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. 2001 Apr 15;29(8):1808–1814. doi: 10.1093/nar/29.8.1808

E2F-dependent transcription of the raf proto-oncogene during Drosophila development

Eun-Jeong Kwon 1,2,3, Eun-Jin Oh 1, Young-Shin Kim 1, Fumiko Hirose 2, Katsuhito Ohno 2,3, Yasuyoshi Nishida 3, Akio Matsukage 2, Masamitsu Yamaguchi 2, Mi-Ae Yoo 1,a
PMCID: PMC31312  PMID: 11292854

Abstract

D-raf, a Drosophila homolog of the raf proto-oncogene, has diverse functions throughout development and is transcribed in a wide range of tissues, with high levels of expression in the ovary and in association with rapid proliferation. The expression pattern resembles those of S phase genes, which are regulated by E2F transcription factors. In the 5′-flanking region of D-raf, four sequences (E2F sites 1–4) similar to the E2F recognition sequence were found, one of them (E2F site 3) being recognized efficiently by Drosophila E2F (dE2F) in vitro. Transient luciferase expression assays confirmed activation of the D-raf gene promoter by dE2F/dDP. Expression of Draf–lacZ was greatly reduced in embryos homozygous for the dE2F mutation. These results suggest that dE2F is likely to be an important regulator of D-raf transcription.

INTRODUCTION

Raf-1, a serine/threonine kinase encoded by the proto-oncogene c-raf-1, has been demonstrated to be a component of the mitogen-activated protein kinase (MAPK) cascade and an important mediator of diverse extracellular signals regulating cellular proliferation, differentiation and survival (1–5). In accordance with its diverse functions, it has been demonstrated by northern blot analyses that c-raf-1 is expressed ubiquitously while other members of the raf gene family, A-raf and B-raf, are expressed in a more restricted pattern, e.g. expression in urogenital organs including kidney, testis, ovary and epididymis (6,7). However, their mechanisms of transcriptional regulation are poorly understood.

Raf is highly conserved during evolution and Drosophila contains a single raf gene, D-raf. Analyses of D-raf mutations have revealed multiple functions during development, such as in regulation of cell proliferation, reading of positional information at embryonic termini, cell fate decision of photoreceptor cells and formation of wing veins (8–11). D-raf is expressed throughout development in a wide range of tissues, with high levels of expression in the ovary and in association with rapid proliferation, such as in imaginal discs (12). This pattern of expression resembles those of S phase genes, which are essential for cell proliferation and DNA replication. We previously demonstrated that transcription of D-raf is regulated by the DNA replication-related element (DRE)/DRE-binding factor (DREF) system (13). This DRE/DREF system is involved in the transcriptional regulation of cell proliferation-related genes, including those encoding DNA polymerase α, proliferating cell nuclear antigen (PCNA), Cyclin A and dE2F in Drosophila (14–19).

E2F transcription factors have been identified as key downstream factors of the Retinoblastoma (Rb) family members, playing essential roles in regulating the correct timing of activation of S phase genes (20,21). Many S phase genes, such as those encoding DNA polymerase α, thymidine kinase, ribonucleotide reductase, dihydrofolate reductase, PCNA, Cyclin A, Cyclin E and Cdc2, contain at least one E2F-binding site (consensus sequence 5′-TTTCGCGC) within their promoter regions (22–25). In mammals at least six E2F family members (E2F1, E2F2, E2F3, E2F4, E2F5 and E2F6) and two dimer partners (DP1 and DP2) have been characterized (20,26–29) and it has been suggested that E2Fs have distinct functions in regulating cell cycle progression. Overexpression of E2F1, E2F2 or E2F3 can efficiently activate the transcription of S phase genes and induce DNA replication (30,31). On the other hand, E2F4 and E2F5 are expressed in quiescent cells (27,29) and overexpressed E2F6, which lacks a Rb-binding domain, has been shown to compete with E2F1, E2F2 and E2F3 for E2F-binding sites in promoters of the target genes and to repress their transcription (28).

Drosophila contains two E2Fs, dE2F and dE2F2, and a dimer partner, dDP (32–34). It has been documented that promoters of Drosophila genes encoding DNA polymerase α and PCNA contain E2F recognition sequences in addition to a DRE. The sequences are essential for promoter activity of these genes in vivo and their activation depends on dE2F (24,25,35).

In the present study we found several sequences similar to the E2F recognition sequence in the 5′-flanking region of the D-raf gene and that transcription of D-raf indeed depends largely on dE2F during development. We also found that dE2F binds strongly to one of the putative E2F recognition sequences and that this sequence is responsible for positive regulation by dE2F.

MATERIALS AND METHODS

Oligonucleotides

The sequence of oligonucleotides used in primer extension experiments for determining the transcription initiation site was 5′-GTGGAGTTCTAATGGTGAGCCACCCACATCCACTT-3′ (PR1)

The following double-stranded oligonucleotides containing a 6 bp linker sequence, recognizable by BglII and BamHI, were chemically synthesized with sequences containing two E2F sites or their base substitutional mutants in the adenovirus E2 promoter as follows: AdE2F wild-type (wt), 5′-gatccTCCGTTTTCGCGCTTAAATTTGAGAAAGGGCGCGAAACTGGa-3′ and 3′-gAGGCAAAAGCGCGAATTTAAACTCTTTCCCGCGCTTTGACCtctag-5′; AdE2F mutant (mut), 5′-gatccTCCGTTGTCGAGCTTAAATTTGAGAAAGGGCTCGACACTGGa-3′ and 3′-gAGGCAACAGCTCGAATTTAAACTCTTTCCCGAGCTGTGACCtctag-5′.

Double-stranded oligonucleotides containing potential E2F sites or their base substitutional mutants in the D-raf promoter were defined as follows: Draf-E2Fsite1wt, 5′-CGAAATGTAGTAAAATTCGCGGAAAGTAAATAAATTGTTA-3′ and 3′-GCTTTACATCATTTTAAGCGCCTTTCATTTATTTAACAAT-5′; Draf-E2Fsite2wt, 5′-agcggtaccGGTGATTTGCCGGAACGC-3′ and 3′-CCACTAAACGGCCTTGCG-5′; Draf-E2Fsite3wt, 5′-agcggtaccTCAAATTTCGCGCCTATG-3′ and 3′-AGTTTAAAGCGCGGATAC-5′; Draf-E2Fsite 3mut, 5′-agcggtaccTCAAATTGAGATCCTATG-3′ and 3′-AGTTTAACTCTAGGATAC-5′; Draf-E2Fsite4wt, 5′-GGATCCTAACTTATCGGCCAAGCCATCATCAACAGCAATT-3′ and 3′-CCTAGGATTGAATAGCCGGTTCGGTAGTAGTTGTCGTTAA-5′.

The 3×DrafE2F3wt and 3×DrafE2F3mut oligonucleotides used for luciferase reporter constructs were as follows: 3×DrafE2F3wt, 5′-gatccTCAAATTTCGCGCCTATGTCAAATTTCGCGCCTATGTCAAATTTCGCGCCTATGa-3′ and 3′-gAGTTTAAAGCGCGGATACAGTTTAAAGCGCGGATACAGTTTAAAGCGCGGATACtctag-5′; 3×DrafE2F3mut, 5′-gatccTCAAATTGAGATCCTATGTCAAATTGAGATCCTATGTCAAATTGAGATCCTATGa-3′ and 3′-gAGTTTAACTCTAGGATACAGTTTAACTCTAGGATACAGTTTAACTCTAGGATACtctag-5′. Mutated bases are underlined and lower case letters indicate the linker sequences recognizable by BglII and BamHI.

The oligonucleotide OEBS (optimum dE2F-binding sequence) determined by sequence selection (to be published elsewhere) contains the nucleotide sequence 5′-TTTGGCGCCAAA.

Plasmid construction

The plasmid p5′-663Drafwt-luc contains the D-raf gene fragment spanning –663 to +302 with respect to the transcription initiation site placed upstream of the plasmid PGVB (Toyo Inc.) (36). The expression plasmids pAct-dE2F and pAct-dDP contain Drosophila E2F and DP full-length cDNAs placed under control of the Drosophila actin 5C gene promoter (32). 3×DrafE2F3wt and 3×DrafE2F3mut were inserted between SmaI and SacI sites of plasmid TATA-PGVB (34) to create 3×DrafE2F3wt-TATA-luc and 3×DrafE2F3mut-TATA-luc.

Expression of GST fusion proteins

Expression of GST–dE2F and GST–dDP fusion proteins in Escherichia coli was carried out as described earlier (37). The E.coli were collected and suspended in a solution containing 25 mM HEPES pH 7.9, 1 mM EDTA, 0.02% 2-mercaptoethanol, 10% glycerol, 0.1% Tween-80 and 0.2 M KCl, sonicated and then centrifuged. The supernatant was loaded onto a glutathione–Sepharose column equilibrated with buffer A (20 mM Tris–HCl pH 7.8, 6% glycerol, 0.1 M KCl, 0.1 mM EDTA, 0.2% Tween-80 and 5 mM DTT) and washed with 20 ml of buffer A and 20 mM WE buffer (20 mM Tris–HCl pH 7.8, 2 mM MgCl2 and 1 mM DTT). Then the proteins were eluted with 20 ml of buffer G [50 mM Tris–HCl pH 9.6 and 5 mM glutathione (reduced form)], concentrated using Centricon 20 (Amicon) and dialyzed against PC buffer (50 mM Tris–HCl, pH 7.8, 1 mM DTT and 0.1 mM EDTA) containing 0.15 M KCl and 50% glycerol. The purified proteins were stored at –20°C.

Preparation of nuclear extracts and band mobility shift assays

Band mobility shift assays were performed as described earlier (15). For the GST fusion protein, 32P-labeled probes (20 000 c.p.m.) were incubated for 15 min on ice in a reaction mixture containing 20 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM EDTA, 10% glycerol, 0.5 mM DTT, 0.1 M KCl, 500 ng sonicated herring sperm DNA and 500 ng poly(dI–dC). For this step unlabeled oligonucleotides were added as competitors. Then purified GST fusion proteins were added and incubation continued for 10 min on ice. For the Kc cell nuclear extract 32P-labeled probes (20 000 c.p.m.) were incubated in a reaction mixture containing 25 mM HEPES pH 7.9, 1 mM EDTA, 10% glycerol, 0.1% Tween-80, 0.02% 2-mercaptoethanol and 1 µg sonicated herring sperm DNA. In the case of antibody treatment Kc cell nuclear extracts were preincubated with antibody for 1 h on ice. Rabbit anti-dE2F serum was kindly supplied by Dr J.Nevins (Duke University Medical Center, Durham, NC) (38). DNA–protein complexes were electrophoretically resolved on 4% polyacrylamide gels in 50 mM Tris–borate pH 8.3, 1 mM EDTA and 2.5% glycerol at 25°C. Gels were dried and autoradiographed or quantified with a BAS2000 (Fuji Film) imaging analyzer.

Primer extension

Primer extension analysis was performed as described earlier (19). Total cellular RNA was extracted from Drosophila 16–20 h embryos and third instar larvae by methods detailed previously (39). A 35mer primer (PR1) complementary to the region downstream of the first ATG codon was chemically synthesized. The reaction product was analyzed by gel electrophoresis under denaturing conditions, followed by autoradiography. 35S-labeled DNA fragments, produced in a dideoxy sequencing reaction with plasmid pBluescript-Draf 1.23 as template using PR1 as a primer, were run in parallel, allowing precise mapping of the transcription initiation site.

Cell culture, DNA transfection and luciferase assay

Drosophila Kc cells were grown at 25°C in M3 (BF) medium (Sigma) (40,41) supplemented with 2% fetal bovine serum and 0.5% penicillin/streptomycin (Gibco BRL). Transfection of various DNA mixtures into Kc cells was performed using Cell-Fectin reagent (Gibco BRL) and cells were harvested 48 h thereafter. The luciferase assay was carried out by means of a Dual-luciferase assay reporter system (Promega), as described previously (18). Firefly luciferase activities were normalized to Renilla luciferase activities and all assays were performed within the range of a linear relation of activity to incubation time and protein amount.

X-gal staining and in situ hybridization

Embryos were collected, dechorinated and incubated in a fixing solution containing 0.5 ml of PEM buffer (0.1 M PIPES, 2 mM EDTA and 1 mM MgSO4 pH 6.9), 0.45 ml of heptane and 0.05 ml of 37% formaldehyde for 30 min. The embryos were briefly treated with 100% methanol, rinsed with PBT (phosphate-buffered saline containing 0.3% Triton X-100) and incubated in a staining solution containing 10 mM sodium phosphate pH 7.2, 150 mM NaCl, 1 mM MgSO4, 6.1 mM potassium ferricyanide, 6.1 mM potassium ferrocyanide and 0.2% X-Gal in the dark at 37°C for 5–16 h.

Whole mount in situ hybridization to detect expression of the endogenous D-raf gene in embryos was conducted essentially as described by Tautz and Pfeifle (42). Embryos from parents with the genotype +/+; +/+; E2F7172/TM3 (35) were collected, aged at 25°C, dechorionated and fixed, then stored in 70% ethanol at –70°C and rehydrated when needed. As a probe the 2.3 kb HincII–SmaI fragment from plasmid pGEM-Draf4.3 (11) was labeled by the random priming method with a Digoxigenin Non-radioactive DNA Labeling and Detection Kit (Boehringer Mannheim). Embryos were developmentally staged using criteria described by Campos-Ortega and Hartenstein (43).

RESULTS

Potential E2F recognition sequences in the 5′-flanking region of the D-raf gene

To determine the transcription initiation site of the D-raf gene we performed a primer extension experiment using a primer complementary to the sequence near the 5′-end of D-raf cDNA (Fig. 1A). A single band was detected with total RNAs from both 16–20 h embryos and third instar larvae. The results located the start point at 440 bp upstream from the translation initiation site. This site is 215 bp upstream from the start point tentatively identified previously based on the location of a sequence similar to the downstream basal promoter element, which is conserved among Drosophila TATA box-less genes (13).

Figure 1.

Figure 1

Figure 1

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Structure of the 5′-upstream region of the D-raf promoter and base substitution mutants in potential E2F sites. (A) The 32P-labeled primer complementary to the 5′-region of D-raf cDNA was hybridized with total RNA isolated from 16–20 h embryos and third instar larvae. To align the extended products with the genomic sequence, a parallel dideoxy sequencing reaction was carried out using the same primer (lanes A, C, G and T). The major transcription initiation site is defined as nucleotide position +1. (B) Four closed boxes indicate the E2F recognition sites. The locations of each site relative to the transcription initiation site are indicated by numbering.

In the 5′-flanking region of D-raf four sequences similar to the mammalian E2F recognition sequence (5′-TTTCGCGC) were found at positions –83 to –76 (E2F site 1), –397 to –390 (E2F site 2), –428 to –421 (E2F site 3) and –652 to –645 (E2F site 4) with respect to the transcription initiation site (Fig. 1B). Among them, E2F site 3 completely matches the consensus sequence while 5–6 of 8 nt match in the other sites.

Affinity of the Draf–E2F sites for recombinant dE2F/dDP

To examine whether the dE2F/dDP heterodimer recognizes the putative E2F sites found in the D-raf promoter region, a band mobility shift assay was performed using recombinant GST–dE2F and GST–dDP fusion proteins. The oligonucleotide AdE2Fwt contains two copies of the E2F recognition sequence from the adenovirus E2 gene promoter and it bound to GST–dE2F/GST–dDP proteins (Fig. 2). Addition of the Draf-E2Fsite3wt oligonucleotide as a competitor greatly reduced the intensity of the shifted bands and the E2F site 4 oligonucleotide also showed significant competitor activity. On the other hand, the E2F site 1 and 2 oligonucleotides showed weak competitor activities against the GST–dE2F/GST–dDP complex and only when a large amount of the oligonucleotides were included in the reaction. These results indicate that E2F site 3 has a strong affinity for the dE2F/dDP heterodimer.

Figure 2.

Figure 2

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Cooperative binding of GST–dE2F and GST–dDP to the oligonucleotide AdE2Fwt and competition by four Draf–E2F oligonucleotides. Radiolabeled double-stranded AdE2Fwt oligonucleotides were incubated with each lysate from bacteria carrying pGST-dE2F or pGST-dDP in the absence or presence of the indicated amounts of competitor oligonucleotides (indicated at the top of each lane). In the last lane, 2 µl of lysate from bacteria carrying pGEX-2T alone was introduced. AdE2Fwt, oligonucleotide containing two wild-type E2F sites from the adenovirus E2 gene promoter; AdE2Fmut, oligonucleotide containing two mutant E2F sites from the adenovirus E2 gene promoter; Draf-E2Fsite1, oligonucleotide containing the E2F site 1 of the D-raf gene promoter; Draf-E2Fsite2, oligonucleotide containing the E2F site 2 of the D-raf gene promoter; Draf-E2Fsite3, oligonucleotide containing the E2F site 3 of the D-raf gene promoter; Draf-E2Fsite4, oligonucleotide containing the E2F site 4 of the D-raf gene promoter.

Affinity of E2F site 3 for endogenous dE2F/dDP

Recently we identified an 11 nt sequence as the OEBS by affinity selection and amplification (K.Ohno, F.Hirose, Y.Nishida, A.Matsukage and M.Yamaguchi, unpublished results). In a band mobility shift assay using the OEBS oligonucleotide as probe and Kc cell nuclear extracts two distinct DNA–protein complexes were detected (Fig. 3A, lane 1). Addition of anti-dE2F antibody to the binding reaction resulted in a supershift of the slower migrating band (Fig. 3A, lane 3). The results indicate that the slower migrating band represents a complex with dE2F.

Figure 3.

Figure 3

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Complex formation between OEBS oligonucleotides and Kc cell nuclear extracts and competition by Draf-E2Fsite3 oligonucleotide. (A) 32P-labeled OEBS oligonucleotide was incubated with Kc cell nuclear extracts in the presence or absence of anti-dE2F antibody or control normal rabbit antiserum. (B) 32P-labeled OEBS oligonucleotides were incubated with Kc cell nuclear extracts in the presence or absence of the indicated amounts of competitor oligonucleotides (indicated at the top of each lane). OEBS, oligonucleotide containing the wild-type OEBS; Draf-E2Fsite3wt, oligonucleotide containing E2F site 3 in the D-raf gene promoter; Draf-E2Fsite3mut, oligonucleotide containing base substitutions in E2F site 3 in the D-raf gene promoter.

The affinity of E2F site 3 for endogenous dE2F protein was tested by inclusion of the E2Fsite3wt oligonucleotide as a competitor in the binding reactions. As shown in Figure 3B, the intensity of the upper band showing the complex between OEBS and dE2F was greatly diminished by the wild-type sequence, indicating that dE2F has strong affinity for E2F site 3. The E2Fsite3mut oligonucleotide, in which nucleotides were replaced at four positions (Fig. 1B), failed to compete for formation of the complex between OEBS and dE2F (Fig. 3B). The results suggest that dE2F recognizes E2F site 3 specifically.

Activation of the D-raf gene promoter by dE2F and dDP

Since E2F site 3 demonstrated a high affinity for dE2F we tested its E2F-dependent promoter activity. The reporter construct 3×DrafE2F3wt-TATA-luc contains three copies of the wild-type E2F site 3, a TATA box and the luciferase gene and was transfected into Kc cells with or without vectors expressing dE2F and dDP proteins. Transfection of the dE2F expression vector increased the luciferase activity ∼2-fold (Fig. 4). Co-expression of dDP remarkably enhanced luciferase activity, suggesting that the moderate enhancement on overexpression of dE2F alone was limited by the amount of endogenous dDP, which is the active form of E2F, available for heterodimer formation. The sequence specificity of the promoter activity of E2F site 3 was confirmed by the lack of dE2F/dDP-dependent promoter activity detected with the 3×DrafE2F3mut reporter construct (Fig. 4).

Figure 4.

Figure 4

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Effects of dE2F and dDP expression on D-raf gene promoter activity. 3×DrafE2F3wt-TATA-luc having three copies of the Draf-E2Fsite3wt oligonucleotide and 3×DrafE2F3mut-TATA-luc having three copies of the Draf-E2Fsite3mut oligonucleotide were co-transfected into Kc cells with the indicated amounts of pAct-dE2F and pAct-dDP. The luciferase activity of the reporter construct alone was scored as 1.00.

dE2F-dependent expression of Draf–lacZ in embryos

The above results strongly suggested that the D-raf promoter is under the regulation of dE2F/dDP. To elucidate the role of dE2F in expression of D-raf during development, we made a w/w; Draf-lacZ/Draf-lacZ; E2F7172/TM3 line by genetic crossing of E2F7172/TM3 flies with transgenic flies carrying p5′-663Drafwt-lacZ, which contains a DNA fragment from the D-raf promoter region (–663 to +302) fuzed to lacZ (12), and examined expression of Draf–lacZ in embryos defective in dE2F by X-gal staining. It has been demonstrated that maternal dE2F activity starts to be extinguished by the begining of the cellular blastoderm stage (cycle 14) while zygotic expression becomes apparent at stage 12 (35). In the stage 13 normal embryo, in which germ band retraction is taking place, high levels of Draf–lacZ expression were seen in the endoderm and central nervous system (Fig. 5A). The Draf–lacZ expression pattern is essentially identical to the pattern of D-raf transcripts detected by in situ hybridization (13) and to those of S phase genes such as PCNA and cyclin E (44–46). On the other hand, Draf–lacZ expression was greatly reduced in embryos with genotype dE2F7172/dE2F7172 as compared to the control dE2F7172/TM3 case (Fig. 5B and C). We also observed by in situ hybridization that D-raf transcripts were greatly diminished in dE2F7172/dE2F7172 mutant embryos compared to control dE2F7172/TM3 embryos (data not shown). The results showed that expression of D-raf during embryogenesis largely depends on dE2F.

Figure 5.

Figure 5

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Reduced expression of the Draf–lacZ gene in embryos homozygous for the dE2F mutation. The w/w; Draf-lacZ/Draf-lacZ; E2F7172/TM3 line was made through genetic crossing of E2F7172/TM3 flies with transgenic flies carrying p5′-663Drafwt-lacZ, which contains the DNA fragment from the D-raf promoter region (–663 to +302) fuzed to lacZ (12). Expression patterns of Draf–lacZ in wild-type (A) and homozygous dE2F mutant embryos (B) were detected by X-gal staining. The embryos are shown at stage 13, the posterior to the left and the dorsal aspect facing upward. Reduced expression of the Draf–lacZ gene in the homozygous dE2F mutant embryo (B) is apparent all over the region. Approximately one-quarter of the stage 13 embryos from parents with the genotype w/w; p5′-663Drafwt-lacZ/p5′-663Drafwt-lacZ; dE2F7172/TM3 were similar to the embryo shown in (B). (C) Wild-type and E2F7172/E2F7172 embryos are shown on the same slide. (D) p5′-663Drafwt-lacZ/p5′-663Drafwt-lacZ; +/+ embryos stained homogenously are shown as a control. The embryo in the box shows that the Draf–lacZ expression pattern is essentially identical to the pattern of D-raf transcripts detected by in situ hybridization (13).

DISCUSSION

D-raf has multiple roles to play during development and one of its major functions is the regulation of cellular proliferation (1,2). It has been well established that growth factor signaling activates mammalian Raf-1 and increases transcription of the cyclin D1 gene through activation of MAPK. This facilitates formation of Cyclin D1/Cdk4 complexes, which then phosphorylate and inactivate Rb protein, an inhibitor of E2F (47–49). D-raf is expected to function similarly in response to growth stimuli and should be placed upstream of dE2F in the pathway regulating cell proliferation. On the other hand, the pattern of D-raf transcription is similar to that of S phase genes, which are regulated by E2Fs and the DRE/DREF system (14,24). We previously reported that D-raf transcription is positively regulated by the DRE/DREF system (13). In the present study we have shown that D-raf is also regulated by dE2F during development. In the 5′-flanking region of D-raf an E2F-binding sequence (E2F site 3) was identified and shown to be responsible for positive regulation by dE2F.

These observations indicate that D-raf is transcriptionally regulated together with S phase genes by dE2F and DREF despite its major function upstream of dE2F. As a consequence, growth signal-stimulated activation of D-raf would result in an increase in D-raf transcription through activation of dE2F. In contrast to the products of S phase genes, whose abundance oscillates during the cell cycle with periodic synthesis and degradation, D-raf should be stable since maternal D-raf activity is sufficient to support development of embryos homozygous for null D-raf mutations (50,51). This suggests the possibility that transcriptional regulation of the D-raf gene by dE2F may result in accumulation of D-raf protein in proliferating cells. This is in line with the elevated transcription of D-raf in rapidly proliferating cells such as imaginal disc cells (12).

Given its multiple functions throughout development, D-raf expression must be regulated by multiple mechanisms (52). dE2F- and DREF-dependent regulation of D-raf transcription may increase the competence to growth signals of proliferating cells, such as imaginal disc cells, allowing them to proliferate rapidly and continuously. It is of interest whether other genes involved in the transduction of growth signals are also under control of the E2F and DREF systems. Since putative E2F-binding sequences are present in the 5′-flanking region of the human c-raf-1 gene, E2F-dependent regulation may be conserved in mammals and may play a significant role in regulation of cell proliferation during development and homeostasis and in tumor formation.

Acknowledgments

ACKNOWLEDGEMENTS

We are grateful to Dr N.Dyson for the Act–dE2F and Act–dDP expression plasmids and Dr J.Nevins for anti-dE2F serum. We thank Dr M.Moore for valuable comments on the manuscript. This work was supported in part by a grant (976-0500-004-2) from the Korea Science and Engineering Foundation to M.-A.Y. and grants-in-aid from the Ministry of Education, Science, Sports and Culture, Japan.

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