Essential role of E2F recognition sites in regulation of the proliferating cell nuclear antigen gene promoter during Drosophila development.

We have found sequences similar to the transcription factor E2F recognition site within the Drosophila proliferating cell nuclear antigen (PCNA) gene promoter. These sequences are located at positions -43 to -36 (site I). and -56 to -49 (site II) with respect to the cap site Glutathione S-transferase (GST)-E2F and GST-DP fusion proteins cooperate and bind to the potential E2F sites in the PCNA promoter in vitro. A binding factor(s) to these sequences that has similar binding specificity to that of E2F was detected in nuclear extracts of Drosophila Kc cells. Furthermore, transient expression of target site for the activation coincided with the E2F sites. These results indicate that the PCNA gene is a likely target gene of E2F. Examination of lacZ expression from PCNA-lacZ fusion genes carrying mutations in either or both of two E2F sites introduced into flies by germ line transformation revealed that site II plays a major role in the PCNA promoter activity during embryogenesis and larval development, although both sites are required for optimal promoter activity. However, for maternal expression in ovaries, either one of the two sites is essentially sufficient to direct optimal promoter activity. These results demonstrate, for the first time, an essential role for E2F sites in regulation of PCNA promoter activity during development of a multicellular organism.

Many lines of evidence have indicated that the expression of genes involved in DNA replication is closely correlated with the proliferation state of cells and repressed in accordance with progression of differentiation in various tissues during development (1,2). In budding yeast, genes involved in DNA replication contain a common promoter element (MluI cell cycle box, 5Ј-ACGCGT) (3), and the specific transcription factor complex DSC1 (MBF) is required for expression at the G 1 -S boundary (4,5).
In mammalian cells, expression of genes involved in DNA replication increases dramatically at late G 1 in response to growth stimulation (6,7). Many of these genes including the proliferating cell nuclear antigen (PCNA) 1 gene contain the transcription factor E2F-binding site (5Ј-TTTCGCGC) within their promoter regions (8 -10) or a first intron (11). Mammalian E2F is a heterogeneous factor representing the combined activity of at least four gene products called E2F-1, E2F-2, E2F-3, and DP-1. E2F-1 and DP-1 associate into stable complexes and activate transcription in a cooperative manner (12,13). The regulation of E2F function also appears to play an important role during muscle terminal differentiation (14).
In Drosophila, we have isolated genes for PCNA (15) and the DNA polymerase ␣ (16) and found a common regulatory element, DRE (5Ј-TATCGATA) and a specific DRE-binding factor, DREF. The DRE-DREF system appears to play a key role in the differentiation-coupled repression of cell proliferation during embryogenesis (17). In addition, cDNAs for Drosophila homologs of E2F-1 and DP-1 have been recently cloned (18,19). These two proteins interact with each other and cooperate to give sequence-specific DNA binding and optimal trans-activation (19). Furthermore, multiple E2F recognition sites have been identified in the promoter of the Drosophila DNA polymerase ␣ gene (18).
To assess the possibility that the Drosophila PCNA gene might have E2F sites, as is the case with mammalian PCNA genes (11), we searched for sequences similar to those in the DNA polymerase ␣ gene and found two such sequences within the PCNA promoter. We have detected a binding factor(s) to these sequences that has similar specificity to that of E2F. Furthermore, expression of E2F in Kc cells activated the PCNA promoter, and the target site for the activation coincided with the E2F sites. Analyses with transgenic flies indicate that the E2F sites are required for PCNA promoter function throughout Drosophila development.

Oligonucleotides
The sequences of double-stranded oligonucleotides containing potential E2F sites or their base-substituted derivatives in the PCNA promoter were defined as follows.

E2F-PmutI&II gatccCACATCCCTcgCaaGCTCATTTctCaaGCCTGAAAGTa gGTGTAGGGAgcGttCGAGTAAAgaGttCGGACTTTCAtctag
The sequences of double-stranded oligonucleotides containing E2F sites in the DNA polymerase ␣ promoter were as follows.

Pol␣site1 attggtaccTCGGATTTCCCGCCAAAATAT ggAGCCTAAAGGGCGGTTTTATA
Pol␣site2ϩ3 gatcCGATATGTTCCCGCCATTCCCGCTTTGA GCTATACAAGGGCGGTAAGGGCGAAACTctag * This work was supported in part by grants-in-aid from the Ministry of Education, Science and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The sequences of double-stranded oligonucleotides containing two E2F sites or their base-substituted derivatives in the adenovirus E2 promoter (20) were as follows.

Plasmid Construction
The plasmid p5Ј-168DPCNACAT contains the PCNA gene fragment spanning from Ϫ168 to ϩ23 placed upstream of the chloramphenicol acetyltransferase (CAT) gene in the plasmid pSKCAT (22). A fragment from Ϫ86 to ϩ57 having a base-substitutional mutation in E2F site I was generated by the polymerase chain reaction (PCR) method (23) using p5Ј-168DPCNACAT as a template with primers CAT-1 and mutI. The PCR product was digested with EcoRV (Ϫ80) and SacII (ϩ23) and then replaced with the fragment between EcoRV and SacII sites of the p5Ј-168DPCNACAT to create the plasmid p5Ј-168E2FmutIDPCNA-CAT. Plasmids p5Ј-168E2FmutIIDPCNACAT and p5Ј-168E2FmutI&-IIDPCNACAT were constructed in the same way except that mutII and mutI&II in addition to CAT-1 were used as PCR primers. The obtained plasmids were verified by nucleotide sequence analysis with synthetic primers (24). A double-stranded oligonucleotide E2F-P was inserted into the BamHI site of the p5Ј-168E2FmutI&IIDPCNACAT in normal or reverse orientation to create plasmids p5Ј-168E2FmutI&IIE2F-P(N)DPCNACAT and p5Ј-168E2FmutI&IIE2F-P(R)DPCNACAT, respectively.
The plasmid p5Ј-607DPCNAlacZW8HS (22) contains the PCNA gene fragment spanning from Ϫ607 to ϩ137 fused with the lacZ in a Pelement vector. The plasmid p5Ј-168DPCNAlacZW8HS (22) contains the PCNA gene fragment spanning from Ϫ168 to ϩ137 fused with the lacZ in a P-element vector. To create mutated derivatives in P-element vector backbones, fragments having various mutations in E2F sites were isolated from CAT plasmids by digestion with SalI (Ϫ168) and SacII (ϩ23) and then inserted between XhoI (Ϫ607) and SacII (ϩ23) sites of the p5Ј-607DPCNAlacZW8HS.
The expression plasmids Act-dE2F (19) and Act-dDP (19), respectively, contain Drosophila E2F and DP full-length cDNAs placed under the control of the Drosophila actin 5C promoter (25). The expression plasmid pdrosE2F1WT (18) contains Drosophila E2F cDNA covering amino acid 77 to the C-terminal end of the E2F protein fused with an N-terminal 11-amino-acid region of the ubx gene. This plasmid is also under control of the actin 5C promoter. The plasmid pDhsp70-L (26) contains the luciferase gene under control of the Drosophila hsp70 promoter (27).
Fusion genes of E2F with glutathione S-transferase (GST) and of DP with GST were prepared by PCR using appropriate primers with BamHI restriction sites at their 5Ј-ends as described (19). The amplified fragments were digested with BamHI and subcloned into pGEX-2T (Pharmacia Biotech Inc.) in frame to create plasmids pGST-dE2F and pGST-dDP. These plasmids produce full-length E2F and DP proteins fused with GST. All plasmids were propagated in Escherichia coli XL-1 Blue and isolated by standard procedures (28).

Preparation of Nuclear Extracts and Gel Mobility Shift Assay
Preparation of nuclear extracts from Kc cells was as described elsewhere (21). Each nuclear extract was incubated in 15 l of reaction mixture containing 15 mM Hepes (pH 7.6), 60 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 12% glycerol, 1 g of poly(dI-dC) for 10 min on ice. Unlabeled competitor DNA fragments were added at this step. Then, 32 P-end-labeled E2F-P oligonucleotides (160 pg) were added, and the mixture was incubated for 10 min on ice. The complex of DNA and a binding protein(s) was electrophoretically separated from free probes in a 4% polyacrylamide gel in 50 mM Tris borate (pH 8.3) and 1 mM EDTA containing 2.5% glycerol at room temperature. The gel was dried and autoradiographed.

Expression of GST Fusion Proteins and Gel Mobility Shift Assay
Expression of GST-E2F and GST-DP fusion proteins was carried out as described elsewhere (29). Lysates of cells were prepared by sonication in buffer D containing 0.6 M KCl, 1 mM phenylmethylsulfonyl fluoride, 1 g/ml each of pepstatin, leupeptin, and aprotinin. Lysates were cleared by centrifugation at 12,000 ϫ g for 20 min at 4°C and used for gel mobility shift assay using a 32 P-end-labeled AdE2Fwt oligonucleotide (117 pg) as a probe. The gel mobility shift assay was carried out as described above except that the reaction mixture for the binding contained 20 mM Hepes (pH 7.5), 120 mM KCl, 10 mM MgCl 2 , 1 mM EGTA, 0.5 mM dithiothreitol, 10% glycerol, 1 g of sonicated salmon sperm DNA.

DNA Transfection into Cells, CAT Assay, and Luciferase Assay
Drosophila Kc cells (30) were grown in M3(BF) medium supplemented with 2% fetal calf serum (31). Cells were plated at about 5 ϫ 10 6 cells/60-mm dish for 16 h before DNA transfection. DNA was transfected into cells by the calcium phosphate coprecipitation technique described elsewhere (32). One or 0.5 g of PCNA promoter-CAT plasmid as a reporter plasmid and 0.1 g of pDhsp70-L as an internal control plasmid were cotransfected with the indicated amount of the effector plasmid. The total amount of effector plasmid was kept constant by the addition of the vector pAcGEM3 (22), and the total amount of DNA was adjusted to 10 g by the addition of pGEM3. Cells were harvested at 48 h after transfection. Cell extracts were prepared, and CAT activity was measured as described (33). The radioactivity of acetylated chloramphenicol on thin-layer plates was quantified with an imaging analyzer BAS2000 (Fuji Film).
The luciferase assay was carried out by means of a PicaGene assay kit (Toyo Inc.) as described previously (9). All assays were performed within the range of linear relation of the activities to incubation time and protein amounts. CAT activity was normalized to the luciferase activity.

Establishment of Transgenic Flies
P-element-mediated germ line transformation was carried out as described (34,35). G 1 transformants were selected on the basis of white eye color rescue. Multiple independent lines were obtained for each of the various fusion genes. Established transgenic strains and their chromosomal linkages are listed in Table I.

Analysis of Expression Patterns for PCNA-lacZ
Quantitative Measurement of ␤-Galactosidase Activity in Extracts (36)-Male transgenic flies were crossed with female wild type flies. Groups of 50 -100 individuals each of embryos, larvae, pupae, and adult flies were homogenized in 500 l of ice-cold assay buffer (50 mM potassium phosphate, pH 7.5, 1 mM MgCl 2 ). Homogenates were centrifuged at 10,000 ϫ g at 4°C for 5 min. For each assay, 50 -200 l of the supernatant was added to give 1 ml of assay buffer containing 1 mM chlorophenol red-␤-D-galactopyranoside substrate (Boehringer Mannheim). Reaction incubations were at 37°C in the dark. Substrate conversion was measured at 574 nm using a spectrophotometer. The ␤galactosidase activity was defined as absorbance units/h/mg of protein.
To correct for endogenous ␤-galactosidase activity, extracts from the wild type strain were included in each experiment, and this background reading was subtracted from readings obtained with each transformant line. Deviation among independent strains was less than 30% (not shown).

RESULTS
Potential E2F Recognition Sequences Located in the Promoter Region of the Drosophila PCNA Gene-Three potential E2F sites have been identified in the Drosophila DNA polym-erase ␣ promoter (Fig. 1A) (18). Site 1 has been demonstrated to be the most effective for binding of Drosophila E2F (18). A search for E2F sites similar to those of the DNA polymerase ␣ gene revealed two such sequences within the PCNA promoter. These sequences are located at positions Ϫ43 to Ϫ36 (site I) and Ϫ56 to Ϫ49 (site II) relative to the cap site (Fig. 1B). Nucleotide sequences of site I and site II, respectively, match seven out of eight and six out of eight nucleotides of the E2F site I in the DNA polymerase ␣ promoter (18) (Fig. 1).
GST-E2F and GST-DP Fusion Proteins Cooperate and Bind to the Potential E2F Recognition Sequences in the PCNA Promoter-Lysates were prepared from bacteria carrying pGST-E2F or pGST-DP, and gel mobility shift assays were carried out. As shown in Fig. 2A, a DNA-protein complex was detected with the AdE2Fwt oligonucleotide containing the two E2F sites of the adenovirus E2 promoter (20) only when both GST-E2F and GST-DP lysates were mixed. Specificity of binding was evident in competition with wild type and mutant E2F sites from the E2 promoter (Fig. 2B, lanes a-d and q-s). E2F-P oligonucleotide (Fig. 1B) containing the two potential E2F sites of the PCNA promoter effectively competed for the binding (Fig. 2B, lanes e-g). The oligonucleotide E2F-PmutI carrying mutations in the E2F site I competed for the binding as effectively as the wild type E2F-P (Fig. 2B, lanes k-m). In contrast, the oligonucleotide E2F-PmutII carrying mutations in the site II much less efficiently competed for the binding (Fig. 2B, lanes  n-p), and the oligonucleotide E2F-PmutI&II carrying mutations in both sites did not compete at all (Fig. 2B, lanes h-j). Thus, the potential E2F sites of the PCNA promoter have high affinity for the complex of GST-E2F and GST-DP fusion proteins, and site II appears to play a major role in the binding.
Detection of a Binding Factor(s) for the Potential E2F Sites in the PCNA Promoter-Nuclear extracts were prepared from Kc  (lanes b-d). B, radiolabeled double-stranded AdE2Fwt oligonucleotides were incubated with or without (Ϫ, lane t) 1 l each of lysates from bacteria carrying pGST-dE2F or pGST-dDP in the presence of the indicated amounts of competitor oligonucleotides (indicated at the top of each lane). AdE2Fwt, oligonucleotides containing two wild type E2F sites from the adenovirus E2 promoter; AdE2Fmut, oligonucleotides containing two mutant E2F sites from the E2 promoter; E2F-P, oligonucleotides containing two wild type E2F sites from the PCNA promoter; E2F-PmutI, oligonucleotides having a mutation in the E2F site I of the PCNA promoter; E2F-PmutII, oligonucleotides having a mutation in the E2F site II of the PCNA promoter; E2F-PmutI&II, oligonucleotides having mutations in both E2F sites I and II of the PCNA promoter.  III  21  X  73  II  89  II  91  III  p5Ј-168E2FmutIDPCNAlacZW8HS  6  II  18  III  25  II  26  II  29  II  40  III  46  II  cells, and gel mobility shift assays were carried out. As shown in Fig. 3A, a specific DNA-protein complex could be detected using an E2F-P oligonucleotide as a probe. The band shifted with 32 P-labeled E2F-P was diminished by adding an excess amount of unlabeled E2F-P as a competitor but not by adding unrelated sequences of similar size such as DRE-P or DRE-PM (Fig. 3A, lanes a-d and k-q). The oligonucleotide containing the wild-type E2F site from the adenovirus E2 promoter (20) competed for the binding when added to the reaction in excess (Fig. 3A, lanes r-t). Similarly, oligonucleotides containing the E2F sites in the DNA polymerase ␣ promoter (18) competed for the binding (Fig. 3A, lanes e-j). In contrast, the oligonucleotide containing the mutant E2F site from the adenovirus E2 promoter did not compete under the examined conditions (Fig. 3A,  lanes u-w). These results indicate that a binding factor(s) to the potential E2F sites in the PCNA promoter has binding specificity indistinguishable from that of Drosophila E2F (18).
As shown in Fig. 3B, the oligonucleotide E2F-PmutI carrying mutations in the E2F site I (Fig. 1B) competed for the binding as effectively as the wild type E2F-P. In contrast, the oligonucleotide E2F-PmutII carrying mutations in the E2F site II (Fig.  1B) only weakly competed for the binding (Fig. 3B, lanes g-k). Therefore, site II appears to play a major role in the binding.
Effects of Mutations in the Potential E2F Sites on PCNA Promoter Activity in Kc Cells-The PCNA promoter carrying mutations in either or both of two E2F sites was placed upstream of the CAT gene in a CAT vector (Fig. 1B). These plasmids were transfected into Kc cells, and CAT expression levels were determined. As shown in Fig. 4, the plasmid carrying mutations in E2F site I showed 41% of CAT expression as compared with that of the original plasmid. Much more extensive reduction of CAT expression was observed with the plasmid p5Ј-168E2FmutIIDPCNACAT carrying mutations in E2F

FIG. 3. Complex formation between E2F-P oligonucleotides and Kc cell nuclear extract and competition by various oligonucleotides.
Radiolabeled double-stranded E2F-P oligonucleotides were incubated with Kc cell nuclear extract (2 g of protein) in the presence or absence (0) of the indicated amounts of competitor oligonucleotides (indicated at the top of each lane). A, E2F-P, oligonucleotides containing two wild type E2F sites from the PCNA promoter; pol␣site2ϩ3, oligonucleotides containing E2F sites 2 and 3 from the DNA polymerase ␣ promoter; pol␣site1, oligonucleotides containing the E2F site 1 from the DNA polymerase ␣ promoter; DRE-P, oligonucleotides containing the DRE sequence from the PCNA promoter; DRE-PM, DRE-P oligonucleotides having a mutation in the DRE sequence; AdE2Fwt, oligonucleotides containing two wild type E2F sites from the adenovirus E2 promoter; AdE2Fmut, oligonucleotides containing two mutant E2F sites from the E2 promoter. B, oligonucleotides having a mutation in E2F site I of the PCNA promoter (E2F-PmutI) and oligonucleotides having a mutation in the E2F site II of the PCNA promoter (E2F-PmutII).
site II (Fig. 4, lanes e and f). Slight further reduction of CAT expression was observed with the plasmid p5Ј-168E2Fmut-I&IIDPCNACAT carrying mutations in both sites (Fig. 4, lanes  g and h). These results indicate that E2F site II plays a major role, and site I might play an additional role in regulation of the PCNA promoter activity. These E2F sites are essential but not sufficient for the promoter activity, since deletion up to position Ϫ86 completely abolished the promoter activity, even when the two E2F sites were kept intact (Fig. 4, lanes i and j). In addition, insertion of the E2F-P downstream of the CAT gene of the plasmid p5Ј-168E2FmutI&IIDPCNACAT did not enhance CAT expression (Fig. 4, lanes m-r), indicating the importance of the position of E2F sites for activation of transcription.
Activation of PCNA Promoter-directed CAT Expression by E2F-To determine whether the PCNA promoter can be activated by E2F, a cotransfection assay using Kc cells was carried out. Expression of the E2F protein activated PCNA promoterdirected CAT expression 2-fold (Fig. 5, upper panel). However, expression of the DP protein did not affect CAT expression. When plasmid p5Ј-116DPCNACAT, carrying the region from Ϫ116 to ϩ23 of the PCNA gene linked to the CAT-coding region, was used as the reporter plasmid, more extensive activation of CAT expression was observed with E2F-expressing plasmids (Fig. 5, lower panel). Here too, the DP-expressing plasmid had no effect on CAT expression. In addition, when the DP-expressing plasmid was cotransfected with the reporter plasmid and the E2F-expressing plasmid, no further activation of CAT expression was observed (not shown). These results indicate that the E2F protein can activate the PCNA promoter (as is the case with the DNA polymerase ␣ promoter (18)), and the level of the E2F protein but not that of the DP protein appears to be limiting for the activation in Kc cells.
Mapping of the Target Region for Activation by E2F Protein-A set of 5Ј-deletion derivatives of the plasmid p5Ј-168DPCNACAT were cotransfected with the E2F-expressing plasmid. Deletions toward position Ϫ116 caused a gradual decrease of CAT expression and a progressive increase of activation by E2F (Fig. 6A). A further deletion to position Ϫ86 completely abolished the CAT expression, and accordingly, the stimulation by E2F was no longer detectable.
To examine the responsibility of E2F sites for the activation by E2F, base substitution derivatives of p5Ј-168DPCNACAT were cotransfected with the E2F-expressing plasmid. As shown in Fig. 6B, the E2F-expressing plasmid still activated CAT expression from plasmids carrying mutations in either of two E2F sites. However, mutations in both sites completely abolished the response to E2F expression (Fig. 6B, lanes m-p). Therefore, at least one of two E2F sites is required for the E2F protein to activate the PCNA promoter.
Role of E2F Sites in Function of the PCNA Promoter in Living Flies-Although the results of CAT transient expression assay in Kc cells clearly demonstrate the essential role of E2F sites in the PCNA promoter activity, these observations have to be confirmed in living flies, and transgenic Drosophila provides an appropriate system to characterize transcriptional regulatory elements in vivo. We have established transgenic flies carrying PCNA (Ϫ168 to ϩ137) and lacZ fusion genes (Fig. 1B) (22). Male transgenic flies were crossed with wild type females to examine zygotic expression of the lacZ. Expression of lacZ was found to be high in embryos, first and second instar larvae, and adult females and low at other stages of development (Fig. 7, top panel) (37).
To examine the role of E2F sites in the PCNA promoter activity during Drosophila development, we generated PCNA-lacZ fusion genes carrying base substitutions in either or both of two E2F sites. These fusion genes were then introduced into flies by germ line transformation. Activities of modified promoters were then monitored by the quantitative ␤-galactosidase assay at various developmental stages of Drosophila. As shown in Fig. 7, mutation in E2F site I resulted in extensive reduction of lacZ expression in embryos and larvae, although high expression of the lacZ was still observed in adult females. Mutation in site II almost completely abolished lacZ expression in embryos, and only a weak expression of the lacZ was observed in larvae. Here, too, high expression of the lacZ in adult females was still observed. When both sites were mutated, no expression of the lacZ was detected throughout development, even in adult females (Fig. 7, bottom panel).
To corroborate the results from colorimetric assays using crude extracts, ␤-galactosidase activity was demonstrated in dissected larval tissues and adult ovaries. Transgenic larvae having mutations in E2F site I had a reduced staining signal in the salivary glands, the brain lobes, and the imaginal discs (Fig. 8, panels B, G, and L). More extensive reduction was observed with the larvae having mutations in site II (Fig. 8,  panels C, H, and M), and mutations in both sites completely abolished the staining signal in these tissues (Fig. 8, panels D,  I, and N). Although results with leg discs are shown in panels K-O, essentially the same results are obtained with other imaginal discs (not shown). In contrast, strong staining was clearly observed in ovaries from the transgenic lines carrying mutations in either one of the E2F sites (Fig. 8, panels Q and R), although mutations in both sites completely abolished the staining signal (Fig. 8, panel S). From these results, taken together, it is concluded that the E2F site II plays a major role in PCNA promoter activity during embryogenesis and larval development, although both sites are required for optimal promoter activity. However, for maternal expression in ovaries, either one of the two E2F sites is essentially sufficient to direct optimal promoter activity. DISCUSSION In mammalian cells, a group of genes that are commonly regulated in late G 1 of the growth response and that encode proteins important for DNA replication appear to be regulated by E2F (8). In Drosophila, multiple E2F sites have been identified in the gene for the 180-kDa catalytic subunit of the DNA polymerase ␣ (18). In the present study, we have identified two E2F recognition sites in the PCNA promoter. It is thus clearly of interest to identify the presence of E2F site(s) in the promoter regions of other DNA replication-related genes in Drosophila.
Molecular cloning of two other Drosophila genes involved in DNA replication has been so far reported. One is the gene for the 73-kDa regulatory subunit of the DNA polymerase ␣ (38), and the other is that for the 50-kDa subunit of the DNA primase (39). The former gene contains three potential E2F sites in its 5Ј-flanking region. Two of them (5Ј-TTTCGCGG and 5Ј-CTTCGCGG) match seven out of eight and six out of eight nucleotides of the binding consensus (5Ј-TTTCGCGC) for mammalian E2F, respectively. The other site (5Ј-TTACCCGC) matches seven out of eight nucleotides of the E2F recognition site I of the DNA polymerase ␣ 180-kDa subunit gene. The   FIG. 8. Demonstration of ␤-galactosidase activity in the salivary glands, the brain lobes, and the imaginal discs of third instar larvae and in the ovaries of adult females. Salivary glands (panels A-D), brain lobes (panels F-J), and leg discs (panels K-O) were dissected from the third instar larvae of male transgenic flies carrying the fusion gene (indicated at the left side of each panel) ϫ wild type females. Ovaries (lanes P-T) were dissected from 3-day-old adult females carrying the transgenes indicated at the left side of each panel. They were then subjected to demonstration of ␤-galactosidase activity. Tissues from Canton S larvae and adult females carrying no transgene were processed in the same way as controls (panels E, J, O, and T).
Ϫ168, strain 73 carrying the p5Ј-168DPCNAlacZW8HS; mutI, strain 29 carrying the p5Ј-168 mutIDPCNAlacZ-W8HS; mutII, strain 5 carrying the p5Ј-168 mutIIDPCNAlacZW8HS; mutI&II, strain 72 carrying the p5Ј-168 mutI&II-DPCNAlacZW8HS. 50-kDa primase gene also contains a potential E2F site in its 5Ј-untranslated region. This site (5Ј-ATTCCCGC) perfectly matches the nucleotide sequence of the E2F site 3 of the DNA polymerase ␣ gene. Although promoter sequence information is not available for other Drosophila genes involved in DNA replication, we predict that they very likely contain E2F sites, as is the case with mammalian DNA replication-related genes.
In our previous studies of Drosophila genes for PCNA and DNA polymerase ␣, we found a common regulatory element, DRE (21), which therefore appeared to be an important element for at least these two genes. DRE is essential for the function of the PCNA promoter both in embryos and in larvae (26). Since DRE was found to be by itself not sufficient to activate the PCNA promoter during larval stages, we searched for another regulatory element and found an upstream regulatory element (URE) located in the region from nucleotide position Ϫ168 to Ϫ119 (to be published elsewhere). Since the URE sequence alone was also not sufficient to activate the PCNA promoter in larvae, both URE and DRE appear to be required to activate the promoter during larval stages.
In the present study, we have identified two E2F sites in the region downstream of DRE of the PCNA gene. Analyses with transgenic flies demonstrated that these sites are essential for PCNA promoter activity throughout development. However, E2F sites alone proved to be insufficient for PCNA gene promoter activity during embryonic and larval stages, since deletion of the upstream region containing URE and DRE sequences completely abolished the promoter activity during these stages (to be published elsewhere). Thus, URE, DRE, and E2F sites likely cooperate to direct optimal PCNA promoter activity during these stages.
A number of studies have been conducted to explore the regulation of E2F during the cell cycle. Critical roles of E2F sites for regulated expression in late G 1 have been demonstrated with the mammalian genes for DHFR (10) and PCNA (11). However, such observations with cultured cell systems have to be confirmed in living organisms, and in this sense transgenic Drosophila provides an appropriate system to characterize E2F sites in vivo. The present study with transgenic flies provides the first evidence for an essential role of E2F sites in regulation of the promoter activity of genes involved in DNA replication during development of a living organism.