Isolation and Characterization of cDNA for DREF, a Promoter-activating Factor for Drosophila DNA Replication-related Genes*

DREF, a transcription regulatory factor which specif- ically binds to the promoter-activating element DRE (DNA replication-related element) of DNA replication- related genes, was purified to homogeneity from nuclear extracts of Drosophila Kc cells. cDNA for DREF was isolated with the reverse-transcriptase polymerase chain reaction method using primers synthesized on the basis of partial amino acid sequences and following screening of cDNA libraries. Deduced from the nucleo- tide sequences of cDNA, DREF is a polypeptide of 701 amino acid residues with a molecular weight of 80,096, which contains three characteristic regions, rich in basic amino acids, proline, and acidic amino acids, respec- tively. Deletion analysis of bacterially expressed DREF fused with glutathione S -transferase (GST-DREF) indi- cated that a part of the N-terminal basic amino acid region (16–115 amino acids) is responsible for the spe- cific binding to DRE. A polyclonal and four monoclonal antibodies were raised against the GST-DREF fusion protein. The antibodies inhibited specifically the transcription of DNA polymerase (cid:97) promoter in vitro . Cotransfection experiments using Kc cells demonstrated that overproduction of DREF protein overcomes the repression of the proliferating cell nuclear antigen gene promoter by the zerknu¨llt gene product. These results confirmed that DREF is a trans-activating factor for DNA replication-related

A number of enzymes involved in DNA replication have been suggested to form an enzyme complex for this purpose (1). Genes for these enzymes are expressed in proliferating cells and repressed in quiescent cells reaching confluency or in association with cellular differentiation (2). Therefore, it is of interest to clarify the genetic mechanisms governing the coordinate induction or repression of DNA replication-related genes in relation to growth or differentiation signals.
In budding yeast, the MluI cell cycle box and the specific binding factor, DSC-1, are responsible for cell cycle-dependent transcription of a number of DNA replication-related genes (3)(4)(5)(6).
The mRNAs for human DNA polymerase ␣, PCNA, 1 murine DNA polymerase ␣-primase complex, and thymidylate synthetase are present throughout the cell cycle and increase slightly prior to the S phase (7)(8)(9)(10). The critical promoter regions of DNA polymerase ␣, dihydrofolate reductase (DHFR), and thymidine kinase genes contain binding sites for the E2F family (11)(12)(13). Mutagenesis of the promoter of the DHFR gene has provided strong evidence that the E2F element is responsible for the promoter activation in late G 1 (14). Furthermore, the E2F-binding site was shown to be involved in activation of the DHFR and thymidine kinase genes following serum stimulation (15,16). The fact that the active form of E2F accumulates in late G 1 toward the S phase (17)(18)(19) provides further evidence that E2F family members are likely candidates for involvement in transcriptional regulation of specific G 1 /S-phase-activated genes that are required for DNA replication. The E2F binding sites of the Drosophila genes for DNA polymerase ␣ and PCNA are also important for their proliferation-related expression (20,21). However, little is known about regulation mechanisms of DNA replication-related genes during the transition from G 0 into the cell cycle or the transition from proliferating to quiescent states.
We have analyzed upstream regulatory regions of Drosophila genes for the DNA polymerase ␣ 180-kDa catalytic subunit (22) and the PCNA (23), and found a novel transcription regulatory sequence consisting of an 8-bp palindromic sequence (5Ј-TATCGATA), called DRE (DNA replication-related element) and a specific binding factor, DREF (DRE-binding factor) (24). Three DREs and one DRE are present in the DNA polymerase ␣ and PCNA genes, respectively. Transient CAT expression and gel mobility shift assay using cultured Kc cells indicated that DRE stimulates the promoter activities and that DREF can bind specifically to DRE (24).
Another aspect is that promoters of Drosophila DNA replication-related genes are repressed by the product of the zerknü llt (zen) gene, a homeobox gene which regulates the differentiation of the optic lobe and the amnioserosa in the dorsal region of the Drosophila embryo (25)(26)(27). Repression of promoter activities by Zen protein has been observed not only in cultured Kc cells but also in transgenic flies carrying the PCNA gene promoter-directed lacZ gene (28,29). Recently we obtained evidence indicating that overexpression of Zen protein results in reduction of DREF activities in the cell (29). There-fore, DREF may be one of the key transcription regulatory factors involved in proliferation-and differentiation-related control of DNA replication genes.
We earlier purified DREF as a homodimer of an approximately 86-kDa polypeptide to near homogeneity (24). In the present study, we isolated cDNA for DREF and demonstrated the involvement of DREF in the high level of expression of DNA replication-related genes with DRE sequences.

EXPERIMENTAL PROCEDURES
Cell Culture-Kc cells derived from Drosophila early embryos were grown in spinner culture at 25°C using M3(BF) medium (30) supplemented with 2% fetal calf serum in the presence of 5% CO 2 .
Purification of DREF-The DREF polypeptide was purified using DRE oligonucleotide-conjugated latex particles and gel filtration column chromatography as described earlier (24). It was then concentrated using a Centricon 30 (Amicon, Inc.) and precipitated by adding trichloroacetic acid to a final concentration of 10%.
Determination of the Partial Amino Acid Sequences-The trichloroacetic acid-precipitated DREF polypeptide (20 g) was washed with ice-cold acetone, dissolved in sample buffer for SDS-polyacrylamide gel electrophoresis, boiled for 1 min, separated on an 8% SDS-polyacrylamide gel, and electrophoretically blotted onto a sheet of Glassybond paper. Membrane-bound protein was subjected to in situ proteolytic cleavage using endopeptidase Lyso-C (Wako) in 0.1 M NH 4 HCO 3 at 37°C for 24 h. The resulting oligopeptide mixture was separated with an HPLC apparatus equipped with a reverse-phase C4 column. Oligopeptide-containing fractions were collected manually into Eppendorf tubes. When necessary, each of the peak fractions was further purified with rechromatography on a reverse-phase C18 column. Samples were stored at Ϫ70°C before analysis. Each sample was adsorbed to a disc of Glassybond, and the amino acid sequence was analyzed with an Applied Biosystems model 470A automated gas-phase protein sequencer.
Oligonucleotides-All oligonucleotides were chemically synthesized using an Applied Biosystems DNA synthesizer. Degenerate primers for reverse transcriptase-PCR were synthesized depending on the partial amino acid sequences of DREF. The synthesized oligonucleotides sequences were as follows: I, 5Ј-TT (C/T I-V were also  synthesized (IR, IIR, IIIR, IVR, and VR).
To amplify a sequence containing a complete open reading frame, a set of PCR primers with a BamHI site was synthesized: 5Ј-specific oligonucleotide, 5Ј-ACAGGATCCAAGATGAGCGAAGGGGTACCA, and 3Ј-specific oligonucleotide, 5Ј-ATCCTAATTGTTGTGATGATGCT, where sequences with underlining indicate the BamHI site, translation initiation codon, and stop codon of the DREF cDNA, in that order.
Full-length cDNA clones for DREF were isolated by screening two kinds of gt10 cDNA libraries constructed from mRNAs obtained from 0 -3-h-old and 3-12-h-old Drosophila embryos under high stringency conditions using a 32 P-labeled 1.8-kb cDNA fragment excised from pDCDREF1.8 as a probe. The obtained cDNA clones (2.7 kb) had a single EcoRI site and, therefore, two EcoRI fragments of 1.0 and 1.7 kb were excised from gt10 DNA. Each EcoRI fragment was subcloned into the EcoRI site of pBluescript II SK(Ϫ) (pDCDREF1.0 and pDC-DREF1.7) and sequenced.
A cDNA containing a complete open reading frame without 5Ј-and 3Ј-untranslated sequences was obtained with 30 cycles of PCR (1 min at 94°C, 1 min at 55°C, and 2 min at 72°C) using 10 ng of DNA of gt10 clone carrying full-length cDNA as a template and 5Ј-and 3Ј-specific oligonucleotides as primers. The resultant DNA fragment was digested with BamHI and subcloned into BamHI-SmaI sites of pBluescript II SK(Ϫ) (pDCDREF2.2).
Expression of GST-DREF Fusion Proteins in Escherichia coli-To construct deletion mutant GST-DREF expressing plasmids, 27 mer PCR primers were synthesized. All primers for coding and complementary strands were designed to contain BamHI and EcoRI sites, respectively.
We produced recombinant DREF fused to GST. A construct containing the full-length DREF (amino acids 1-701; pGST-DREF1-701) was created by inserting a 2.2-kb cDNA fragment from pDCDREF2.2 with BamHI and EcoRV into BamHI and SmaI sites of pGEX-2T. A construct containing amino acid residues 16 -608 (pGST-DREF16 -608) was obtained by excising a 1.8-kb cDNA fragment from pDCDREF1.8 with EcoRV and BamHI, filling the protruding-end with Klenow fragment and inserting it into the SmaI site of pGEX-3X. This expression plasmid was used for large scale preparation of the DREF polypeptide, which was then applied as an antigen to raise antibodies. Extraction and purification of recombinant protein were carried out as described previously (35).
Constructs containing the N-terminal basic region (amino acids 16 -242; pGST-DREF16 -242) and C-terminal acidic region (amino acid 240 -608; pGST-DREF240 -608) were prepared by partially digesting pGST-DREF16 -608 with EcoRI and religating the plasmid DNA with T4 DNA ligase. cDNAs of the other GST-DREF mutants (see Fig. 2) were amplified by PCR with EcoRI-BamHI sites and inserted into pGEX-2T. All constructs were sequenced. DREF-fusion proteins were produced in E. coli essentially as described earlier (36). Lysates of cells were prepared by sonication in buffer D containing 0.6 M KCl, 1 mM phenylmethylsulfonyl fluoride, and 1 g/ml each of pepstatin, leupeptin, and aprotinin. Lysates were clarified by centrifugation at 12,000 ϫ g for 20 min at 4°C, and used for analysis by SDS-polyacrylamide gel electrophoresis, gel mobility shift assay, or footprinting.
Gel Mobility Shift Assay-Preparation of nuclear extracts from Kc cells and conditions of gel mobility shift assay were as described elsewhere (24).
o-Phenanthroline-Copper Footprinting-The probe for o-phenanthroline-copper footprinting was prepared as described earlier (37). The DNA fragment excised from plasmid pHP3 (Ϫ292 to ϩ45 region of the DNA polymerase ␣ gene) (24) was labeled at the 5Ј-end of either the upper or the lower strand using T4 polynucleotide kinase and [ 32 P]ATP. The probe (1 ng, 5 ϫ 10 4 cpm) was incubated with an extract (1 g of protein) of E. coli producing GST-DREF16 -242 in 30 l of solution containing 15 mM Hepes (pH7.6), 100 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 12% glycerol, 1 g of poly(dI-dC), and 1 g of sonicated calf thymus DNA on ice for 20 min. The resulting complexes were resolved on a 3% polyacrylamide gel. After electrophoresis, one of the glass plates was removed. The gel attached to the other glass plate was transferred to a glass dish containing 200 ml of 10 mM Tris-HCl (pH 8.0), and equilibrated for 1 h at room temperature. Then, 20 ml of 0.9 mM CuSO 4 , 4 mM 1,10-phenanthroline solution were added to the equilibrated gel. The cleavage reaction was initiated by adding 20 ml of 0.5% 3-mercaptopropionic acid, allowed to proceed for 1 min at room temperature, and stopped by adding 10 ml of 1.2% 2,9-dimethyl-1,10-phenanthroline. The gel was rinsed twice with distilled water and exposed to x-ray film for 4 h at 4°C. Portions of gel containing DNA-protein complexes or free DNA probe were excised and incubated in 500 l of DNA elution buffer containing 1% SDS, 0.2 M NaCl, 20 mM EDTA, and 1 mg/ml tRNA overnight at 37°C to elute the DNA. The elution buffer was removed from the gel fragment and extracted once with phenol/ chloroform, and the DNA was ethanol-precipitated. The DNA was then dissolved in formamide-loading dye solution and resolved on a 6% denaturing polyacrylamide gel in parallel with products of Maxam-Gilbert sequencing reactions (38) using the same DNA used as a probe for sequencing controls. After electrophoresis, gels were dried and autoradiographed.
Preparation of Antibodies against DREF-Rabbit antiserum and mouse monoclonal antibodies against DREF polypeptide were raised against the bacterially produced recombinant DREF consisting of 16 -608 amino acids fused with GST. Monoclonal antibodies were screened by enzyme-linked immunoassay using the recombinant GST-DREF polypeptide. If necessary, each of monoclonal antibodies was purified from culture medium of hybridomas using E-Z-SEP (Pharmacia Biotech Inc.). Polyclonal antibody specific for DREF polypeptide was purified from antiserum using a GST-DREF-conjugated Sepharose column after passing through a GST-conjugated-Sepharose column. The obtained IgG was precipitated at 50% ammonium sulfate saturation, dissolved in phosphate-buffered saline, and dialyzed against phosphate-buffered saline.
Expression Plasmid, DNA Transfection, and CAT Assay-The expression plasmid pUAS-DREF1-701 contained the full-length DREF coding sequence placed under the promoter consisting of five Gal-4 binding sites and the hsp70 TATA box (pUAST vector). The plasmid pAct-Gal-4 plasmid expressed full-length yeast Gal-4 protein under control of Drosophila actin 5C promoter (pAcT-GEM3 vector). DNA transfection was carried out by the calcium phosphate coprecipitation technique described earlier (23). Two g of p5ЈϪ168DPCNACAT as a reporter plasmid and 2 g of pAct-Gal-4 were cotransfected with indicated amounts of pUAS-DREF1-701 and pAct5C-zen as expression plasmids. Total amount of effector plasmid was kept constant by addition of the expression vectors pAcGEM3 and pUAST. At 48 h after transfection, cell extracts were prepared and CAT activity was measured as described previously (23). The CAT activity was quantified with an imaging analyzer BAS2000 (Fuji film). CAT activity was normalized to the protein amount.
In Vitro Transcription Reaction-The template used in in vitro transcription assay was the plasmid Ϫ1107DPOLACAT containing Ϫ1107 to ϩ45 of the DNA polymerase ␣ gene in the plasmid pOLCAT as described earlier (24). In vitro transcription was performed with Kc cell nuclear extracts prepared as detailed previously (24). Kc nuclear extract (40 g of protein) was preincubated with 2 g of control or anti-DREF IgG in 10 l of solution containing 50 mM Hepes (pH 7. The open reading frame with initiation and stop codons, at nucleotide numbers 571 and 2683, respectively, encodes a protein of 701 amino acid residues (shown in single letter code below the nucleotide sequence). Peptide sequences determined by microsequencing after lysyl-endopeptidase digestion of DREF are underlined. A putative polyadenylation signal is shown by bold letters. B, schematic structure of DREF. The region rich in basic amino acids (61-203 amino acids) and the region rich in acidic amino acids (218 -390 amino acids) are shown by the hatched and shaded boxes, respectively. The proline-rich region (99 -217 amino acids) is also indicated. NLS represents a putative nuclear localizing sequence (143-151 amino acids; RRRRTPPRK). The amino acids essential for DRE-binding (16 -105 amino acids) are also indicated by a bracket. M NaCl. After phenol/chroloform extraction, RNA was precipitated with ethanol. Primer extension analysis was carried out essentially as described earlier (39) using an oligonucleotide (28 mer) synthesized based on the nucleotide sequence of the CAT gene as a primer, 5Ј-GTGG-TATATCCAGTGATTTTTTTCTCCA. Quantification was achieved with an image analyzer BAS2000 (Fuji film).
Northern Analysis-Total RNA was extracted from Kc cells by the acid guanidinium thiocyanate-phenol-chloroform method (31). Twenty micrograms of total RNA were separated on a 1% agarose gel containing formaldehyde and blotted onto a sheet of GeneScreen Plus membrane (DuPont). Probes were radiolabeled using the random primer method (32). Hybridization and washing conditions were the same as described elsewhere (22). Blots were exposed to Kodak X-Omat XAR films and also to a Fuji-imaging plate, and relative radioactivity was quantified using a Fuji BAS 2000 imaging analyzer.
Immunocytochemical Staining of Embryos-Canton S embryos (0 -3 h old) were collected, dechorionated, fixed, and devitellinized as described earlier (40). Embryos were blocked with Tris-buffered saline containing 20% normal donkey serum, and 0.15% Triton X-100, and then incubated in Tris-buffered saline with either 5 g/ml of rabbit anti-DREF IgG (used at a dilution 1:100) or hybridoma culture supernatant. Secondary antibodies were used at a dilution of 1:500 and color development was performed as for the Western blotting. Samples were examined under a microscope (Olympus BX-50) and photographed using TMAX 100 films (Eastman Kodak Co.).

RESULTS
Cloning of cDNA for DREF-DREF was affinity-purified from the nuclear extract of Kc cells using DRE-conjugated latex particles and chromatography on a gel filtration column as described earlier (24). The purity of the DREF polypeptide, which has an apparent molecular mass of 86 kDa, was greater than 90%. This polypeptide specifically bound to the DREs from DNA polymerase ␣ and PCNA genes as described previously (24). Yield of the DREF polypeptide was about 1 g from 1 g of Kc cells. Twenty g of purified DREF were subjected to SDS-polyacrylamide gel electrophoresis, and the 86-kDa polypeptide was isolated from the gel. Automated Edman degradation revealed that the N-terminal amino acid was blocked. Thus, we determined amino acid sequences of oligopeptides obtained by digesting the DREF polypeptide with endopeptidase Lyso-C. Oligopeptides were separated by reverse-phase HPLC and subjected to microsequence analysis. Three oligopeptide sequences were obtained as indicated with underlining in Fig. 1.
Reverse-transcriptase-PCR with all possible combinations of degenerated primers deduced from amino acid sequences was performed to isolate cDNA fragments. PCR products of 1.7 and 1.8 kb were obtained with primer III in combination with primers VR and IIR, respectively. The 1.8-kb cDNA fragment was used as a probe to screen cDNA libraries (gt10) prepared with mRNAs extracted from 0 -3-h or 3-6-h embryos. Eight clones were isolated from 4 ϫ 10 5 phages.
Southern blot analysis of Drosophila genomic DNA using the 1.8-kb cDNA fragment as a probe indicated that the DREF gene is present as a single copy (data not shown).
Structure of DREF-The nucleotide sequence containing a complete DREF-coding region which was determined from three cDNA clones is shown in Fig. 1. This 2.7-kb cDNA fragment appears to be nearly full-length since Northern analysis demonstrated a single mRNA species of approximately 2.8 kb (see Fig. 6), and nucleotide sequence analysis revealed that 5Ј-ends of eight cDNA clones independently isolated were mapped within 200 bp. In a primer extension experiment, the major transcription start site was mapped to 570 bp upstream from the ATG translation initiation codon (data not shown).
The open reading frame encodes a polypeptide of 701 amino acid residues with a predicted molecular weight of 80,096 and pI of 8.5. A data base search using the FASTA program did not cover any significant homology with reported conserved motifs.
DREF contains three characteristic regions, 1) rich in basic amino acids (28.7% between amino acid residues 61 and 203), 2) rich in proline (17.9% between amino acid residues 99 and 217), and 3) rich in acidic amino acids (18.0% between amino acid residues 218 and 390), as shown in Fig. 1B. DREF also contains a putative consensus sequence for the nuclear localization signal (RRRRTPPRK) at 143-151 residues. However, typical structures reported to be responsible for DNA binding, transactivation, and dimerization were not found.
DRE Binding Potential of DREF Produced in E. coli-To ascertain the DRE binding activity, we overexpressed DREF protein in the form of a fusion protein with GST in E. coli. We tested DNA binding using E. coli extracts carrying expression plasmids GST-DREF16 -242 and GST-DREF230 -608 by a gel mobility shift assay in which the DRE-P oligonucleotide was used as a probe. DRE binding activity was found to be associ-

FIG. 2. DRE binding of deletion mutants of GST-DREF.
A, expression of deletion mutants of the GST-DREF fusion protein. E. coli carrying plasmids expressing deleted DREF fused to GST were inoculated in 2 ml of LB medium containing 50 g/ml ampicillin, and synthesis of GST-DREF was induced at 37°C for 1 h by adding 1 mM ␤-D-isopropyl-thiogalactopyranoside. E. coli were pelleted by centrifugation from 100 l of culture medium, resuspended in SDS sample buffer, boiled for 3 min and subjected to electrophoresis on an 8% SDS-polyacrylamide gel. B, DRE binding activities of deletion mutants of GST-DREF fusion protein. E. coli lysates expressing GST-DREF were prepared as described under "Experimental Procedures." A gel mobility shift assay with 32 P-labeled DRE-P oligonucleotide (DRE-containing sequence from the PCNA gene) (24) was performed using Kc cell nuclear extract (Kc), lysates of E. coli containing GST-DREF fusion proteins and E. coli lysate containing GST (GST). ated with the fragment of 16 -242 amino acid residues but not with that of 230 -608 amino acid residues (see Fig. 2B). Prior to fine mapping the DNA binding domain, the binding specificity of the recombinant DREF polypeptide to the DRE sequence was examined by the o-phenanthroline-copper footprinting method (Fig. 2). We used the 5Ј-upstream region of the Drosophila DNA polymerase ␣ gene containing three DRE sequences (DRE-␣I, ␣II, and ␣III) as a probe (24) and the E. coli extract containing GST-DREF16 -242. The footprinting patterns containing the DRE-sequence at the centers were indistinguishable from those obtained using Kc cell nuclear extract (24) and purified DREF (data not shown).
Determination of the DNA Binding Region of DREF-Plasmids expressing a set of deletion mutants of GST-DREF were constructed using PCR to examine the specificity of DRE binding activity. Analysis of extracts of E. coli carrying expression plasmids by SDS-polyacrylamide gel electrophoresis showed that similar amounts of GST-DREF polypeptides were produced ( Fig. 2A).
The DRE binding activities of these mutant polypeptides were examined by gel mobility shift assay using DRE-P as a probe (Fig. 2B). Truncated polypeptides encoding from the 16th amino acid residue up to the C termini at 242, 230, 205, 185, 165, 145, and 125 amino acid residues exhibited DNA binding activities as effective as GST-DREF16 -608. C-terminal deletion up to amino acid residue 105 resulted in a reduction of DNA binding activity to approximately half that of the 16 -125 construct, and deletion up to amino acid residue 85 resulted in only weak activity, less than 1% that of the 16 -125 form. Further C-terminal deletion up to residue 49 caused complete loss of DNA binding activity. These results indicate that the region essential for strong DRE binding activity is located between amino acid residues 16 and 115, although amino acid residues 16 -85 are sufficient for weak DRE binding.
Deletion of 32 amino acid residues from the N terminus resulted in an almost complete ablation of DNA binding activity, although we detected very weak signals of DRE-protein complexes with N-terminal deletion mutants, 32-230, 32-145, 32-125, and 32-105 after long-term exposure of gels to the imaging plate. Interestingly, their mobilities in the gel were clearly faster than those of DREF mutants with similar sizes carrying the 16 -32 region. One possible explanation is that amino acid residues 16 -32 are required for dimer formation and also for the strong DRE binding activity.
Characterization of Antibodies against DREF-We generated a rabbit antiserum and four mouse hybridoma lines producing antibodies against DREF polypeptide using GST-DREF16 -608 fusion protein as an antigen. The specificities of these antibodies were confirmed by immuno-Western blotting  1, 3, and 5) and GST-DREF240 -608 (C) (lanes 2, 4, and 6) were fractionated by SDSpolyacrylamide gel electrophoresis, transferred onto a poly(vinylidene fluoride) membrane sheet, and immunostained with anti-DREF antiserum (lanes 1 and 2), culture supernatants of hybridoma lines 1 (lanes 3 and 4) and 2 (lanes 5 and 6) as primary antibodies and alkaline phosphatase-conjugated second antibodies. B, effects of antibodies on DRE-DREF complex formation. A gel mobility shift assay was performed using 32 P-labeled DRE-P oligonucleotide as the probe incubated without (lanes 1, 3, 5, 7, and 9) or with (lanes 2, 4, 6, 8, and 10) Kc cell nuclear extract in the absence (lanes 1 and 2) or presence (lanes 3, 4,5,6,7,8,9, and 10) of various antibodies. Used antibodies were: lanes 3 and 4, culture supernatant of a mouse hybridoma cell line (4-2D) which produces anti-chick primase antibody (6 l); lanes 5 and 6, culture supernatant of mouse hybridoma cell line 1 (6 l); lanes 7 and 8, culture supernatant of mouse hybridoma cell line 4 (6 l); lanes 9 and 10, rabbit anti-DREF polyclonal antibody (50 g/ml IgG, 6 l). Nuclear extract from Kc cells (2 g) was mixed with each antibody, incubated for 2 h on ice, added to mixtures containing 32 P-labeled DRE-P oligonucleotides (10 4 cpm) and 1 g of poly(dI-dC), incubated for 15 min on ice, and analyzed on a 4% polyacrylamide gel. analysis and gel mobility shift assay. An example of the results of the former is shown in Fig. 4A. E. coli extracts expressing GST-DREF16 -242 (N) and GST-DREF240 -608 (C) were electrophoretically separated and blotted, followed by analysis of their immunoreactivities. The antiserum reacted with both N and C polypeptides, whereas culture supernatants with monoclonal antibodies 1 and 2 reacted only with N and C, respectively. Immunoblotting analysis using culture supernatants of the other hybridoma lines revealed that both 3 and 4 monoclonal antibodies specifically recognize GST-DREF240 -608 (data not shown).
Effects of antibodies on DRE-DREF complex formation were examined by gel mobility shift assay, in which the Kc cell nuclear extract was incubated with each antibody prior to adding the probe (Fig. 4B). Monoclonal antibody 1 inhibited complex formation. Western blotting analysis with a set of truncated GST-DREF proteins indicated that the epitope for monoclonal antibody 1 is located within the DNA binding region between amino acid residues 72 and 125 (data not shown), confirming that this region plays a role in DNA binding. Addition of monoclonal antibody 4 as well as the polyclonal antiserum resulted in supershifts of the DRE⅐DREF complexes. Monoclonal antibodies 2 and 3 had no effect on DRE⅐DREF complex formation (data not shown). The results confirmed that the cDNA isolated in this study encodes the DREF polypeptide.
Trans-activating Property of the DREF Protein-To confirm the trans-activation property of DREF, cotransfection experiments were carried out using the DREF expression plasmid as an effector and CAT plasmids directed by various promoters with or without DRE elements as reporters. In such experiments, overproduction of DREF neither enhanced nor reduced CAT expression (data not shown). We supposed that the amount of DREF might be almost saturated in Kc cells so that the effect of additional DREF expression is minimal. Therefore, we employed an in vitro transcription assay using the DNA polymerase ␣ gene promoter as a template with the Kc cell nuclear extract to investigate the DREF requirement. Nuclear extracts were preincubated with control or anti-DREF antibodies and added to the reaction mixture for in vitro transcription, and transcripts were analyzed by the primer extension method (Fig. 5). The sizes of the products revealed that transcription faithfully started at the cap site on the DNA polymerase ␣ gene as determined previously (22). Preincubation with the preimmune rabbit IgG or the control mouse IgG (lanes 3 and 4) slightly stimulated transcription activity, while treatment of the nuclear extract with the anti-DREF polyclonal antibody (lane 5) or monoclonal antibody 1 (lane 6) resulted in almost complete and 60% loss of transcription activity, respectively. The results clearly confirm the contribution of DREF polypeptide to transcription on DNA polymerase ␣ promoter.
DREF Overproduction Overcomes the DRE/DREF-mediating Repression by zen-Previously we suggested that the Zen protein represses PCNA and DNA polymerase ␣ gene promoters by reducing DREF in the transfected cells (29). We investigated whether overproduction of DREF protein suppresses the repression of the promoter by Zen protein. As expected, the DREF expression plasmid overcame the repression of PCNA promoter by Zen protein in a dose-dependent manner (Fig. 6). However, the CAT activity never increased beyond that without zen and DREF expression plasmids, supporting our idea that the DREF amount in Kc cells is almost saturated, as mentioned above.
Changes in the Amount of DREF mRNA during Development-We measured the level of DREF mRNA with Northern hybridization analysis using RNA extracted from Drosophila bodies at various developmental stages. Using DREF cDNA as a probe, a single transcript of 2.9 kb was detected. A cDNA for the DNA polymerase ␣ was also used as a reference probe, since this gene (and also the PCNA gene) is expressed in correlation with cell proliferation during development and is suggested to be under the control of the DRE/DREF system (22,23). As shown in Fig. 7, DREF mRNA was detected at the highest level in 4 -8 h embryos, and at a relatively high level in unfertilized eggs, 2-4 h embryos and adult female flies. mRNA at a low level was detected in larvae, pupae, and adult male flies. Fluctuations of DREF mRNA were roughly similar to those of DNA polymerase ␣ mRNA and PCNA mRNA through most developmental stages. However, discrepancies appeared in 12-16-h embryos and second larvae and adult male flies.
Immunocytochemical Localization of the DREF Polypeptide in Early Embryos-Expression of DREF was determined by immunocytochemical study in embryos during the first 13 nuclear division cycles (Fig. 8). Embryos were also stained with 4,6-diamidino-2-phenylidole (DAPI) to judge their nuclear division cycles. No significant staining was observed in any stage when normal rabbit IgG was used as a primary antibody (Fig.  8I, and data not shown).
The ooplasm of unfertilized eggs was weakly stained with the antibody (Fig. 8A), confirming maternal storage of DREF. Up to nuclear division cycle 7, the nuclei and the surrounding cytoplasmic region were stained weakly, but still more strongly than other regions of the syncytial cytoplasm (Fig. 8C). The signals differed from those observed with anti-PCNA antibody, which was uniformly strong in nuclei at these cycles (39). After cycle 8, strong and uniform nuclear staining was observed (Fig.  8E). By cycle 11, staining in the cortical cytoplasm underneath surface nuclei had mostly faded, suggesting that most maternally stored DREF had been translocated into nuclei from the cytoplasm by this point. DISCUSSION In our previous studies, promoters of DNA replication-related genes such as those encoding PCNA and DNA polymerase ␣ 180-kDa catalytic subunit) were found to be positively regulated by DRE and a specific binding factor, DREF (24,41). We have searched for the TATCGATA sequence in a Drosophila data DNA base and found 60 genes carrying this sequence within 600-bp upstream regions from the transcription initiation sites. Interestingly, more than 30 of these genes are related to cell proliferation, suggesting that DRE is a common regulatory element responsible for the coordinated expression of many proliferation-related genes (42). Furthermore, overexpression of the zerknullt gene product results in repression of promoters of both PCNA and DNA polymerase ␣ genes by impeding the DREF activity in cells (29). These findings suggest that DNA replication related genes are both positively and negatively regulated via DRE and DREF. The present isolation of cDNA for DREF, and preparation of specific antibodies should provide important clues toward further understanding how DREF functions in this regulation.
The DREF polypeptide contains three characteristic domains, respectively rich in basic amino acids, proline, and acidic amino acids. Although no significant similarity with any other proteins in data bases was found, these characteristic regions may be required for the function of DREF as a transcription regulatory factor. The DNA binding domain was mapped between 16 -105 amino acid residues in the basic amino acid-rich region of 90 amino acid residues, but no characteristic feature similar to those previously reported could be identified. Structural analysis with nuclear magnetic resonance imaging is now under way using the DNA binding domain of the recombinant DREF polypeptide.
Transactivation of DRE-containing promoters by DREF overproduction was not observed in the co-transfection experiment with DREF expression and CAT plasmids. We suppose that DREF might be almost saturated in Kc cells. However, anti-DREF antibodies inhibited in vitro transcription activity of Kc cell nuclear extract, indicating that DREF is required for the high level of transcription from the DNA polymerase ␣ gene promoter. In addition, over-expression of DREF protein in Kc cells overcame repression of the PCNA gene promoter by Zen protein. We have obtained similar results with the DNA polymerase ␣ gene promoter (data not shown). The evidence indicates that DREF is one of the positive factors required for DNA replication-related genes and that it might be an important transcription regulatory factor involved in proliferationand differentiation-related control. We have isolated the gene for DREF and analysis of its promoter region is in progress. We preliminary obtained results suggesting that zen protein represses the DREF promoter.
In the present study, the fluctuation of DREF mRNA content during development was similar to those of DNA polymerase ␣ and PCNA, providing further evidence that DREF is an important transcription regulatory factor for DNA replication-related genes. The DREF polypeptide is distinctly localized in nuclei after the nuclear division cycle 8. A pulse-labeling experiment of zygotic-transcribed RNA demonstrated that nuclei become competent for transcriptional activation at cycle 10 while synthesis of rRNA, tRNAs, 5sRNA, snRNA, and poly(A) RNA is first detectable during cycles 11 or 12 (43). Transcripts for Krü ppel, hunchback, and hairy genes, members of the gap gene and pair-rule gene families, are first detected at cycle 11 and their products begin to be expressed at cycles 13-14, (44 -46). The histone genes become transcriptionally competent during cycle 10 (43). Northern hybridization analysis has revealed that the amounts of mRNAs for both PCNA and DNA polymerase ␣ start to increase significantly in embryos by about 2 h after fertilization (cycles 12-15) (22). Therefore, the appearence of DREF in nuclei at and after cycle 8 might contribute to such zygotic transcription of DNA replication-related genes.