Oncogene (2001) 20, 1379 ± 1387
ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00
www.nature.com/onc
Expression analysis using DNA microarrays demonstrates that E2F-1
up-regulates expression of DNA replication genes including replication
protein A2
Yael Kalma1, Lea Marash1, Yocheved Lamed1 and Doron Ginsberg*,1
1
Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel
The transcription factor E2F-1 plays a pivotal role in the
regulation of G1/S transition in higher eukaryotes cell
cycle. We used a cell line containing an inducible E2F-1
and oligonucleotide microarray analysis to identify novel
E2F target genes. We show that E2F-1 up-regulates the
expression of a number of genes coding for components
of the DNA replication machinery. Among them is the
gene coding for the 32 Kd subunit of replication protein
A (RPA2). Replication protein A is the most abundant
single strand DNA binding complex and it is essential for
DNA replication. We demonstrate that RPA2 is a novel
E2F target gene whose expression can be directly
regulated by E2F-1 via E2F binding sites in its promoter.
In addition, expression of Topoisomerase IIa and subunit
IV of DNA polymerase a is also up-regulated upon E2F1 induction. Taken together, these results provide novel
links between components of the DNA replication
machinery and the cell growth regulatory pathway
involving the Rb tumor suppressor and E2F. Oncogene
(2001) 20, 1379 ± 1387.
Keywords: E2F; cell cycle; DNA chips; RPA
Introduction
E2F transcription factors play a pivotal role in the
control of cell cycle progression and regulate the
expression of genes required for G1/S transition
(Dyson, 1998; Muller and Helin, 2000). The DNA
binding complex named E2F is a heterodimeric
complex consisting of an E2F component and a
dimerization partner, DP. To date, six E2F genes and
two DP genes have been isolated (Dyson, 1998). E2F
activity is modulated by multiple mechanisms including negative regulation by interaction with the
product of the Rb tumor suppressor gene, RB, and
its related proteins p107 and p130 (reviewed in
Dyson, 1998). Binding of RB family members to
E2F results in active transcriptional repression of
E2F regulated genes and growth suppression. During
*Correspondence: D Ginsberg
Received 11 October 2000; revised 19 December 2000; accepted 3
January 2001
late G1 phosphorylation of RB family members by
cyclin dependent kinases results in release of E2Fs
from the complex, leading to activation and/or
derepression of E2F target genes (Kaelin, 1999).
This, in turn, leads to cell cycle progression. The
importance of the RB/E2F pathway in controlling
cell growth is emphasized by frequent inactivation of
the Rb gene or its upstream regulators, cyclin D,
Cdk4 and p16, in human tumors (Hall and Peters,
1996; Sherr, 1996; Weinberg, 1995).
Known E2F regulated genes are involved in
nucleotide synthesis, recognition of origins of replication, DNA replication and control of cell cycle
progression (Helin, 1998). In agreement with the
regulation of many growth related genes by E2F,
ectopic expression of E2F-1, 2 or 3 is sucient to
induce S phase entry of quiescent immortal rodent
cells (Johnson et al., 1993; Qin et al., 1994; Shan and
Lee, 1994; Vigo et al., 1999). Furthermore, overexpression of E2F-1 and its heterodimeric partner
DP-1 together with activated ras leads to transformation of primary rodent cells (Johnson et al., 1994).
More recent studies show that double transgenic mice
over-expressing E2F-1 and activated ras in their
epidermis develop skin tumors (Pierce et al., 1998).
Thus, E2F-1 contributes to tumorigenesis in vivo and
it therefore functions, at least in some settings, as an
oncogene.
DNA replication is a complex process involving
numerous proteins yet only four of them, DNA
polymerase a subunit I and II, proliferating cell
nuclear antigen (PCNA) and DNA polymerase d
catalytic subunit have so far been recognized as the
products of E2F target genes (Lee et al., 1995;
Nishikawa et al., 2000; Pearson et al., 1991; Zhao
and Chang, 1997).
In this study we used a cell line containing an
inducible E2F-1 and DNA array hybridization to
identify additional E2F target genes. We show that
E2F-1 up-regulates the expression of six new genes
encoding proteins that participate in DNA replication,
among them are the genes encoding for the 32 Kd
subunit of replication protein A (RPA2), Topoisomerase IIa and subunit IV of DNA polymerase a. Their
expression is E2F responsive and in the case of RPA2
this E2F responsiveness depends on the E2F binding
sites in its promoter.
E2F-1 up-regulates expression of DNA replication genes
Y Kalma et al
1380
Results
Analysis of up-regulated genes after E2F-1 induction
In an attempt to identify new E2F target genes we
used a Rat-1a derived cell line containing a stably
integrated Zinc inducible E2F-1, Rat-1-MT-wtE2F-1
(Qin et al., 1994). Upon addition of ZnCl2 (100 mM)
to quiescent Rat-1-MT-wtE2F-1 cells, E2F-1 protein
level is elevated and the cells enter S phase and later
undergo apoptosis (Qin et al., 1994). When these cells
were kept for 48 h in medium with 0.1% serum they
entered quiescence, as determined by FACS analysis
(data not shown). Under these conditions, addition of
ZnCl2 (100 mM) resulted in a rapid and signi®cant
elevation of E2F-1 protein level. E2F-1 protein level
was increased 3 h after ZnCl2 addition and it
remained high for at least 12 h (Figure 1). Total
RNA was extracted from Rat-1-MT-wtE2F-1 arrested
cells before and after E2F-1 induction in two
independent experiments. In one experiment the
RNA was extracted 12 h after E2F-1 induction and
in the other at two time points after E2F-1 induction:
12 and 16 h. These RNAs were used to prepare
probes to screen an A€ymetrics Rat DNA microarrays (Rat Genome U34A array) containing 8700
cDNAs and ESTs. RNA extracted from arrested cells
before and 12 h after E2F-1 induction was also used
to probe an Atlas membrane containing 588 rat
cDNAs (Clontech, 7738-1). In the screen of the
A€ymetrics Rat DNA microarrays, out of the 8700
genes present on each chip expression of 35 genes
and 10 ESTs was reproducibly increased more than
twofold upon E2F-1 induction. Seven of the 35
induced genes were known E2F target genes ± Cyclin
E, PCNA, Thymidylate synthase, DNA polymerase a
subunit II, DNA polymerase d catalytic subunit,
MCM6 and MCM2. Among the other induced genes,
Table 1
Gene
Topoisomerase IIa
DNA replication
DNA polymerase a subunit IVa
DNA replication
DNA polymerase a subunit II
DNA polymerase d catalytic subunit
Replication factor C (RF-C4) (37 Kd)a
DNA replication
DNA replication
DNA replication
Replication protein A (RPA2) (32 Kd)
PCNA
Fen1
DNA polymerase a subunit IIIa
DNA
DNA
DNA
DNA
Cyclin Ea
Cell cycle
Thymidylate synthase
MCM6
MCM2
Nucleotide synthesis
Origin recognition
Origin recognition
a
Figure 1 The kinetic of expression of inducible E2F-1 in
quiescent rat cells. Rat-1a-1093 (1093) and Rat-1a-MT-wtE2F-1
(wt E2F-1) cells were kept for 48 h in medium containing 0.1%
serum and then 100 mM ZnCl2 was added. At the times indicated
at the top of each lane (in hours), cells were harvested and cell
extracts were used for Western blot analysis with an anti E2F-1
monoclonal antibody (SQ41). Molecular size markers in kilodaltons are shown on the left
DNA replication related genes induced by E2F-1
Function
a
not described previously as E2F target genes, a most
evident functional group of genes included six genes
involved in DNA replication, a cellular process
known to be regulated by E2F-1 (Table 1).
In the screen of Atlas membrane expression of two
known E2F target genes present on the membrane,
cyclin E and PCNA, was increased upon E2F-1
induction (Figure 2). In addition, expression of
several genes including the gene encoding for the
32 Kd subunit of replication protein A, RPA2, was
similarly induced (Figure 2). Computerized analysis
of the data, performed using AtlasImage 1.01 software (Clontech, USA), demonstrated that the induction of expression of RPA2, cyclin E and PCNA was
3.48-, 2.14- and 1.2-fold, respectively.
replication
replication
replication
replication
0 ± 12 h (A)
Fold of induction
0 ± 12 h (B)
0 ± 16 h (B)
3.4
2.7
3
2.1
2.8
2.8
2.7
1.8
2.6
2.5
2.3
2
19.4
4.6
2.8
2.3
2.1
2.1
3.1
1.9
3.6
2.1
3.2
9.1
3.4
2.1
3
2.2
2.6
2.6
3.7
4.9
2.7
3.2
2
2.2
2.9
2
4.2
NI
2.6
7.5
2.8
1.9
2.2
1.9
2.2
1.8
3.4
3.2
2
3.3
2
2.1
These genes were present twice on the DNA chip. Fold of induction in both cases is presented. (A) and (B) are two independent experiments.
NI, not informative
Oncogene
E2F-1 up-regulates expression of DNA replication genes
Y Kalma et al
1381
Figure 2 Di€erential hybridization of atlas rat array. Total
RNA prepared from growth arrested Rat-1a-MT-wtE2F-1 cells
before and after 12 h induction with 100 mM ZnCl2 was used to
generate cDNA. Radiolabeled cDNA was hybridized to the
Clontech Atlas membranes (7738-1). An enlargement of part of
the membranes is presented. Each cDNA is present on the ®lter
on two adjacent spots. Three of the genes which were upregulated
upon induction of E2F-1 are indicated by arrows
Analysis of RPA2 induction by E2F-1
Replication protein A (RPA) is the most abundant
ssDNA binding protein in eukaryotic cells and it plays
an essential role in DNA replication (Erdile et al.,
1990; Kenny et al., 1990). RPA is a heterotrimer
composed of 70 (RPA1), 32 (RPA2) and 14 Kd
(RPA3) subunits (Fairman and Stillman, 1988; Wold
and Kelly, 1988). The DNA chip analysis indicated
that expression of the RPA2 gene was reproducibly
elevated upon E2F-1 induction (Table 1) and similar
elevation was detected using the Atlas membrane.
Therefore we further investigated the regulation of its
expression by E2F-1. Elevation of RPA2 mRNA levels
upon E2F-1 induction was detected also by RT ± PCR
analysis (Figure 3a) and Northern blot analysis (data
not shown). To investigate whether this E2F-1 induced
activation of RPA2 expression requires a transcriptionally active E2F-1 we utilized a Rat-1 derived cell line
containing a stably integrated Zinc inducible E2F-1
mutant, Rat-1-MT-E2F-1dlTA. This E2F-1 mutant
lacks the transactivation domain and, therefore, can
not transactivate gene expression. Upon addition of
Zinc to this cell line protein levels of E2F-1dlTA were
comparable to those of wtE2F-1 in Rat-1-MT-wtE2F-1
following Zinc addition (Figure 3b). However, this did
not lead to an increase in RPA2 expression (Figure 3a).
This result demonstrates that RPA2 gene expression is
not induced in response to the Zinc addition itself and
that a transcriptionaly functional E2F-1 is required for
this induction. To test whether expression of RPA2
changes as arrested cells resume growth, cells of Rat-11093 (containing the empty vector), Rat-1-MT-wtE2F1 cell lines and the human glioblastoma cell line T98G
were growth arrested at G1 by serum starvation and
then allowed to resume growth by addition of serum.
Growth arrest and resumption of growth were veri®ed
Figure 3 RPA2 expression is upregulated by E2F-1 and by
serum. (a) RPA2 mRNA levels are upregulated by E2F-1. Rat-1MT-wtE2F-1 (wtE2F-1) and Rat-1-MT-E2F-1dlTA (E2F-1dlTA)
cells were kept in medium with 0.1% serum for 48 h and then
cells were treated with 100 mM ZnCl2 for 12 h (+) or not treated
(7) prior to RNA extraction. Total RNA was used for RT ± PCR
analysis using RPA2 and GAPDH speci®c oligonucleotides. (b)
Cells were treated as in a and then lysed and subjected to Western
blot analysis using an anti E2F-1 monoclonal antibody (Santa
Cruz, sc-251). (c) RPA2 mRNA levels are upregulated by serum
in both Rat and Human cells. Rat-1a-1093 (1093), Rat-1a-MTwtE2F-1 (wt E2F-1) and T98G cells were kept in medium with
0.1% FCS for 48 h and then serum was added to a ®nal
concentration of 15%. Cells were lysed at the indicated times (in
hours) after serum addition, RNA was extracted and subjected to
RT ± PCR analysis using RPA2 and GAPDH speci®c oligonucleotides
by FACS analysis (data not shown). Under these
conditions, RPA2 mRNA levels were induced in all
three cell lines 12 h after serum addition (Figure 3c),
demonstrating that RPA2 gene expression is growth
regulated.
Induction of E2F-1 expression in quiescent Rat-1MT-wtE2F-1 cells leads to S phase entry ((Qin et al.,
1994) and Figure 4). Therefore, up-regulation of RPA2
expression may be an indirect consequence of E2F-1induced cell cycle progression. To address this possibility we tested whether RPA2 up-regulation precedes
E2F-1-induced S phase entry. As can be seen in Figure
4, RPA2 mRNA levels are increased already 6 h after
E2F-1 induction while cell cycle distribution of the cells
at this time does not di€er signi®cantly than that of uninduced cells. At 12 h post induction S phase entry is
evident and RPA2 mRNA levels are further increased.
Thus, RPA2 induction precedes S phase entry indicating that the former is not an indirect consequence of the
latter. The RPA2 gene and its upstream sequence are
available as part of the Human Genome project. To
further study the regulation of RPA2 expression by
E2F-1 we determined the position of its transcription
start site by performing a 5' rapid ampli®cation of
cDNA ends (race) using Gibco ± BRL 5'RACE kit
(18374-054). As shown in Figure 5a, a PCR reaction
using an oligonucleotide located 588 bp downstream to
Oncogene
E2F-1 up-regulates expression of DNA replication genes
Y Kalma et al
1382
Figure 4 RPA2 induction precedes S phase entry. Rat-1-MTE2F-1 cells were serum starved (0.1% serum) for 48 h prior to the
addition of 100 mM ZnCl2. At the times indicated at the top of
each graph (in hours), cells were harvested. Cell cycle distribution
was measured by the Cell-Cycle Flow Cytometry Assay. The
graphs depict cell cycle distribution of living cells. The lower right
panel depicts PRA2 mRNA levels. Cells were treated same as for
FACS analysis and then lysed and total RNA was extracted.
RNA was used for RT ± PCR analysis using RPA2 and GAPDH
speci®c oligonucleotides
the 5' end of the previously published RPA2 cDNA
(Erdile et al., 1990) gave rise to a single fragment of
631 bp. Sequencing of this PCR product demonstrated
that it contains 53 additional bases upstream to the 5'
end of the published human RPA2 cDNA. Comparison
of the sequence of the PCR product to the genomic
fragment containing the RPA2 gene clearly indicated
that the additional 53 bases are positioned immediately
upstream to the 5' end of the published cDNA. The 5'
end of our 5'-race product is therefore used as the +1
reference point (Figure 5) and the RPA2 promoter lies
upstream to it. An examination of the RPA2 promoter
region revealed four putative E2F DNA binding sites at
positions 7125/7118,7320/7313,7309/7316 and
7406/7399. The site at 7320/7313 matches perfectly
with the E2F consensus binding sequence and the other
three sites di€er from the consensus at one position but
are identical to E2F sites in known E2F responsive
promoters (Figure 5). Computer analysis of the 700 bp
sequence upstream to the transcription start site
identi®ed additional potential binding sites of SP1 and
ATF1. As for many of the known E2F target genes, the
RPA2 promoter is TATA-less. A 682 bp fragment
spanning the RPA2 promoter from +55 to 7627 was
synthesized by PCR using human placenta genomic
DNA as a template and was cloned in pA3Luc resulting
in RPA2(7627/+55)Luc. Transient transfection of
RPA2(7627/+55)Luc into cells of the human lung
carcinoma cell line H1299 resulted in a luciferase
activity which was 34-fold higher than that of the
empty vector, pA3Luc, indicating that the cloned
Oncogene
Figure 5 Sequence and schematic representation of the human
RPA2 promoter. (a) 5' race employed primers at position +588
of the longest available cDNA (left lane). RNA was extracted
from MCF-7 cells and subjected to 5' race analysis. Sizes of DNA
markers, in base pairs, are depicted on the right. (b) Nucleotide
sequence of the human RPA2 5' end and upstream region. The
transcription start site, as determined by the 5' RACE is marked
by an arrow. The E2F binding sites are boxed. (c) Schematic
representation of putative binding sites of transcription factors in
the promoter of human RPA2. The transcription start site is
marked by an arrow. The four E2F binding sites (including a bidirectional E2F binding site at 7313) and putative sites for
binding of ATF-1 and SP-1 (GC Box) are indicated
fragment is indeed a functional promoter (Figure 6).
The role of E2F-1 in controlling the RPA2 promoter
activity was then assessed by co-transfection of
RPA2(7627/+55)Luc and E2F-1 and DP1 expression
plasmids, into H1299 cells. This co-transfection resulted
in an additional 2.5-fold increase in luciferase activity
over the basal promoter activity (Figure 6), indicating
that the RPA2 promoter is an E2F responsive
promoter. No such increase was detected upon cotransfection of pA3Luc together with E2F-1 and DP1
(Figure 6). To test whether the E2F DNA binding sites
present in the RPA2 promoter are required for its E2F
responsiveness, three of these sites were mutated using
PCR-site directed mutagenesis to generate RPA2(Mut)Luc. Unlike the result obtained using RPA2(7627/
E2F-1 up-regulates expression of DNA replication genes
Y Kalma et al
(Isaacs et al., 1998; Woessner et al., 1991) and we
demonstrated that this is true also for Rat-1-1093 and
Rat-1-MT-E2F-1 cells. When these cell lines were
growth arrested at G1 by serum starvation and then
allowed to resume growth by addition of 15% serum,
Topoisomerase IIa mRNA levels were induced in both
cell lines 12 h after serum addition (Figure 7b)
demonstrating that Topoisomerase IIa gene expression
is growth regulated.
1383
Discussion
Figure 6 The RPA2 promoter is activated by E2F-1. H1299 cells
were transiently transfected with 0.5 mg of pA3Luc (vector) or a
RPA2(7627/+55)Luc (wtRPA2) or RPA2(Mut)Luc (mutRPA2),
together with 0.5 mg of pCMV-b-GAL. 0.2 mg pCMV-E2F-1 and
0.2 mg pCMV-DP1 were added where indicated. Forty-two hours
post transfection cell extracts were prepared and used for b-GAL
and Luciferase assays. Fold of activation in the luciferase assay,
after normalization for b-GAL activity, are depicted in the bar
graph. Fold of activation is relative to the sample transected with
pA3Luc
Since the identi®cation of E2F-1 as an RB target a
large body of evidence accumulated to support the idea
that E2F-1 plays a critical role in regulating S phase
entry. First, overexpression of E2F-1 in quiescent cells
leads to S phase entry (Johnson et al., 1993; Kowalik
et al., 1995). Second, E2F regulates the expression of
genes coding both for enzymes involved in nucleotide
synthesis, such as thymidine kinase, thymidilate
synthase, dihydrofolate reductase (DHFR), and for
proteins which are components of the origin recognition complex such as Orc1, cdc6 and MCM2 to
+55)Luc, when RPA2(Mut)Luc was transfected into
H1299 cells its activity was not changed by coexpression of E2F-1 and DP1 (Figure 6) demonstrating
that the e€ect of E2F-1 on the RPA2 promoter activity
is, most likely, a direct e€ect mediated by the E2F DNA
binding sites. Similar results were obtained using
another cell line, MCF-7 (data not shown). Interestingly, the basal activity of RPA2(Mut)Luc was somewhat higher than that of RPA2(7627/+55)Luc (Figure
6) suggesting that the mutated sequences mediate also
transcriptional repression.
Induction of Topoisomerase IIa and subunit IV of DNA
polymerase a expression by E2F-1
Two of the DNA replication related genes whose
expression was upregulated upon E2F-1 induction are
the genes encoding for Topoisomerase IIa and subunit
IV of DNA polymerase a. These genes were present
twice on the DNA microarray, and they were induced
in both cases (Table 1), further validating their
regulation by E2F-1. Elevation of mRNA levels of
both genes upon E2F-1 induction was detected also by
RT ± PCR analysis (Figure 7a). Northern blot analysis
demonstrated that mRNA levels of Topoisomerase IIa
were elevated 12 h after addition of Zinc in Rat-1-MTwtE2F-1 cells but not in Rat-1-1093 cells (Figure 7b)
thus demonstrating that Topoisomerase IIa gene
expression is not induced in response to the Zinc
addition itself. It has been previously reported that
mRNA levels of Topoisomearse IIa increase in S phase
Figure 7 Expression of Topoisomerae IIa and DNA polymerase
a subunit IV is up-regulated by E2F-1 (a) Topoisomerase IIa and
DNA polymerase a subunit IV mRNA levels are upregulated by
E2F-1. Rat-1a-MT-wtE2F-1 cells were kept in medium with 0.1%
serum for 48 h and then cells were treated with (+) or without
(7) 100 mM ZnCl2 for 12 h prior to RNA extraction. Total RNA
was used for RT ± PCR analysis using Topoisomerase IIa, DNA
polymeraes a subunit IV and GAPDH speci®c oligonucleotides.
(b) Topoisomerase IIa mRNA levels are upregulated by serum in
Rat cells. Rat-1a-1093 (1093) and Rat-1a-MT-wtE2F-1 (wtE2F-1)
cells were kept in medium with 0.1% serum for 48 h and then
treated by addition of either 100 mM ZnCl2 (upper panel) or
serum to a ®nal concentration of 15% (lower panel). Total RNA
was prepared from cells at the indicated times (in hours) after
ZnCl2 or serum addition and subjected to Northern analysis using
a Topoisomerase IIa probe. Acidic Ribosomal Protein (ARPP
PO) was used as a control probe
Oncogene
E2F-1 up-regulates expression of DNA replication genes
Y Kalma et al
1384
Oncogene
MCM7 (Leone et al., 1998; Yan et al., 1998). In
addition, genes coding for components of the DNA
replication machinery itself, such as DNA polymerase
a subunits I and II, PCNA, and DNA polymerase d
catalytic subunit are also E2F targets (Lee et al., 1995;
Nishikawa et al., 2000; Pearson et al., 1991; Zhao and
Chang, 1997). DNA replication is a complex process
and the proper expression and function of many
proteins is required for its initiation as well as its
successful completion. Taking into consideration the
pivotal role played by E2F in controlling S phase entry
it is reasonable to assume that many other genes
coding for DNA replication proteins will be identi®ed
as E2F targets.
Using RNA from a cell line with inducible E2F-1 as
a probe for di€erential screening of both an Atlas
membrane and DNA chips we detected an increase in
the expression of seven known E2F target genes: DNA
polymerase a subunit II, DNA polymerase d catalytic
subunit, PCNA, cyclin E, Thymidylate synthase,
MCM6 and MCM2. In addition, we detected an
increase in the expression of six replication related
genes which were not described previously as E2F
target genes. These are: (1) Topoisomerase IIa whose
primary role is to disentangle interwined chromosomes
during anaphase (reviewed in Isaacs et al., 1998); (2,3)
Subunits III and IV of DNA polymerase a which
constitute the DNA primase (reviewed in Foiani et al.,
1997); (4) RF-C4, coding for the 37 Kd subunit of
replication factor C (RF-C). RF-C is a complex
consisting of ®ve proteins which binds the templateprimer junction thus recruiting DNA polymerases to
the site of DNA synthesis (Mossi and Hubscher, 1998);
(5) RPA2 encoding for the 32 Kd subunit of
replication protein A which is the most abundant
single strand DNA binding complex (reviewed in Iftode
et al., 1999) and (6) Fen1, a 5'-3' exo/endonuclease
essential for Okazaki fragments maturation (reviewed
in Bambara et al., 1997). These genes are depicted in
Table 1.
A comparison of our results with the comprehensive analysis of cell cycle-regulated genes of the yeast
Saccharomyces cerevisiae (Spellman et al., 1998) show
that, in agreement with our ®ndings, most of the
yeast homologues of the Rat genes presented in
Table 1 are up-regulated in G1 phase of the cell
cycle. These include the yeast homologues of DNA
polymerase a subunit II, III and IV, DNA polymerase d catalytic subunit, PCNA, RF-C4, RPA2
and Fen1.
The E2F-dependent regulation of expression of three
of the genes shown in Table 1 was further studied,
resulting in the clear identi®cation of RPA2 as a new
E2F-1 target gene. We also demonstrated that E2F-1
plays a role in the regulation of Topoisomerase IIa and
DNA polymerase a subunit IV. RPA2 mRNA levels
are induced both by E2F-1 and by serum and the
RPA2 promoter contains four putative E2F binding
sites. Our data clearly show that this promoter is
activated by E2F-1 and mutations in three of the E2F
binding sites abolish this activation.
The RPA2 gene codes for the 32 Kd subunit of
replication protein A (RPA) which is a heterotrimeric
protein complex (Fairman and Stillman, 1988; Wold
and Kelly, 1988). RPA is the most abundant ssDNA
binding protein in eukaryotic cells. It can unwind
dsDNA and it stabilizes DNA in the single strand
conformation (Georgaki et al., 1992; Seroussi and
Lavi, 1993; Treuner et al., 1996) and stimulates DNA
synthesis by DNA polymerase a (Kenny et al., 1990).
RPA plays an essential role in DNA replication and
mutations in RPA2 were found to be lethal to yeast
(Philipova et al., 1996). Importantly, an anti RPA2
antibody blocks the stimulation of DNA polymerase a
by RPA and two distinct anti RPA2 monoclonal
antibodies strongly inhibit SV40 DNA replication
(Erdile et al., 1990; Kenny et al., 1990). These data
clearly demonstrate that the 32 Kd subunit of RPA,
encoded by RPA2, is essential for DNA replication.
Taken together with our results, these data strongly
suggest that induction of RPA2 expression by E2F-1
contributes to E2F-1 induced DNA synthesis.
Little is known about the regulation of expression
of the three RPA genes. However, experiments using
S. cerevisia demonstrate that all three RPA genes are
expressed in a cell cycle dependent manner and their
mRNA levels peak at the G1/S boundary (Brill and
Stillman, 1991). The data presented here demonstrates
that expression of at least one component of the RPA
heterotrimer, RPA2, is cell cycle regulated also in
higher eukaryotes. In addition to its regulation at the
transcriptional level, RPA2 is phosphorylated in a cell
cycle dependent fashion at the G1/S transition (Din et
al., 1990; Fang and Newport, 1993), and RPA
association with origins of replication is regulated by
cyclin and Dbf-4 dependent kinases (Tanaka and
Nasmyth, 1998). Thus, RPA2 is regulated in a cell
cycle dependent manner both at the transcriptional
and at the post-translational levels. Such a dual
regulation is reminiscent of other E2F target genes
such as cdc6 (Hateboer et al., 1998; Petersen et al.,
1999; Yan et al., 1998). The RPA1 gene was present
neither on the Atlas membrane nor on the DNA chip
used for this study. The RPA3 gene was also present
on the DNA chip, however, its expression was only
mildly increased upon E2F-1 induction: 2.6, 1.8 and
1.6 in experiments 0-12A, 0-12B and 0-16B depicted in
Table 1. Thus, it is not clear whether the co-regulation
of the three RPA subunits exist also in higher
eukaryotes and understanding the regulation of
expression of the three RPA subunits requires
additional studies.
It is noteworthy that RPA has been shown to
recognize some types of damaged DNA, to interact
with a number of proteins involved in DNA repair and
to play a role in the response to DNA damage (He et
al., 1995; Li et al., 1995; Nagelhus et al., 1997). Recent
reports demonstrate that E2F-1 protein level and DNA
binding activity are induced upon DNA damage
(Blattner et al., 1999; Ho€erer et al., 1999; O'Connor
and Lu, 1999). Therefore, it is possible that E2F-1
regulates RPA2 expression not only when DNA
E2F-1 up-regulates expression of DNA replication genes
Y Kalma et al
replication is required, i.e. before S phase, but also as
part of the DNA damage response.
Two other genes are demonstrated in the present
report to be E2F responsive, namely Topoisomerase
IIa and subunit IV of DNA polymerase a.
The latter encodes for the 48 Kd subunit of DNA
polymerase a, which is the catalytic DNA primase
subunit. It is sucient to synthesize in vitro the short
RNA primers required to provide the 3' ends for
elongation by DNA polymerase a (reviewed in Foiani
et al., 1997). DNA polymerase a has four subunits and
its subunits I and II are known E2F target genes
(Pearson et al., 1991; Nishikawa et al., 2000). The gene
encoding for subunit I was present on the DNA chip,
however, its hybridization results were not informative.
The genes encoding for subunits II and III were also
present on the DNA chip and their expression was
increased upon E2F-1 induction (Table 1). This raises
the possibility that E2F co-regulates the four subunits
of DNA pol a, that plays a major role in DNA
replication and is the only enzyme that can initiate
DNA chains de novo.
The Topoisomerase IIa gene codes for a 170 Kd
protein whose primary role is to disentangle interwined
chromosomes during anaphase (Isaacs et al., 1998).
The Topoisomerase IIa promoter shares some structural similarities with other cell cycle-regulated promoters
such as Thymidine kinase, DHFR and DNA polymerase a subunit I. However, it does not contain E2F
binding sites, and previous studies have shown that,
unlike many E2F target genes, its mRNA expression
peaks in late S rising to about 10-fold higher than in
G1 (Isaacs et al., 1998; Woessner et al., 1991). These
data suggest that the E2F dependent increase detected
in expression of Topoisomerase IIa is most probably
indirect. This is in agreement with the presence of a
myb binding site within the Topoisomerase IIa
promoter (Isaacs et al., 1998) and the ability of Bmyb, a known E2F target gene (Lam and Watson,
1993), to up-regulate expression of Topoisomerase IIa.
Irrespectively of the mechanism by which Topoisomerase IIa is up-regulated in response to E2F induction,
this is an interesting phenomenon, especially since
Topoisomerase IIa is the primary cellular target of the
most widely used anti-neoplastic agents including
doxorubicin, etoposide and mitoxantrone. It has been
documented that Topoisomerase IIa mRNA levels are
higher in a variety of tumors in comparison to adjacent
normal tissue (Hasegawa et al., 1993; Isaacs et al.,
1998). It would be interesting to test whether tumorrelated mutations in the RB/E2F pathway result in
changes in the levels of Topoisomerase IIa, and
whether this is re¯ected in the responsiveness of the
tumor to anti-neoplastic treatment with inhibitors of
Topoisomerase IIa.
The ability of E2F-1 to up-regulate the expression of
a number of genes coding for components of the DNA
replication machinery provide novel links between
components of the DNA replication machinery and
the cell growth regulatory pathway involving RB and
E2F. DNA replication is a complex process involving
numerous proteins and the genes coding for many of
them were not present on the DNA chips analysed
here. The full spectrum of replication related genes that
are regulated, either directly or indirectly, by E2Fs
awaits additional studies.
1385
Materials and methods
Cell culture
Rat-1a ®broblasts that had stably integrated the p1093,
p1093-E2F-1 or p1093-E2F-1d1TA were grown in Dulbecco's
modi®ed Eagle's medium (DMEM) supplemented with 10%
(vol/vol) fetal calf serum (FCS) and G418 (500 mg/ml). T98G
and MCF-7 cells were grown in DMEM supplemented with
10% (vol/vol) fetal bovine serum (FBS). H1299 cells were
grown in RPMI supplemented with 10% (vol/vol) FCS.
Western blotting
Cells were lysed in lysis bu€er [20 mM HEPES pH 7.8,
450 mM NaCl, 25% glycerol, 0.2 mM EDTA, 0.5 mM DTT,
phenylmethylsulfonyl ¯uoride (43 mg/ml), aprotinin (17 mg/
ml), leupeptin (22 mg/ml)]. Twenty mg of protein from each
lysate, as determined by Bradford assay, were resolved by
electrophoresis in a SDS 10% polyacrylamide gel and
transferred to ®lters (Protran BA, 85, S&S). Filters were
incubated with the indicated antibodies over night after 2 h
incubation in PBS with 0.05% Tween and 5% dry milk.
Binding of the primary antibodies was detected using an
enhanced chemiluminescence kit (ECL Amersham).
Screening of atlas cDNA expression array
Rat-1-MT-E2F-1 cells were kept for 48 h in DMEM
containing 0.1% FCS. Half of the cells were treated with
100 mM ZnCl2 for 12 h and then cells were harvested and total
RNA was extracted using Tri Reagent method (Molecular,
Research Center). After DNase treatment radiolabeled cDNA
was prepared from 9 mg of total RNA and hybridized to the
membranes (Clontech, 7738-1) according to the manufacturer's instructions. Computerized analysis of the data was
performed using AtlasImage 1.01 software (Clontech, USA).
The signal intensities were normalized globally using the sum
method (AtlasImage Clontech, USA).
DNA chip
RNA was extracted as described for the Atlas cDNA
expression array and double strand cDNA was synthesized
by Reverse Transcription. Biotin-labeled cRNA was produced from the cDNA and used to probe A€ymetrics Rat
DNA micro-array (Rat Genome U34A array). Hybrydization
and washes were performed using A€ymetrics gene chip
system according to manufacturer's instructions.
RT ± PCR
Reverse transcription-PCR (RT ± PCR) was performed on
total RNA prepared by the Tri Reagent method. 7.5 mg of
RNA were employed for cDNA synthesis using M-MLV
reverse transcriptase (Promega, 200 u) and oligo dT (0.5 mg,
Pharmacia). PCR was performed on 1 ml of the 20 ml cDNA
sample. Below are indicated, respectively, the number of
cycles and the sequences of 5' and 3' primers used for each of
the tested genes: for the gene encoding RPA2, 30
Oncogene
E2F-1 up-regulates expression of DNA replication genes
Y Kalma et al
1386
cycles, using 5'-CCGGATCCGCAAGAGCCCGAGCCCAGC-3' and 5'-CGGAATTCGCACGGTGAGGCCATTTGCTGGC-3'; for the gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 19 cycles using 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3'. The annealing temperature for both genes
was 508C. For the gene encoding topoisomerase II a, 26
cycles, using 5'-CAATGCTCAGCCCTTTGGCTCG-3' and
5'-GTAGTCCCACTCTCTCTGCC-3'; for the gene encoding
DNA polymerase a subunit IV, 26 cycles, using 5'CACAACACAGTGAAGCTGGG-3' and 5'-GGGTGAACACTAAAAGGGC-3'. The annealing temperature for both
genes was 588C.
Northern blot analysis
RNA was extracted from cells by the Tri Reagent method.
Twenty mg of total RNA from each sample were separated on
a formaldehyde gel and then blotted to GeneScreen Plus
membrane (DU PONT) according to manufacturer's instructions. Acidic Ribosomal Protein (ARPP PO) was used as a
control probe.
Cell-cycle flow cytometry assays
Bromodeoxyuridine (BrdU, ®nal concentration, 10 mM) was
added to cells 30 min prior to harvesting. Cells were
trypsinized and ®xed with 70% ethanol (720). After
®xation, cells were centrifuged for 5 min at 1200 r.p.m.
and incubated for 30 min at room temperature in 1 ml of
2 M HCl/0.5% Triton X-100. After recentrifugation, cells
were washed with 1 ml of 0.1 M Na2B4O7 (pH 8.5) and
reacted with anti BrdU antibody (Becton Dickinson,
347580, 15 ml per test) and then with Fluorescein
isothiocyanate-conjugated anti mouse antibody. Then cells
were resuspended in PBS containing 5 mg/ml propidium
idodide for 30 min at room temperature. Fluorescence
intensity was analysed on a Becton Dickinson ¯ow
cytometer.
5' Rapid amplification of cDNA ends (5' RACE)
Total RNA was extracted from growing MCF-7 cells using
Tri Reagent method and subjected to 5'-race analysis
according to the manufacture instructions (Gibco ± BRL,
18374-058). The primer used for the ®nal step of ampli®ca-
tion of the 5' end of human RPA2 mRNA was 5'GGGAATTCGCGCTTAGTACCATGTGTGC-3'.
Isolation of human RPA2 promoter
A 682 bp fragment of the RPA2 promoter from the +55 to
7627 was generated by PCR using 100 ng human genomic
placenta DNA as template and 60pm each from the 5' and
3' primers: 5'-CGGGGTACCGGATGAGACTGGGATGGGG-3' and 5'-CGGAAGCTTCCACTGCGCCGCTCTGGC-3'.
Plasmids
The luciferase reporter plasmid RPA2(7627/+55) Luc was
constructed by subcloning the 682 bp KpnI/HindIII fragment
of the RPA2 promoter in PA3Luc. Mutation in the E2F
DNA binding sites were generated by PCR-sitedirected mutagenesis which changed TTTGGCGC(7320
to 313) to TTTGAAGC, GTTCGCGC(7309 to 7316)
to GTTCGCTT, TTTGGAGC(7125 to 7118) to
TTTGAAGC to generate RPA2(mut)Luc. pCMV-b-Gal,
pCMV-E2F-1 and pCMV-DP1 were described previously
(Krek et al., 1993; Lindeman et al., 1997).
Transfection assay
H1299 cells were transfected by the calcium phosphate
method. Cells were harvested 42 h after transfection, and
lysates were tested for luciferase and b-galactosidase activities
as described previously (Lindeman et al., 1997).
Acknowledgments
We thank Drs WG Kaelin Jr and PD Adams for cells lines
and Dr D Givol for reagents and critical reading of the
manuscript. We thank Dr Shirley Horn-Saban for excellent
technical assistance with DNA microarray analysis. This
work was supported in part by grants from the Israel
cancer Association (ICA), Minerva Foundation (Germany), the Israel Cancer Research Fund (ICRF) and Yad
Abraham Research Center for Diagnostics and Therapy. D
Ginsburg is an incumbent of the Recanati Career Development chair of cancer research.
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