SNAIL and miR-34a feed-forward regulation of ZNF281/ZBP99 promotes epithelial-mesenchymal transition
2013; Springer Nature; Volume: 32; Issue: 23 Linguagem: Inglês
10.1038/emboj.2013.236
ISSN1460-2075
AutoresStefanie Hahn, René Jackstadt, Helge Siemens, Sabine Hünten, Heiko Hermeking,
Tópico(s)RNA modifications and cancer
ResumoArticle1 November 2013free access SNAIL and miR-34a feed-forward regulation of ZNF281/ZBP99 promotes epithelial–mesenchymal transition Stefanie Hahn Stefanie Hahn Experimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-University Munich, Munich, Germany Search for more papers by this author Rene Jackstadt Rene Jackstadt Experimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-University Munich, Munich, Germany Search for more papers by this author Helge Siemens Helge Siemens Experimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-University Munich, Munich, Germany Search for more papers by this author Sabine Hünten Sabine Hünten Experimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-University Munich, Munich, Germany Search for more papers by this author Heiko Hermeking Corresponding Author Heiko Hermeking Experimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-University Munich, Munich, Germany German Cancer Consortium (DKTK), Heidelberg, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Stefanie Hahn Stefanie Hahn Experimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-University Munich, Munich, Germany Search for more papers by this author Rene Jackstadt Rene Jackstadt Experimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-University Munich, Munich, Germany Search for more papers by this author Helge Siemens Helge Siemens Experimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-University Munich, Munich, Germany Search for more papers by this author Sabine Hünten Sabine Hünten Experimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-University Munich, Munich, Germany Search for more papers by this author Heiko Hermeking Corresponding Author Heiko Hermeking Experimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-University Munich, Munich, Germany German Cancer Consortium (DKTK), Heidelberg, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Author Information Stefanie Hahn1, Rene Jackstadt1, Helge Siemens1, Sabine Hünten1 and Heiko Hermeking 1,2,3 1Experimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-University Munich, Munich, Germany 2German Cancer Consortium (DKTK), Heidelberg, Germany 3German Cancer Research Center (DKFZ), Heidelberg, Germany *Corresponding author. Experimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-University Munich, Thalkirchner Strasse 36, 80337 Munich, Germany. Tel.:+49 89 2180 73685; Fax:+49 89 2180 73697; E-mail: [email protected] The EMBO Journal (2013)32:3079-3095https://doi.org/10.1038/emboj.2013.236 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Here, we show that expression of ZNF281/ZBP-99 is controlled by SNAIL and miR-34a/b/c in a coherent feed-forward loop: the epithelial–mesenchymal transition (EMT) inducing factor SNAIL directly induces ZNF281 transcription and represses miR-34a/b/c, thereby alleviating ZNF281 mRNA from direct down-regulation by miR-34. Furthermore, p53 activation resulted in a miR-34a-dependent repression of ZNF281. Ectopic ZNF281 expression in colorectal cancer (CRC) cells induced EMT by directly activating SNAIL, and was associated with increased migration/invasion and enhanced β-catenin activity. Furthermore, ZNF281 induced the stemness markers LGR5 and CD133, and increased sphere formation. Conversely, experimental down-regulation of ZNF281 resulted in mesenchymal–epithelial transition (MET) and inhibition of migration/invasion, sphere formation and lung metastases in mice. Ectopic c-MYC induced ZNF281 protein expression in a SNAIL-dependent manner. Experimental inactivation of ZNF281 prevented EMT induced by c-MYC or SNAIL. In primary CRC samples, expression of ZNF281 increased during tumour progression and correlated with recurrence. Taken together, these results identify ZNF281 as a component of EMT-regulating networks, which contribute to metastasis formation in CRC. Introduction The ZNF281/ZBP-99 protein has characteristics of a transcription factor and contains four Krüppel-type zinc-finger domains (Law et al, 1999; Lisowsky et al, 1999). ZNF281 is related to ZBP-89, which has been implicated in the regulation of cell proliferation (Bai and Merchant, 2001), apoptosis (Bai et al, 2004), differentiation (Li et al, 2006) and tumorigenesis (Law et al, 2006). ZNF281 mediates transcriptional repression and activation (Wang et al, 2008). For example, ZNF281 directly regulates the expression of gastrin and represses ornithine decarboxylase (ODC) by binding to GC-rich sequences in their promoters (Law et al, 1999; Lisowsky et al, 1999). Previously, we detected a direct interaction between ZNF281 and the c-MYC oncoprotein (Koch et al, 2007). Moreover, ZNF281 participates in the regulation and maintenance of pluripotency by interacting with transcription factors controlling stemness, such as Nanog, Oct4 and Sox2 (Wang et al, 2006, 2008). Furthermore, ZNF281 directly regulates Nanog expression and contributes to its auto-regulation by recruiting the NuRD complex in mouse embryonic stem cells (Fidalgo et al, 2011, 2012). The Sox4 transcription factor directly induces ZNF281 transcription (Scharer et al, 2009). Interestingly, Sox4 has been implicated in the regulation of differentiation, proliferation, epithelial–mesenchymal transition (EMT) and shows increased expression in many human cancers (Zhang et al, 2012). After DNA damage, the ZNF281 protein is phosphorylated by ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) kinases (Matsuoka et al, 2007). However, other signals regulating ZNF281 activity and expression have remained elusive. As a morphogenic programme, EMT is involved in the formation of tissue and organs during embryonic development and wound healing. During EMT, epithelial cells acquire mesenchymal features, such as decreased cell–cell contacts and loss of polarity, which promote increased motility and invasiveness. Thereby, EMT contributes to the progression of early-stage tumours to invasive malignancies (Thiery, 2002; Lee et al, 2006; Hugo et al, 2007). So far only a few transcription factors, such as SNAIL, SLUG, TWIST1/2 and ZEB1/2, are thought to constitute the central regulatory core of EMT (Peinado et al, 2007; Sanchez-Tillo et al, 2012). EMT has also been shown to promote stemness of cancer cells that may endow tumour initiating cells with traits necessary for metastasis formation, as shown for immortalized human mammary epithelial cells undergoing EMT (Brabletz et al, 2005b; Mani et al, 2008). Accordingly, SNAIL, TWIST and ZEB1 share the ability to induce both stemness and EMT (Mani et al, 2008; Wellner et al, 2009). Furthermore, tumour cells undergoing EMT show accumulation of active β-catenin in the nucleus (reviewed in Brabletz et al, 2005a). Recently, microRNAs (miRNAs) have emerged as major regulators of EMT (Brabletz, 2012; Hermeking, 2012). For example, members of the miR-200 family and miR-205 promote MET by inhibiting EMT inducing factors like ZEB1 and ZEB2 (Gregory et al, 2008; Park et al, 2008), and miR-34a/b/c achieve the same effect by downregulating SNAIL expression (Kim et al, 2011; Siemens et al, 2011). Moreover, SNAIL directly represses miR-34a/b/c transcription (Siemens et al, 2011). The resulting double-negative feedback loop represents a bistable switch, which can be locked in the mesenchymal state by inactivation of miR-34 genes by CpG methylation, which is often found in cancer cells (Hermeking, 2012; Siemens et al, 2013). Interestingly, the genes encoding the miR-200 and miR-34 families are direct p53 targets and their mediation of MET presumably contributes to tumour suppression by p53 (Hermeking, 2012). Here, we show that ZNF281 expression is regulated by a feed-forward loop involving SNAIL and miR-34a. Taken together, we show that ZNF281 is an integral part of the EMT-regulating transcriptional network and controls processes relevant to colorectal cancer (CRC) progression, such as migration, invasion, stemness and metastasis. Results SNAIL regulates ZNF281 expression We previously identified an interaction between c-MYC and ZNF281 proteins in a systematic analysis of c-MYC-associated protein complexes (Koch et al, 2007). ZNF281 was among the proteins represented by the highest number of mass-spectrometric sequence reads, indicating that it is associated with a large fraction of cellular c-MYC and presumably represents a significant regulator or effector of c-MYC. However, so far it is largely unknown how ZNF281 expression itself is regulated and whether it participates in regulatory pathways, which might by relevant for c-MYC function and tumour biology. In order to identify upstream regulators of ZNF281, we inspected the ZNF281 promoter sequence for binding sites of transcription factors, which might hint towards cancer-relevant functions of ZNF281. Thereby, we identified several E-Box motifs (CACCTG) in the ZNF281 promoter, which represent putative SNAIL binding sites (SBSs; Figure 1A). SNAIL is a known regulator of EMT (Mauhin et al, 1993; Batlle et al, 2000) and, similar to ZNF281, a zinc-finger-containing transcription factor (Nieto, 2002; Sanchez-Tillo et al, 2012; see also comparison in Supplementary Figure S1). Two of the SBSs were located ∼500 and ∼700 bp upstream of the transcription start site (TSS; Figure 1B). SBS2 and SBS4 are conserved between the human and mouse ZNF281 promoters, indicating functional relevance (Figure 1B). When SNAIL was ectopically expressed in DLD-1 CRC cells using a Doxycycline (DOX) inducible episomal vector system an increase in the SNAIL occupancy of the ZNF281 promoter was detected by chromatin immunoprecipitation (ChIP) analysis at SBS2 and SBS3, whereas SBS1, 4 and 5 did not display increased binding of SNAIL (Figure 1C). Also endogeneous SNAIL protein selectively occupied SBS2 and SBS3 in SW620 CRC cells (Supplementary Figure S2A). Furthermore, ectopic SNAIL enhanced the expression of ZNF281 at the protein and the mRNA level in DLD-1 cells (Figure 1D and E). SNAIL also induced ZNF281 expression in SKBR3 breast cancer and MiaPaCa2 pancreatic cancer cells (Supplementary Figure S2B). Therefore, the induction of ZNF281 by SNAIL is not restricted to a specific cell type. In order to determine whether ZNF281 is induced by SNAIL via SBS motifs, a region encompassing ∼2 kbp upstream of the ZNF281 transcriptional start site was subjected to a dual reporter assay (Figure 1F). Indeed, the wild-type reporter was induced by SNAIL, whereas mutation of SBS2 abolished and SBS3 mutation decreased the responsiveness to SNAIL. Also a reporter with combined mutation of SBS2 and SBS3 resulted in complete loss of responsiveness to SNAIL. A CDH1 promoter reporter was repressed by SNAIL in an SBS-dependent manner in this assay. ZNF281 displayed the highest expression level in CRC cell lines with mesenchymal features, such as Colo320, SW480 and SW620, whereas HT29, DLD-1 and HCT-15 cells, which display an epithelial phenotype, showed comparatively low ZNF281 expression levels (Figure 1G). ZNF281 expression positively correlated with SNAIL and Vimentin and inversely with E-cadherin expression (Figure 1G). Moreover, analysis of publicly available mRNA expression profiles obtained from seven CRC cell lines (COLO205, HCC2998, HCT116, HCT15, HT29, KM12, SW620) within the NCI-60 panel (Shoemaker, 2006) confirmed a significant correlation between ZNF281 and the mesenchymal markers SNAIL, Vimentin and Fibronectin-1 (Supplementary Table S1). Taken together, these results suggested that the induction of ZNF281 by SNAIL may be an important component of the EMT programme induced by SNAIL. Indeed, when ZNF281 was downregulated using two different siRNAs the induction of EMT by SNAIL was prevented in DLD-1 cells (Figure 1H and I; Supplementary Figure S2C). Also the loss of E-cadherin from the outer membrane, which is typical for EMT, was prevented by simultaneous siRNA-mediated downregulation of ZNF281. Therefore, ZNF281 is required for SNAIL-induced EMT. Figure 1.ZNF281 is a direct SNAIL target required for SNAIL-induced EMT. For the following analyses (besides F and G) DLD-1 cells harbouring a pRTR-SNAIL-VSV vector were treated with DOX for the indicated periods to activate SNAIL-VSV expression. (A) Scheme of the ZNF281 promoter and SNAIL binding sites (SBSs). Grey arrows indicate potential SNAIL binding sites; black rectangles exons and the bar a qChIP amplicon. TSS: transcription start site. (B) Sequence alignment of the indicated SBS in the indicated species. (C) ChIP analysis 24 h after addition of DOX or left untreated using anti-VSV and anti-rabbit-IgG antibodies for ChIP. Results are given as the mean ±s.d. (n=3). (D) Western blot detection of the indicated proteins at the indicated time points. (E) qPCR analysis with values representing the mean±s.d. (n=3). (F) Luciferase assay in DLD-1 cells 48 h after transfection of pcDNA3-VSV (Ctrl.) or pcDNA3-SNAIL-VSV (SNAIL) vectors and the indicated pBV-ZNF281 promoter constructs or pXP2-E-cadherin/CDH-1 vectors as controls (wt: wild type, mut: mutated). (G) Western blot analysis of the indicated proteins in CRC cell lines. 'epi.'=cells with epithelial, 'mes.'=cells with mesenchymal phenotype. (H) Cells were treated with DOX (+) or left untreated (−) for 96 h and simultaneously transfected with the indicated siRNAs. The indicated proteins were detected by western blot analysis. (I) Two upper panels: representative phase-contrast pictures (P/C) of the cells described in (H). × 200 magnification. Two lower panels: detection of E-cadherin by indirect immunofluorescence and confocal microscopy. Nuclear DNA was visualized by DAPI staining. × 200 magnification. Scale bars represent 25 μm. In (D, H), detection of α-Tubulin and in (G), detection of β-Actin served as a loading control. In (D, H), relative densitometric quantifications are indicated. ZNF=ZNF281; E-cad=E-cadherin; α-Tub=α-Tubulin. In (C, E and F), a Student's t-test was used. *P<0.05, **P<0.01 and ***P<0.001.Source data for this figure is available on the online supplementary information page. Source data for Figure 1 [embj2013236-sup-0001-SourceData-S1.pdf] Download figure Download PowerPoint miR-34a directly regulates ZNF281 expression The differences between the pronounced increase in ZNF281 protein levels and minor increase in mRNA levels after ectopic SNAIL expression suggested the possibility of an additional translational regulation mediated by miRNAs. Inspection of the ZNF281 3′-UTR using the TargetSCAN and Miranda algorithms (John et al, 2004; Grimson et al, 2007) revealed a conserved miR-34 seed-matching sequence (Figure 2A). Since we had previously shown that the miR-34a and miR-34b/c genes are directly repressed by SNAIL (Siemens et al, 2011), we hypothesized that at least part of the increase in ZNF281 expression observed after SNAIL induction might be due to a repression of miR-34 genes. Indeed, ectopic miR-34a expression resulted in the downregulation of endogeneous ZNF281 expression at the protein and mRNA level in SW480 CRC cells (Figure 2B and C). This was also observed in MiaPaCa2 pancreatic cancer cells (Supplementary Figure S3A and B). Therefore, the regulation of ZNF281 by miR-34a is not restricted to CRC cells. Furthermore, reporter constructs containing the complete 3′-UTR of ZNF281 (720 bp) or a 77-bp fragment including the seed-matching sequence were repressed by co-transfection of pre-miR-34a, but not when the seed-matching sequence was mutated, demonstrating that it mediates repression by miR-34a (Figure 2D and E). The induction of ZNF281 by SNAIL was prevented by concomitant transfection of pre-miR-34a (Figure 2F). Therefore, the previously documented repression of the miR-34a gene by SNAIL (Siemens et al, 2011) is presumably necessary for the SNAIL-mediated increase in ZNF281 expression. In summary, these results demonstrate that ZNF281 is directly regulated by miR-34a and that SNAIL induces ZNF281, at least in part, by repressing miR-34a. Figure 2.Direct regulation of ZNF281 by miR-34a. (A) Schematic representation of the ZNF281 3′-UTR indicating seed-matching sequences (in red) and miR-34 seed sequences (blue letters) (adapted from www.targetscan.org). The black vertical bars indicate possible base pairing. (B) Western blot analysis of endogeneous ZNF281 protein levels in SW480 cells harbouring a pRTR-pri-miR-34a vector after treatment with DOX for the indicated periods. Relative densitometric quantifications are indicated. ZNF=ZNF281; α-Tub=α-Tubulin. (C) Analysis of ZNF281 mRNA levels in cells corresponding to (B). (D) Mutagenesis of the ZNF281 3′-UTR. Black vertical bars indicate the remaining matches of the miR-34a seed (shaded black) with the miR-34 seed-matching sequence (shaded grey) in the ZNF281 3′-UTR sequence (wt: wild type, mut: mutated). (E) Dual luciferase reporter assay in SW480 cells 72 h after transfection with pre-miR-34a or control oligonucleotides and the empty pGL3 vector or pGL3 harbouring the indicated 3′-UTR-reporter constructs (fl: full length). A 3′-UTR reporter of the known miR-34a target TPD52 served as a positive control. (F) Western blot analysis of the indicated proteins in DLD-1 cells harbouring a pRTR-SNAIL-VSV vector transfected with the indicated oligonucleotides for 60 h and treated with DOX or left untreated for 36 h prior to cell lysis. In (B, F), detection of α-Tubulin served as a loading control. In (C, E), data represent the mean±s.d. (n=3). A Student's t-test was used. *P<0.05 and ***P<0.001.Source data for this figure is available on the online supplementary information page. Source data for Figure 2 [embj2013236-sup-0002-SourceData-S2.pdf] Download figure Download PowerPoint p53 represses ZNF281 via miR-34a Since the miR-34 genes represent direct p53 targets, we asked whether p53 represses ZNF281 expression via inducing miR-34a. Indeed, ectopic expression of p53 resulted in a decrease in ZNF281 at the protein and mRNA level (Figure 3A and B). As expected, miR-34a/b/c levels were increased upon p53 activation, which is likely to mediate the decrease in ZNF281 protein expression (Figure 3C and D). Since miR-34b/c is expressed at least at 10-fold lower levels in CRC and CRC cell lines compared to miR-34a (Toyota et al, 2008; Siemens et al, 2013) we focussed on miR-34a in the further analyses. The recovery of ZNF281 mRNA expression by 72 h of ectopic p53 expression is presumably due to the declining expression of ectopic p53 and therefore reduced pri-miR-34 induction at this time point (Figure 3A, B and D). Nonetheless, ZNF281 protein was still downregulated 72 h after activation of p53 (Figure 3A). In order to determine whether downregulation of ZNF281 is a result of reduced SNAIL expression caused by direct interaction of SNAIL with p53 (Lim et al, 2010) or due to p53-induced miR-34, we directly interfered with miR-34a function using antagomirs. Indeed, miR-34a-specific antagomirs largely abolished the downregulation of ZNF281 after p53 induction, whereas a control antagomir did not affect the repression of ZNF281 by p53 (Figure 3E). The remaining minor repression of ZNF281 may be due to p53-induced miR-34b and -c, which are presumably not affected by the miR-34a-specific antagomir used here. Additionally, we analysed the expression of ZNF281 in HCT116 p53+/+ cells and an isogenic clone with homozygous deletion of p53 resembling p53 inactivation in tumours. HCT116 p53+/+ cells expressed lower endogeneous levels of ZNF281 protein and mRNA than p53-deficient cells (Figure 3F and G). As previously described (Siemens et al, 2011), the expression of the SNAIL protein was elevated in the HCT116 p53−/− cells (Figure 3F). When SNAIL was downregulated using a SNAIL-specific siRNA, the expression of ZNF281 protein was only decreased to a minor extent in p53-deficient HCT116 cells (Figure 3H). Therefore, the increase in ZNF281 expression is presumably mainly due to the decrease in miR-34a expression in p53-deficient cells (Figure 3G). Taken together, these results show that miR-34a represents an important mediator for the repression of ZNF281 by p53. Figure 3.p53 regulates the expression of ZNF281 via induction of miR-34a. In (A–E), SW480 cells harbouring a pRTR-p53-VSV vector treated with DOX for the indicated periods or left untreated were used. (A) Western blot analysis of endogenous ZNF281 expression at the indicated time points. (B) Ectopic p53 was expressed for the indicated periods before RNA was harvested and subjected to qPCR analysis. (C) Analysis of mature miR-34a/b/c expression levels 48 h after addition of DOX to induce p53 or left untreated. (D) Analysis of pri-miR-34a mRNA levels at the indicated time points. (E) Cells were transfected with the indicated oligonucleotides for 72 h in the presence or absence of DOX for the last 48 h prior to lysis of the cells. The indicated proteins were detected by western blot analysis. (F) Detection of the indicated proteins by western blot analysis in p53+/+ and p53−/− HCT116 cells. (G) qPCR analysis of the ZNF281 mRNA expression in p53+/+ and p53−/− HCT116 cells. (H) Western blot analysis of the indicated proteins in HCT116 p53−/− cells 96 h after transfection of a SNAIL-specific siRNA. In (A, E), detection of β-Actin and in (F, H), detection of α-Tubulin served as a loading control and were used for relative densitometric quantifications in (E, H). ZNF=ZNF281; α-Tub=α-Tubulin. In (B–D and G), values represent the mean±s.d. (n=3). A Student's t-test was used. *P<0.05, **P<0.01 and ***P 90% of the cells were positive for eGFP, which is expressed from a bidirectional promoter also driving the expression of ZNF281 (Supplementary Figure S4A). After induction of ectopic ZNF281 expression DLD-1 cells changed from an epithelial morphology (dense islands of cobblestone-shaped cells) to a mesenchymal morphology with spindle-shaped cells forming protrusions and displaying a scattered growth pattern (Figure 4A). This was reminiscent of the effect of ectopic SNAIL expression observed in DLD-1 cells before (Siemens et al, 2011). Also molecular markers of EMT were regulated by the expression of ZNF281 (Figure 4B). The distinct membrane-bound expression of E-cadherin in DLD-1 cells was lost upon ZNF281 activation. Furthermore, ZNF281-expressing cells displayed an increased cytoplasmic expression of the mesenchymal marker Vimentin. In addition, F-actin, which forms stress fibres (Moreno-Bueno et al, 2009), was relocated from the membrane to the cytoplasm. Figure 4.Ectopic ZNF281 induces EMT, migration and invasion in DLD-1 cells. DLD-1 cells harbouring a pRTR-ZNF281-VSV vector treated with DOX or left untreated were analysed. (A) Representative phase-contrast (P/C) pictures of the cells treated with DOX or left untreated for 96 h. × 200 magnification. (B) Confocal laser-scanning microscopy of E-cadherin, Vimentin and F-actin proteins detected by indirect immunofluorescence 96 h after addition of DOX. Nuclear DNA was visualized by DAPI staining. × 200 magnification. (C) Cells were treated with DOX or left untreated for 48 h before the scratch was applied. Upper panel: representative pictures of the wound areas at the indicated time points after scratching. × 100 magnification. Lower panel: results represent the average (%) of wound closure determined by the final width of the scratch in three independent wells. Error bars represent±s.d. (n=3). Boyden-chamber assays of cellular migration (D) or invasion (E). Cells were cultivated in the presence or absence of DOX for 72 h with serum starvation for the last 48 h. To analyse invasion, membranes were coated with Matrigel. After 48 h, cells were fixed and stained with DAPI. The average number of cells per well was counted in three different inserts. Relative invasion or migration is expressed as the value of treated cells to control cells with control set as one. (F) Cells were subjected to a soft-agar assay and treated with DOX or left untreated. Two weeks after seeding the resulting colonies were stained with crystal violet. Results represent the mean number of colonies in soft agar per well±s.d. (n=3). (C–F) A Student's t-test was used. **P<0.01 and ***P<0.001. In (A, B), scale bars represent 25 μm. Download figure Download PowerPoint Subsequently, we determined whether ectopic ZNF281 expression influences cellular migration and invasion, since EMT has been previously linked to increased migration and invasion (reviewed in Christiansen and Rajasekaran, 2006). In a wound-healing assay, ectopic ZNF281 expression resulted in a minor, but reproducible increase in the closure of a scratch in a confluent layer of DLD-1 cells compared to controls (Figure 4C; Supplementary Figure S4H). When migration and invasion were examined in Boyden-chamber assays the effect of ectopic ZNF281 expression was more pronounced (Figure 4D and E). Furthermore, ectopic expression of ZNF281 significantly enhanced the ability of DLD-1 cells to form colonies in soft agar (Figure 4F). The addition of DOX to DLD-1 cells harbouring an empty vector control did not result in EMT-related morphological changes or significant effects in the above-mentioned assays (Supplementary Figure S4E–H). The effects of ectopic ZNF281 were not due to increased proliferation, since ZNF281 activation had a slight anti-proliferative effect (Supplementary Figure S5), which has also been described for other EMT-TFs, such as SNAIL (Peinado et al, 2007). Taken together, these results show that ectopic expression of ZNF281 is sufficient to mediate EMT and enhances migration, invasion and anchorage-independent growth. Transcriptional regulation of EMT markers by ZNF281 Next, we determined whether ZNF281 also induces changes in the expression of genes previously implicated in the transcriptional programme of EMT. After activation of ectopic ZNF281 expression in DLD-1 cells, an upregulation of SNAIL was observed at the protein and mRNA level (Figure 5A and B). In addition, the mesenchymal markers SLUG, ZEB1 and Fibronectin-1 were induced after ectopic ZNF281 expression in DLD-1 cells (Figure 5B). In line with the indirect immunofluorescence results shown in Figure 4B, E-cadherin/CDH-1 was repressed at the protein level after induction of ZNF281 (Figure 5A), whereas expression of CDH1 mRNA was not significantly affected by ZNF281 (Figure 5C). However, when ZNF281 was expressed in HT29 CRC cells E-cadherin was repressed at both the protein and mRNA level (Supplementary Figure S6A and C). Therefore, the regulation of EMT markers by ZNF281 is at least partially dependent on the cellular context. Other epithelial markers, such as OCLN and CLDN-7, were repressed at the mRNA level in DLD-1 and HT29 cells (Figure 5C; Supplementary Figure S6C), which is characteristic for EMT (Ikenouchi et al, 2003; Martinez-Estrada et al, 2006). Furthermore, ectopic ZNF281 expression resulted in the downregulation of a number of additional epithelial marker genes encoding components of tight junctions (ZO-1/3, CLDN-1) and adherens junctions (CDH-3), as well as desmosomes (PKP2, DSP) (Figure 5D), as previously shown for ZEB2 (Vandewalle et al, 2005). Figure 5.ZNF281 activates a transcriptional EMT programme that includes activation of SNAIL. Unless mentioned otherwise, DLD-1 cells harbouring a pRTR-ZNF281-VSV vector treated with DOX or left untreated were analysed. (A) Western blot detection of the indicated proteins after ectopic expression of ZNF281 for the indicated periods. Relative densitometric quantifications are indicated. ZNF=ZNF281, E-cad=E-cadherin, α-Tub=α-Tubulin. (B) Expression of the indicated mRNAs was determined by qPCR analyses. Results represent the mean±s.d. (n=3). (C) Cells were induced with DOX for the indicated periods or left untreated and qPCR analyses were performed to determine the indicated mRNA expression levels. (D) Cells were treated with DOX for 96 h or left untreated and the indicated mRNAs were analysed by qPCR. In (C, D) results represent the mean±s.d. (n=3). (E) Schematic depiction of the SNAIL promoter. Amplicons (black bars) used for ChIP analysis, exons (black rectangles) and the TSS (transcription start site) are indicated. (F) ChIP assay of DLD-1/pRTR (Ctrl.) or DLD-1/pRTR-ZNF281-VSV (ZNF281) cells 24 h after addition of DOX using anti-VSV and anti-rabbit-IgG antibodies. The previously described ZNF281 occupancy at the ODC1 promoter served as a positive control. Results represent the percentage of input chroma
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