PLAG1, the Main Translocation Target in Pleomorphic Adenoma of the Salivary Glands, Is a Positive Regulator of IGF-II1

Home , Pleomorphic adenoma

[CANCER RESEARCH 60, 106–113, January 1, 2000] PLAG1, the Main Translocation Target in Pleomorphic Adenoma of the Salivary Glands, Is a Positive Regulator of IGF-II1

Marianne L. Voz,2 Nancy S. Agten, Wim J. M. Van de Ven, and Koen Kas Laboratory for Molecular Oncology, Center for Human Genetics, University of Leuven and Flanders Interuniversity Institute for Biotechnology, Herestraat 49, B-3000 Leuven, Belgium

ABSTRACT The PLAG1 protein contains seven canonical C2H2 zinc finger domains and a serine-rich COOH terminus that exhibits transactiva- PLAG1, a novel developmentally regulated C H zinc finger gene, is 2 2 tion capacities when fused to the Gal4 DNA binding domain (4), consistently rearranged and overexpressed in pleomorphic adenomas of suggesting that it may act as a transcriptional regulator. the salivary glands with 8q12 translocations. In this report, we show that PLAG1 is a nuclear protein that binds DNA in a specific manner. The To extend our knowledge on the function of the PLAG1 gene and consensus PLAG1 binding site is a bipartite element containing a core the mechanisms by which it causes salivary gland adenomas, we sequence, GRGGC, and a G-cluster, RGGK, separated by seven random decided to further investigate functional characteristics of PLAG1 and nucleotides. DNA binding is mediated mainly via three of the seven zinc in particular its potential transcriptional role. We determined in which fingers, with fingers 6 and 7 interacting with the core and finger 3 with the subcellular compartment PLAG1 exerts its function by immunofluo- G-cluster. In transient transactivation assays, PLAG1 specifically acti- rescence studies; determined whether PLAG1 could bind DNA in a vates transcription from its consensus DNA binding site, indicating that sequence-specific manner and identified its consensus DNA binding PLAG1 is a genuine transcription factor. Potential PLAG1 binding sites site by performing CASTing experiments. The zinc fingers required were found in the promoter 3 of the human insulin-like growth factor II for sequence-specific DNA binding were determined by deletion/ (IGF-II) gene. We show that PLAG1 binds IGF-II promoter 3 and stim- mutation analysis; and we used the PLAG1 consensus was used to ulates its activity. Moreover, IGF-II transcripts derived from the P3 promoter are highly expressed in salivary gland adenomas overexpressing screen the eukaryotic promoter databank. Possible target genes were PLAG1. In contrast, they are not detectable in adenomas without abnor- studied regarding the capacity of PLAG1 to bind and activate their mal PLAG1 expression nor in normal salivary gland tissue. This indicates promoter. Finally, we analyzed the expression of such target genes in a perfect correlation between PLAG1 and IGF-II expression. All of these normal salivary gland tissue and in pleomorphic adenomas with or results strongly suggest that IGF-II is one of the PLAG1 target genes, without PLAG1 overexpression to determine whether these genes providing us with the first clue for understanding the role of PLAG1 in could be PLAG1 targets in salivary gland adenomas. salivary gland tumor development. MATERIALS AND METHODS INTRODUCTION Construction and Production of GST-PLAG1 Fusion Proteins. The 3 Activation of the PLAG1 gene on chromosome 8q12 is the most PLAG1 NH2-terminal region (N2-C244) as well as parts of it (N84-C244; frequent gain-of-function mutation found in pleomorphic adenomas of N101-C244; N159-C244) were fused in-frame to GST by inserting in pGEX- the salivary glands (1, 2). This mainly results from recurrent chromo- 5X-2 (Pharmacia) the DNA fragments obtained by the PCR with full-length somal translocations that lead to promoter substitution between PLAG1 cDNA as template. The NH2-terminal oligonucleotides used to gen- Ј PLAG1, a gene mainly expressed in fetal tissue, and more broadly erate the various constructs were: G8N2, 5 -CCCGAATTCTGGCCACTGT- CATTCCTGGT-3Ј; G8N84, 5Ј-CCCGAATTCTGGCTACTCATTCTCCT- expressed genes. The three translocation partners characterized thus Ј Ј Ј ␤ GAGA-3 ; G8N101, 5 -CCCGAATTCTGTTTCACCGGAAAGATCATC-3 ; far are the -catenin gene on 3p21 found in the most common G8N159, 5Ј-CCCGAATTCCTTTTGAAAGCACGGGAGTG-3Ј; and as translocation, the t(3;8)(p21;q12), the leukemia inhibitory factor re- COOH-terminal oligonucleotide G8C244, 5Ј-GGGCTCGAGCTATTTGAC- ceptor gene on 5p13 found in a recurrent (5;8)(p13;q12) translocation CTTCAGAAGCTCTTGA-3Ј. Pfu-amplified fragments were gel purified, di- (2), and the elongation factor SII gene (3). Breakpoints invariably gested with EcoRI and XhoI, and ligated in the EcoRI-XhoI-digested pGEX- occur in the 5Ј noncoding part of the PLAG1 gene, leading to an 5X-2 vector. The construct GST-PLAG1 (N2-C203) was obtained by digesting exchange of the regulatory control elements while preserving PLAG1 GST-PLAG1 (N2-C244) by DraIII and XhoI, blunt ending, and recirculariza- coding sequence. The replacement of the PLAG1 promoter, inactive in tion. All of the fusion proteins were expressed in Escherichia coli BL-21 cells adult salivary glands, by a strong promoter derived from the translo- and purified on Glutathione Sepharose 4B (Pharmacia) according to the cation partner, leads to ectopic expression of PLAG1 in the tumoral manufacturer’s protocol. The protein sizes were estimated by SDS-PAGE, followed by Coomassie blue staining; concentrations were determined by cells. This abnormal PLAG1 expression presumably results in a comparison to a well-defined concentration marker. deregulation of PLAG1 target genes, causing salivary gland tumori- CASTing. To prepare a pool of random double-strand oligomers for the genesis. first round of CASTing, 400 pmol of CAST25 (5Ј-CTGTCGGAATTCGCT- GACGT-(N)25-CGTCTTATCGGATCCTACGT-3Ј) were incubated in 100 ␮l Received 7/20/99; accepted 10/28/99. of polymerase reaction buffer containing 1200 pmol of CAST-LOW (5Ј- The costs of publication of this article were defrayed in part by the payment of page ACGTAGGATCCGATAAGACG-3Ј), 200 ␮M of each deoxynucleoside charges. This article must therefore be hereby marked advertisement in accordance with triphosphate, 2.5 ␮l[␣-32P]dCTP (DuPont NEN), and 10 units of Amplitaq 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the “Geconcerteerde Onderzoekacties 1997–2001” and (Perkin-Elmer-Cetus) and treated as follows: 5 min at 94°C, 20 min at 65°C, the “Fonds voor Wetenschappelijk Onderzoek Vlaanderen” (FWO). M. V. is a post-doc of and 20 min at 72°C. Fifty ␮l were incubated with 500 ng of GST-PLAG1 the Flanders Interuniversity Institute for Biotechnology. K. K. is a post-doc of the FWO. (N2-C244) bound to Glutathione Sepharose beads, 50 ␮g of poly polydeoscyi- 2 To whom requests for reprints should be addressed, at Laboratory for Molecular nosinic-deoscycytidylic acid (Sigma), and 50 ␮g of BSA in 500 ␮l of binding Oncology, Center for Human Genetics, University of Leuven and Flanders Interuniversity ␮ Institute for Biotechnology, Herestraat 49, B-3000 Leuven, Belgium. Phone: 32-16- buffer [10 mM Tris (pH 7.5), 200 mM NaCl, 50 M ZnCl2, 10% glycerol, 1 mM 346041; Fax: 32-16-346073; E-mail: [email protected]. MgCl2,and1mM DTT]. After a 30-min incubation on a rotator at room 3 The abbreviations used are: PLAG1, pleomorphic adenoma gene 1; CASTing, cyclic temperature the beads were washed four times with cold binding buffer, and amplification and selection of target sequences; GST, glutathione S-transferase; EMSA, the radioactivity still present on the beads was counted to monitor the level of electrophoretic mobility shift assay; ATCC, American Type Culture Collection; PDGF, platelet-derived growth factor; EPD, Eukaryotic Promoter Databank; IGF, insulin-like enrichment in each step. The oligonucleotides were eluted from the beads by growth factor. resuspending in 100 ␮l of water, followed by phenol extraction and ethanol 106

precipitation. An aliquot was used for the subsequent amplification reaction in have been obtained by inserting into pTK81luc (12) six copies of 100 ␮l of polymerase reaction buffer containing 200 pmol of each amplimer the corresponding ds oligonucleotides: WT, 5Ј-CTAGAAGGGGCTCTA- CAST-UP (5Ј-CTGTCGGAATTCGCTGACG-3Ј) and CAST-LOW, 200 ␮M GAAAGGGTAA-3Ј; mCO, 5Ј-CTAGAATGCACTCTAGAAAGGGTAA-3Ј; deoxynucleotide triphosphates, and 2.5 units of Amplitaq (Perkin-Elmer Ce- mCLU, 5Ј-CTAGAAGGGGCTCTAGAAA-TACTAA-3Ј; and mCOmCLU, tus) with 1 ␮lof[␣-32P]-dCTP (DuPont NEN) with 25 cycles of 1 min at 94°C, 5Ј-CTAGAATGCACTCTAGAAATACTAA-3Ј. 1 min at 65°C, and 1 min at 72°C. The amplified products were subsequently Transfections and Luciferase Assay. The human fetal kidney epithelial used for a second round of selection performed as described above. After four cell line 293 (ATCC; CRL 1573) was cultured according to the suppliers’ rounds of selection, the subsequent three steps of selection were performed by protocols. Cells (6-well plates) were transiently cotransfected in triplicate with EMSA with 100 ng of eluted GST-PLAG1 (N2-C244). After X-ray exposure 200 ng of the expression vector DNA, 200 ng of reporter plasmid, and 200 ng of the dried gel, the shifted bands were cut out of the gel, and the double- of internal control Rous sarcoma virus ␤-galactosidase DNA using 3 ␮lof stranded DNA was eluted3hat50°C in 200 ␮l of polymerase reaction buffer. FuGENE 6 Transfection Reagent (Boehringer Mannheim) according to the An aliquot of the eluate was used for amplification. After a total of seven manufacturer’s protocol. Cells were harvested 40 h after the transfection, and amplification cycles, the oligonucleotides were cloned into the pGEM-T Easy luciferase reporter enzyme activity was measured using a Monolight 2010 vector according to the manufacturer’s protocol (Promega), and 23 indepen- luminometer (Analytical Luminiscence Laboratory) and performing end point dent clones were sequenced. assays. EMSA. The different probes were synthesized as complementary oligonu- Preparation of RNA and Northern Blot Analysis. Total RNA was ex- cleotides with 4-bp sticky ends, annealed, subsequently end-labeled with tracted from primary tumors and analyzed by Northern blot analysis as de- [␣-32P]dCTP and Klenow enzyme, and finally purified with the QIAquick scribed previously (2). The human IGF-II exon 9 probe, common to the four Nucleotide removal kit (Qiagen). DNA-protein binding reactions were carried different transcripts P1, P2, P3, and P4, was generated by PCR and contained out for 10 min at room temperature in 30 ␮l of EMSA binding buffer [10 mM nucleotides 7970 to 8774 of the published gene sequence (Ref. 13; GenBank/ ␮ Tris (pH 7.5), 100 mM NaCl, 50 M ZnCl2, 10% glycerol, 1 mM MgCl2,1mM EMBL, accession number X03562). A human IGF-II exon 5 probe specific for DTT, 1 ␮g polydeoxyadenylic acid-polythymidylic acid (Sigma), 1 ␮gof the P3 transcript was a kind gift of Dr. P. E. Holthuizen. The human GOS24 salmon sperm DNA, and 300 ng of BSA] with 10,000 dpm of probes (about 0.1 probe was generated by PCR and contained nucleotides 1828 to 2345 of the ng of oligonucleotides) and an equimolar amount of proteins. DNA-protein sequence submitted to GenBank (accession number M92844). The human complexes were analyzed on nondenaturing polyacrylamide gels [6% acryl- c-Ha-Ras probe was generated by isolation of 1.2-kb HindIII coding fragments bisacrylamide (19:1), 0.5ϫ TBE (1ϫ TBE ϭ 89 mM Tris base, 89 mM boric from RSV-ras(leu61) vector (14). The human PDGF-B probe was generated by acid, 2 mM EDTA pH 8.0) and 5% glycerol]. Electrophoresis was performed purification of the 1.7-kb BamHI fragment isolated from the pAO73 plasmid at 4°C at 14 V/cm. (15). Plasmid Constructions. The PLAG1 expression vector pCDNA3-PLAG1 Immunofluorescence Analyses. COS-1 kidney fibroblast cells (ATCC, was constructed by inserting into EcoRI-XhoI-digested pCDNA3 (Invitrogen) CRL1650) were grown on glass chamber slides (Nunc) and transfected with 1 the complete open reading frame of PLAG1 preceded by its own Kozak ␮g of pCAGGS-PLAG1 and 1 ␮g of PM3 plasmid, which encodes the Gal4 consensus translation start site. This fragment was generated by PCR using Pfu DNA binding domain (amino acids 1–147; Ref. 16). Twenty-four h after polymerase (Stratagene) with the 5Ј primer G8N-3 (5Ј-CCCGAATTCTAG- transfection, the cells were washed twice with PBS and fixed with 4% GCTGCGATGGCCACTGT-3Ј) and the 3Ј primer G8C500 (5Ј-GGGCTC- paraformaldehyde in PBS for 30 min at room temperature, followed by two GAGCTACTGAAAAGCTTGATGGAAAC-3Ј). The same blunt-ended frag- washes with PBS. Slides were then incubated in PBS-BT [PBS/0.5% blocking ment was also cloned in the blunt-ended EcoRI site of the pCAGGS vector (5) reagent (Boehringer Mannheim)/0.2% Triton X-100] for 30 min at room to get a second expression construct for PLAG1 (pCAGGS-PLAG1) with temperature. To simultaneously detect the PLAG1 and Gal4 proteins, cotrans- higher levels of expression in transfected cells. The three mutant PLAG1 fected cells were incubated at room temperature for1hinPBS-BT containing proteins (PLAG1-F2mut, PLAG1-F3mut, and PLAG1-F7mut) were produced the rabbit polyclonal anti-PLAG1 together with the mouse monoclonal anti-

by replacing in pCDNA3-PLAG1 the first histidine in the C2H2 motif of the GAL4 (SC-510; Santa-Cruz). The polyclonal anti-PLAG1 was obtained by corresponding zinc finger (His81, His110, and His231, respectively) with an immunizing rabbits with the peptide FSSTSYAISIPEKEQPL (amino acids alanine. For this, we applied the QuickChange Site-directed Mutagenesis kit 336–352 in PLAG1) and the specificity of the antibody was verified by (Stratagene) according to the instructions of the supplier. All constructs were Western blot analysis (data not shown). After three washes with PBS-T sequenced to confirm the fidelity of the PCR and the site-specific mutagenesis. (PBS/0.2% Triton X-100), the slides were incubated in PBS-BT with FITC- The full-length PLAG1 protein as well as the three mutants were expressed by labeled swine anti-rabbit (DAKO, F0205) and Texas red-labeled sheep anti- in vitro transcription and translation using the TnT kit (Promega). Quality of mouse (Amersham, N2031). This allowed simultaneous visualization of the translation was monitored by SDS gel analysis of [35S]Met-labeled proteins. PLAG1 (FITC, green) and GAL4 (Texas red) proteins. After three washes in For the EMSA experiments, 3 ␮l of translation reaction products were used per PBS-T, slides were mounted in Citifluor containing 0.5 ␮g/␮lof4Ј,6-dia- lane. midino-2-phenylindole (DAPI) and analyzed with a Zeiss Axiophot micro- The PDGF-B (Ϫ389/ϩ22) luciferase reporter construct has been made by scope equipped with UV optics. Images were recorded with a CE200A CCD digesting the vector pA0166 (6) by SstI and HindIII and inserting this fragment camera (Photometrics), using Smart capture (Digital Scientific) and Iplab in SstI/HindIII-digested pGL2basic (Promega). The GOS24 (Ϫ384/ϩ27) Spectrum (Signal Analytics) software. luciferase reporter construct has been obtained by cloning in PGL2basic (Promega) the fragment obtained by PCR amplification on genomic DNA RESULTS using the primers 5Ј-GGCGAGCTCTCCCCGCCCCCATCCGTCT-3Ј and 5Ј- CCGCTCGAGAGTGGGAGCGCTGAAGTC-3Ј derived from the sequence PLAG1 Is Localized in the Nucleus. The presence of seven retrieved from Genbank (accession number M92844). The IGF-II-P3 (Ϫ1229/ canonical C2H2 zinc fingers and the transactivation capacities of the ϩ140) luciferase reporter construct [called Hup3 (7)] is a generous gift of Dr. COOH-terminal domain suggest that PLAG1 may act as a transcrip- P. E. Holthuizen (1. Universiteit, Utrecht, the Netherlands) and has been tional regulator. A prerequisite for such a role is that the protein be obtained by cloning the IGF-II promoter 3 into the pSLA3 luciferase vector localized, at least in some conditions, in the nucleus. To determine the Ϫ ϩ (8). The c-Ha-Ras ( 325 to 58 in the first intron) luciferase reporter subcellular localization of PLAG1, we have made an eukaryotic construct was obtained by inserting the 384-bp fragment XmaIII blunt-ended/ expression construct (pCAGGS-PLAG1) directing the synthesis of a SacI deriving from the pbc-N1 vector (Ref. 9; ATCC) into the pGL2basic with full-length PLAG1 protein in transfected cells. Immunofluorescence a splice acceptor site added. The prohormone convertase 2 (Ϫ789/ϩ137) luciferase reporter construct (10) and the somatostatin (Ϫ192/ϩ50) luciferase staining of transfected COS-1 cells indicates that PLAG1 protein is reporter construct [called pSRIF-Luc (11)] are generous gifts of Dr. E. Jansen confined to the nucleus as demonstrated by the green staining ob- (University of Leuven and Flanders Interuniversity Institute for Biotechnology, served with the PLAG1-specific antibody (Fig. 1A), which perfectly Belgium) and Dr. B. Peers (University of Liege, Belgium), respectively. coincides with the DAPI nuclear DNA staining (Fig. 1B). The PLAG1

(WT)6-TK-luc, (mCO)6-TK-luc, (mCLU)6-TK-luc, and mCLUmCO)6-TK-luc protein also colocalizes with the well-characterized nuclear protein 107

the binding as the destruction of the core. As expected, no competition could be observed, with mCOmClu2 presenting a mutation in both motifs. Fingers 6 and 7 of PLAG1 Bind to the Core, Whereas Finger 3 Interacts with the G-Cluster. To determine which of the zinc fingers contributes to the binding to the consensus sequence, we exam- ined the binding of bacterially expressed GST fusion proteins containing different combinations of zinc fingers (Fig. 3B, Lanes 6–25). A similar binding pattern is observed with the protein containing fingers 3 to 7 compared with the protein F1–F7, which contains the complete zinc finger region (compare Lanes 6–10 with Lanes 1–5), suggesting that fingers 1 and 2 are not directly involved in the binding. In contrast, the protein containing fingers 4–7 binds 3-fold less to the WT2 probe (Fig. 3B, Lane 11). More importantly, protein F4-F7 binds nearly as well to the probe, presenting a mutation in the G-cluster Fig. 1. Nuclear localization of the PLAG1 protein. Immunofluorescence of Cos-1 cells (mCLU2; Fig. 3B, Lane 12) as to the WT2 probe, indicating that the cotransfected with the PLAG1 expression vector construct, pCAGGS-PLAG1 and with G-cluster is not important for the binding of F4–F7. This is a good the pM3 expression vector expressing the DNA binding domain of Gal4 (16). The cells were costained with the PLAG1 antibody, 4Ј,6-diamidino-2-phenylindole and the GAL4 indication for the requirement of finger 3 for the interaction with this antibody (see “Materials and Methods”). This allows the visualization in the same cells of cluster. The F6–F7 protein binds nearly with the same affinity as PLAG1 (A), nuclear DNA (B), GAL4 (C), or all three (D). F4–F7 to all of the different probes (Fig. 3B, compare Lanes 16–20 to Lanes 11–15), suggesting that fingers 4 and 5 are not directly involved in the binding. This F6–F7 protein still binds clearly to all of Gal4 (Fig. 1, C and D). Nuclear localization of the endogenous the probes containing an intact core motif (Fig. 3B, Lanes 16, 17, and PLAG1 protein was also established by immunofluorescence analysis 20), indicating that fingers 6 and 7 are sufficient for the interaction of the fetal kidney 293 cell line and independently confirmed by with the core. Finally, the protein F1–F5 does not show any clear Western blot analysis of 293 nuclear extracts (data not shown). binding (Fig. 3B, see Lanes 21–25), indicating the absolute require- The PLAG1 Binding Site Is Composed of Two Essential Parts, ment of the two last fingers F6 and F7. All of these results suggest that a GRGGC Core and a G-Cluster. The presence of a zinc finger finger 3 of PLAG1 interacts with the G-cluster, whereas the core is domain in PLAG1 as well as its nuclear localization suggests that recognized by fingers 6 and 7. PLAG1 is a DNA binding protein. To test this hypothesis and to To confirm this model, we performed additional EMSAs using identify the putative PLAG1 binding consensus sequence, CASTing full-length PLAG1 protein translated in vitro in reticulocyte lysates was performed as described in “Materials and Methods.” The protein instead of the GST fusion proteins expressed in bacteria. Three used for this study was a chimeric protein containing the complete mutant PLAG1 proteins (F2mut, F3mut, and F7mut) were also zinc finger domain (amino acids 2–244) fused in-frame to the GST. The fusion protein GST-PLAG1 (N2-C244) was immobilized on Glutathione-Sepharose beads and incubated with a pool of oligonucleotides containing a central region of 25 random nucleotides. The pool of oligonucleotides selected by seven rounds of PLAG1 binding was cloned, and 23 independent clones were sequenced. The alignment of all the sequences with the program Macaw (National Center for Biotechnology Information) clearly revealed a consensus sequence composed of a core GRGGC followed 6–8 nucleotides further by a cluster of at least three guanidines (Fig. 2). To assess the importance of the two motifs in the consensus, we performed EMSAs on a double-strand probe containing these two motifs and on four probes presenting mutations in the consensus (Fig. 3A). We found that the fusion protein GST-PLAG1 (N2-C244) binds strongly to the consensus (Fig. 3B, Lane 1), whereas mutations in the G-cluster (Fig. 3B, Lane 2) reduce drastically the binding (ϳ8-fold) and mutations in the Core nearly completely abolish it (ϳ37-fold reduction; Fig. 3B, Lane 3) as well as mutations in both (Fig. 3B, Lane 4). The distance between the G-cluster and the core is also important because PLAG1 binds weakly to the probe containing the G-cluster separated by 2 bp instead of 7 bp from the core (WT2ml; Fig. 3B, Lane 5). These results indicate that both motifs in the consensus are important for the binding of PLAG1, with an importance more pro- nounced for the core compared with the G-cluster. The relative importance of the two motifs was also tested in a series of competition EMSAs using WT2 as probe. As shown in Fig. 3C, WT2 competes much more efficiently than mCLU2 or mCO2, confirming the importance of the two motifs in the consensus. mCLU2 Fig. 2. Determination of the PLAG1 binding site. Alignment of the 23 oligonucleotides selected by seven cycles of CASTing using GST-PLAG1 (N2-C244). The frequency of competes poorly but nevertheless better than mCO2, confirming by each of the bases at each position is shown at the bottom of the figure, and N represents this way that the destruction of the G-cluster is not so deleterious for the number of oligonucleotides that carried a base at that position. 108

Fig. 3. The G-cluster is recognized by finger 3 and the core by fingers 6 and 7 of PLAG1. A, nucleotide sequences of the different oligonucleotides used in EMSA analysis. Mutations are underlined. B, EMSAs performed with equimolar amount (0.6 pmol) of bacterially expressed GST-PLAG1 proteins. GST-PLAG1 (N2-C244) (Lanes 1–5), (N84-C244) (Lanes 6–10), (N101-C244) (Lanes 11–15), (N159-C244) (Lanes 16–20) and (N2-C203) (Lanes 21–25) were incubated with the probes WT2 (Lanes 1, 6, 11, 16, and 21), mCLU2 (Lanes 2, 7, 12, 17, and 22), mCO2 (Lanes 3, 8, 13, 18, and 23), mCLUmCO2 (Lanes 4, 9, 14, 19, and 24), and WT2ml (Lanes 5, 10, 15, 20, and 25) as described in “Materials and Methods”. The percentage of binding of all these mutants on the different probes were compared with the binding of F1–F7 to the probe WT2 and is the mean of at least six experiments. C, competition experiments performed on the probe WT2 in presence of increasing amounts of unlabeled double-strand oligonucleotides (10, 30, 100, 300, and 1000 ng) using recombinant full-length PLAG1 expressed in vitro in reticulocytes lysates; D, EMSAs performed with recombinant PLAG1 proteins produced in vitro in reticulocytes lysates. Wild-type PLAG1 (Lanes 1–5), F2mut (Lanes 6–10), F3mut (Lanes 11–15), and F7mut (Lanes 16–20) were incubated with the probes WT2 (Lanes 1, 6, 11, and 16), mCLU2 (Lanes 2, 7, 12, and 17), mCO2 (Lanes 3, 8, 13, and 18), mCLUmCO2 (Lanes 4, 9, 14, and 19), and WT2ml (Lanes 5, 10, 15, and 20) as described in “Materials and Methods”. Equal efficiency of protein expression was obtained for the different constructs as demonstrated by SDS-PAGE of proteins labeled with [35S]methionine (data not shown). The percentages of binding of all of these mutants on the different probes were compared to the binding of the wild-type PLAG1 to the probe WT2 and is the mean of at least three experiments. 109

Table 1 PLAG1 can stimulate transcription through its consensus binding site expression vector (8-fold). In contrast, a weaker stimulation was Two hundred ng of the indicated reporter luciferase (luc) construct were cotransfected obtained on the GOS24 promoter, and the promoters c-Ha-Ras and into the fetal kidney cell line 293 together with 200 ng of the expression vector pCAGGS-PLAG1 or the empty vector pCAGGS. PLAG1 induction levels are expressed PDGF-B did not respond significantly to PLAG1. As expected, no as the ratio of luciferase activity obtained in the cell transfected with pCAGGS-PLAG1 stimulation was observed of promoters that do not contain any expression vector versus the activity obtained in cells transfected with the empty vector PLAG1 binding sites. We thus focused our attention on IGF-II and pCAGGS. The data are means Ϯ SE of at least two independent transfection experiments, each performed in triplicate. investigated whether we could really observe a binding of PLAG1 to PLAG1 induction the putative PLAG1 binding sites in the promoter 3 of IGF-II. For Ϫ Ϫ Ϯ that, EMSAs were performed on a probe located from 192 to 172 (WT)6-TK-luc 18.9 2.6 Ϯ (mCLU)6-TK-luc 1.0 0.2 in the P3 promoter, which contains one putative PLAG1 binding sites. Ϯ (mCO)6-TK-luc 0.9 0.2 Ϯ This element called P3-4 has been shown to be essential for P3 (mCLUmCO)6-TK-luc 0.6 0.1 TK-luc 1.7 Ϯ 0.6 activity (19). As shown in Fig. 4A, a clear binding of PLAG1 is seen on the probe P3-4. The affinity of PLAG1 for this site is comparable with the one for WT2 because approximately the same level of competition is obtained with P3-4 and WT2 in EMSA competition produced by replacing with an alanine the first histidine of the experiments (Fig. 4B). corresponding C2H2 motif. This mutation hinders the coordination of the zinc and has been shown to prevent the formation of a functional zinc finger (17). The full-length PLAG1 protein ex- Table 2 Identification of human promoters with potential PLAG1 binding sites and pressed in reticulocyte lysate binds with the same specificity as the determination of their capacity to be induced by PLAG1 bacterial F1–F7 protein (compare Fig. 3D with 3B, Lanes 1–5). The EPD (18) has been screened for the presence of the PLAG1 consensus The destruction of the zinc finger 2 (PLAG1/F2mut) does not GRGGC(N)7 RGGK with the pattern-matching algorithm implemented in the program affect the binding specificity of this protein but decreases its findpatterns of the GCG software package (32). The list includes all of the human promoters present in the EPD containing at least two PLAG1 DNA binding consensus in affinity ϳ3-fold (Lanes 6–10). In contrast, the finger 3 destruction their promoter. The ability of each promoter to be induced by PLAG1 has been estimated decreases drastically the affinity of this protein as F3mut binds by cotransfection of the fetal kidney 293 cell line with pCAGGS-PLAG1 or pCAGGS expression vectors, together with reporter constructs in which each promoter has been 17-fold less to the WT2 probe than the natural PLAG1 protein cloned in front of a luciferase gene (see “Materials and Methods”). PLAG1 induction (compare Lane 11 with Lane 1). The specificity was also com- levels are expressed as the ratio of luciferase activity obtained in the cell transfected with pletely modified since F3mut binds equally well to WT2 (Lane 11), pCAGGS-PLAG1 expression vector versus the activity obtained in cells transfected with the empty vector pCAGGS. The data are means Ϯ SE of at least two independent mCLU2 (Lane 12), and WT2ml (Lane 15). Thus, the presence or transfection experiments, each performed in triplicate. absence of a G-cluster does not affect the binding of F3mut, PLAG1 induction indicating that finger 3 is actually the finger required for the 5 consensus : IGF-II promoter 3 7.9 Ϯ 0.8 interaction with this motif. This conclusion is in agreement with 4 consensus : GOS24/TTP/Zfp36 3.0 Ϯ 0.2 the conclusions drawn from the EMSA experiments performed 3 consensus : c-Ha-Ras promoter 3 0.9 Ϯ 0.2 with the bacterial protein. Finally, destruction of finger 7 com- 2 consensus : PDGF-B/c-sis 1.4 Ϯ 0.6 TnI slow ND pletely prevents any binding (Lanes 16–20), confirming the abso- Gastrin ND lute requirement of this finger. VLDL ND SOD-1 ND PLAG1 Can Stimulate Transcription Through Its Consensus Ia-ass. gЈ ND Binding Site. To investigate whether PLAG1 binding sites could H19 ND mediate a transcriptional activation by PLAG1, six copies of the 0 consensus : pGL2 basic 0.7 Ϯ 0.2 pSLA3 1.2 Ϯ 0.1 minimal consensus (WT) were cloned upstream the herpes simplex RSV 1.1 Ϯ 0.2 virus thymidine kinase promoter, followed by the luciferase reporter prohormone convertase 2 0.5 Ϯ 0.03 gene. This reporter construct was then transfected into the fetal kidney somatostatin 1.0 Ϯ 0.3 293 cell line in the presence or absence of the expression vector pCAGGS-PLAG1. Table 1 shows that PLAG1 stimulates expression of this reporter construct ϳ19-fold. This stimulation is completely abolished by mutations in the G-cluster or in the core since no stimulation was detectable with six copies of mCLU or mCO. This demonstrates that the activation is completely dependent of the presence of the two motifs. PLAG1 Binds IGF-II Promoter 3 and Up-Regulates Its Pro- moter Activity. A computer search in the EPD (18) with the PLAG1 binding consensus GRGGC(N)7 RGGK revealed potential PLAG1 binding sites in the promoter region of many genes (about 176 of the 1280 screened). Table 2 presents the list of all of the human promoters present in the EPD containing at least two potential binding sites for PLAG1. This list includes several proto-oncogenes and growth factors. Four promoters, i.e., IGF-II promoter 3, GOS24, c-Ha-ras, and PDGF-B, have been selected for further investigation. By transient transfection experiments, we next investigated whether these promoters are responsive to PLAG1. To that end, we generated four reporter Fig. 4. PLAG1 binds effectively to the P3 promoter of IGF-II. A, EMSAs performed plasmids where each promoter has been cloned independently in front with wild-type PLAG1 protein produced in vitro in reticulocyte lysates and incubated with the probes WT2 and P3-4. B, competition experiments performed on the probe WT2 in the of the luciferase gene. Table 2 shows that the activity of the promoter presence of increasing amounts of unlabeled double-strand oligonucleotides (10, 30, 100, 3 of IGF-II was highly stimulated by cotransfection of a PLAG1 300, and 1000 ng) using wild-type PLAG1 expressed in reticulocyte lysates. 110

Fig. 5. IGF-II P3 transcript is up-regulated in tumors with PLAG1 overexpression. A, Northern blot analysis of normal salivary gland (n.s.g.) tissues and pleomorphic adenomas hybridized se- quentially with a 3.7-kb PLAG1 cDNA probe, an IGF-II exon 9 probe, and a 2-kb ␤-actin probe. RNAs tested included samples from three different normal salivary gland tissue specimens (Lanes 1, 5, and 8) and from adenomas c895 (Lane 2), c904 (Lane 3), cg650 (Lane 4), cg644 (Lane 6), and cg580 (Lane 7). B, recapitulation of the Northern blot analysis of normal salivary gland tissues and pleomorphic adenomas hybridized with probes specific for the genes encoding PLAG1, IGF-II, PDGF-B, GOS24, or c-Ha-Ras. The karyotype of the tumors has been described elsewhere (1, 2), and in tumors cg650 and cg601, the breakpoint occurs outside the PLAG1 region (20, 21).

IGF-II Transcript Is Up-Regulated in Salivary Gland Tumors with PLAG1 Overexpression. The fact that PLAG1 is able to bind and activate the promoter 3 of IGF-II suggested that IGF-II could be a target for PLAG1 in salivary gland tumors. To test this hypothesis, we analyzed the expression of these two genes in specimens of primary salivary gland tumors and of normal glands. As described previously (1), PLAG1 expression could not be detected by Northern blot analysis in normal salivary gland tissue (Fig. 5, Lanes 1, 5, and 8). In contrast, PLAG1 expression is readily detectable in tumors carrying a chromosomal translocation affecting the PLAG1 gene (Fig. 5, Lanes 2, 3, 6, and 7). This overexpression is the result of promoter substitution between PLAG1 and ubiquitously expressed genes such as those coding for ␤-catenin (cg644 and cg682) or the leukemia inhibitory factor receptor (c895 and c904; Refs. 1 and 2). The two tumors carrying chromosome aberrations outside the PLAG1 locus (cg650 and cg601; Refs. 20 and 21) do not show any PLAG1 expression (Fig. 5, Lane 4 and Fig. 5B). We next investigated IGF-II expression in these tumors using a probe specific for exon 9 of IGF-II, which is a common part of all the IGF-II transcripts. No IGF-II expression could be detected in normal salivary gland tissues (Fig. 5, Lanes 1, 5, and 8) and in tumors without PLAG1 overexpression (lane 4 and cg601 in Fig. 5B). In contrast, a drastic up-regulation of IGF-II was observed in tumors with PLAG1 overexpression (Fig. 5, Lanes 2, Fig. 6. Prediction and schematic representation of PLAG1 binding consensus site. A, 3, 6, and 7). The detected 6-kb IGF-II transcript corresponds to the amino acids at position Ϫ1, 2, 3, and 6 (numbering with respect to the start of the ␣-helix) transcript deriving from the P3 promoter, because hybridization with within the PLAG1 zinc fingers are shown in the first column. Bases predicted to be a probe specific for the P3 transcript (exon 5 probe) detects exactly the preferred for binding by these amino acids are shown in the second column (22, 23). The consensus found by CASTing is shown in the third column, and thick lines indicate that same band (data not shown). Thus, we have a perfect correlation the predicted base matches with the selected one. B, comparison between the PLAG1 between PLAG1 and IGF-II gene expression, suggesting that IGF-II is binding site and other reported consensus binding sites like the Zac1 consensus (27), one of the characterized WT-1 binding sites (33), the consensus sequence for Sp1 binding one of the PLAG1 targets. In contrast, no significant expression of described as the decanucleotide 5Ј-(G/T)GGGCGG(G/A)(G/A)(C/T)-3Ј (34) and the c-Ha-Ras and PDGF-B could be detected in tumors and in normal Egr-1/Zif268 consensus binding sequence (35). 111

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2000 American Association for Cancer Research. TRANSCRIPTIONAL REGULATION OF IGF-II BY PLAG1 salivary gland tissue, whereas no alteration in GOS24 expression was proteins to determine whether the opposite functions are the result of observed (Fig. 5B). different target genes or different actions on the same set of genes. The PLAG1 DNA binding consensus is highly GC-rich, which is a hallmark of most promoters of genes controlling cell growth. It is thus DISCUSSION tempting to speculate that PLAG1 will exert its oncogenic effect via the activation of growth factors. The IGF-II is an excellent candidate In this report, we show that PLAG1, the major translocation target because IGF-II is a peptide growth factor that plays an important role gene in pleomorphic adenoma of the salivary glands, codes for a in embryonic development and also in carcinogenesis (28). The hu- nuclear protein that binds DNA in a sequence-specific manner. The man IGF-II is a complex transcription unit that is regulated by minimal PLAG1 binding site is composed of two essential parts, a activation of multiple promoters designated P1 to P4. Promoter 1 GRGGC core separated by seven random nucleotides from a RGGK activity has been demonstrated only in adult liver, whereas promoters cluster. This bipartite binding site is quite unusual and can be ex- 2, 3, and 4 are coexpressed in a variety of fetal tissues, notably fetal plained by the particular way PLAG1 binds DNA. Indeed, we show liver, and at a lower level, in many adult tissues with the exception of that two noncontiguous regions in PLAG1 are essential for DNA adult liver. Promoters 3 and 4 are also highly active in numerous recognition, finger 3 interacting with the G-cluster and fingers 6 and tumor tissues, suggesting that transcriptional up-regulation of P3 and 7 recognizing the core. This model of interaction we found by dele- P4 activities may be importantly involved in tumorigenesis. Our study tion/mutation analysis (see Fig. 3) is corroborated by the stereochem- is the first demonstration of IGF-II up-regulation in tumors of the ical rules governing the DNA contacts of individual zinc fingers (22). salivary glands. This up-regulation is the result of a drastic up- These rules are based on different types of studies: (a) structural regulation of promoter 3 activity, as demonstrated by the hybridiza- studies have shown that each finger module folds to form a compact tion performed with a P3-specific probe. Several lines of evidence ␤␤␣ ␣ structure with the -helix fitting into the major groove (reviewed strongly suggest that IGF-II up-regulation in salivary gland tumors Ϫ in Ref. 23). Residues 1, 2, 3, and 6 (numbering with respect to the results from transcriptional activation by PLAG1: (a) five potential ␣ start of the -helix) typically make key base contacts that are respon- binding sites were found in promoter 3 of IGF-II;(b) PLAG1 binding sible for defining sequence specificity; (b) by phage-display selections was effectively demonstrated on the site P3-4; (c) IGF-II promoter 3 and site-directed mutagenesis, correlations were established between activity is up-regulated by PLAG1; (d) IGF-II is highly expressed in the amino acids present in key positions Ϫ1, 2, 3, and 6 and the tumor cells that overexpress PLAG1 but could not be detected in nucleotide sequences of their optimal binding sites. This led to a tumors without PLAG1 up-regulation or in normal salivary gland recognition code governing zinc-finger/DNA interactions. As de- tissues. Our study suggests that the oncogenic activity of PLAG1 picted in Fig. 6A, the consensus we found by CASTing shows good results from its positive regulation of IGF-II expression, known to agreement with the binding site predicted by such a recognition code. potently stimulate cell proliferation in human tumors through auto- It is interesting to note that the fingers that are not involved in DNA crine or paracrine mechanisms (28–30). recognition (e.g., fingers 1, 4, and 5) do not present in key position The PLAG1 consensus sequence is also reminiscent of G-rich amino acids known to interact with DNA. The distance between the sequences recognized by an important group of zinc finger proteins G-cluster and the core is also in good agreement with the presence of that include Sp1, Zif268/Egr1, and WT-1. As shown in Fig. 6B, the two noninteracting zinc fingers that are predicted to cover six nucle- PLAG1 consensus binding motif overlaps with the consensus binding otides. However, alignment of all the sequences selected by seven sequence for the transcription factor Sp1 and the tumor suppressor rounds of CASTing indicates that the distance between the G-cluster WT-1, suggesting that these proteins could at least partly regulate the and the core seems to vary from six to eight nucleotides (see Fig. 2). same set of genes. In fact, the growth factor gene IGF-II has been One explanation to this variability could be the presence of a longer shown to be also one of the targets of the tumor suppressor WT-1. linker region between fingers 5 and 6 (14 amino acids instead of the High levels of IGF-II in Wilms’ tumor were attributed to the loss of usual 7 amino acids). This longer linker may generate sufficient function of WT-1, which normally represses IGF-II transcription (31). flexibility to allow an interaction with a cluster at different positions. The WT-1 protein binds within the IGF-II P3 promoter to multiple Recently, we and others identified two PLAG1-related proteins, sites with some overlapping with potential PLAG1 binding sites. This PLAGL1 [also called Lot1 (24, 25) or Zac1 (26)] and PLAGL2 (4). suggests that one control for IGF-II expression during development PLAGL1 and PLAGL2 are highly homologous to PLAG1 in their could be provided by a balance between activators (like PLAG1) and

NH2-terminal zinc finger domain (73 and 79% identity, respectively), repressors (like WT-1) acting on overlapping sequences. Deregulation whereas the COOH-terminal region is much more divergent. Strik- of one of these factors is probably an important step in tumor forma- ingly, the best homology is found in fingers 6 and 7, suggesting that tion. PLAGL1 and PLAGL2 should also recognize a core motif analogous to the PLAG1 core. In contrast, fingers 2–5 are much less conserved but still present conservation for the amino acids present in key ACKNOWLEDGMENTS positions (Ϫ1, ϩ2, ϩ3 and ϩ6). This suggests that the three PLAG proteins would interact with similar consensus sequences. The con- We gratefully acknowledge G. Stenman for the specimens of primary salivary gland tumors and P. E. Holthuizen for her kind gift of the IGF-II exon sensus binding site for Zac1/PLAGL1 has been defined recently as 5 probe and for (IGF-II-P3)luc plasmid. We thank J. Remacle, S. Tejpar, and GGGGGGCCCC through a CASTing assay (27). Actually, the B. Peers for critical review of the manuscript. PLAG1 core is present in this large consensus sequence (Fig. 6B). However, no G-cluster was identified, suggesting that Zac1/PLAGL1 does not interact in the same way as PLAG1. Both PLAG1 and REFERENCES Zac1/PLAGL1 are possibly involved in tumorigenesis, PLAG1 as a 1. Kas, K., Voz, M. L., Roijer, E., Astrom, A. K., Meyen, E., Stenman, G., and Van de putative oncogene that contributes to pleomorphic adenomas whereas Ven, W. J. Promoter swapping between the genes for a novel zinc finger protein and Zac1/PLAGL1 as a tumor suppressor candidate that regulates apo- ␤-catenin in pleiomorphic adenomas with t(3;8)(p21;q12) translocations. Nat. Genet., 15: 170–174, 1997. ptosis and cell cycle arrest (26). It is thus interesting to further 2. Voz, M. L., Astrom, A. K., Kas, K., Mark, J., Stenman, G., and Van de Ven, W. J. investigate the differences of specificities between these two related The recurrent translocation t(5;8)(p13;q12) in pleomorphic adenomas results in up- 112

regulation of PLAG1 gene expression under control of the LIFR promoter. Oncogene, 20. Roijer, E., Kas, K., Behrendt, M., Van de Ven, W., and Stenman, G. Fluorescence in 16: 1409–1416, 1998. situ hybridization mapping of breakpoints in pleomorphic adenomas with 8q12–13 3. Astrom, A. K., Voz, M. L., Kas, K., Roijer, E., Wedell, B., Mandahl, N., Van de Ven, abnormalities identifies a subgroup of tumors without PLAG1 involvement. Genes W., Mark, J., and Stenman, G. Conserved mechanism of PLAG1 activation in salivary Chromosomes Cancer, 24: 78–82, 1999. gland tumors with and without chromosome 8q12 abnormalities: identification of SII 21. Kas, K., Roijer, E., Voz, M., Meyen, E., Stenman, G., and Van de Ven, W. J. A 2-Mb as a new fusion partner gene. Cancer Res., 59: 918–923, 1999. YAC contig and physical map covering the chromosome 8q12 breakpoint cluster 4. Kas, K., Voz, M. L., Hensen, K., Meyen, E., and Van de Ven, W. J. M. Transcrip- region in pleomorphic adenomas of the salivary glands. Genomics, 43: 349–358, tional activation capacity of the novel PLAG family of zinc finger proteins. J. Biol. 1997. Chem., 273: 23026–23032, 1998. 22. Choo, Y., and Klug, A. Selection of DNA binding sites for zinc fingers using 5. Niwa, H., Yamamura, K., and Miyazaki, J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene (Amst.), 108: 193–199, 1991. rationally randomized DNA reveals coded interactions. Proc. Natl. Acad. Sci. USA, 6. Van den Ouweland, A. M., Roebroek, A. J., Schalken, J. A., Claesen, C. A., 91: 11168–11172, 1994. Bloemers, H. P., and Van de Ven, W. J. Structure and nucleotide sequence of the 5Ј 23. Choo, Y., and Klug, A. Physical basis of a protein-DNA recognition code. Curr. Opin. region of the human and feline c-sis proto-oncogenes. Nucleic Acids Res., 14: Struct. Biol., 7: 117–125, 1997. 765–778, 1986. 24. Abdollahi, A., Godwin, A. K., Miller, P. D., Getts, L. A., Schultz, D. C., Taguchi, T., 7. Holthuizen, P. E., Cleutjens, C. B., Veenstra, G. J., van der Lee, F. M., Koonen- Testa, J. R., and Hamilton, T. C. Identification of a gene containing zinc-finger motifs Reemst, A. M., and Sussenbach, J. S. Differential expression of the human, mouse based on lost expression in malignantly transformed rat ovarian surface epithelial and rat IGF-II genes. Regul. Pept., 48: 77–89, 1993. cells. Cancer Res., 57: 2029–2034, 1997. 8. van Dijk, M. A., van Schaik, F. M., Bootsma, H. J., Holthuizen, P., and Sussenbach, 25. Abdollahi, A., Roberts, D., Godwin, A. K., Schultz, D. C., Sonoda, G., Testa, J. R., J. S. Initial characterization of the four promoters of the human insulin-like growth and Hamilton, T. C. Identification of a zinc-finger gene at 6q25: a chromosomal factor II gene. Mol. Cell. Endocrinol., 81: 81–94, 1991. region implicated in development of many solid tumors. Oncogene, 14: 1973–1979, 9. Pulciani, S., Santos, E., Lauver, A. V., Long, L. K., and Barbacid, M. Transforming 1997. genes in human tumors. J. Cell. Biochem., 20: 51–61, 1982. 26. Spengler, D., Villalba, M., Hoffmann, A., Pantaloni, C., Houssami, S., Bockaert, J., 10. Jansen, E., Ayoubi, T. A., Meulemans, S. M., and Van de Ven, W. J. Regulation of and Journot, L. Regulation of apoptosis and cell cycle arrest by Zac1, a novel zinc human prohormone convertase 2 promoter activity by the transcription factor EGR-1. finger protein expressed in the pituitary gland and the brain. EMBO J., 16: 2814– Biochem. J., 328: 69–74, 1997. 2825, 1997. 11. Goudet, G., Delhalle, S., Biemar, F., Martial, J. A., and Peers, B. Functional and cooperative interactions between the homeodomain PDX1, Pbx, and Prep1 factors on 27. Varrault, A., Ciani, E., Apiou, F., Bilanges, B., Hoffmann, A., Pantaloni, C., Bock- the somatostatin promoter. J. Biol. Chem., 274: 4067–4073, 1999. aert, J., Spengler, D., and Journot, L. hZAC encodes a zinc finger protein with 12. Nordeen, S. K. Luciferase reporter gene vectors for analysis of promoters and antiproliferative properties and maps to a chromosomal region frequently lost in enhancers. Biotechniques, 6: 454–458, 1988. cancer. Proc. Natl. Acad. Sci. USA, 95: 8835–8840, 1998. 13. Dull, T. J., Gray, A., Hayflick, J. S., and Ullrich, A. Insulin-like growth factor II 28. Toretsky, J. A., and Helman, L. J. Involvement of IGF-II in human cancer. J. precursor gene organization in relation to insulin gene family. Nature (Lond.), 310: Endocrinol., 149: 367–372, 1996. 777–781, 1984. 29. El-Badry, O. M., Romanus, J. A., Helman, L. J., Cooper, M. J., Rechler, M. M., and 14. Medema, R. H., Wubbolts, R., and Bos, J. L. Two dominant inhibitory mutants of Israel, M. A. Autonomous growth of a human neuroblastoma cell line is mediated by p21ras interfere with insulin-induced gene expression. Mol. Cell. Biol., 11: 5963– insulin-like growth factor II. J. Clin. Investig., 84: 829–839, 1989. 5967, 1991. 30. Daughaday, W. H. The possible autocrine/paracrine and endocrine roles of insulin- 15. van den Ouweland, A. M., Breuer, M. L., Steenbergh, P. H., Schalken, J. A., like growth factors of human tumors. Endocrinology, 127: 1–4, 1990. Bloemers, H. P., and Van de Ven, W. J. Comparative analysis of the human and feline 31. Drummond, I. A., Madden, S. L., Rohwer-Nutter, P., Bell, G. I., Sukhatme, V. P., and Ј c-sis proto-oncogenes. Identification of 5 human c-sis coding sequences that are not Rauscher, F. J. d. Repression of the insulin-like growth factor II gene by the Wilms homologous to the transforming gene of simian sarcoma virus. Biochim. Biophys. tumor suppressor WT1. Science (Washington DC), 257: 674–678, 1992. Acta., 825: 140–147, 1985. 32. Devereux, J., Haeberli, P., and Smithies, O. A comprehensive set of sequence analysis 16. Sadowski, I., Bell, B., Broad, P., and Hollis, M. GAL4 fusion vectors for expression programs for the VAX. Nucleic Acids Res., 12: 387–395, 1984. in yeast or mammalian cells. Gene (Amst.), 118: 137–141, 1992. 17. Ikeda, K., and Kawakami, K. DNA binding through distinct domains of zinc-finger- 33. Rauscher, F. J. d., Morris, J. F., Tournay, O. E., Cook, D. M., and Curran, T. Binding homeodomain protein AREB6 has different effects on gene transcription. Eur. J. Bio- of the Wilms’ tumor locus zinc finger protein to the EGR-1 consensus sequence. chem., 233: 73–82, 1995. Science (Washington DC), 250: 1259–1262, 1990. 18. Cavin Perier, R., Junier, T., and Bucher, P. The Eukaryotic Promoter Database EPD. 34. Bucher, P. Weight matrix descriptions of four eukaryotic RNA polymerase II pro- Nucleic Acids Res., 26: 353–357, 1998. moter elements derived from 502 unrelated promoter sequences. J. Mol. Biol., 212: 19. van Dijk, M. A., Holthuizen, P. E., and Sussenbach, J. S. Elements required for 563–578, 1990. activation of the major promoter of the human insulin-like growth factor II gene. Mol. 35. Christy, B., and Nathans, D. DNA binding site of the growth factor-inducible protein Cell. Endocrinol., 88: 175–185, 1992. Zif268. Proc. Natl. Acad. Sci. USA, 86: 8737–8741, 1989.

113

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2000 American Association for Cancer Research. PLAG1, the Main Translocation Target in Pleomorphic Adenoma of the Salivary Glands, Is a Positive Regulator of IGF-II

Marianne L. Voz, Nancy S. Agten, Wim J. M. Van de Ven, et al.

Cancer Res 2000;60:106-113.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/60/1/106

Cited articles This article cites 32 articles, 12 of which you can access for free at: http://cancerres.aacrjournals.org/content/60/1/106.full#ref-list-1

Citing articles This article has been cited by 24 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/60/1/106.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/60/1/106. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.