Distinct roles of E2F recognition sites as positive or negative elements in regulation of the DNA polymerase [alpha] 180 kDa catalytic subunit gene promoter during Drosophila development
Distinct roles of E2F recognition sites as positive or negative elements in regulation of the DNA polymerase [alpha] 180 kDa catalytic subunit gene promoter during Drosophila developmentMasamitsu Yamaguchi*, Yuko Hayashi, Fumiko Hirose, Yoshio Nishimoto and Akio Matsukage
Laboratory of Cell Biology, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya 464, Japan
Received June 27, 1997;Revised and Accepted August 12, 1997
ABSTRACT
The transcription factor E2F plays a key role in transcriptional control during the growth cycle of higher eukaryotic cells. The promoter region of the Drosophila DNA polymerase [alpha] 180 kDa catalytic subunit gene contains three E2F recognition sequences located at positions -353 to -342 (E2F site 1), -21 to -14 (E2F site 2) and -12 to -5 (E2F site 3) with respect to the transcription initiation site. Various base substitutions were generated in each or all of the three E2F sites in vitro to allow examination of their effects on E2F binding and promoter function in cultured Kc cells as well as in living flies. Glutathione S-transferase (GST)-E2F and GST-DP fusion proteins were found to cooperate in binding to the three E2F sites in the DNA polymerase [alpha] gene promoter in vitro. In contrast, an E2F-specific activity detected in nuclear extracts of Kc cells showed little affinity for E2F site 1 but strong binding to sites 2 and 3. Transient expression of Drosophila E2F in Kc cells activated the DNA polymerase [alpha] gene promoter and the target sites for activation coincided with E2F sites 2 and 3. However, analyses with transgenic flies indicate that E2F site 3 functions positively in terms of DNA polymerase [alpha] gene promoter activity, while E2F sites 1 and 2 rather have a negative control function. Thus E2F sites play distinct roles as positive or negative elements in regulation of the DNA polymerase [alpha] gene promoter during Drosophila development.
INTRODUCTION
Regulation of cell proliferation is essential for accurate generation of body structures in multicellular organisms. Switching between the non-proliferation and proliferation states of cells is closely associated with a coordinated shift in transcription of proliferation-related genes. DNA replication is clearly of prime importance for this process and there is much evidence that expression of the involved genes is finely regulated in accordance with progression of differentiation during development (1 ,2 ).
In budding yeast genes involved in DNA replication contain a common promoter element (MluI cell cycle box, 5'-ACGCGT) (3 ) and the specific transcription factor complex DSC1 (MBF) is required for expression at the G1/S boundary (4 ,5 ).
In mammalian cells expression of genes involved in DNA replication increases dramatically at late G1 in response to growth stimulation (6 ,7 ). Those encoding DNA polymerase [alpha], thymidine kinase, ribonucleotide reductase, dihydrofolate reductase (DHFR) and proliferating cell nuclear antigen (PCNA) contain at least one transcription factor E2F binding site (5'-TTTCGCGC) within their promoter regions (8 -10 ) or the first intron (11 ). Mammalian E2F is a heterogeneous factor representing the combined activity of at least seven gene products, called E2F-1, E2F-2, E2F-3, E2F-4, E2F-5, DP-1 and DP-2. The E2F and DP subtypes associate to form stable complexes and activate transcription in a cooperative manner (12 ,13 ). Regulation of E2F function also appears to play an important role during muscle terminal differentiation (14 ).
In Drosophila promoter regions of genes for the DNA polymerase [alpha] 180 kDa catalytic subunit (15 ), the 73 kDa regulatory subunit (16 ) and PCNA (17 ) contain a common 8 bp palindromic sequence (5'-TATCGATA), named the DNA replication-related element (DRE) (18 ). We have also found a specific DRE binding factor (DREF) consisting of an 80 kDa polypeptide homodimer (18 ) and cloned its cDNA (19 ). The DRE-DREF system appears to play a key role in differentiation-coupled repression of cell proliferation during Drosophila embryogenesis (20 ). cDNAs for Drosophila homologs of E2F-1 and DP-1 have been cloned (21 -23 ), these two proteins interacting with each other to effect sequence-specific DNA binding and optimal transactivation (22 ). Multiple E2F recognition sequences have been identified in the promoters of the Drosophila DNA polymerase [alpha] (21 ) and PCNA genes (24 ) and transcription of these genes is completely lost in E2F mutant embryos after division cycle 16 (25 ). We have reported that E2F recognition sites are essential for PCNA gene promoter activity in vivo (24 ). However, in the case of the DNA polymerase [alpha] gene the precise role(s) of each E2F recognition sequence for promoter activity in vivo has yet to be determined.
In the present study we therefore generated mutations in each or all of the three E2F sites in the DNA polymerase [alpha] gene promoter and subsequently examined their effects on E2F binding and promoter function in cultured Kc cells as well as in living flies. Although we detected a binding factor(s) for E2F sites 2 and 3, its binding specificity was found to be different from that of recombinant Drosophila E2F and DP. Transient expression of Drosophila E2F in Kc cells activated the DNA polymerase [alpha] gene promoter and the target site for activation coincided with E2F recognition sites 2 and 3. However, analyses with transgenic flies indicated that whereas E2F site 3 functions positively to stimulate DNA polymerase [alpha] gene promoter activity, sites 1 and 2 rather have a negative control function throughout Drosophila development.
MATERIALS AND METHODS
Oligonucleotides
The sequences of double-stranded oligonucleotides containing E2F recognition sites in the Drosophila DNA polymerase [alpha] gene promoter were as follows:
Pol[alpha]site1
attggtaccTCGGATTTCCCGCCAAAATAT
ggAGCCTAAAGGGCGGTTTTATA
Pol[alpha]site2+3
gatcCGATATGTTCCCGCCATTCCCGCTTTGA
GCTATACAAGGGCGGTAAGGGCGAAACTctag
Pol[alpha]mut2
gatcCGATATGggCaaGCCATTCCCGCTTTGA
GCTATACccGttCGGTAAGGGCGAAACTctag
Pol[alpha]mut3
gatcCGATATGTTCCCGCCAggCaaGCTTTGA
GCTATACAAGGGCGGTccGttCGAAACTctag
Pol[alpha]mut2+3
gatcCGATATGggCaaGCCAggCaaGCTTTGA
GCTATACccGttCGGTccGttCGAAACTctag
The sequences of double-stranded oligonucleotides containing two E2F sites or their base substituted derivatives in the adenovirus E2 gene promoter (26 ) were as follows:
AdE2Fwt
gatccTCCGTTTTCGCGCTTAAATTTGAGAAAGGGCGCGAAACTGGa
gAGGCAAAAGCGCGAATTTAAACTCTTTCCCGCGCTTTGACCtctag
AdE2Fmut
gatccTCCGTTgTCGaGCTTAAATTTGAGAAAGGGCtCGAcACTGGa
gAGGCAAcAGCtCGAATTTAAACTCTTTCCCGaGCTgTGACCtctag
Substituted nucleotides are shown by small letters and the E2F recognition sequences are underlined. The double-stranded 30 bp oligonucleotide DRE-P contains the 24 bp DRE sequence of the PCNA gene promoter and the 6 bp linker sequence (18 ) and DRE-PM contains a 2 bp substitution in the DRE (18 ). The other oligonucleotides used were:
The plasmid pDgpol[alpha]2.4BB contains a 2.4 kb BamHI fragment of the DNA polymerase [alpha] gene (15 ). A DNA polymerase [alpha] gene fragment from -358 to +15 was generated by the PCR method (27 ) using pDgpol[alpha]2.4BB as template with Pol[alpha]KpnIwt and Pol[alpha]SacIIwt as primers. The PCR product was digested with KpnI and SacII, then inserted between KpnI and SacII sites of plasmid pSKCAT (28 ) to create plasmid p5'-358Dpol[alpha]CAT. Plasmid p5'-358Dpol[alpha]mut1CAT was constructed in the same way except that Pol[alpha]KpnImut and Pol[alpha]SacIIwt were used as PCR primers. Plasmids p5'-358Dpol[alpha]mut2CAT, p5'-358Dpol[alpha]mut3CAT and p5'-358Dpol[alpha]mut2+3CAT were constructed in the same way except that Pol[alpha]SacIImut2, Pol[alpha]SacIImut3 and Pol[alpha]SacIImut2+3 in addition to Pol[alpha]KpnIwt were used as PCR primers. Plasmid p5'-358Dpol[alpha]mut1+2+3CAT was constructed in the same way except that Pol[alpha]KpnImut and Pol[alpha]SacIImut2+3 were used as PCR primers. The obtained plasmids were verified by nucleotide sequence analysis with synthetic primers (29 ).
To construct plasmid p5'-358Dpol[alpha]lacZW8HS, p5'-358Dpol[alpha]CAT was digested with KpnI (-358), blunt-ended using T4 DNA polymerase and then digested with SacII (+15). A 373 bp fragment was isolated and inserted between the blunt-ended XhoI (-607) and SacII (+23) sites of p5'-607DPCNAlacZW8HS. Plasmid p5'-607DPCNAlacZW8HS (28 ) contains the PCNA gene fragment spanning -607 to +137 fused with lacZ in a P element vector. To create mutated derivatives, fragments having various mutations in E2F sites were isolated from CAT plasmids in the same way, then inserted between the blunt-ended XhoI (-607) and SacII (+23) sites of p5'-607DPCNAlacZW8HS. The plasmids obtained were verified by nucleotide sequence analysis with synthetic primers.
The expression plasmids Act-dE2F (22 ) and Act-dDP (22 ) respectively contain Drosophila E2F and DP full-length cDNAs placed under control of the Drosophilaactin 5C gene promoter (30 ). Expression plasmid pdrosE2F1WT (21 ) contains Drosophila E2F cDNA covering amino acids 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 Drosophilaactin 5C gene promoter. Plasmid pDhsp70-L (31 ) contains the luciferase gene under control of the Drosophilahsp70 gene promoter (32 ).
Fusion genes of E2F with glutathione S-transferase (GST) and of DP with GST were prepared as described earlier (24 ) to produce full-length E2F and DP proteins fused with GST.
All plasmids were propagated in Escherichia coli XL-1 Blue, isolated by standard procedures (33 ) and further purified through two cycles of ethidium bromide/CsCl density gradient centrifugation.
Preparation of nuclear extracts and band mobility shift assay
Preparation of nuclear extracts from Kc cells was as described elsewhere (18 ). 32P-end-labeled Pol[alpha]site2+3 oligonucleotides (160 pg) and unlabeled competitor DNA fragments were incubated in 15 [mu]l reaction mixture containing 20 mM HEPES, pH 7.6, 150 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 12% glycerol, 0.5 [mu]g poly(dI[middot]dC) for 5 min on ice. Then nuclear extracts were added and the mixtures incubated for 15 min on ice. Complexes of DNA and binding protein(s) were electrophoretically separated from free probes in 4% polyacrylamide gels in 50 mM Tris-borate, pH 8.3, and 1 mM EDTA containing 2.5% glycerol at room temperature. Gels were then dried and autoradiographed.
Expression of GST fusion proteins and band mobility shift assay
Expression of GST-E2F and GST-DP fusion proteins was carried out as described elsewhere (34 ). Lysates of cells were prepared by sonication in buffer D containing 0.6 M KCl, 1 mM PMSF, 0.5% Triton X-100 and 1 [mu]g/ml each pepstatin, leupeptin and aprotinin, cleared by centrifugation at 12 000 g for 20 min at 4oC and used for band mobility shift assays using a 32P-end-labeled AdE2Fwt oligonucleotide (117 pg) as probe. The assay was carried out as described above except that the reaction mixture for binding contained 20 mM HEPES, pH 7.5, 120 mM KCl, 10 mM MgCl2, 1 mM EGTA, 0.5 mM dithiothreitol, 10% glycerol, 1 [mu]g sonicated salmon sperm DNA.
DNA transfection into cells, CAT assay and luciferase assay
Drosophila Kc cells (35 ) were grown in M3(BF) medium supplemented with 2% fetal calf serum (36 ) and plated at ~5 * 106 cells/60 mm dish for 16 h before DNA transfection by the calcium phosphate co-precipitation technique as described elsewhere (24 ). Aliquots of 1 or 0.5 [mu]g DNA polymerase [alpha] gene promoter-CAT plasmid as a reporter plasmid and 0.1 [mu]g pDhsp70-L as an internal control plasmid were co-transfected with the indicated amounts of effector plasmid. The total amount of effector plasmid was kept constant by addition of expression vector pAcGEM3 (28 ) and the total amount of DNA was adjusted to 10 [mu]g by addition of pGEM3. Cells were harvested at 48 h after transfection. Cell extracts were prepared and CAT activity was measured as described previously (37 ). 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 Ink) as described previously (9 ). All assays were performed within the range of linear relation of activity to incubation time and protein amount. CAT activity was normalized to luciferase activity. The obtained values were essentially similar to those normalized to protein amount determined by BioRad protein assay.
Establishment of transgenic flies
Fly stocks were maintained at 25oC on standard food. Canton S flies were used as the wild-type strain. P element-mediated germline transformation was carried out as described earlier (38 ) and G1 transformants were selected on the basis of white eye color rescue (39 ). Multiple independent lines were obtained for each of the various fusion genes. The established transgenic fly strains and their chromosomal linkages are listed in Table 1 .
Analysis of expression patterns of DNA polymerase [alpha]-lacZ fusion genes
Quantitative measurement of [beta]-galactosidase activity in extracts was performed (40 ). Male transgenic flies were crossed with female wild-type flies. Groups of 50-100 dechorionated embryos, larvae, pupae and adult flies were homogenized in 500 [mu]l ice-cold [beta]-galactosidase assay buffer (50 mM potassium phosphate, pH 7.5, 1 mM MgCl2). Homogenates were centrifuged at 10 000 g at 4oC for 5 min. For each assay 50-200 [mu]l supernatant were added to give 1 ml assay buffer containing 1 mM chlorophenol red [beta]-D-galactopyranoside substrate (CPRG; Boehringer Mannheim). Reaction incubations were at 37oC in the dark. Substrate conversion was measured at 574 nm using a spectrophotometer 0.25, 0.5, 0.75, 1 and 1.5 h after addition of extract and the rate of color development was linear. [beta]-Galactosidase specific activity was defined as absorbance units/h/mg protein. To correct for endogenous [beta]-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. The protein concentrations of the extracts were determined by BioRad protein assay.
. Transformants carrying lacZ fused to the DNA polymerase [alpha] gene 5'-flanking sequence
P element plasmids
Strains
Chromosome linkage
p5'-358Dpol[alpha]lacZW8HS
1
III
20
II
27
II
44
II
57
X
61
III
76
II
93
II
100
X
101
III
103a
X
p5'-358Dpol[alpha]mut1lacZW8HS
6
III
18
II
20
II
23
II
40
X
p5'-358Dpol[alpha]mut2lacZW8HS
3
III
23
II
p5'-358Dpol[alpha]mut3lacZW8HS
31
II
37
III
52
II
59
II
84
II
p5'-358Dpol[alpha]mut2+3lacZW8HS
12
III
26
III
28a
III
34
II
97
X
p5'-358Dpol[alpha]mut1+2+3lacZW8HS
63
III
64
III
83
II
85
II
127
II
aLines whose lacZ expression patterns are different from those of other lines carrying the same fusion gene.
RESULTS
GST-E2F and GST-DP fusion proteins cooperate to bind to the E2F recognition sequences in the DNA polymerase [alpha] gene promoter
Detection of a binding factor(s) in Drosophila Kc cell nuclear extracts that discriminates between the E2F sites
Nuclear extracts were prepared from Kc cells and band mobility shift assays were carried out. As shown in Figure 3 A, lane a, a DNA-protein complex could be detected using oligonucleotide pol[alpha]site2+3 as probe. The band shifted with 32P-labeled pol[alpha]site2+3 was diminished by adding an excess amount of unlabelled pol[alpha]site2+3 as competitor (Fig. 3 A, lanes b-d). The oligonucleotide containing the wild-type E2F site from the adenovirus E2 promoter (26 ) competed for binding when added to the reaction in excess (Fig. 3 A, lanes h-j), but this was not the case with the mutant E2F site from the adenovirus E2 promoter (Fig. 3 A, lanes k-m). Unexpectedly, oligonucleotide pol[alpha]site1 did not compete for binding under the examined conditions (Fig. 3 A, lanes e and f) and no formation of the sequence-specific DNA-protein complex was observed using a pol[alpha]site1 oligonucleotide as probe (data not shown). As shown in Figure 3 B, the oligonucleotides carrying base substitutions in either or both of E2F sites 2 and 3 did not compete for binding, so that both appear to be required.
Effects of mutations in the E2F recognition sequences on the DNA polymerase [alpha] gene promoter activity in cultured Kc cells
The DNA polymerase [alpha] gene promoter carrying mutations in either or both of two E2F recognition sequences was placed upstream of the CAT gene in a CAT vector (Fig. 1 ). Plasmids carrying these constructs were transfected into Kc cells and CAT expression levels determined. As shown in Figure 4 , the plasmid carrying base substitutions in E2F site 1 showed only a marginal reduction of CAT expression as compared with that of the original plasmid, p5'-358Dpol[alpha]CAT. The plasmid carrying base substitutions in E2F site 3 showed 66% of the CAT expression of p5'-358Dpol[alpha]CAT (Fig. 4 , lanes g and h). More extensive reduction of CAT expression was observed with plasmid p5'-358Dpol[alpha]mut2CAT carrying base substitutions in E2F site 2 (Fig. 4 , lanes e and f). A slight further reduction in CAT expression was observed with plasmid p5'-358Dpol[alpha]mut1+2+3CAT carrying base substitutions in all E2F sites (Fig. 4 , lanes k and l). These results indicate that E2F site 2 plays a major role and sites 1 and 3 might play additional roles in regulation of DNA polymerase [alpha] gene promoter activity.
Mapping of the target region in the DNA polymerase [alpha] gene promoter for activation by E2F protein
Roles of E2F recognition sites in function of the DNA polymerase [alpha] gene promoter during fly development
Although the results of CAT transient expression assay in Kc cells clearly demonstrated an important role for E2F recognition sites in DNA polymerase [alpha] promoter activity, these observations have to be confirmed in living flies and transgenic Drosophila provide an appropriate system to characterize transcriptional regulatory elements in vivo. We have established transgenic flies carrying DNA polymerase [alpha] (-358 to +15) and lacZ fusion genes. Established transgenic lines and their chromosomal linkages are listed in Table 1 . Male transgenic flies were crossed with wild-type females to examine zygotic lacZ expression. This 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). This pattern of expression is very similar to the levels of zygotically expressed DNA polymerase [alpha] mRNA during development (15 ).
DISCUSSION
Three E2F recognition sites have been identified in the gene for the 180 kDa catalytic subunit of Drosophila DNA polymerase [alpha] (21 ). Generation of various base substitutions singly or in combination of the recognition sequences in vitro allowed examination of their effects on E2F binding and promoter function in cultured Kc cells as well as in transgenic flies. As summarized in Table 2 , we have confirmed binding of complexes of GST-E2F and GST-DP fusion proteins to all three E2F sites, with sites 1 and 2 appearing to be more effective for binding than site 3. A binding factor(s) for E2F sites 2 and 3 was detected in nuclear extracts of Drosophila Kc cells. Although the factor(s) required both E2F sites 2 and 3 for binding, it showed little affinity for E2F site 1 and no detection of binding activity to E2F site 1 in nuclear extracts has so far been possible. One possibility to interpret this difference is that the GST tag might modify the DNA binding properties of the E2F-DP complex. Another possibility is that complexes of E2F and DP in nuclear extracts are likely to be associated with other protein(s), like RBF (RB family protein) (23 ,41 ), and therefore might behave differently from complexes of GST-E2F and GST-DP fusion proteins. Further analyses using specific antibodies to E2F, DP and RBF might make it possible to identify each component in the complexes formed in nuclear extracts.
. Summary of the roles of E2F recognition sites of the DNA polymerase [alpha] gene
E2F sites
1
2
3
Binding activity
GST-E2F/GST-DP
+++
++
+
Kc cell nuclear extract
-
++
Promoter stimulation
Kc cell
[up arrow]
[up arrow][up arrow][up arrow]
[up arrow][up arrow]
Embryo
<=> <=>
<=> <=>
[up arrow][up arrow][up arrow]
Larva
<=> <=>
<=> <=>
[up arrow][up arrow]
Ovary
<=>
<=>
No
[up arrow], [up arrow][up arrow], [up arrow][up arrow][up arrow], weak moderate and strong stimulation; <=> , <=> <=> , weak and moderate repression. No, no significant effect
A number of studies have been conducted to explore E2F effects during the cell cycle. In mammalian cells expression of a group of genes that are commonly regulated in late G1 of the growth response and that encode proteins important for DNA replication appear to be regulated by E2F (8 ). Thus critical roles of E2F sites for regulated expression in late G1 have been demonstrated with the 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 provide an appropriate system to characterize E2F recognition sites in vivo. In the present study of the DNA polymerase [alpha] gene promoter function we have observed several differences in results between in vitro and transgenic fly analyses (Table 2 ). It is known that high copy numbers (>105) of plasmid DNA are incorporated into cells by the calcium phosphate co-precipitation method and most of them exist in an episomal state in cells. In contrast, the P element method provides only one copy of the transgene integrated into the chromosome. Therefore, transgenic fly analysis is very likely to more faithfully represent regulation in vivo.
Previously we identified two E2F sites in the promoter region of the PCNA gene (24 ). Analyses with transgenic flies demonstrated that these two sites are essential for PCNA gene promoter activity throughout development (24 ). However, E2F sites alone proved to be insufficient for PCNA gene promoter activity during the embryonic and larval stages, since deletion of the upstream region containing the URE and DRE sequences completely abolished the associated promoter activity (42 ). Thus the URE, DRE and E2F sites presumably cooperate to direct optimal promoter activity of the PCNA gene.
In the case of the DNA polymerase [alpha] gene promoter two of the three E2F recognition sites were here found to act rather negatively on promoter activity during development with only the E2F site 3 stimulating promoter activity. E2F sites of several mammalian genes have been reported to act as negative elements in the G0 and G1 phases of the cell cycle, but release of RB protein from the binding complex effects a switch to a positive influence (43 ,44 ). Therefore, protein complexes interacting with E2F sites 1 and 2 in vivo might contain negative regulators, like RBF, and those interacting with E2F site 3 might not.
The observation that even when all E2F sites 1-3 were mutated lacZ expression in larvae, pupae and adults was still observed (Fig. 7 ) demonstrates that the requirement for DNA polymerase [alpha] gene promoter activity is less stringent than for the PCNA gene (24 ). Since the DNA polymerase [alpha] gene promoter contains three DRE sequences, while the PCNA gene promoter contains just one, they might be able to substitute at least partly the function of E2F sites during development. Alternatively, some unidentified additional E2F sites might play roles in activation of the DNA polymerase [alpha] gene promoter in vivo.
ACKNOWLEDGEMENTS
We are grateful to Drs K.Ohtani and J.Nevins for providing pdrosE2F1WT, N.Dyson for Act-dE2F and Act-dDP and M.Asano, J.Nevins and M.Moore for critical reading of the manuscript. This work was supported in part by grants-in-aid from the Ministry of Education, Science and Culture, Japan.
27 Kawasaki,E.S. (1990) In Innis,M.A., Gelfand,D.H., Sninsky,J.J. and White,T.J. (eds), PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, pp. 21-27.
