was reported that proteins of tight junctions, desmosomes and gap the SIP1 protein can be sumoylated, which attenuates gene junctions were found to be transcriptionally regulated repression by disruption of CtBP recruitment (6). We reported previously that binding of the E-cadherin pro- by the transcriptional repressor SIP1. Moreover, moter by SIP1 downregulates E-cadherin expression (7). In study of the promoter regions of selected genes by epithelial MDCK cells, this suppression of E-cadherin expres- luciferase reporter assays and chromatin immuno- sion was accompanied by loss of aggregation and acquisition precipitation shows that repression is directly medi- of invasive properties. An inverse correlation between SIP1 ated by SIP1. These data indicate that, during epithelial and E-cadherin expression levels was observed in several epi- dedifferentiation, SIP1 represses in a coordinated thelial tumor cell lines, such as MDA-MB-435S1 and MDA- manner the transcription of genes coding for junc- MB-231; high levels of SIP1 mRNA are observed in these tional proteins contributing to the dedifferentiated cells while E-cadherin transcripts are not detectable. Vice state; this repression occurs by a general mechanism versa, a transformed breast cancer cell line, MCF7/AZ, still mediated by Smad Interacting Protein 1 (SIP1)- expresses E-cadherin but lacks SIP1 expression (7). In the binding sites. intestinal type of gastric carcinomas, the downregulation of *To whom correspondence should be addressed at Department for Molecular Biomedical Research, VIB-Ghent University, Technologiepark 927, 9052 Ghent (Zwijnaarde), Belgium. Tel: +32.0 9.3313740; Fax: +32.0 9.3313609; Email: [email protected] The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors The Author 2005. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact [email protected] Nucleic Acids Research, 2005, Vol. 33, No. 20 6567 E-cadherin expression was again shown to be inversely doxycycline (Dox). The cells were cultured in RPMI with 10% correlated with SIP1 mRNA expression levels (8). SIP1 was fetal calf serum (FCS), 100 U/ml penicillin and 100 mg/ml also identified in a large-scale screen for cancer related genes, streptomycin. The cDNAs for wild-type SIP1 and for mutant which demonstrates its putative role in oncogenic transforma- SIP1 (mutated in both zinc finger clusters) (1) were cloned in tion (9). In addition, SIP1 expression is involved in neurogenesis the pcDNA4/TO vector (Invitrogen). To this end, the wild- of Xenopus laevis (10,11). SIP1 deletions as well as nonsense type and mutant SIP1 fragments (1), both of which contain an and frameshift mutations were demonstrated to play a role in N-terminal myc-tag, were cut from the pCS3 vectors, using Hirschsprung disease, a syndrome characterized by mental XbaI and ClaI, and blunted. Fragments were cloned into the retardation and multiple congenital anomalies (12–15). EcoRV digested pcDNA4/TO vector. Both constructs were In the adherens junction, E-cadherin complexes contain stably transfected in DLD1Tr21 cells by electroporation several catenins, through which E-cadherin is linked to the using 30 mg of the SIP1 expression plasmid. Transfectants actin cytoskeleton. Intercellular interactions between the were selected in 500 mg/ml Zeocin (Invitrogen) and 10 mg/ml E-cadherin proteins on adjacent cells result in strong cell– blasticidin (Invitrogen) for 2 weeks. Clones were isolated cell adhesion and explicit epithelial cell polarity. Abnormal- using cloning cylinders and designated DLD1Tr21/WTSIP1 ities in epithelial cells are at the root of the majority of human and DLD1Tr21/mutSIP1. SIP1 expression was induced using cancers. In these cells, E-cadherin fulfills the role of a major Dox (2 mg/ml, Sigma). As the transfected vectors encode cell–cell adhesion molecule and potently suppresses invasion. myc-tagged SIP1 fusion proteins, resistant colonies were Epithelial mesenchymal transition (EMT) occurs in patholo- tested for induction of SIP1 expression by immunofluorescent gical situations, such as wound healing, fibrosis and the staining with the anti-myc antibody 9E10 (22). A squamous acquisition of an invasive phenotype in epithelial tumors epidermoid carcinoma cell line A431 with Tet-on Dox- (16). This EMT allows cells to dissociate from epithelial tissue regulated SIP1 expression was constructed (J. Mejlvang et al., and become more motile. Furthermore, EMT also participates manuscript submitted). These cells, designated A431/WTSIP1 in mesoderm and neural crest formation during normal devel- were cultured in DMEM with 10% FCS, 100 mg/ml penicillin opment. The putative role of SIP1 in EMT processes was and 100 mg/ml streptomycin. suggested by the phenotype of the Zfhx1b-knock-out mouse, which displays delamination arrest of cranial neural crest cells SDS–PAGE and immunoblotting resulting in the loss of migratory behavior of these cells (17). Cells were rinsed with phosphate-buffered saline (PBS) and This delamination is normally mediated by the triggering of proteins were extracted with 1· Laemmli lysis buffer. Total EMT (16), indicating that SIP1 is a key player in EMT pro- protein lysates of cultured cells were loaded on an 8% one- cesses during development. The adherens junctions are not the dimensional SDS–PAGE gel and the separated proteins were only cell–cell junctions nullified during EMT processes. For transferred on to Immobilon-P membranes (Millipore Corp.). tight junctions, which are adjacent to the adherens junctions, it After blocking with 5% non-fat dry milk in Tris buffered saline was recently shown that the transcriptional repressor Snail (TBS, pH 7.4) containing 0.01% Tween-20, the membranes directly represses claudin and occludin expression, and were incubated with primary antibody. After several washing induces EMT concomitantly with the disappearance of tight steps in TBS, the membranes were incubated with secondary junctions (18,19). Disappearance of the desmosome, another horseradish peroxidase (HRP)-conjugated antibodies (dilution cell junction complex, was also reported in several EMT 1:3000; Amersham Pharmacia Biotech). Proteins were detec- events (19,20). ted using the enhanced chemiluminescence (ECL) detection It is unlikely that E-cadherin is the only SIP1 target gene system (Amersham Pharmacia Biotech). that is involved in the triggering of SIP1-induced conversion of epithelial cells to a more fibroblast-like morphology. Using Antibodies and immunofluorescence human epithelial cell lines with conditional SIP1 expression, we examined the molecular mechanism involved in the SIP1- Fixation and immunofluorescence were performed following induced EMT-like process. This study revealed that SIP1 standard procedures (23). Primary antibodies used for both expression downregulates several cell junctional genes. immunofluorescence and immunoblotting were mouse mono- Furthermore, we illustrate that this downregulation is caused clonal antibody HECD1, raised against human E-cadherin by SIP1-induced repression of promoter activity. Our results (1:75; Takara), polyclonal anti-aE-catenin antibody (1:500; demonstrate that by binding promoter regions containing Sigma), polyclonal anti-b-catenin antibody (1:1000; Sigma), SIP1 recognition sites, SIP1 performs its role in EMT-like mouse anti-myc antibody 9E10 [1:500; (22)], rabbit anti-myc processes by altering in a coordinated fashion the functionality antibody (1:100; Upstate), mouse anti-p120ctn antibody of adherens junctions, tight junctions, desmosomes and gap pp120 (1:200; Transduction) and mouse anti-N-cadherin anti- junctions. body (1:1000; Transduction). Mouse anti-plakophilin 2 anti- body (1:10; Progen), mouse anti-desmoplakin I + II antibody (1:10; Boehringer Mannheim) and mouse anti-claudin 4 anti- body (1:300; Zymed) were used for immunofluorescence only. MATERIALS AND METHODS A specific monoclonal antibody for SIP1 was generated Cell culture and transfection by immunizing C57/BL6 mice with a fusion protein composed The colon cancer cell line DLD1 was provided with the of glutathione S-transferase (GST) coupled to mouse SIP1 T-REX system (Invitrogen) by Van de Wetering et al. (21) amino acids 26–129, according to a described protocol (24). to yield DLD1Tr21 Tet-on cells. This Tet-on system activates After characterization of the antibody, supernatant from transcription of the gene of interest in the presence of hybridoma 7F7 was purified on a protein-G Sepharose column 6568 Nucleic Acids Research, 2005, Vol. 33, No. 20 0 0 0 (Amersham Pharmacia Biotech, Rainham, UK). Secondary were: 5 -CCCCCAGCAGGTGTG-3 and 5 -AAGGGG- antibodies were Alexa-488 and Alexa-594 coupled anti- GAAACTGATAGGAT-3 . Primers for a distal region of mouse, anti-rat or anti-rabbit Ig (1:500; Molecular Probes). the E-cadherin promoter (4834 to 4779) were: 5 - 0 0 TGCCAGGTGACAGGGTCTCT-3 and 5 -AGAGGCCTT- GCCCTTCAGAT-3 ; primers for a distal region of the plako- Real-time quantitative RT–PCR (Q-RT–PCR) philin 2 promoter (6039 to 5974) were: 5 -GGCAGCTG- 0 0 0 Primers and probes used for amplification were designed TGGTCATCCAT-3 and 5 -GGGCATGCAGAAGCACAGTAC-3 using Primer Express 1.0 software (Perkin-Elmer Applied primers for a distal region of the tight junction protein 3 pro- Biosystems). cDNA synthesis and PCR amplification were moter (4416 to 4337) were: 5 -CCGTGAAACATGTCC- 0 0 0 described previously (25). The average threshold cycle of CAGATT-3 and 5 -ACCTCACAGCCCACCTCATC-3 and triplicate reactions was used for all subsequent calculations primers for a distal region of the connexin 26 promoter (4294 0 0 using the delta Ct method. Sequences of primers for mouse to 4232) were: 5 -AAAAGCTACTGCCGTCCATCA-3 and 0 0 0 SIP1 cDNA amplification were: 5 -AGGCATATGGTGACG- 5 -ACAAGGGCAATAGAGCGATGA-3 . 0 0 0 CACAA-3 and 5 -CTTGAACTTGCGGTTACCTGC-3 . The Taqman probe sequence was: 5 -FAM-CAGATCAGCAC- Collagen invasion and fast aggregation assay CAAATGCTAACCCAAGG-TAMRA-3 (Eurogentec). For human E-cadherin, the primers were: 5 -GTCACTGACAC- For the collagen invasion assay, cells were seeded on gelified 0 0 CAACGATAATCCT-3 and 5 -TTTCAGTGTGGTGATTA- Collagen S (type I, 0.22%) solution (Seromed, Biochrom KG, CGACGTTA-3 . The E-cadherin Taqman probe sequence Berlin, Germany) and the invasion assay was performed as was: 5 -FAM-TTCAATCCCACCACGTACAAGGGTCAG- described (26). For a fast cell aggregation assay, single cell TAMRA-3 . For human N-cadherin, the primers were: suspensions were prepared according to an E-cadherin protein 0 0 0 5 -AGCCTGACACTGTGGAGCCT-3 and 5 -TCAGCGTG- saving procedure (27). Cells were incubated in an isotonic 2+ GATGGGTCTTTC-3 and the Taqman probe sequence was: buffer containing 1.25 mM Ca under continuous shaking. 5 -FAM-ATGCCATCAAGCCTGTGGGAATCCG-TAMRA- E-cadherin was functionally blocked using DECMA-1 0 0 3 . For human claudin 4, the primers were: 5 -GGCCGG- (Sigma; 1:500). Particle diameters were measured in a Coulter 0 0 CCTTATGGTGATA-3 and 5 -GCCACCAGCGGATTGTA- particle size counter LS200 (Coulter, Electronics Ltd) at the GA-3 ; for human tight junction protein 3 (ZO-3), the primers start (N0) and after 30 min of incubation (N30); these results 0 0 0 were: 5 -CGTCGCCTCTACGCACAAG-3 and 5 -TGAAG- were plotted against the percentage of volume distribution AGGTGGCTGCTGTGTT-3 ; for human P-cadherin (CDH3), (expressed as percent of the total cell volume). 0 0 the primers were: 5 -ATGACGTGGCACCAACCAT-3 and 0 0 5 -GTTAGCCGCCTTCAGGTTCTC-3 ; for human plako- Isolation of promoter fragments and reporter assays philin 2 (PKP2), the primers were: 5 -CGGAAATCTTCACC- 0 0 0 GAACCA-3 and 5 -AACGGCCTCCAACAAAATCAT-3 ; The human P-cadherin, claudin 4 and connexin 26 promoter for human desmoplakin (DSP), the primers were: 5 -CAG- sequences were identified by screening the public human 0 0 TGGTGTCAGCGATGATGT-3 and 5 -TGACGCTGGATA- genomic DNA database (http://genome.ucsc.edu) with the TGGTGGAA-3 ; for human connexin 26 (GJB2), the primers respective cDNA fragments. These promoter sequences 0 0 0 were: 5 -CTGGCTCACCGTCCTCTTCA-3 and 5 -GCAGC- were amplified by PCR from genomic DNA isolated from CACAACGAGGATCA-3 and for human connexin 31 MDA-MB-435S1 cells. Primers used for P-cadherin were: 0 0 0 0 (GJB3), the primers were: 5 -TCTGGCATGGCTTCAATA- 5 -ACGGGAGGTGGAGAAAGAG-3 and 5 -AGAGAGAG- 0 0 0 0 0 TGC-3 and 5 -GGCAATGTAGCAGTCCACGAT-3 . For GGGTGAAGCAG-3 ; for claudin 4: 5 -GGGGTACCTTCT- 0 0 human TBP (TATA-box binding protein), the primers were GGGGGACCTGTTCA-3 and 5 -CCCAAGCTTCTTAACG- 0 0 0 0 0 5 -CGGCTGTTTAACTTCGCTTC-3 and 5 -CACACGCC- TTCGCAGAGTG-3 and for connexin 26: 5 -GGGGTACC- 0 0 0 AAGAAACAGTGA-3 and the Taqman probe sequence GGGCGCCAATTTTTCAAG-3 and 5 -CCCAAGCTTGGC- 0 0 was: 5 -FAM-CATAGTGATCTTTGCAGTGACCCAG- CGCAACACCTGTCTC-3 . Amplified fragments were CAGC-TAMRA-3 ; for human UBC (Ubiquitin C), the cloned in the pGL3basic vector (Promega, Madison, WI), in 0 0 0 primers were: 5 -ATTTGGGTCGCGGTTCTTG-3 and 5 - which the multicloning site was exchanged for the promoter TGCCTTGACATTCTCGATGGT-3 and for human GAPD fragments. The 1554 bp connexin 26 promoter, containing (Glyceraldehyde-3-phosphate dehydrogenase), the primers 5 SIP1-binding sites, was shortened to a 1294 bp fragment 0 0 0 were: 5 -TGCACCACCAACTGCTTAGC-3 and 5 -GGCAT- by removing the most distal SIP1-binding element using KpnI GGACTGTGGTCATGAG-3 . Primers for PCR analysis for and NruI. Transient transfection with the luciferase reporter chromatin immunoprecipitation (ChIP) of a proximal frag- constructs and cotransfection with the pCS3SIP1FS expres- ment of the human E-cadherin promoter (86 to +60) sion vector in MCF7/AZ cells was performed using FuGENE 0 0 0 were: 5 -GGCCGGCAGGTGAAC-3 and 5 -GGGCTGGAG- 6 reagent (Roche). Approximately 200 000 cells were seeded 0 2 TCTGAACTGAC-3 ; primer sequences for the human plako- per 10 cm well. After 24 h, 500 ng of each plasmid DNA philin 2 proximal promoter (529 to 391) were: 5 -GCGA- was transfected. Luciferase activity was measured with a 0 0 CAAAGCCTGACTAACCA-3 and 5 -GGATGGATTTCC- Galacto-Star kit (Tropix) 48 h after transfection. Transfection GCTCGAT-3 ; primer sequences for the human tight junction was normalized by measuring b-galactosidase (Galacto-Star protein 3 proximal promoter (784 to 563) were: 5 -CTG- kit; Tropix), encoded by the co-transfected pUT651 plasmid 0 0 CAACTCAGGCGCTGTTC-3 and 5 -CCTGAGTAGCTGG- (Eurogentec). Mutagenesis of the SIP1-binding sites in the GCTCCTGAG-3 and primer sequences for the human human connexin 26 promoter segment was performed connexin 26 proximal promoter (1088 to 1017) with the QuickChange Multi Site-Directed Mutagenesis Kit Nucleic Acids Research, 2005, Vol. 33, No. 20 6569 Figure 1. The stable transfectants DLD1Tr21/WTSIP1 and A431/WTSIP1 expressing SIP1. (A) Phase contrast images demonstrate the morphological changes that follow induction of SIP1 in vitro. The non-induced DLD1Tr21/WTSIP1 and A431/WTSIP1 cell lines display a general epithelial phenotype. WTSIP1 induces a clear morphological change in these cells whereas mutant SIP1 does not. (B) Immunofluorescence analysis with a monoclonal antibody directed against the myc-tag confirmed the absence of SIP1 protein from the non-induced cell lines and its presence in the induced cell lines. (Stratagene) using 4 primers, each mutated in the SIP1-binding E-cadherin repressing transcription factors Snail or Slug, as sequence: Mut 1: 5 -AAGTGGGTGCCCGAGATGGGG- they showed no detectable mRNA expression in the 0 0 CGGGGGTTG-3 ; Mut 2: 5 -CCAGAAAGCCCCCAGCA- DLD1Tr21 cell line conditionally expressing SIP1 (data not 0 0 GATGTGCAGTGCAGAGC-3 ; Mut 3: 5 -CCTCACCCCG- shown). The status of the E-cadherin–catenin complex was 0 0 AAAGGAGTCATCTCCTTGCAGTTCC-3 ; Mut 4: 5 -CC- studied in detail by immunofluorescence and western blot ACGGCGGGAGACAGATGTTGCGGCCAAGC-3 . analysis (Figure 2). Expression of SIP1 resulted in the loss of membranous E-cadherin and aE-catenin expression and in ChIP assays their internalization. Western blot analysis revealed that pro- tein levels of E-cadherin and aE-catenin were significantly DLD1Tr21/WTSIP1 and A431/WTSIP1 cells were grown for decreased only after 4 days of SIP1 expression. However, Q- 24 h up to 80% confluency in the absence or presence of Dox. RT–PCR experiments showed that extensive repression of Cells were then crosslinked with 1% formaldehyde and pro- endogenous E-cadherin mRNA was already evident after 12 cessed using the ChIP-IT kit from Active Motif as described h of SIP1 protein expression (Figure 3). On the other hand, the (28). Purified immunoprecipitated DNA was used for real-time aE-catenin mRNA levels remained unchanged after SIP1 quantitative PCR. expression (data not shown). b-catenin protein was relocal- ized, but unlike aE-catenin, it was not strongly downregu- lated. It should be noted that APC is mutated in the RESULTS DLD1Tr21 cell line, and so b-catenin cannot be degraded Exogenous SIP1 expression induces morphological by the ubiquitin-proteasome machinery in the DLD1Tr21 changes in human epithelial cells derivatives. b-catenin was translocated to the cytoplasm and We reported previously that SIP1 induces downregulation of putatively also to the nucleus, even though nuclear b-catenin endogenous E-cadherin in the MDCK cell line (7). One way to staining was barely detected (Figure 2A). The SIP1-induced further elucidate the functional role of SIP1 in dedifferenti- changes in p120ctn protein levels are also remarkable. ation and invasion of epithelial cells is to analyze SIP1- Immunofluorescence analysis indicated that expression of mediated differential gene expression. The human p120ctn decreased at the cell contacts (Figure 2A). Due to DLD1Tr21 cell line is an E-cadherin positive colon cancer alternative splicing of internal exons and multiple translation cell line expressing high levels of the tetracycline repressor initiation sites, several p120ctn isoforms can be expressed (TetR) protein. Using stable transfection with expression vec- from a single gene (29,30). For the induced SIP1- tors under control of the Tet responsive promoter for myc- expressing DLD1Tr21/WTSIP1 cell line, western blot analysis revealed an upregulation of a protein of 120 kDa, represent- tagged wild-type and mutant SIP1, we created the inducible model cell systems DLD1Tr21/WTSIP1 and DLD1Tr21/ ing p120ctn isoform 1 (Figure 2B). On the other hand, isoform mutSIP1, respectively. Furthermore, a similar Tet-on indu- 3 was clearly downregulated upon SIP1 expression. This cible human epidermoid cancer cell line, A431/WTSIP1 inverse regulation of p120ctn isoforms by SIP1 was also detec- was constructed (J. Mejlvang et al., manuscript submitted). ted in A431/WTSIP1 (data not shown). Addition of Dox to the cell cultures resulted in a dose- dependent expression of SIP1. Nuclear expression of SIP1 Wild-type SIP1 induces loss of aggregation and invasion was detectable by immunofluorescence after 4 h of Dox treat- but mutant SIP1 does not ment. The cells expressing SIP1 underwent a dramatic morphological conversion, from an epithelial cell state to a The adhesion function of the E-cadherin–catenin complex is fibroblast-like phenotype, which was most apparent after lost in the induced SIP1-expressing cells. In a fast aggregation 4 days of SIP1 expression (Figure 1). This conversion was assay, induced DLD1Tr21/WTSIP1 cells showed loss of not due to changes in the expression status of the other cell–cell aggregation, whereas expression of the mutant 6570 Nucleic Acids Research, 2005, Vol. 33, No. 20 Figure 2. Behavior of the different proteins of the cadherin–catenin complex upon SIP1 induction. (A) Immunofluorescence microscopy of non-induced and induced DLD1Tr21/WTSIP1 cells using antibodies specific for adherens junction components. E-cadherin as well as aE-catenin, p120ctn and b-catenin became nearly undetectable at cell–cell contacts in the SIP1-induced cells. (B) Western blot analysis of the non-induced and induced DLD1Tr21/WTSIP1 cell line. E-cadherin and aE-catenin were downregulated at the protein level in the SIP1-expressing cells. b-catenin protein levels were unaltered in the SIP1-induced compared to non-induced cells. Protein expression of p120ctn isoform 1 was upregulated and that of isoform 3 downregulated after SIP1 induction (*: addition of Dox every 2 days, **: washing away Dox from the cell culture medium). Nucleic Acids Research, 2005, Vol. 33, No. 20 6571 Figure 3. Downregulation of E-cadherin mRNA due to induction of SIP1 expression in DLD1Tr21/WTSIP1 cells. Q-RT–PCR for mRNA expression of SIP1 and E-cadherin. E-cadherin downregulation is already clear after 12 h of SIP1 induction. Maximum inhibition was observed after 24 h of induction. TBP amplification was used to normalize the values. Figure 4. Wild-type SIP1 induces loss of cell aggregation and invasion whereas mutant SIP1 does not. (A) Fast aggregation assay. No cell aggregates were detected in liquid cell suspensions at time 0 (N0). After 30 min, cell–cell aggregation was detected for the DLD1Tr21 cell line expressing mutant SIP1, but no aggregates were detected when the cell line expressed wild-type SIP1. (B) Invasion into type I collagen was induced by induction (+Dox) of expression of wild-type SIP1 but it was not induced by mutant SIP1. SIP1 had no effect on the aggregation capacity compared to gene expression analysis using cDNA microarrays was per- the non-induced cells (Figure 4A). SIP1 expression in formed (will be reported elsewhere) 12, 24 and 48 h after SIP1 DLD1Tr21/WTSIP1 results in the induction of an invasive induction. Besides E-cadherin, a distinct but large set of genes phenotype (Figure 4B). Invasion into collagen type I was encoding proteins localized in the different epithelial cell junc- induced as efficiently by SIP1 as by the E-cadherin- tions showed modified expression. The differential expression blocking antibody DECMA-1 (data not shown). This is in of transcripts encoding tight junction, adherens junction, des- line with the demonstrated invasive behavior of MDCK mosome and gap junction proteins was confirmed by Q-RT– cells upon SIP1 expression (7). In contrast, the induction of PCR in the DLD1Tr21/WTSIP1 versus DLD1Tr21/mutSIP1 SIP1 protein, mutated in the zinc finger domains, in cell line (Figure 5). SIP1-mediated downregulation was shown DLD1Tr21/mutSIP1 cells had no influence on the in vitro for transcripts encoding E-cadherin, P-cadherin, claudin 4, invasive behavior of these cells (Figure 4B). These data indic- tight junction protein 3 (ZO-3), plakophilin 2, desmoplakin, ate that functional DNA-binding of the SIP1 protein is needed connexin 26 (GJB2) and connexin 31 (GJB3). None of the to convert the cells to a more mesenchymal phenotype. genes that were downregulated by wild-type SIP1 were repressed by mutant SIP1. To further verify the authenticity Wild-type SIP1 downregulates expression of tight of the observations made on the transcript level, we performed junction, adherens junction, desmosome and gap immunofluorescence analysis for proteins encoded by junction proteins at the mRNA level SIP1-repressed genes from both the non-induced and SIP1- In order to gain better insight into the functional impact of induced DLD1Tr21/WTSIP1 and A431/WTSIP1 cell lines. SIP1 expression in epithelial cells, a comparative differential The desmosomal proteins desmoplakin and plakophilin 2 6572 Nucleic Acids Research, 2005, Vol. 33, No. 20 Figure 5. SIP1 downregulates the mRNA expression levels of constituents of tight, adherens, desmosomal and gap junctions in epithelial cells. Total RNA was isolated from DLD1Tr21/WTSIP1 and DLD1Tr21/mutSIP1 cell lines after induction with Dox (at 12 h, 24 h and 48 h) or without Dox. Q-RT–PCR analysis for the genes indicated was compared with the microarray results. It is clear that all these transcripts are downregulated by WTSIP1, but the expression of none of them was affected in the cell line expressing mutant SIP1. Amplification of TBP, UBC and GAPD was used to normalize the values. were clearly downregulated (Figure 6). The tight junction SIP1 regulated genes in the SIP1-induced cell line were protein claudin 4 was also repressed at the protein level by repressed only by the wild-type SIP1, not by the mutated induction of SIP1. SIP1 protein. This indicates that the SIP1-induced morpholo- gical changes observed are caused by direct functional pro- moter regulation of target genes. Until now, E-cadherin is the SIP1 induces cadherin switching in A431/WTSIP1 cells only known target gene of SIP1 which is known to be involved in EMT and invasion processes (7). Therefore, we investigated In contrast to E-cadherin, N-cadherin is believed to promote whether the promoters of several putative SIP1 target genes cell migration and tumor progression, and has been shown are directly regulated by SIP1. Initially, the promoters of previously to be upregulated in invasive cancer cell lines P-cadherin, claudin 4 and connexin 26 (GJB2) were screened (31,32). Q-RT–PCR revealed upregulation of N-cadherin for the presence of SIP1-binding sites [CACCT(G)], and suit- mRNA in A431/WTSIP1 cells 48 h after induction of SIP1, able fragments were cloned in the pGL3basic vector upstream while E-cadherin expression was strongly downregulated of the luciferase reporter gene. In the P-cadherin promoter (Figure 7A). This cadherin switching was confirmed at the (531 bp) 1 AGGTG and 3 CACCTG sequences are present. protein level (Figure 7B). One CACCT and two AGGTG sequences were identified in the claudin 4 promoter (635 bp) (Figure 8A). Finally, the Effect of SIP1 expression on promoter activities isolated connexin 26 promoter fragment that was cloned After induction of SIP1, morphological changes were (1294 bp) contains 1 CACCT, 1 AGGTG and 2 CAGGTG observed only in the DLD1Tr21/WTSIP1 cell line, not in sequences. To elucidate whether SIP1-binding affects the the DLD1Tr21/mutSIP1, in which SIP1 is mutated in both transcriptional activity of these cloned promoters, we transi- zinc finger clusters and is therefore unable to bind promoter ently co-transfected the reporter plasmids together with a SIP1 sequences (2,7). Indeed Q-RT–PCR analysis revealed that the expression vector in the E-cadherin positive epithelial cell line Nucleic Acids Research, 2005, Vol. 33, No. 20 6573 Figure 6. SIP1 downregulates different proteins of the tight junctions and desmosomes. Immunofluorescence microscopy of non-induced and induced DLD1Tr21/ WTSIP1 and A431/WTSIP1 cells using specific antibodies for tight junctional and desmosomal components. SIP1 expression decreases expression of desmoplakin and PKP2 at contact regions. Claudin 4 expression at the tight junctions was affected by SIP1 expression. DAPI staining was done in DLD1Tr21/WTSIP1 and PI (propidium iodide) staining in A431/WTSIP1 to visualize nuclei of the same cell fields. MCF7/AZ. SIP1 expression caused significant decreases in the repressive activity was diminished (derepressed) only when promoter activities of P-cadherin, claudin 4 and connexin 26 all 4 SIP1-binding sites were mutated (Figure 8C). Mutating 1, (Figure 8C). To address the specificity of SIP1 action, the 4 2 or 3 SIP1 recognition sequences did not have a large impact SIP1-binding sites present in the human connexin 26 promoter on the repressed promoter activity. These data show that the were mutated, either separately or in different combinations integrity of a single SIP1-binding element in the connexin 26 (Figure 8B). When these mutant connexin 26 promoter promoter is sufficient for recruitment of SIP1 to the promoter constructs were co-transfected with SIP1 cDNA, the SIP1 and significant repressive activity. These findings are in line 6574 Nucleic Acids Research, 2005, Vol. 33, No. 20 Figure 7. Cadherin switching in A431/WTSIP1 upon SIP1 induction. (A) Q-RT–PCR for E-cadherin and N-cadherin mRNAs in A431/WTSIP1. E-cadherin was clearly downregulated after 48 h of SIP1 induction, while N-cadherin mRNA was upregulated. TBP and GAPD amplification was used to normalize the values. (B) Western blot analysis showed a similar inverse correlation between E-cadherin and N-cadherin protein levels after induction of SIP1. with the results obtained previously with the E-cadherin pro- DISCUSSION moter in which both SIP1-binding sites had to be mutated in order for SIP1 to lose its repressive activity EMT occurs frequently during normal development in pro- (Figure 8C) (7). cesses such as mesoderm and neural crest cell formation. During tumor progression, EMT is also crucial for loss of cell polarity of epithelial cells, thus facilitating migratory SIP1 associates at the chromatin level with promoters and invasive behavior. The involvement of the transcription containing SIP1-binding sites factor SIP1/ZEB2 during EMT in developmental processes We wanted to investigate whether SIP1 associates directly, at was indicated by the phenotype of the SIP1 knock-out the chromatin level, with these new target genes via their SIP1- mouse (17). Loss of SIP1 expression was correlated with binding sites. Therefore, we performed chromatin immuno- loss of the migratory capacities of neural crest cells. Retroviral precipitation (ChIP) assays in both the DLD1Tr21/WTSIP1 insertion mutagenesis suggested that SIP1 could contribute to and the A431/WTSIP1 inducible cell systems. Cells were oncogenic transformation (9). Furthermore, the upregulation grown to 80% confluency in the absence or presence of of ZEB-family members during EMT was recently demon- Dox. After 24 h the cells were crosslinked with 1% formal- strated (33). To study the role of SIP1 in more detail in EMT- dehyde and harvested. A SIP1-specific mouse monoclonal like processes, we generated human cell lines with conditional antibody was used to pull down any chromatin fragment phys- SIP1 expression. In these Tet-on cell systems, adding Dox to ically bound by SIP1. Background was determined using an the cell culture medium resulted in nuclear expression of SIP1. irrelevant IgG antibody. Quantitative PCR performed with A drastic morphological change was induced in these epithe- primers specific for SIP1-binding site containing promoter lial cells as a consequence of exogenous SIP1 expression. As fragments of E-cadherin (CDH1), plakophilin 2 (PKP2), the transmembrane cell adhesion protein E-cadherin is a direct tight junction protein 3 (ZO-3) and connexin 26 (CX26) target of SIP1, we analyzed the expression of the different revealed an enrichment of these sequences after induction components of the cadherin–catenin complex. Downregula- of SIP1 (Figure 9), whereas distal promoter sequences for tion of protein and mRNA levels was only detected for these genes showed no significant enrichment. This indicates E-cadherin. The complexing aE-catenin was altered only at that SIP1 can directly downregulate expression of epithelial the protein expression level, probably as a consequence of loss cell junctional genes in a direct manner by physically inter- of E-cadherin expression (34). Different p120ctn isoforms acting with the promoter regions containing SIP1-binding were inversely regulated during the SIP1-induced EMT-like sites. process. The upregulation of p120ctn isoform 1 (120 kDa) Nucleic Acids Research, 2005, Vol. 33, No. 20 6575 Figure 8. SIP1 transcriptionally downregulates genes of several intercellular junctional complexes. (A) Schematic overview of the cloned promoter regions of E-cadherin, P-cadherin, claudin 4 and connexin 26. The putative SIP1-binding sites are indicated. (B) Mutations generated in the SIP1-binding sites of the human E-cadherin promoter (7) and the human connexin 26 promoter. E2-boxes are shaded gray. Mut 4 carries the mutation CAGGTG ! CAGATG; Mut 2 + 4 carries 2 identical CAGGTG! CAGATG mutations; Mut 1 + 2 + 4 carries an additional AGGTG! AGATG mutation; Mut 1 + 2 + 3 + 4 carries the 3 mutations described above and has a CACCT ! CATCT mutation in SIP1-binding site 3. (C) Promoter activity assays on extracts of transfected MCF7/AZ cells. Cells were co- transfected with a SIP1 expression vector and luciferase promoter constructs for E-cadherin, P-cadherin, claudin 4 or connexin 26. Co-expression of SIP1 with the promoter constructs resulted in downregulation of promoter activities. Mutation of all 4 SIP1-binding elements in the connexin 26 promoter (see B) relieved the repressive activity of SIP1. Mutation of less than 4 SIP1-binding sequences preserved the repressive effect of SIP1. Luciferase values are normalized with b-galactosidase activities. and the downregulation of isoform 3 (100 kDa) indicate The E-cadherin promoter was previously identified as a putative specific roles for different isoforms in epithelial direct target of SIP1. SIP1 binds to the E2-boxes (CACCTG) and mesenchymal states. A similar shift in p120ctn isoform present in the E-cadherin promoter, resulting in downregula- expression was seen in FosER cells, in which EMT is induced tion of the promotor’s activity (7). Global gene expression as a consequence of FosER activation by estradiol addition analysis using the in vitro SIP1-induced cell models revealed (35). This shift is in line with the previously observed pre- that SIP1 expression results in downregulation of major con- dominant expression of 100 kDa and 120 kDa isoforms in stituents of different cell junctional complexes, such as tight epithelial cells and in highly motile fibroblastoid cells, respect- junctions, adherens junctions, desmosomes and gap junctions. ively (36). The functional difference between these isoforms Interestingly we found by Q-RT–PCR analyis that SIP1 down- remains to be elucidated. The other E-cadherin-binding Arma- regulates several cell junction genes on the transcript level. dillo protein b-catenin showed no decrease in mRNA and The fact that expression of a SIP1 mutant, with one missense protein expression levels, but b-catenin was no longer mutation in each of the zinc finger clusters, has no effect on expressed at the cell–cell contacts. SIP1 expression resulted mRNA expression levels of these regulated genes, suggests in the redistribution of b-catenin to the cytoplasm and possibly that downregulation by SIP1 is mediated mainly via promoter also to the nucleus. regulation. Both zinc finger clusters are indeed needed for 6576 Nucleic Acids Research, 2005, Vol. 33, No. 20 Figure 9. SIP1 associates with the promoter regions of cell junction genes at the chromatin level. (A) Promoter regions of junctional genes repressed by SIP1. The 0 0 promoter region, 5 untranslated region (5 -UTR), open reading frame (ORF) and intron were defined using the sequence information derived from the Database for Transcriptional Start Sites (http://dbtss.hgc.jp) and the public human genomic DNA database (http://genome.ucsc.edu) using the Refseq Ids as mentioned under the gene symbol. CACCT(G) and (C)AGGTG boxes were mapped. Amplicons analyzed in the chromatin immunoprecipitation are shown as black bars. (B) DLD1Tr21/ WTSIP1 and A431/WTSIP1 cells were either not induced or induced with Dox for 24 h. In vivo binding of SIP1 to proximal promoter sequences in DLDTr21/ WTSIP1 and A431/WTSIP1 cells, as determined by ChIP analysis. Enrichment of bound sequences was quantified by quantitative real-time PCR and is depicted as the fold increase of association of SIP1 detected with a SIP1-specific antibody in the Dox induced cells versus non-induced cells. Background was determined using an irrelevant IgG antibody and is also depicted as the fold increase in the induced versus non-induced cells. Irrelevant sequences (Irr. seq.) are amplified distal promoter sequences, 4 to 7 kb upstream of the transcription start site of E-cadherin, plakophilin 2, tight junction protein 3 and connexin 26. SIP1-dependent promoter repression via E-box-binding (7). in mesenchymal cells expressing EMT inducers such as Snail Cloning of the promoter regions of the regulated genes con- cannot restore the epithelial phenotype (19,40,41). Moreover, nexin 26 (in the gap junctions), P-cadherin (at the adherens downregulation of the tight junction components, occludins junction) and claudin 4 (in the tight junctions) revealed the and claudins, by Snail was linked to repression of their pro- presence of several SIP1-binding sequences in each of them. moter activity (18,19). Hence, we have to conclude that Mutation of these elements in the cloned connexin 26 pro- E-cadherin and other junctional genes are simultaneously moter showed the importance of the integrity of these downregulated as part of the SIP1 driven reprogramming dur- sequences in the SIP1-dependent suppressive activity. Further- ing EMT. It remains enigmatic though why these different more, we could demonstrate physical interaction at the junctional genes are repressed in a coordinated fashion. We chromatin level between SIP1 and the promoter regions of do know that some of these genes can be regarded as NACos, E-cadherin, plakophilin 2, connexin 26 and ZO-3, all of proteins that can localize both to the nucleus and adhesion which contain SIP1-binding sites. complexes (42). Such proteins have the intriguing potential to The change in expression and distribution of those SIP1 coordinate the regulation of cell adhesion and transcription. target genes during EMT could be explained as a secondary The function of several NACos proteins belonging to the consequence of repression of E-cadherin. A crucial role for Armadillo family such as PKP2, b-catenin and p120ctn E-cadherin in epithelial cell polarity has been well docu- seem to be affected in epithelial cells with SIP1 expression. mented (37–39). However, exogenous E-cadherin expression The desmosomal PKP2 has been reported to be present also in Nucleic Acids Research, 2005, Vol. 33, No. 20 6577 the nucleus at all times. This protein seems to be part of 3. Postigo,A.A. and Dean,D.C. (2000) Differential expression and function of members of the zfh-1 family of zinc finger/homeodomain repressors. particles containing RNA polymerases (43). Moreover the Proc. Natl Acad. Sci. USA, 97, 6391–6396. transcription factor Snail is also able to repress PKP2 expres- 4. Van Grunsven,L.A., Michiels,C., Van De Putte,T., Nelles,L., sion very potently (19). This strong downregulation of PKP2 Wuytens,G., Verschueren,K. and Huylebroeck,D. (2003) Interaction suggests that inhibiting the potential role of PKP2 in adhesion between Smad-interacting protein-1 and the corepressor C-terminal binding protein is dispensable for transcriptional repression of E-cadherin. and/or transcriptional regulation could be essential in the J. Biol. Chem., 278, 26135–26145. process of EMT. It is at present not clear if the SIP1-induced 5. Shi,Y., Sawada,J.I., Sui,G., Affar,E.B., Whetstine,J.R., Lan,F., isoform switching has functional consequences for a particular Ogawa,H., Luke,M.P., Nakatani,Y. and Shi,Y. (2003) Coordinated nuclear role of p120ctn (23). P120ctn can interact with Kaiso histone modifications mediated by a CtBP co-repressor complex. and has as such the potential to influence beta-catenin/ Nature, 422, 735–738. 6. Long,J., Zuo,D. and Park,M. (2005) Pc2-mediated sumoylation of Smad- TCF signaling (44). On the other hand, downregulation of interacting protein 1 attenuates transcriptional repression of E-cadherin. E-cadherin could result in loss of b-catenin sequestration to J. Biol. Chem., 280, 35477–35489. sites of cell–cell adhesion, enabling b-catenin/TCF mediated 7. Comijn,J., Berx,G., Vermassen,P., Verschueren,K., van Grunsven,L., transcription. However SIP1 did not induce clear nuclear Bruyneel,E., Mareel,M., Huylebroeck,D. and van Roy,F. (2001) The two-handed E-box-binding zinc finger protein SIP1 downregulates b-catenin localization nor enhanced WNT signaling (data E-cadherin and induces invasion. Mol. Cell, 7, 1267–1278. not shown). 8. Rosivatz,E., Becker,I., Specht,K., Fricke,E., Luber,B., Busch,R., Enhanced SIP1 expression has so far been reported in a Hofler,H. and Becker,K.F. (2002) Differential expression of the distinct set of cancers comprising gastric, hepatocellular, ovar- epithelial-mesenchymal transition regulators snail, SIP1, and twist ian and breast carcinomas (8,45,46). Here, the described in gastric cancer. Am. J. Pathol., 161, 1881–1891. 9. Mikkers,H., Allen,J., Knipscheer,P., Romeyn,L., Hart,A., Vink,E. and candidate SIP1 target genes have been documented to show Berns,A. (2002) High-throughput retroviral tagging to identify abberant expression in a variety of human cancer types components of specific signaling pathways in cancer. Nature Genet., (47–50). This suggests that repression of these genes could 32, 153–159. be due to enhanced SIP1 expression, although this has to be 10. Eisaki,A., Kurosda,H., Fukui,A. and Asashima,M. (2000) XSIP1,a member of two-handed zinc finger proteins, induced anterior neural examined in detail in the near future. markers in Xenopus Laevis animal cap. Biochem. Biophys. Res. Commun., Taken together, the present results identify the SIP1 protein 271, 151–157. as an important mediator of epithelial dedifferentiation 11. van Grunsven,L.A., Papin,C., Avalosse,B., Opdecamp,K., through direct downregulation of a distinct set of constituents Huylebroeck,D., Smith,J.C. and Bellefroid,E.J. (2000) XSIP1,a Xenopus of adherens junctions, tight junctions, desmosomes and gap zinc finger/homeodomain encoding gene highly expressed during early neural development. Mech. Dev., 94, 189–193. junctions, which are key determinants of the epithelial pheno- 12. Amiel,J., Espinosa-Parrilla,Y., Steffann,J., Gosset,P., Pelet,A., Prieur,M., type, including epithelial cell polarity. Boute,O., Choiset,A., Lacombe,D., Philip,N. et al. (2001) Large-scale deletions and SMADIP1 truncating mutations in syndromic Hirschsprung disease with involvement of midline structures. Am. J. Hum. Genet., 69, 1370–1377. ACKNOWLEDGEMENTS 13. Cacheux,V., Dastot-Le Moal,F., Kaariainen,H., Bondurand,N., We thank Dr Hans Clevers for providing us with the DLDTr21 Rintala,R., Boissier,B., Wilson,M., Mowat,D. and Goossens,M. (2001) Loss-of-function mutations in SIP1 Smad interacting protein 1 result in a cell line. We are grateful to B. Gilbert for expert assistance. J.C. syndromic Hirschsprung disease. Hum. Mol. Genet., 10, 1503–1510. was supported by the ‘Vlaams Instituut voor de Bevordering 14. Wakamatsu,N., Yamada,Y., Yamada,K., Ono,T., Nomura,N., van het Wetenschappelijk-Technologisch Onderzoek in de Taniguchi,H., Kitoh,H., Mutoh,N., Yamanaka,T., Mushiake,K. et al. industrie’ en the ‘Stichting Emmanuel Van der Schueren’. (2001) Mutations in SIP1, encoding Smad interacting protein-1, cause a form of Hirschsprung disease. Nature Genet., 27, 369–370. C.V. is a research assistant with the Fund for Scientific 15. Yamada,K., Yamada,Y., Nomura,N., Miura,K., Wakako,R., Research, Flanders (FWO). G.B. is a postdoctoral fellow Hayakawa,C., Matsumoto,A., Kumagai,T., Yoshimura,I., Miyazaki,S. with the FWO. The research was supported by the et al. (2001) Nonsense and frameshift mutations in ZFHX1B, encoding Association for International Cancer Research (AICR-UK), Smad-interacting protein 1, cause a complex developmental disorder with the Geconcerteerde Onderzoeksacties of Ghent University, a great variety of clinical features. Am. J. Hum. Genet., 69, 1178–1185. 16. Hay,E.D. (1995) An overview of epithelio-mesenchymal transformation. FWO-Flanders, Fortis Insurances (Belgium), and Acta Anat., 154, 8–20. Interuniversity Attraction Poles Programme (IUAP, Belgian 17. Van de Putte,T., Maruhashi,M., Francis,A., Nelles,L., Kondoh,H., Science Policy). Funding to pay the Open Access publication Huylebroeck,D. and Higashi,Y. (2003) Mice lacking Zfhx1b, the gene that charges for this article was provided by IUAP. codes for Smad-interacting protein-1, reveal a role for multiple neural crest cell defects in the etiology of Hirschsprung disease-mental Conflict of interest statement. None declared. retardation syndrome. Am. J. Hum. Genet., 72, 465–470. 18. Ikenouchi,J., Matsuda,M., Furuse,M. and Tsukita,S. (2003) Regulation of tight junctions during the epithelium-mesenchyme transition: direct repression of the gene expression of claudins/occludin by Snail. J. Cell REFERENCES Sci., 116, 1959–1967. 1. Verschueren,K., Remacle,J.E., Collart,C., Kraft,H., Baker,B.S., 19. De Craene,B., Gilbert,B., Stove,C., Bruyneel,E., Van Roy,F. and Berx,G. Tylzanowski,P., Nelles,L., Wuytens,G., Su,M.T., Bodmer,R., Smith,J.C. (2005) The transcription factor Snail induces tumor cell invasion through and Huylebroeck,D. (1999) SIP1, a novel zinc finger/homeodomain modulation of the epithelial cell differentiation program. Cancer Res., repressor, interacts with Smad proteins and binds to 5 -CACCT sequences 65, 6237–6244. in candidate target genes. J. Biol. Chem., 274, 20489–20498. 20. Savagner,P., Yamada,K.M. and Thiery,J.P. (1997) The zinc-finger 2. Remacle,J.E., Kraft,H., Lerchner,W., Wuytens,G., Collart,C., protein slug causes desmosome dissociation, an initial and necessary step Verschueren,K., Smith,J.C. and Huylebroeck,D. (1999) New mode of for growth factor-induced epithelial-mesenchymal transition. J. Cell DNA binding of multi-zinc finger transcription factors: dEF1 family Biol., 137, 1403–1419. members bind with two hands to two target sites. EMBO J., 18, 21. van de Wetering,M., Sancho,E., Verweij,C., deLau,W., Oving,I., 5073–5084. Hurlstone,A., van der Horn,K., Batlle,E., Coudreuse,D., Haramis,A.P. 6578 Nucleic Acids Research, 2005, Vol. 33, No. 20 et al. (2002) The beta-catenin/TCF-4 complex imposes a crypt progenitor involves nuclear translocation of beta-catenin and upregulation of phenotype on colorectal cancer cells. Cell, 111, 241–250. beta-catenin/lymphoid enhancer binding factor-1 transcriptional activity. 22. Evan,G.I., Lewis,G.K., Ramsay,G. and Bishop,J.M. (1985) Isolation of J. Cell Biol., 148, 173–188. ctn monoclonal antibodies specific for human c-myc proto-oncogene 36. Reynolds,A.B. and Daniel,J.M. (1997) p120 : A Src-substrate turned product. Mol. Cell Biol., 5, 3610–3616. catenin. In Cowin,P. and Klymkowsky,M.W. (eds), Cytoskeletal- 23. van Hengel,J., Vanhoenacker,P., Staes,K. and van Roy,F. (1999) Nuclear Membrane Interactions and Signal Transduction. Landes Company and localization of the p120ctn Armadillo-like catenin is counteracted by a Chapman & Hall, Austin, TX, pp. 31–48. nuclear export signal and by E-cadherin expression. Proc. Natl Acad. 37. Takeichi,M. (1991) Cadherin cell adhesion receptors as a morphogenetic Sci. USA, 96, 7980–7985. regulator. Science, 251, 1451–1455. 24. Scha ¨fer,S., Stumpp,S. and Franke,W.W. (1996) Immunological 38. Thiery,J.P. (2002) Epithelial-mesenchymal transitions in tumour identification and characterization of the desmosomal cadherin Dsg2 progression. Nature Rev. Cancer, 2, 442–454. in coupled and uncoupled epithelial cells and in human tissues. 39. Rodriguez-boulan,E. and Nelson,W.J. (1989) Morphogenesis of the Differentiation, 60, 99–108. polarized epithelial cell phenotype. Science, 245, 718–725. 25. Bieche,I., Laurendeau,I., Tozlu,S., Olivi,M., Vidaud,D., Lidereau,R. and 40. Ohkubo,T. and Ozawa,M. (2004) The transcription factor Snail Vidaud,M. (1999) Quantitation of MYC gene expression in sporadic downregulates the tight junction components independently of breast tumors with real-time reverse transcription-PCR assay. E-cadherin downregulation. J. Cell Sci., 117, 1675–1685. Cancer Res., 59, 2759–2765. 41. Yang,J., Mani,S.A., Donaher,J.L., Ramaswamy,S., Itzykson,R.A., 26. Vleminckx,K., Vakaet,L.Jr, Mareel,M., Fiers,W. and van Roy,F. (1991) Come,C., Savagner,P., Gitelman,I., Richardson,A. and Weinberg,R.A. Genetic manipulation of E-cadherin expression by epithelial tumor (2004) Twist, a master regulator of morphogenesis, plays an essential cells reveals an invasion suppressor role. Cell, 66, 107–119. role in tumor metastasis. Cell, 117, 927–939. 27. Bracke,M.E., Vyncke,B.M., Bruyneel,E.A., Vermeulen,S.J., 42. Balda,M.S. and Matter,K. (2003) Epithelial cell adhesion and the De Bruyne,G.K., Van Larebeke,N.A., Vleminckx,K., Van Roy,F.M. and regulation of gene expression. Trends Cell Biol., 13, 310–318. Mareel,M.M. (1993) Insulin-like growth factor I activates the invasion 43. Mertens,C., Hofmann,I., Wang,Z., Teichmann,M., Sepehri Chong,S., suppressor function of E-cadherin in MCF-7 human mammary Schnolzer,M. and Franke,W.W. (2001) Nuclear particles containing RNA carcinoma cells in vitro. Br. J. Cancer, 68, 282–289. polymerase III complexes associated with the junctional plaque protein 28. Vanpoucke,G., Goossens,S., De Craene,B., Gilbert,B., van Roy,F. and plakophilin 2. Proc. Natl Acad. Sci. USA, 98, 7795–7800. Berx,G. (2004) GATA-4 and MEF2C transcription factors control the 44. Park,J.I., Kim,S.W., Lyons,J.P., Ji,H., Nguyen,T.T., Cho,K., tissue-specific expression of the alphaT-catenin gene CTNNA3. Barton,M.C., Deroo,T., Vleminckx,K., Moon,R.T. and McCrea,P.D. Nucleic Acids Res., 32, 4155–4165. (2005) Kaiso/p120-catenin and TCF/beta-catenin complexes 29. Keirsebilck,A., Bonne ´,S., Staes,K., van Hengel,J., Nollet,F., Reynolds,A. coordinately regulate canonical Wnt gene targets. Dev. Cell, 8, and van Roy,F. (1998) Molecular cloning of the human p120ctn catenin 843–854. gene (CTNND1): expression of multiple alternatively spliced isoforms. 45. Elloul,S., Elstrand,M.B., Nesland,J.M., Trope,C.G., Kvalheim,G., Genomics, 50, 129–146. Goldberg,I., Reich,R. and Davidson,B. (2005) Snail, Slug, and Smad- 30. Mo,Y.Y. and Reynolds,A.B. (1996) Identification of murine p120(cas) interacting protein 1 as novel parameters of disease aggressiveness in isoforms and heterogeneous expression of p120(cas) isoforms in human metastatic ovarian and breast carcinoma. Cancer, 103, 1631–1643. tumor cell lines. Cancer Res., 56, 2633–2640. 46. Miyoshi,A., Kitajima,Y., Sumi,K., Sato,K., Hagiwara,A., Koga,Y. and 31. Hazan,R.B., Kang,L., Whooley,B.P. and Borgen,P.I. (1997) N-cadherin Miyazaki,K. (2004) Snail and SIP1 increase cancer invasion by promotes adhesion between invasive breast cancer cells and the stroma. upregulating MMP family in hepatocellular carcinoma cells. Br. J. Cell Adhes. Commun., 4, 399–411. Cancer, 90, 1265–1273. 32. Tran,N.L., Nagle,R.B., Cress,A.E. and Heimark,R.L. (1999) N-cadherin 47. Reynolds,A.B. and Carnahan,R.H. (2004) Regulation of cadherin stability expression in human prostate carcinoma cell lines. An epithelial- and turnover by p120ctn: implications in disease and cancer. Semin. mesenchymal transformation mediating adhesion with stromal cells. Cell Dev. Biol., 15, 657–663. Am. J. Pathol., 155, 787–798. 48. Aasen,T., Hodgins,M.B., Edward,M. and Graham,S.V. (2003) The 33. Jechlinger,M., Grunert,S., Tamir,I.H., Janda,E., Ludemann,S., relationship between connexins, gap junctions, tissue architecture and Waerner,T., Seither,P., Weith,A., Beug,H. and Kraut,N. (2003) tumour invasion, as studied in a novel in vitro model of HPV-16- Expression profiling of epithelial plasticity in tumor progression. associated cervical cancer progression. Oncogene, 22, 7969–7980. Oncogene, 22, 7155–7169. 49. Mertens,C., Kuhm,C., Moll,R., Schwetlick,I. and Franke,W.W. (1999) 34. Nagafuchi,A., Takeichi,M. and Tsukita,S. (1991) The 102 kd cadherin- Desmosomal plakophilin 2 as a differentiation marker in normal associated protein: similarity to vinculin and posttranscriptional and malignant tissues. Differentiation, 64, 277–290. regulation of expression. Cell, 65, 849–857. 50. Kanczuga-Koda,L., Sulkowski,S., Koda,M. and Sulkowska,M. (2005) 35. Eger,A., Stockinger,A., Schaffhauser,B., Beug,H. and Foisner,R. (2000) Alterations in connexin26 expression during colorectal carcinogenesis. Epithelial mesenchymal transition by c-Fos estrogen receptor activation Oncology, 68, 217–222.

Journal

Nucleic Acids Research – Oxford University Press

Published: Jan 1, 2005

There are no references for this article.