(Received for publication, June 12, 1995; and in revised form, August 9, 1995)
From the
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 E2F in Kc cells activated the PCNA promoter, and the 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, 5`-ACGCGT)(3) , and the specific transcription
factor complex DSC1 (MBF) is required for expression at the
G-S boundary(4, 5) .
In mammalian
cells, expression of genes involved in DNA replication increases
dramatically at late G in response to growth
stimulation(6, 7) . Many of these genes including the
proliferating cell nuclear antigen (PCNA) (
)gene contain the
transcription factor E2F-binding site (5`-TTTCGCGC) within their
promoter regions (8, 9, 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 (5`-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.
The sequences of double-stranded oligonucleotides containing E2F
sites in the DNA polymerase promoter were as
follows.
The sequences of double-stranded oligonucleotides containing two E2F sites or their base-substituted derivatives in the adenovirus E2 promoter (20) were as follows.
Nucleotides with substitution for the wild type sequence are shown by lowercase letters. The double-stranded oligonucleotide, DRE-P contains the 24-base pair DRE sequence of the PCNA gene promoter and the 6-base pair linker sequence(21) . DRE-PM contains a 2-base pair substitution in the DRE sequence of the DRE-P(21) . The other oligonucleotides used were as follows: CAT-1, 5`-GCTCCTGAAAATCTCGCCAAGCTCGAGC; mutI, 5`-GGCGATATCGCCTGTGGCTTTTCACATCCCTATCCCGCTCATTTctCaaGCCTGAAAGT; mutII, 5`-GGCGATATCGCCTGTGGCTTTTCACATCCCTcgCaaGCTCATTTAGCC; mutI&, 5`-GGCGATATCGCCTGTGGCTTTTCACATCCCTcgCaaGCTCATTTctCaaGCCTGAAAGT.
The plasmid p5`-607DPCNAlacZW8HS (22) contains the PCNA gene fragment spanning from -607 to +137 fused with the lacZ in a P-element vector. The plasmid p5`-168DPCNAlacZW8HS (22) contains the PCNA gene fragment spanning from -168 to +137 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(-168) and SacII (+23) and then inserted between XhoI(-607) and SacII (+23) sites of the p5`-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 5`-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) .
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.
Figure 1:
Nucleotide sequences of potential E2F
recognition sites in the Drosophila DNA polymerase and
PCNA genes. A, site I in the DNA polymerase
promoter
contains an overlapping pair of E2F recognition sequences as indicated
by horizontallines. Locations of each site relative
to the cap site are indicated by numbers with verticallines. B, constructs of wild type PCNA-lacZ (p5`-168DPCNAlacZW8HS) and PCNA-CAT (p5`-168DPCNACAT)
fusion genes are shown. The verticallines with horizontalarrows indicate the cap site. The open and closedboxes indicate the 5`-untranslated
and coding sequences of the PCNA gene, respectively. The darkstippledboxes indicate the DRE sequence. The shaded and the hatchedboxes indicate the lacZ coding and CAT coding sequences, respectively.
Nucleotide sequences in and around the two E2F sites of wild type and
mutant PCNA genes are shown. Nucleotides with substitution for the wild
type sequence are shown by lowercaseletters.
Nucleotide sequences of potential E2F recognition sites I and II are
indicated by boxes.
Figure 2: Cooperative binding of E2F and DP to the oligonucleotide AdE2Fwt and competition by wild type and mutant E2F-P oligonucleotides. A, radiolabeled double-stranded AdE2Fwt oligonucleotides were incubated with or without (-, lanee) the indicated amounts of lysates from bacteria carrying pGEX-2T (lanesa-c), pGST-dE2F (lanesb and d), or pGST-dDP (lanesc and d), individually (lanea) or in combination (lanesb-d). B, radiolabeled double-stranded AdE2Fwt oligonucleotides were incubated with or without (-, lanet) 1 µl 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.
Figure 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 µg 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; polsite2+3,
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).
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, lanesg-k). Therefore, site II appears to play a major role in the binding.
Figure 4:
Effects of mutations in E2F sites on PCNA
promoter activity in Kc cells. One µg each of CAT plasmids
harboring wild type or mutant PCNA promoters (indicated at the top of each lane) were cotransfected with 0.1 µg of
pDhsp70-L plasmid into Kc cells. 48 h after the transfection,
cell extracts were prepared to determine the CAT expression levels,
normalized to the luciferase activity. Averaged values obtained from
two independent dishes with standard deviations are given as CAT
activity relative to that of p5`-168DPCNACAT (-168, lanesa and b). Promoterless CAT (pSKCAT)
plasmids were included as controls (lanesk and l). Acetylated forms of
[C]chloramphenicol were undetectable in lanesi-l. Acetylated and
nonacetylated forms of [
C]chloramphenicol are
marked by Ac and CM, respectively. -168, p5`-168DPCNACAT; -168mutI, p5`-168
mutIDPCNACAT; -168mutII, p5`-168 mutIIDPCNACAT; -168mutI&II, p5`-168 mutI&; -86, p5`-86DPCNACAT; -168
mutI&IIE2F-P(N), p5`-168 mutI&-P(N)DPCNACAT; -168 mutI&IIE2F-P(R), p5`-168
mutI&-P(R)DPCNACAT.
These E2F sites are essential but not sufficient for the promoter activity, since deletion up to position -86 completely abolished the promoter activity, even when the two E2F sites were kept intact (Fig. 4, lanesi and j). In addition, insertion of the E2F-P downstream of the CAT gene of the plasmid p5`-168E2FmutI& did not enhance CAT expression (Fig. 4, lanesm-r), indicating the importance of the position of E2F sites for activation of transcription.
Figure 5: Effect of cotransfecting E2F or DP expression plasmid on the CAT activity directed by the regulatory region of the PCNA gene. 0.5 µg each of plasmid p5`-168DPCNACAT (upperpanel) or p5`-116DPCNACAT (lowerpanel) was cotransfected into Kc cells with 0.1 µg of pDhsp70-L plasmid and the indicated amounts of Act-dE2F (opencircles), pdrosE2F1WT (closedcircles) or Act-dDP (closedsquares). 48 h after the transfection, cell extracts were prepared to determine the CAT expression levels, normalized to the luciferase activity and plotted against activity in the absence of the effector plasmid. Averaged values obtained from three independent transfections are shown.
Figure 6:
Mapping of the target region in the PCNA
gene for activation by E2F protein. 0.5 µg each of the indicated
5`-deletion (A) or base substitution derivatives (B)
of plasmid p5`-168DPCNACAT were cotransfected into Kc cells with
(+) or without(-) 1 µg of Act-dE2F plasmid. 0.1 µg
of pDhsp70-L plasmid was also included to normalize CAT
activity to the luciferase activity. 48 h after the transfection, cell
extracts were prepared to determine the CAT expression levels. Averaged
values obtained from two independent dishes are given as -fold
stimulation relative to those obtained by transfections without
Act-dE2F effector plasmid. A and B show independent
experiments, and wild type PCNA-CAT (-168) was included
as a control. Acetylated forms of
[C]chloramphenicol were undetectable in lanesq-x of panelA.
Acetylated and nonacetylated forms of
[
C]chloramphenicol are marked by Ac and CM, respectively. -168, p5`-168DPCNACAT; -149, p5`-149DPCNACAT; -119,
p5`-119DPCNACAT; -116, p5`-116DPCNACAT; -86, p5`-86DPCNACAT; -168mutI, p5`-168
mutIDPCNACAT; -168mutII, p5`-168 mutIIDPCNACAT; -168mutI&II, p5`-168
mutI&.
To examine the responsibility of E2F sites for the activation by E2F, base substitution derivatives of p5`-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, lanesm-p). Therefore, at least one of two E2F sites is required for the E2F protein to activate the PCNA promoter.
Figure 7:
Effects of base substitution mutations in
E2F sites on PCNA promoter activity in transgenic flies. Male
transgenic flies (indicated in each panel) were crossed with
female wild type flies, and extracts were prepared from Drosophila bodies at various stages of development. The -galactosidase
activities in the extracts are expressed as absorbance units/h/mg of
protein. Closedbars indicate the average values for
independent transgenic strains carrying the indicated fusion gene.
Numbers (n) of independent strains carrying the same fusion
gene are given in each panel.
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, bottompanel).
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, panelsB, G, and L). More extensive reduction was observed with the larvae
having mutations in site II (Fig. 8, panelsC, H, and M), and mutations in both sites completely
abolished the staining signal in these tissues (Fig. 8, panelsD, I, and N). Although
results with leg discs are shown in panelsK-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, panelsQ and R), although mutations in both sites completely abolished the
staining signal (Fig. 8, panelS). 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.
Figure 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 (panelsA-D), brain
lobes (panelsF-J), and leg discs (panelsK-O) were dissected from the third instar larvae of
male transgenic flies carrying the fusion gene (indicated at
the leftside of each panel)
wild
type females. Ovaries (lanesP-T) were
dissected from 3-day-old adult females carrying the transgenes
indicated at the leftside 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 (panelsE, J, O, and T). -168, strain 73 carrying the
p5`-168DPCNAlacZW8HS; mutI, strain 29 carrying the
p5`-168 mutIDPCNAlacZW8HS; mutII, strain 5 carrying
the p5`-168 mutIIDPCNAlacZW8HS; mutI&II,
strain 72 carrying the p5`-168
mutI&lacZW8HS.
In mammalian cells, a group of genes that are commonly
regulated in late G 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 5`-flanking region. Two of them (5`-TTTCGCGG and
5`-CTTCGCGG) match seven out of eight and six out of eight nucleotides
of the binding consensus (5`-TTTCGCGC) for mammalian E2F, respectively.
The other site (5`-TTACCCGC) matches seven out of eight nucleotides of
the E2F recognition site I of the DNA polymerase
180-kDa subunit
gene. The 50-kDa primase gene also contains a potential E2F site in its
5`-untranslated region. This site (5`-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 -168 to
-119 (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 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.