Zinc Finger Proteins Act as Transcriptional Repressors of Alkaloid Biosynthesis Genes in Catharanthus roseus
2004; Elsevier BV; Volume: 279; Issue: 51 Linguagem: Inglês
10.1074/jbc.m404391200
ISSN1083-351X
AutoresBea Pauw, Frédérique Hilliou, Virginia Martin, Guillaume Chatel, Cocky J.F. de Wolf, Antony Champion, Martial Pré, Bert van Duijn, Jan W. Kijne, Leslie van der Fits, Johan Memelink,
Tópico(s)Plant and fungal interactions
ResumoIn Catharanthus roseus cell suspensions, the expression of several terpenoid indole alkaloid biosynthetic genes, including two genes encoding strictosidine synthase (STR) and tryptophan decarboxylase (TDC), is coordinately induced by fungal elicitors such as yeast extract. To identify molecular mechanisms regulating the expression of these genes, a yeast one-hybrid screening was performed with an elicitor-responsive part of the TDC promoter. This screening identified three members of the Cys2/His2-type (transcription factor IIIA-type) zinc finger protein family from C. roseus, ZCT1, ZCT2, and ZCT3. These proteins bind in a sequence-specific manner to the TDC and STR promoters in vitro and repress the activity of these promoters in trans-activation assays. In addition, the ZCT proteins can repress the activating activity of APETALA2/ethylene response-factor domain transcription factors, the ORCAs, on the STR promoter. The expression of the ZCT genes is rapidly induced by yeast extract and methyljasmonate. These results suggest that the ZCT proteins act as repressors in the regulation of elicitor-induced secondary metabolism in C. roseus. In Catharanthus roseus cell suspensions, the expression of several terpenoid indole alkaloid biosynthetic genes, including two genes encoding strictosidine synthase (STR) and tryptophan decarboxylase (TDC), is coordinately induced by fungal elicitors such as yeast extract. To identify molecular mechanisms regulating the expression of these genes, a yeast one-hybrid screening was performed with an elicitor-responsive part of the TDC promoter. This screening identified three members of the Cys2/His2-type (transcription factor IIIA-type) zinc finger protein family from C. roseus, ZCT1, ZCT2, and ZCT3. These proteins bind in a sequence-specific manner to the TDC and STR promoters in vitro and repress the activity of these promoters in trans-activation assays. In addition, the ZCT proteins can repress the activating activity of APETALA2/ethylene response-factor domain transcription factors, the ORCAs, on the STR promoter. The expression of the ZCT genes is rapidly induced by yeast extract and methyljasmonate. These results suggest that the ZCT proteins act as repressors in the regulation of elicitor-induced secondary metabolism in C. roseus. Perception of stress signals or of pathogen-derived molecules, called elicitors, activates a number of signal transduction steps in plants, eventually leading to the transcriptional activation of numerous genes, and consequently to de novo synthesis of a variety of defense proteins and protective secondary metabolites (1Nimchuk Z. Eulgem T. Holt B.F. II I Dangl J.F. Annu. Rev. Genet. 2003; 37: 579-609Crossref PubMed Scopus (441) Google Scholar). The biosynthesis of one or more secondary signals, such as jasmonic acid (JA), 1The abbreviations used are: JA, jasmonic acid; MeJA, methyljasmonate; BPF, box P-binding factor; GBF, G-box-binding factor; YE, yeast extract; STR, strictosidine synthase; TDC, tryptophan decarboxylase; ORCA, octadecanoid-responsive Catharanthus AP2 domain; AP2, APETALA2; ERF, ethylene-response-factor; TFIIIA, transcription factor IIIA; EMSA, electrophoretic mobility shift assay; GUS, β-glucuronidase; ZCT, zinc finger Catharanthus transcription factor. salicylic acid, and ethylene, plays a crucial role in this stress response (2Kunkel B.N. Brooks D.M. Curr. Opin. Plant Biol. 2002; 5: 325-331Crossref PubMed Scopus (1160) Google Scholar). In elicitor-induced accumulation of secondary metabolites, jasmonic acid and its volatile methyl-ester methyljasmonate (MeJA), have been shown to act as intermediate signals (3Memelink J. Verpoorte R. Kijne J.W. Trends Plant Sci. 2001; 6: 212-219Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). Knowledge about the molecular mechanisms regulating elicitor-responsive expression of secondary metabolite biosynthesis genes is limited. In parsley, a fungal elicitor induces the expression of the MYB-like transcription factor box P-binding factor (BPF)-1, which interacts with the promoter of a gene encoding the phenylpropanoid biosynthesis enzyme phenylalanine ammonia-lyase (4da Costa e Silva O. Klein L. Schmelzer E. Trezzini G.F. Hahlbrock K. Plant J. 1993; 4: 125-135Crossref PubMed Scopus (82) Google Scholar). Terpenoid indole alkaloid biosynthesis in Catharanthus roseus is one of the best studied elicitor-induced secondary metabolic pathways. In suspension cells, the perception of yeast extract (YE) leads to the activation of terpenoid indole alkaloid biosynthesis (5Moreno P.R.H. van der Heijden R. Verpoorte R. Plant Cell Tissue Org. Cult. 1995; 42: 1-25Crossref Scopus (202) Google Scholar). Two genes involved in terpenoid indole alkaloid biosynthesis, encoding strictosidine synthase (STR) and tryptophan decarboxylase (TDC), are coordinately regulated and their mRNAs accumulate transiently after YE treatment (6Pasquali G. Goddijn O.J.M. de Waal A. Verpoorte R. Schilperoort R.A. Hoge J.H.C. Memelink J. Plant Mol. Biol. 1992; 18: 1121-1131Crossref PubMed Scopus (197) Google Scholar, 7Roewer I.A. Cloutier C. Nessler C.L. De Luca V. Plant Cell Rep. 1992; 11: 86-89Crossref PubMed Scopus (49) Google Scholar). Induction of these genes by YE is mediated by protein phosphorylation, the influx of calcium, and the biosynthesis of JA via the octadecanoid pathway (3Memelink J. Verpoorte R. Kijne J.W. Trends Plant Sci. 2001; 6: 212-219Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 8Menke F.L.H. Parchmann S. Mueller M.J. Kijne J.W. Memelink J. Plant Physiol. 1999; 119: 1289-1296Crossref PubMed Scopus (214) Google Scholar). In the STR promoter, two elicitor- and jasmonate-responsive sequences have been identified; the so-called BA region and a sequence close to the TATA box, called jasmonate- and elicitor-responsive element, located in the RV region (see Fig. 8). The BA region was found to bind to a homologue of parsley PcBPF-1, called CrBPF1 (9van der Fits L. Zhang H. Menke F.L.H. Deneka M. Memelink J. Plant Mol. Biol. 2000; 44: 675-685Crossref PubMed Scopus (111) Google Scholar). The jasmonate- and elicitor-responsive element interacts with two JA-responsive transcription factors called ORCA2 and ORCA3 (10Menke F.L.H. Champion A. Kijne J.W. Memelink J. EMBO J. 1999; 18: 4455-4463Crossref PubMed Scopus (370) Google Scholar, 11van der Fits L. Memelink J. Plant J. 2001; 25: 43-53Crossref PubMed Google Scholar). Both ORCAs belong to the APETALA2/ethylene response-factor (AP2/ERF) family of transcription factors. ORCA3 was shown to regulate multiple genes involved in primary and secondary metabolism, including the TDC and STR genes (3Memelink J. Verpoorte R. Kijne J.W. Trends Plant Sci. 2001; 6: 212-219Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 11van der Fits L. Memelink J. Plant J. 2001; 25: 43-53Crossref PubMed Google Scholar, 12van der Fits L. Memelink J. Science. 2000; 289: 295-297Crossref PubMed Scopus (764) Google Scholar). The NR region of the STR promoter, which is not required for responsiveness to elicitor or jasmonate (10Menke F.L.H. Champion A. Kijne J.W. Memelink J. EMBO J. 1999; 18: 4455-4463Crossref PubMed Scopus (370) Google Scholar), interacts with two G-box binding basic leucine zipper proteins (CrGBFs; Ref. 13Sibéril Y. Benhamron S. Memelink J. Giglioli-Guivarc'h N. Thiersault M. Boisson B. Doireau P. Gantet P. Plant Mol. Biol. 2001; 45: 477-488Crossref PubMed Scopus (117) Google Scholar). The TDC promoter also contains a YE-responsive element, the so-called DB element (14Ouwerkerk P.B.F. Memelink J. Plant Mol. Biol. 1999; 39: 129-136Crossref PubMed Scopus (38) Google Scholar). The ORCA transcription factors 2J. Memelink, unpublished results. or the MYB-related protein CrBPF1 (9van der Fits L. Zhang H. Menke F.L.H. Deneka M. Memelink J. Plant Mol. Biol. 2000; 44: 675-685Crossref PubMed Scopus (111) Google Scholar) do not bind to the DB element, whereas CrGBFs have a weak affinity for a G-box-like sequence in the DB element in vitro (13Sibéril Y. Benhamron S. Memelink J. Giglioli-Guivarc'h N. Thiersault M. Boisson B. Doireau P. Gantet P. Plant Mol. Biol. 2001; 45: 477-488Crossref PubMed Scopus (117) Google Scholar). To isolate transcription factors that interact with the DB element, a yeast one-hybrid screening was performed. This screening identified three members of the transcription factor IIIA (TFIIIA-type; Cys2/His2-type) zinc finger protein family from C. roseus, ZCT1, ZCT2, and ZCT3. In vitro DNA binding studies showed that these proteins bind in a sequence-specific manner to the TDC and STR promoters. Furthermore, these zinc finger proteins were shown to act as transcriptional repressors of STR and TDC promoter activity in trans-activation assays. Finally, expression of these zinc finger genes is rapidly induced by YE and MeJA. Together these data show that TFIIIA-type zinc finger transcription factors can act as repressors in the regulation of YE-induced secondary metabolism. Isolation of Zinc Finger Clones—cDNA fragments encoding zinc finger proteins ZCT1, ZCT2, and ZCT3 were isolated by a one-hybrid screening of a C. roseus cDNA library with the DB element of the TDC promoter as bait. Tetramerization of the DB element from the TDC promoter was described in (14Ouwerkerk P.B.F. Memelink J. Plant Mol. Biol. 1999; 39: 129-136Crossref PubMed Scopus (38) Google Scholar). The DB tetramer was fused to the yeast HIS3 reporter gene in plasmid p601 (15Grueneberg D.A. Natesan S. Alexandre C. Gilman M.Z. Science. 1992; 257: 1089-1095Crossref PubMed Scopus (256) Google Scholar). The tetramer-HIS3 fusion was transferred as a BamHI fragment into the BclI site of integration vector pJP04, which is essentially similar to pINT1 (16Meijer A.H. Ouwerkerk P.B. F Hoge J.H.C. Yeast. 1998; 14: 1407-1416Crossref PubMed Scopus (49) Google Scholar). The resulting plasmid was linearized with NcoI and introduced into yeast strain Y187 (17Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5250) Google Scholar). Recombinants were selected on YPD (yeast extract/peptone/dextrose) medium containing 150 μg/ml G418, and the occurrence of single recombination events between the pJP04 derivative and the chromosomal PDC6 locus was verified by Southern blot analysis. The pACTII cDNA library with a complexity of 3.5 × 106 independent transformants was prepared from elicitor-treated C. roseus cell suspension line MP183L as described by Ref. 10Menke F.L.H. Champion A. Kijne J.W. Memelink J. EMBO J. 1999; 18: 4455-4463Crossref PubMed Scopus (370) Google Scholar. After transformation of the cDNA library into the yeast strain, cells were plated on minimal medium lacking leucine and histidine. Screening of an estimated total number of 2.4 × 106 yeast transformants resulted in 188 colonies containing plasmids conferring His/Leu-independent growth upon isolation/retransformation. Plasmid cross-hybridizations and sequencing of representative members of each class resulted in the identification of three C2H2 zinc finger classes. Construction of Full-length cDNA Clones—To construct full-length clones, 5′ sequences were isolated by PCR with a gene-specific primer and the vector primer 5′-CCCCACCAAACCCAAAAAAAG-3′ using the pACTII cDNA library as a template. ZCT1 appeared to be a full-length clone. To confirm this notion, 5′ sequences amplified with the gene-specific primer 5′-CTAAAGATTGATGGAGTAGATC-3′ were digested with BamHI/HindIII and cloned in pBluescript SK+. Sequencing of the longest PCR fragment yielded additional sequence information of 10 nucleotides. ZCT2 5′ sequences amplified with the gene-specific primer 5′-CATCAACAATATTCGACTTCTTCACC-3′ were digested with BamHI/NdeI and cloned in pUC28. The insert from the pACTII-ZCT2 clone was excised with BamHI/XhoI and first cloned into the vector pIC-19R (18Marsh J.L. Erfle M. Wykes E.J. Gene (Amst.). 1984; 32: 481-485Crossref PubMed Scopus (522) Google Scholar) digested with BglII/SalI, after which it was transferred as a NdeI/SmaI fragment to the pUC28 plasmid containing the PCR fragment digested with NdeI/EcoRV, resulting in a full-length ZCT2FL cDNA. ZCT3 5′ sequences amplified with the gene-specific primer 5′-CTAAAGATTGATGGAGTA-3′ were digested with BamHI/SacI and cloned in pBluescript SK+. The insert from the pACTII-ZCT3 clone was excised with EcoRI/EcoRV, and first cloned into the vector pIC-19H after which it was transferred as a SacI fragment to the pBluescript SK+ plasmid containing the PCR fragment, resulting in a full-length ZCT3FL cDNA. Construction of Escherichia coli Expression Plasmids—The ZCT1 insert was excised from the pACTII vector with SmaI/XhoI and inserted in pIC-19R digested with EcoRV/SalI. The resulting plasmid was used as template with primers 5′-CGGGATCCTCGAGATGGGCGTGAA-GAGATTCAGAG-3′ and M13–40 in a PCR, and the product was digested with BamHI and cloned in pACTII. From there it was excised with XhoI and introduced in pGEX-KG (19Guan K.L. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1641) Google Scholar). The ZCT2FL insert was amplified with the primers 5′-CGCGGATCCGCGATGGTGATGATTA-ATATA-3′ and 5′-CCCAAGCTTGGGT15-3′, and after digestion with BamHI/HindIII, was introduced in pGEX-KG. The ZCT3FL insert was amplified with the primers 5′-CGCGGATCCGCGATGGCACTT-GAAGCTTTG-3′ and T3, and following digestion with BamHI/XhoI introduced in pGEX-KG. Expression plasmids were introduced in E. coli strain BL21 (DE3) pLysS, and proteins isolated using glutathione-Sepharose 4B beads (Amersham Biosciences) according to the manufacturer's instructions were dialyzed against electrophoretic mobility shift assay (EMSA) binding buffer. EMSAs—STR promoter fragments, RV wild-type and mutant fragments (10Menke F.L.H. Champion A. Kijne J.W. Memelink J. EMBO J. 1999; 18: 4455-4463Crossref PubMed Scopus (370) Google Scholar), and TDC promoter fragments (20Ouwerkerk P.B.F. Trimborn T.O. Hilliou F. Memelink J. Mol. Gen. Genet. 1999; 261: 610-622Crossref PubMed Scopus (40) Google Scholar) were isolated and labeled as described. DNA-binding reactions contained 0.1 ng of end-labeled DNA probe, 500 ng of poly(dA-dT)-poly(dA-dT), binding buffer (25 mm HEPES-KOH, pH 7.2, 100 mm KCl, 0.1 mm EDTA, 10% glycerol), and protein extract in a 10-μl volume. For analysis of the requirement of zinc for binding, ZCT proteins were pre-incubated for 5 min in binding buffer containing 3 mm EDTA, 3 mm EGTA, 10 mm 1,10-phenanthroline (Sigma)/1% ethanol or 1% ethanol before addition of probe DNA. Binding reactions were incubated for 30 min at room temperature before loading on 5% acrylamide/bisacrylamide (37:1)-0.5× Tris-borate-EDTA gels under tension. After electrophoresis at 125 V for 1 h, gels were dried on Whatman DE81 paper and autoradiographed. Construction of Plant Expression Vectors—The ZCT1 insert was excised from pACTII with SmaI/XhoI, cloned in pIC-19R digested with EcoRV/SalI, and then cloned in pMOG463 as a BamHI fragment. The ZCT2 insert was excised from pUC28-ZCT2FL with BamHI and cloned in pMOG183. The pMOG vectors are pUC18 derivatives carrying a double-enhanced CaMV 35 S promoter and the nos terminator separated by a BamHI site. The full-length ZCT3FL cDNA was cloned as a BamHI/BglII fragment in SK+-35 S-nos. This pBluescript derivative carries a double-enhanced CaMV 35 S promoter and the nos terminator separated by a BamHI site. Cell Cultures—C. roseus cell suspension line MP183L was grown as described (6Pasquali G. Goddijn O.J.M. de Waal A. Verpoorte R. Schilperoort R.A. Hoge J.H.C. Memelink J. Plant Mol. Biol. 1992; 18: 1121-1131Crossref PubMed Scopus (197) Google Scholar). Transient Expression Assays—Cells of C. roseus cell line MP183L were co-transformed with plasmids carrying different promoter parts fused to GUSA and overexpression vectors carrying ZCT1, ZCT2, ZCT3, and/or ORCA2 or ORCA3 cDNAs fused to the CaMV 35 S promoter. Co-transformations of the promoter-GUS constructs with an empty overexpression vector (pMOG184) served as controls. Cells were transformed with a total of 10 μg of plasmid DNA through particle bombardment as described before (21van der Fits L. Memelink J. Plant Mol. Biol. 1997; 33: 943-946Crossref PubMed Scopus (59) Google Scholar), using the two constructs in a ratio of 1:4 (GUS:ZCT/ORCA). In the case of co-bombardment with both ORCA and zinc finger cDNAs, the ratio was 1:4:4 (GUS:ZCT:ORCA). Each plasmid combination was bombarded in triplicate, where each replicate consisted of an independent DNA coating of tungsten particles. Twenty-four hours after transformation, cells were harvested and frozen in liquid nitrogen. β-Glucuronidase (GUS) activity assays were performed as described (21van der Fits L. Memelink J. Plant Mol. Biol. 1997; 33: 943-946Crossref PubMed Scopus (59) Google Scholar). GUS reporter activity was related to total protein amounts to correct for the amount of cells used in each transformation. GUS activity was depicted as relative activity compared with the vector control. Statistical analysis of the results was done using the nonparametric Wilcoxon-Mann-Whitney test. Elicitor and Jasmonate Treatment—Partially purified elicitor was prepared from yeast extract (YE) (Difco), through ultrafiltration and a number of chromatographic steps, as described in Ref. 8Menke F.L.H. Parchmann S. Mueller M.J. Kijne J.W. Memelink J. Plant Physiol. 1999; 119: 1289-1296Crossref PubMed Scopus (214) Google Scholar. The amount of purified elicitor used for induction experiments was calibrated to correspond to a final concentration of 400 μg/ml of crude YE using a semi-quantitative alkalinization response assay as described before (8Menke F.L.H. Parchmann S. Mueller M.J. Kijne J.W. Memelink J. Plant Physiol. 1999; 119: 1289-1296Crossref PubMed Scopus (214) Google Scholar). Methyljasmonate (Bedoukian Research Inc.) was diluted in dimethyl sulfoxide (Me2SO). RNA Extraction and Northern Blot Analysis—RNA extraction and Northern blot analysis were performed as described before (8Menke F.L.H. Parchmann S. Mueller M.J. Kijne J.W. Memelink J. Plant Physiol. 1999; 119: 1289-1296Crossref PubMed Scopus (214) Google Scholar), loading 20-μg RNA samples onto the gels. All Northern blots were probed using 32P-labeled cDNA fragments. ORCA2, ORCA3, RPS9, and STR probes were described before (8Menke F.L.H. Parchmann S. Mueller M.J. Kijne J.W. Memelink J. Plant Physiol. 1999; 119: 1289-1296Crossref PubMed Scopus (214) Google Scholar). Isolation of Zinc Finger Proteins ZCT1, ZCT2, and ZCT3—To identify DNA-binding proteins that interact with the YE-responsive DB element of the TDC promoter, a yeast one-hybrid screening was performed with this element. A derivative of yeast strain Y187, containing a tetramer of DB fused to the HIS3 selection marker, was used in a screen to isolate DNA-binding proteins from a cDNA library of C. roseus cloned in a fusion with the GAL4 activation domain in yeast expression vector pACTII. In total, 2.4 million Y187–4DB transformants were screened for reporter gene activation. A total of 188 cDNA clones, belonging to several classes, were isolated from yeast colonies that showed growth on medium lacking histidine. No cDNAs encoding ORCA or CrBPF1 proteins were recovered, which is consistent with the fact that these proteins do not bind DB in vitro. In addition, no clones encoding CrGBFs were found, despite the fact that CrGBFs have a weak affinity for a G-box-like sequence in the DB element in vitro (13Sibéril Y. Benhamron S. Memelink J. Giglioli-Guivarc'h N. Thiersault M. Boisson B. Doireau P. Gantet P. Plant Mol. Biol. 2001; 45: 477-488Crossref PubMed Scopus (117) Google Scholar). Comparison of the DNA sequences to sequences in the NCBI data base revealed that three cDNA classes encoded proteins with two Cys2/His2-type (TFIIIA-type) zinc fingers. In a TFIIIA-type zinc finger protein, two cysteines and two histidines, in a conserved sequence motif (CX2–4CX3FX5LX2HX3–5H), tetrahedrally coordinate a zinc atom to form a compact structure that interacts with the major groove of DNA in a sequence-specific manner (22Pavletich N. Pabo C. Science. 1991; 252: 809-817Crossref PubMed Scopus (1763) Google Scholar, 23Choo Y. Klug A. Curr. Opin. Struct. Biol. 1997; 7: 117-125Crossref PubMed Scopus (212) Google Scholar). All three Catharanthus classes possess the typical characteristics of plant TFIIIA-type two-fingered proteins (24Takatsuji H. Plant Mol. Biol. 1999; 39: 1073-1078Crossref PubMed Scopus (221) Google Scholar). Both fingers have the QALGGH sequence in the putative DNA-contacting surfaces, and the two fingers are separated by a long spacer (Fig. 1). We called the three encoded proteins ZCT1, ZCT2, and ZCT3, for zinc finger Catharanthus transcription factor. The ZCT3 class was isolated 14 times, the ZCT1 class 8 times, and ZCT2 was a single clone. The longest clone from the ZCT1 class was full-length, whereas all ZCT2 and ZCT3 clones appeared to be partial. The missing portions of ZCT2 and ZCT3 were isolated via PCR and fused to the partial cDNAs, to construct complete clones. An alignment of the deduced amino acid sequences of ZCT1, ZCT2, and ZCT3 is shown in Fig. 1. The ZCT1, ZCT2, and ZCT3 proteins have predicted molecular masses of 19.6, 21, and 27.4 kDa, respectively. Comparison of the deduced ZCT1 and ZCT2 amino acid sequences to sequences in the NCBI data base showed highest homology to ZPT2-5, ZPT2-14, ZPT2-12, and ZPT2-13 from Petunia hybrida. One of the closest homologues of ZCT3 is the SCOF-1 protein from soybean, which is involved in cold tolerance (25Kim J.C. Lee S.H. Cheong Y.H. Yoo C.M. Lee S.I. Chun H.J. Yun D.J. Hong J.C. Lee S.Y. Lim C.O. Cho M.J. Plant J. 2001; 25: 247-259Crossref PubMed Google Scholar). Besides the two zinc fingers, the ZCT proteins contain several conserved regions. Near their N termini, they contain a short basic region (B-box; Ref. 26Sakamoto H. Araki T. Meshi T. Iwabuchi M. Gene (Amst.). 2000; 248: 23-32Crossref PubMed Scopus (152) Google Scholar), which may function as a nuclear localization signal (Fig. 1). Between the B-box and the first zinc finger, the ZCT proteins contain a short region of hydrophobic residues rich in leucines (L-box). The motif has been found in several other Cys2/His2 zinc finger proteins, and has been suggested to play a role in protein-protein interactions or in maintaining the folded structure of the proteins (26Sakamoto H. Araki T. Meshi T. Iwabuchi M. Gene (Amst.). 2000; 248: 23-32Crossref PubMed Scopus (152) Google Scholar, 27Meissner R. Michael A.J. Plant Mol. Biol. 1997; 33: 615-624Crossref PubMed Scopus (58) Google Scholar). In their C-terminal region, the ZCT proteins have an LxLxL motif (Fig. 1), which is a potent repression domain found in most TFIIIA-type zinc finger, several AP2/ERF (28Ohta M. Matsui K. Hiratsu K. Shinshi H. Ohme-Takagi M. Plant Cell. 2001; 13: 1959-1968Crossref PubMed Scopus (690) Google Scholar), and in all Arabidopsis AUX/IAA (29Tiwari S.B. Hagen G. Guilfoyle T.J. Plant Cell. 2004; 16: 533-543Crossref PubMed Scopus (433) Google Scholar) transcriptional repressors. In AP2/ERF proteins this motif has also been called the ERF-associated amphiphilic repression domain (28Ohta M. Matsui K. Hiratsu K. Shinshi H. Ohme-Takagi M. Plant Cell. 2001; 13: 1959-1968Crossref PubMed Scopus (690) Google Scholar). The ZCT Proteins Bind to Several Regions of the TDC and STR Promoters—The ability of the ZCT proteins to activate HIS3 gene expression via the DB region in yeast and the presence of two zinc finger DNA-binding domains, indicated that they are DNA-binding proteins. To directly test the DNA binding of the zinc finger proteins, recombinant GST-ZCT fusion proteins were isolated from E. coli and EMSAs were performed. Incubation of the ZCT proteins with labeled DB fragment from the TDC promoter showed that they can bind to this fragment (Fig. 2C). ZCT1 and ZCT2 showed a similar binding pattern consisting of two bands, whereas ZCT3 formed a single shifted band. To test whether the ZCT proteins can also bind to other parts of the TDC promoter, EMSAs were performed with probes covering a 535-bp region of the TDC promoter upstream of the TATA box (Fig. 2A). ZCT1 and ZCT2 bound with highest affinity to the HS and DB regions of the TDC promoter, with little binding to the other fragments tested (Fig. 2D). However, ZCT3 bound to all fragments of the TDC promoter with highest affinity for HS and DB (Fig. 2D). Recombinant GST did not bind to any of the fragments used in EMSAs (data not shown). Because the TDC and STR genes are coordinately regulated by YE and MeJA, the binding of the ZCT proteins to the STR promoter was also determined. Transformation of the zinc finger clones in pACTII to a yeast strain carrying a tetramer of the RV region of the STR promoter fused to the HIS3 selection gene (10Menke F.L.H. Champion A. Kijne J.W. Memelink J. EMBO J. 1999; 18: 4455-4463Crossref PubMed Scopus (370) Google Scholar) indicated that the ZCT proteins were also able to bind to the elicitor- and jasmonate-responsive RV region of the STR promoter in vivo (results not shown). Incubation of the ZCT proteins with probes covering a 583-bp region of the STR promoter in vitro (Fig. 2B) showed that they indeed bound to the RV region and additionally to the BA and VH regions (Fig. 2D). ZCT3 bound additionally to the XD and DB fragments of the STR promoter (Fig. 2D). The RV region of the STR promoter contains the binding site for the ORCA transcriptional activators. In a previous study, a mutation scanning of the RV fragment, which comprised changing blocks of six adjacent nucleotides into their complementary nucleotides (Fig. 2B; Ref. 10Menke F.L.H. Champion A. Kijne J.W. Memelink J. EMBO J. 1999; 18: 4455-4463Crossref PubMed Scopus (370) Google Scholar), demonstrated that the ORCA binding site is located in the M2-M3-M4 region. To determine the specific binding site of the ZCT proteins in the RV fragment, the different RV mutant fragments were used as probes in EMSAs. Because the ZCT proteins showed little or no binding to mutated RV fragment M2, but did bind to the other mutated RV fragments, it can be concluded that the main binding determinant for the ZCT proteins is located in the M2 region (Fig. 2E). The ZCT binding site is therefore distinct from but overlapping with the binding site for the ORCA proteins. To determine whether the interaction of the ZCT proteins with DNA requires the binding of a zinc atom to their zinc fingers, the DNA binding affinity of the ZCT proteins was analyzed in the presence of the zinc-chelating agents EDTA or 1,10-phenanthroline. Fig. 3 shows that under standard experimental conditions the ZCT proteins can bind to the RV fragment. However, the presence of EDTA or 1,10-phenanthroline inhibits the binding of the ZCT proteins to the RV fragment, indicating that zinc is required for binding (Fig. 3). The presence of EGTA, which has a chemical structure similar to EDTA but specifically binds calcium, or the solvent ethanol did not influence the binding of the ZCT proteins to RV (Fig. 3) indicating the specificity of the inhibition by EDTA and 1,10-phenanthroline. A similar experiment using the DB fragment of the TDC promoter showed that zinc is also essential for the binding of the ZCT proteins to this fragment (results not shown). The ZCT Proteins Act as Transcriptional Repressors of STR and TDC Promoter Activity—Binding of the zinc finger proteins to both the TDC and STR promoters suggested that these proteins might be involved in the coordinated regulation of the expression of these genes. To test whether the ZCT proteins can regulate these promoters in vivo, C. roseus cells were co-transformed with a TDC-promoter-GUSA construct and an overexpression vector carrying a ZCT cDNA fused to the CaMV 35 S promoter. Co-expression of any of the ZCT proteins reduced TDC promoter activity ∼2-fold compared with the vector control (Fig. 4A). Co-expression of any of the ZCT proteins reduced STR promoter activity at least 5-fold (Fig. 4A). These results show that the ZCT proteins can act as transcriptional repressors of both the TDC and STR promoters. The repressor activity of the ZCT proteins is consistent with the presence of the LxLxL motif within these proteins. We focused our in vivo trans-regulatory studies on the STR promoter, because its structure with regard to cis-acting elements and their interaction with trans-acting factors has been elucidated in more detail than for the TDC promoter (30Gantet P. Memelink J. Trends Pharmacol. Sci. 2002; 23: 563-56931Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). As shown above, the ZCT proteins can bind to the BA and RV regions of the STR promoter in vitro. To test whether the in vitro binding affinities are reflected in in vivo repressor activities, Catharanthus cells were co-transformed with GUS reporter plasmids carrying tetramers of the BA or RV fragments fused to the minimal CaMV 35 S promoter (–47 to +27), and an overexpression vector carrying a ZCT cDNA fused to the CaMV 35 S promoter. All three ZCT proteins could repress the activity of both the RV and BA promoter fragments (Fig. 4B). A repressor protein can inhibit transcription via different mechanisms, requiring promoter binding (e.g. competition with activators for DNA binding sites or recruitment of chromatin-modifying or remodeling complexes) or not requiring promoter binding (e.g. sequestration of basal transcription factors or activators). To determine whether the repression by the ZCT proteins occurs via a direct interaction with the DNA, co-bombardment experiments were performed using the different RV mutants fused to a minimal promoter-GUS gene. The RV mutants affected in the ORCA binding site have reduced basal transcriptional activity (11van der Fits L. Memelink J. Plant J. 2001; 25: 43-53Crossref PubMe
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