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(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 In mammalian
cells, expression of genes involved in DNA replication increases
dramatically at late G In Drosophila, we have
isolated genes for PCNA (15) and the DNA polymerase 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
The sequences of double-stranded oligonucleotides containing E2F
sites in the DNA polymerase
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
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; pol
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
[
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
[
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
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
To corroborate the results from
colorimetric assays using crude extracts,
Figure 8:
Demonstration of
In mammalian cells, a group of genes that are commonly
regulated in late G 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 In our previous studies of Drosophila genes for PCNA and DNA polymerase 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
Volume 270,
Number 42,
Issue of October 20, 1995 pp. 25159-25165
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-S boundary(4, 5) . 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) .
(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) .
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.
promoter were as
follows.
Plasmid Construction
The plasmid p5`-168DPCNACAT contains the PCNA gene fragment
spanning from -168 to +23 placed upstream of the
chloramphenicol acetyltransferase (CAT) gene in the plasmid
pSKCAT(22) . A fragment from -86 to +57 having a
base-substitutional mutation in E2F site I was generated by the
polymerase chain reaction (PCR) method (23) using
p5`-168DPCNACAT as a template with primers CAT-1 and mutI. The PCR
product was digested with EcoRV(-80) and SacII
(+23) and then replaced with the fragment between EcoRV
and SacII sites of the p5`-168DPCNACAT to create the plasmid
p5`-168E2FmutIDPCNACAT. Plasmids p5`-168E2FmutIIDPCNACAT and
p5`-168E2FmutI& were constructed in the same way except
that mutII and mutI& 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
p5`-168E2FmutI& in normal or reverse orientation to
create plasmids p5`-168E2FmutI&-P(N)DPCNACAT and
p5`-168E2FmutI&-P(R)DPCNACAT, respectively.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 µl of reaction mixture containing 15 mM Hepes (pH 7.6),
60 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol,
12% glycerol, 1 µg of poly(dI-dC) for 10 min on ice. Unlabeled
competitor DNA fragments were added at this step. Then, 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 µg/ml each of pepstatin,
leupeptin, and aprotinin. Lysates were cleared by centrifugation at
12,000 
g for 20 min at 4 °C and used for gel
mobility shift assay using a 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
, 1 mM EGTA, 0.5 mM dithiothreitol, 10% glycerol, 1 µg 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 10
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 µg of PCNA promoter-CAT plasmid as a reporter plasmid
and 0.1 µg 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 µg 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).Establishment of Transgenic Flies
P-element-mediated germ line transformation was carried out
as described(34, 35) . G 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 1.
Analysis of Expression Patterns for PCNA-lacZ
Quantitative Measurement of
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
µl of ice-cold assay buffer (50 mM potassium phosphate, pH
7.5, 1 mM MgCl
-Galactosidase
Activity in Extracts(36). Homogenates were centrifuged at
10,000 g at 4 °C for 5 min. For each assay,
50-200 µl 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).Demonstration of
-Galactosidase
Activity-Galactosidase activity of larval and adult
tissues was visualized as described elsewhere(36) . After
dissection, tissues were incubated in fixative (12 mM sodium
cacodylate buffer, pH 7.3, 26% glutaraldehyde) for 15 min at room
temperature. Treated tissues were then incubated with a staining
solution containing 0.2% 5-bromo-4-chloro-3-indoyl
-D-galactoside in the dark at 37 °C for 5-16 h.
For photography, tissues were immersed in glycerol, mounted on slides,
and photographed with an Olympus microscope (BX-50) using Tri-X pan 400
films (Kodak).
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 polymerase
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 -43 to
-36 (site I) and -56 to -49 (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).
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.
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, lanesa-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, lanesk-m). In
contrast, the oligonucleotide E2F-PmutII carrying mutations in the site
II much less efficiently competed for the binding (Fig. 2B, lanesn-p), and the
oligonucleotide E2F-PmutI& carrying mutations in both sites did
not compete at all (Fig. 2B, lanesh-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 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 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, lanesa-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, lanesr-t). Similarly, oligonucleotides
containing the E2F sites in the DNA polymerase
promoter (18) competed for the binding (Fig. 3A, lanese-j). In contrast, the oligonucleotide
containing the mutant E2F site from the adenovirus E2 promoter did not
compete under the examined conditions (Fig. 3A, lanesu-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) .
site2+3,
oligonucleotides containing E2F sites 2 and 3 from the DNA polymerase
promoter; polsite1, 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).
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 p5`-168E2FmutIIDPCNACAT carrying mutations in E2F site
II (Fig. 4, lanese and f). Slight
further reduction of CAT expression was observed with the plasmid
p5`-168E2FmutI& carrying mutations in both sites (Fig. 4, lanesg 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.
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.
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 promoter-directed CAT expression
2-fold (Fig. 5, upper panel). However, expression of
the DP protein did not affect CAT expression. When plasmid
p5`-116DPCNACAT, carrying the region from -116 to +23 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, lowerpanel).
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 5`-deletion derivatives of the plasmid
p5`-168DPCNACAT were cotransfected with the E2F-expressing plasmid.
Deletions toward position -116 caused a gradual decrease of CAT
expression and a progressive increase of activation by E2F (Fig. 6A). A further deletion to position -86
completely abolished the CAT expression, and accordingly, the
stimulation by E2F was no longer detectable.
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&.
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
(-168 to +137) 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, toppanel)(37) .
-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.
-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).-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.
-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.
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. (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., 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. 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.
)
We are grateful to K. Ohtani and J. Nevins for
providing pdrosE2F1WT, N. Dyson for Act-dE2F and Act-dDP, Y. Nishimoto
for technical assistance, and M. Moore for comments on the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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