Cloning of Cutinase Transcription Factor 1, a Transactivating Protein Containing Cys6Zn2 Binuclear Cluster DNA-binding Motif*

(Received for publication, January 29, 1997, and in revised form, March 4, 1997)

Daoxin Li and Pappachan E. Kolattukudy Dagger

From the Neurobiotechnology Center, Ohio State University, Columbus, Ohio 43210

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

Hydroxy fatty acids from plant cutin were shown previously to induce the expression of the cutinase gene via a palindromic sequence located at -159 base pairs of the cutinase gene in Fusarium solani f. sp. pisi (Nectria hematococca mating type VI). Of the two overlapping palindromes in this sequence, palindrome 2 was found to be essential for the inducibility of cutinase by hydroxy fatty acids. Screening of a phage expression library with the concatenated palindrome 2 as probe detected a distinct cDNA clone encoding a polypeptide designated cutinase transcription factor 1alpha (CTF1alpha ) with a calculated molecular weight of 101,109. This protein contains a Cys6Zn2 binuclear cluster motif sharing homology to the Cys6Zn2 binuclear cluster DNA-binding domains of transcription factors from Saccharomyces cerevisiae, S. carlsbergensis, Kluyveromyces lactis, Neurospora crassa, Aspergillus nidulans, and A. flavus. CTF1alpha , expressed in Escherichia coli, showed specific binding to the palindrome 2 DNA fragment but not to palindrome 1 or mutant palindrome 2 DNA fragments, suggesting specific binding of CTF1alpha to palindrome 2. When CTF1alpha was expressed as a fusion protein with the nuclear localization sequence of SV40 in yeast, it transactivated the native cutinase promoter fused to the chloramphenicol acetyl transferase (cat) gene. Mutation of palindrome 2 but not palindrome 1 abolished this transactivation. Thus, CTF1alpha positively acts in vivo by binding selectively to palindrome 2 of the cutinase gene promoter.


INTRODUCTION

Extracellular cutinases can help fungi to penetrate through the outermost cuticular barrier of the host plant and, thus, play a role in pathogenesis (1). When conidia of highly virulent fungal strains that carry low levels of cutinase (2) contact the host cuticular surface, small amounts of cutin hydrolysate would be produced, and the monomers thus released are known to trigger expression of the cutinase gene in the germinating conidia, leading to targeted secretion of cutinase at the contact site (2). It is known that 10,16-dihydroxy C16 fatty acid and 9,10,18-trihydroxy C18 fatty acid, the unique monomers of cutin, are the best inducers of cutinase (3). The cis-elements responsible for this inducible expression of the cutinase gene were identified previously using Fusarium solani f. sp. pisi transformants containing segments of the 5'-flanking regions of the cutinase gene and their mutants fused to the chloramphenicol acetyl transferase (cat) gene (4). The element essential for the inducible expression of cutinase gene was found to be located at -159 bp1 (4). Of the two overlapping palindromes in this region, palindrome 2 was the element necessary for the inducible expression of the cutinase gene. Gel retardation studies showed that glucose-depleted cultures contained a protein factor that specifically bound palindrome 2, and this factor was designated cutinase transcription factor 1 (CTF1) (4).

Our efforts to identify transacting factors involved in the regulation of cutinase gene transcription led to the isolation of a zinc finger protein that specifically bound to the overlapping palindromic sequence designated palindrome-binding protein (PBP) (5). Here we report that further examination of the specificity of this binding by PBP revealed that this protein bound only to palindrome 1 but not to palindrome 2. We, therefore, further examined expression libraries for a transacting factor that would specifically bind palindrome 2 and report here the isolation of a clone that encodes a distinct protein that specifically binds to palindrome 2 but not to palindrome 1. This protein, designated CTF1alpha , shows a low degree of sequence identity to GenBank sequences, but it contains a Cys6Zn2 binuclear cluster motif similar to those found in positively acting transcription factors in other fungi. That CTF1alpha is a transcriptional activator in vivo for palindrome 2 of the cutinase gene promoter was further demonstrated by its ability to transactivate the cutinase promoter fused to the cat gene in yeast and by the observation that this transactivation was abolished by mutation of palindrome 2 but not palindrome 1.


EXPERIMENTAL PROCEDURES

Materials and Bacterial Strains

Restriction and modification enzymes were from Life Technologies, Inc. Chemicals were from Sigma or Amresco. Poly(deoxyinosinic-deoxycytidylic) acid was from Boehringer Mannheim and labeled nucleotides were from Amersham Corp. Nitrocellulose filter discs were from Schleicher & Schuell. Duralon-UV membranes were from Stratagene. Escherichia coli DH5alpha (Life Technologies, Inc.) was used for propagating all plasmids (6).

Isolation of Phage Clones for CTF1alpha

The 27 discrete clones identified in the original tertiary screening for PBP (5) were tested by polymerase chain reaction, and those homologous to PBP clone were not tested further. Phage DNAs were purified from the remaining clones and double digested with either EcoRI and BamHI, or EcoRI and SalI, or EcoRI and SstI, or EcoRI and XhoI. Subsequently, those phage clones that differed in restriction patterns were probed (7) with 32P-labeled palindrome 2 fragment that was concatenated to an average size of 316 bp or 17 copies of the palindrome 2 fragment as described (5). A polypeptide encoded by one of the phage clones (designated lambda ctf-15) that bound the concatenated palindrome 2 was designated CTF1alpha . The cDNA insert from this phage clone was subcloned into pBluescript KS- vector (Stratagene) to produce pCTF1-15. Sequence analyses indicated that this clone was not full-length. Additional clones for CTF1alpha were isolated by screening a lambda gt11 library constructed previously with random primers (5) and another lambda gt11 library constructed similarly with oligo(dT) (5)2 with the cDNA inserts from pCTF1-15 or its terminus as probe using standard procedures (6).

Subcloning and Sequence Analysis

DNA inserts in lambda gt11 phage clones were subcloned into KS- vector. Plasmid DNAs were prepared (6) and used for automated sequencing with a model 373A sequencer (Applied Biosystems). The putative subcellular location for the polypeptide was predicted with PSORT (8) (version 6.3) in Pedro's Biomolecular Research Tools from the World Wide Web using Netscape. Protein homology search was conducted with the Blast program from NCBI (9).

Production of Recombinant CTF1alpha

The cDNA insert from lambda ctf-15 was directly cloned into the EcoRI sites of a glutathione S-transferase fusion vector, pGEX-4T-1 (Pharmacia Biotech Inc.) to generate pGEX-4T-1/CTF1alpha (1-526). The resulting plasmid was introduced into BL21 cells for expressing glutathione S-transferase-CTF1alpha (1-526) fusion protein. Additionally, a BamHI fragment from pGEX-4T-1/CTF1alpha (1-526) encoding the DNA-binding domain of CTF1alpha was subcloned into a thioredoxin fusion vector, pET-32a(+) (Novagen) to generate pET-32a/CTF1alpha (DBD), which was introduced into BL21(DE3) to express TRX-CTF1alpha (DBD) fusion protein.

Partial Purification and Renaturation of TRX-CTF1alpha (DBD) Fusion Protein

TRX-CTF1alpha (DBD) fusion protein was induced (5), partially purified with a Ni2+-NTA agarose column (Qiagen), under denaturing conditions (10) and renatured in a dialysis bag as described (7). The dialyzed protein samples were concentrated with Centricon-30 filtration units pretreated overnight with 5% Tween 20 (Amicon).

DNA-binding Assay

The 37-mer palindromic element (-159 to -178 bp) containing both overlapping palindromes 1 and 2 was prepared as described (5). To prepare palindrome 1 fragment (pal 1), oligonucleotides aat tcG GAT CGC GAG CCg and aat tcG GCT CGC GAT CCg were annealed. To prepare palindrome 2 fragment (pal 2), oligonucleotides aat tCG AGC CGA GGC TCG and aat tCG AGC CTC GGC TCG were annealed. The annealed fragments contained palindrome 1 of 12 nucleotides and palindrome 2 of 14 nucleotides, each flanked on both ends by EcoRI sites (shown in lowercase). These EcoRI sites served for concatenation and fill-in labeling with [alpha -32P]dATP. DNA-binding assays by gel retardation with about 0.5 µg of partially purified recombinant PBP (5) and CTF1alpha were done essentially as described (4, 5).

Construction of Plasmids and Yeast Transformation

For expression of the full-length polypeptide of CTF1alpha (1-909), fragments for full-length cDNA clone were first amplified by polymerase chain reaction as separate 5'-end and 3'-end fragments with Pfu polymerase (Stratagene). These two fragments were then joined by SOEing polymerase chain reaction (11) and cloned in-frame into the PstI site of the yeast expression vector pGAD424, a GAL4 DNA-activation domain hybrid cloning vector (CLONTECH), to produce pGAD424/CTF1alpha . To delete the GAL4 DNA-activation domain from this chimeric plasmid, pGAD424/CTF1alpha was first digested with KpnI and SmaI. The large fragment containing the DNA segment for CTF1alpha was gel-purified by Geneclean (Bio 101) and ligated to a KpnI-SmaI linker that was prepared by annealing two oligomers, CGC CGC CGC and GCG GCG GCG GTA C. The resultant plasmid was designated pLEU/CTF1alpha . This plasmid allows for leucine selection in yeast and would produce a fusion polypeptide of SV40 NLS and CTF1alpha . As a negative control vector, the fragment for GAL4 DNA-activation domain in pGAD424 was also replaced as above with the KpnI-SmaI linker to produce pLEU.

For expression of the N-terminal 526 amino acids of CTF1alpha , an expression vector that allows for tryptophan selection in yeast was first modified from pGAD424 as follows. pGBT9, a GAL4 DNA-binding domain hybrid cloning vector from CLONTECH, was digested with PstI and PvuII. The small fragment coding for tryptophan was isolated and ligated to PstI and EcoRV-cut pGAD424 to produce expression vector pTRP-AD that allows for tryptophan selection in yeast. To remove the GAL4 DNA-activation domain from pTRP-AD, the plasmid was digested with KpnI and EcoRI. The large fragment was isolated and ligated to a KpnI-EcoRI linker that was prepared by annealing two oligomers, CGC GGC GGC GG and AAT TCC GCC GCC GCG GTA C. The resultant plasmid was designated pTRP, and it allows for tryptophan selection in yeast. To express the N-terminal 526 amino acids of CTF1alpha , the EcoRI fragment of lambda ctf1-15 was directly cloned into the EcoRI site of pTRP to generate pTRP/CTF1alpha (1-526).

To introduce into yeast the wild-type and mutant cutinase gene promoter/cat gene fusions, three yeast centromeric plasmids were constructed from pCAT360, pCAT433Delta pal1, and pCAT433Delta pal2 (4). The plasmid pCAT360 is a pBluescript KS vector that contains the E. coli gene for hygromycin phosphotransferase (hph) under the regulation of a Cochliobolus heterostrophus promoter and the gene for chloramphenicol acetyl transferase (cat) under the control of the wild-type cutinase gene promoter. pCAT433Delta pal1 and pCAT433Delta pal2 are similar to pCAT360 with the exception that in pCAT433Delta pal1, the sequence GGATCG in the first half of palindrome 1 was mutated to ATGAGC, and in pCAT433Delta pal2, the sequence GGCTCG in the second half of palindrome 2 was mutated to TATGGC (4). pCAT360 was digested with SalI and NotI to release the DNA fragment for the wild-type cutinase gene promoter/cat gene, and this fragment was cloned into the SalI and NotI sites of pRS413, a yeast centromere plasmid (YCp) that allows for histidine selection in yeast (Stratagene). The resultant plasmid is designated pYCAT.

Similarly, DNA fragments for mutant cutinase gene promoter/cat gene fusions were isolated from pCAT433Delta pal1 and pCAT433Delta pal2 by digesting the plasmids with XbaI and SalI. The isolated DNA fragments were then cloned into XbaI- and SalI-digested pRS413 to generate pYDelta pal1 and pYDelta pal2, respectively.

Plasmids were introduced with manufacturer's protocols (CLONTECH) into yeast strain YPH499 (MATa ura3-52 lys2-801amber ade2-101orche trp1-Delta 63 his3-Delta 200 leu1-Delta 1) (Stratagene) in the following combinations, pLEU and pYCAT, pTRP and pYCAT, pTRP/CTF1alpha (1-526) and pYCAT, pLEU/CTF1alpha and pYCAT, pLEU/CTF1alpha and pYDelta pal1, and pLEU/CTF1alpha and pYDelta pal2. Yeast transformants were selected on tryptophan- and histidine-lacking or leucine- and histidine-lacking minimal medium (CLONTECH) according to the above combinations of plasmids. For long-term storage at -80 °C, the resultant transformants were scraped from plates and resuspended in 15% glycerol in screw-capped tubes.

Growth of Yeast Transformants and CAT Assay

Yeast transformants were grown in 6.5-ml minimal medium in 50-ml conical tubes for 36-48 h (CLONTECH). Cells were harvested by centrifugation, washed with 10 ml of H2O once, and resuspended in 0.4 ml of 0.1 mM Tris·HCl, pH 7.8, and cells were disrupted for 3 min in a Mini-Beadbeater (Biospec Products, Bartlesville, OK) in the presence of 100 mg of glass beads (710-1180 µm) (Sigma). The mixture was transferred to a new tube, incubated for 10 min at 65 °C, and then centrifuged at 16,000 × g for 10 min at ambient temperature. Protein concentration of the supernatant was determined with a Bio-Rad protein assay kit (Bio-Rad) with bovine serum albumin as standard. CAT assays were done as described with D-threo-[dichloroacetyl-1,2-14C]chloramphenicol (DuPont NEN) as substrate (4). To obtain the linear rate of acetylation of chloramphenicol, the yeast protein supernatant was diluted appropriately, and different aliquots were assayed for CAT activity. The products were separated and quantitated with a PhosphorImager (Molecular Dynamics) (4). Relative CAT activity was expressed as a percentage of chloramphenicol acetylated.


RESULTS

Previously, we isolated a zinc finger protein, PBP, that specifically bound to the palindromic fragment (5) containing both of the overlapping palindromes present in the cutinase gene promoter. Because palindrome 2 is the one essential for induction of cutinase by hydroxy fatty acids, we tested to determine its binding specificity of PBP to the individual palindromes by gel retardation assay. PBP bound only to the palindrome 1 DNA fragment but not to palindrome 2 DNA fragment (data not shown). Therefore, additional protein factors that specifically bind to this palindrome 2 were sought. We screened lambda gt11 expression library again to search for clones encoding CTF1 that specifically binds palindrome 2 (4). Because the phage clones encoding PBP were isolated using the fragment containing both palindromes, the phage clones obtained during that screening may also contain clones that would encode CTF1. During the tertiary screening for PBP clones, 27 discrete clones were obtained. Polymerase chain reaction tests indicated that 10 of the phage clones belonged to those encoding PBP (data not shown). The remaining 17 clones showed five distinctly different restriction patterns, and the representative clones from each group were designated as lambda ctf1-8, lambda ctf1-11, lambda ctf1-15, lambda ctf1-22, and lambda ctf1-26. When tested by Southwestern hybridization (7), all five clones showed binding to the concatenated palindrome 2 fragment (data not shown). DNA inserts from these phage clones were subcloned into pBS KS- to generate pCTF1-8, pCTF1-11, pCTF1-15, pCTF1-22, and pCTF1-26. Initial sequencing of these clones indicated that pCTF1-8 was a partial clone of pCTF1-15, pCTF1-22 was a partial clone of pCTF1-26, and pCTF1-11 was a distinct clone.

The inserts in pCTF1-15, pCTF1-11, and PCTF1-26 were completely sequenced, and the deduced polypeptides revealed the presence of Cys6Zn2 binuclear cluster DNA-binding motifs. We chose to characterize lambda ctf1-15 here because it produced the strongest signal in the Southwestern hybridization (7) (data not shown). The polypeptide encoded by the DNA insert in pCTF1-15 was designated CTF1alpha (Fig. 1). The lack of in-frame stop codon for the DNA insert indicated that this clone represented a partial open reading frame. Further screening of lambda gt11 libraries identified four additional overlapping clones for CTF1alpha (Fig. 1). The complete sequencing of the five overlapping clones for CTF1alpha revealed a contiguous cDNA sequence of 3271 bp containing a complete open reading frame (Fig. 1).


Fig. 1. Characterization of clones for CTF1alpha . In A, the open reading frame is shown as a thick solid line. The full-length cDNA clone for CTF1alpha was determined from five overlapping clones (shown below the thick solid line). B, deduced amino acid sequence of CTF1alpha . The putative DNA-binding domain is underlined, and the six conserved cysteine residues are in boldface. C, putative motifs and conserved sequence consensus for modifications by protein kinases in CTF1alpha .
[View Larger Version of this Image (47K GIF file)]

The 3271-bp contiguous sequence for CTF1alpha contained a single open reading frame of 2727 bp that would encode a protein of 909 amino acids with a calculated molecular weight of 101,109 (Fig. 1). CTF1alpha contains 11 potential consensus sequences for phosphorylation by casein kinase II (12), 10 potential sites for phosphorylation by the cyclic AMP-dependent protein kinase A (13), and 6 potential phosphorylation sites for protein kinase C (14). Two consensus sites, PQTP and PATP, for potential phosphorylation by mitogen-activated protein kinase were also present (15). Six potential asparagine-glycosylation sites (16) were observed. Three plausible nuclear localization signals with the residue patterns of KRKK, KRHRK, and PKRK(17) were identified in CTF1alpha , with a certainty of 0.7 (on a scale of 1) of it being localized in the nucleus as predicted by PSORT. Homology searches identified only a Cys6Zn2 binuclear cluster domain located at the N terminus from amino acid residues 59 to 92 of CTF1alpha (Fig. 2).


Fig. 2. Homologies of the Cys6Zn2 binuclear cluster motif of CTF1alpha of F. solani f. sp. pisi; GAL4, ARGRII, PPR1, PDR1, PUT3, HAP1, and UGA3 of S. cerevisiae; LAC9 of K. lactis; MAL63 of S. carlsbergensis; NIT4 of N. crassa; NIRA, UAY, QUTA, and AMDR of A. nidulans; and AFLR of A. flavus. The conserved cysteine residues are indicated with asterisks (*), and the conserved proline residue is indicated with a dot (bullet ).
[View Larger Version of this Image (72K GIF file)]

As an initial step to test the function of the identified CTF1alpha , segments of the DNA fragment coding for CTF1alpha were cloned into expression vectors. When the glutathione S-transferase fusion vector was used to express the first 526 amino acids of CTF1alpha , no protein was detected (data not shown). When thioredoxin (TRX) fusion vector was used to express the Cys6Zn2 binuclear cluster DNA-binding domain of CTF1alpha , fusion protein of TRX-CTF1alpha (DBD) was detected (data not shown). Even though the expressed TRX-CTF1alpha (DBD) fusion protein was insoluble, solubilization with 6 M guanidine hydrochloride, partial purification, and renaturation yielded a soluble protein preparation. DNA-binding assays by gel retardation were performed with this renatured protein. As shown in Fig. 3A, a single retarded band of palindrome fragment-CTF1alpha complex was observed. That the binding of CTF1alpha to the palindrome was specific was indicated by the observation that unlabeled palindromic fragment but not an unrelated DNA fragment competed for the binding by the labeled palindromic fragment. In addition, CTF1alpha showed binding only to palindrome 2 but not to palindrome 1 (Fig. 3B). Mutations in the first half of palindrome 2 that overlaps with palindrome 1 drastically reduced CTF1alpha binding (Fig. 3C). Mutations in the second half of palindrome 2 abolished CTF1alpha binding. Substitution of the G-nucleotides previously found to be at the binding site for the naturally occurring palindrome 2 (4) also abolished binding of palindrome 2 to the recombinant CTF1alpha (Fig. 3C). These results demonstrate the sequence specificity of palindrome 2 involved in binding CTF1alpha .


Fig. 3. Binding of expressed CTF1alpha to palindrome 2 fragment. In A, partially purified and renatured TRX-CTF1alpha (DBD) (0.5 µg) was incubated with 32P-labeled palindromic fragment in the presence of molar excesses of either nonspecific competitor DNA fragments or specific unlabeled palindromic DNA fragments. In B, TRX-CTF1alpha (DBD) (0.5 µg) was incubated with 32P-labeled DNA fragments (pal 1, palindrome 1; pal 2, palindrome 2; pal (1+2), the palindromic fragment). In C, TRX-CTF1alpha (DBD) (0.5 µg) was incubated with 32P-labeled mutant pal 2 fragments or the wild-type pal 2 fragment: pal2M1/2, aat tGC CTC CGA GGC TCG; pal2M1, aat tCG AGC CGA TAT GGC; pal2M2, aat tCA AAT TAA AAT TTG; and wild-type pal2, aat tCG AGC CGA GGC TCG. Only the sense strands are listed here with the mutated or substituted nucleotides underlined and the flanking EcoRI site in lowercase. All samples were then separated on a 6% polyacrylamide gel. C, DNA-protein complex; F, free DNA probe.
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The above tests indicated that CTF1alpha is a protein factor that binds the cutinase promoter; therefore, we tested whether it could function as a cutinase transcriptional activator in vivo. Plasmid constructs were made to express a hybrid fusion protein in which CTF1alpha was fused to the nuclear localization sequence of SV40. The CTF1alpha hybrid protein was tested for activation of transcription of the cat gene fused to the cutinase promoter. Its transactivating capability was indicated by the production of high levels of CAT activity when the full-length cDNA clone for CTF1alpha was introduced into yeast cells carrying the wild-type cutinase gene promoter/cat gene fusion cassette (Fig. 4B, lanes WT and pLEU/CTF1alpha ). Mutations in the first half of palindrome 1 did not change the CAT activity appreciably (Fig. 4B, lane Delta pal 1), whereas mutations in the second half of palindrome 2 resulted in a drastic decrease of CAT activity (Fig. 4B, lane Delta pal 2). That the C-terminal segment of CTF1alpha was essential for the transactivation was shown by the observation that expression of the hybrid protein containing the N-terminal 526 amino acids of CTF1alpha fused to the nuclear localization sequence of SV40 failed to activate the cutinase gene promoter/cat gene (Fig. 4B, lane WT and pTRP/CTF1alpha (1-526)).


Fig. 4. Activation of the cutinase gene promoter/cat gene fusion cassette by CTF1alpha in vivo in yeast. A, mutations (in boldface) in the first half of palindrome 1 (pYDelta pal1) and the second half of palindrome 2 (pYDelta pal2, carrying the same mutations as those in the fragment pal 2M1 used for gel retardation assay in Fig. 3C). pYCAT refers to the yeast centromere plasmid that carried the wild-type cutinase gene promoter/cat gene fusion cassette. B, CAT activities were determined with yeast whole-cell extract (5 µg of protein). CAT Act. (CAT activity) indicates the percentage of chloramphenicol acetylated for the assay shown. Ave. ± SD refers to the mean and S.D. of three independent experiments with three duplicates in each experiment. WT, Delta pal1, and Delta pal2 refer to pYCAT, pYDelta pal1, and pYDelta pal2, respectively. Control, no protein CAT assay.
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DISCUSSION

Of the two overlapping palindromes in the palindromic DNA segment, palindrome 2 is the one that confers inducibility of the cutinase gene expression by plant cutin monomers. Therefore, we sought a transcription factor specific for palindrome 2 by screening phage clones originally obtained during the screening of the lambda gt11 expression library for PBP clones (5). This screening identified three protein factors, and one of them was designated CTF1alpha .

Mutations in the first half of palindrome 2 that overlaps with palindrome 1 reduced the inducibility of the promoter by over 60-fold, and mutations in the second half of palindrome 2 abolished any inducibility of the promoter in vivo (4). The specific binding of the expressed CTF1alpha to palindrome 2 indicated by the gel shift assays suggest that the cloned protein is one of the factors involved in the regulation of cutinase gene expression in vivo. In further support of this conclusion is the finding that substitution of the G residues in palindrome 2, previously found to be involved in the binding of the naturally occurring cutinase transcription factor, abolished the binding of the expressed CTF1alpha .

CTF1alpha was demonstrated to be capable of functioning in vivo as a positively acting transcription factor of the cutinase gene promoter in yeast. CTF1alpha fused to the nuclear localization sequence was able to activate transcription of the cat gene (Fig. 4B). That this transactivation involved specific binding to palindrome 2 was strongly suggested by the observation that mutation of palindrome 2, but not palindrome 1, abolished the transactivation (Fig. 4B). The notion that the Cys6Zn2 DNA-binding domain in the cutinase transcription factor is in the N terminus and the activation domain would be expected to be in the C terminus is consistent with the finding that expression of the hybrid protein containing the N-terminal 526 amino acids of CTF1alpha fused to the nuclear localization sequence of SV40 failed to activate the cutinase gene promoter/cat gene (Fig. 4B). These results demonstrate that CTF1alpha functions as a transcriptional activator for palindrome 2 of the cutinase gene promoter in vivo. Mutations in palindrome 1 elevated inducibility of the cutinase gene promoter by 2-fold in F. solani f. sp. pisi in vivo (4), suggesting that PBP may function as a repressor by competing for binding the overlapping palindromes.

The presence of putative nuclear localization signals suggested that CTF1alpha may be a nuclear protein. The N-terminal Cys6Zn2 binuclear cluster motifs found in CTF1alpha is probably involved in the binding to the palindrome. Such DNA-binding motif is characteristic of other regulatory proteins such as GAL4 (18), ARGRII (19), PPR1 (20), PDR1 (21), PUT3 (22), HAP1 (23), and UGA3 (24) of Saccharomyces cerevisiae; LAC9 of Kluyveromyces lactis (25); MAL63 of S. carlsbergensis (26); NIT4 of Neurospora crassa (27); NIRA (28), UAY (29), QUTA (30), and AMDR (31) of Aspergillus nidulans; and AFLR of A. flavus (32). However, CTF1alpha does not share homology to any other regions of those factors. Functionally, GAL4 is a positive activator that regulates the transcription of the galactose-inducible genes GAL1, GAL2, GAL7, GAL10, and MEL1 (18). PPR1 positively regulates transcription of the genes URA1, URA3, and URA4 involved in controlling pyrimidine levels (20). Some of these protein factors recognize a DNA sequence with two inverted repeats of CGG elements separated by a spacing characteristic of the specific protein factor (33). For example, PPR1 recognizes 5'-CGG(n6)CCG with a spacer of 6 nucleotides, whereas the spacing for GAL4, PUT3, PPR1, and LEU3 is 11, 10, 6, and 4 nucleotides, respectively (34). HAP1, on the other hand, binds a direct repeat of CGG triplet with a spacer of six nucleotides (33). Interestingly, CTF1alpha binds to a palindrome with an oppositely oriented 5'-GCC(n2)GGC (4).

The presence of nuclear localization signals, sequence-specific binding to the region of the cutinase gene that is involved in the induction of cutinase gene by hydroxy fatty acid, and the demonstrated capability of the cloned factor to function as a transcription factor suggests that CTF1alpha is a DNA-binding transcriptional activator involved in mediating the inducibility of the cutinase gene promoter by plant cutin.

Phosphorylation of DNA-binding protein factors play an important role in nuclear localization (35), DNA binding, and transactivation (36, 37). Previously, it was shown that cutin monomers caused phosphorylation of nuclear proteins (38) and when nuclear extracts from cutin monomer-induced culture were treated with immobilized phosphatase binding to the cutinase gene promoter was abolished (38). In addition, protein phosphorylation was detected only in the presence of cutin monomers. Furthermore, transcription of the cutinase gene induced by hydroxy fatty acids in a nuclear preparation from F. solani f. sp. pisi showed a 30-min lag period during which phosphorylation of a 50-kDa protein occurred and inhibition of phosphorylation by protein kinase inhibitors strongly inhibited cutinase gene transcription induced by the hydroxy fatty acids (38). On the basis of such results, it was suggested that protein phosphorylation may be involved in the activation of cutinase gene transcription by hydroxy fatty acids. In fact, CTF1alpha contains numerous putative consensus sites for phosphorylation by CKII, protein kinase C, and two possible consensus sites for mitogen-activated protein kinase. Whether any of these consensus sites are phosphorylated in vivo remains to be elucidated.

Activation of the cutinase gene transcription by hydroxy fatty acids occurs only when glucose in the culture medium is depleted (39). This catabolite repression could involve cyclic AMP; experimental evidence had suggested that the involvement of cyclic AMP in cyclic AMP levels in the fungus was elevated almost 3-fold upon glucose depletion, and exogenous cyclic AMP could derepress the glucose effects.2 CTF1alpha contains several potential sites for phosphorylation by protein kinase A, although it is not known whether they are actually involved in cutinase gene activation.


FOOTNOTES

*   This work was supported in part by National Science Foundation Grant IBN-9318544.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U51671[GenBank].


Dagger    To whom correspondence should be addressed: Neurobiotechnology Center, Ohio State University, 206 Rightmire Hall, 1060 Carmack Rd., Columbus, OH 43210. Tel.: 614-292-5682; Fax: 614-292-5379; E-mail: Kolattukudy.2{at}osu.edu.
1   The abbreviations used are: bp, base pair(s); CTF, cutinase transcription factor; PBP, palindrome-binding protein; CAT, chloramphenicol acetyltransferase; TRX, thioredoxin.
2   U. Kämper and P. E. Kolattukudy, unpublished data.

ACKNOWLEDGEMENT

We thank Kristi Rupert for help with DNA preparations.


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