Two E2F elements regulate the proliferating cell nuclear antigen promoter differently during leaf development.
ABSTRACT E2F transcription factors regulate genes expressed at the G1/S boundary of the cell division cycle in higher eukaryotes. Although animal E2F proteins and their target promoters have been studied extensively, little is known about how these factors regulate plant promoters. An earlier study identified two E2F consensus binding sites in the promoter of a Nicotiana benthamiana gene encoding proliferating cell nuclear antigen (PCNA) and showed that the proximal element (E2F2) is required for the full repression of PCNA expression in mature leaves. In this study, we examined the distal element (E2F1) and how it interacts with the E2F2 site to regulate the PCNA promoter. Gel shift assays using plant nuclear extracts or purified Arabidopsis E2F and DP proteins showed that different complexes bind to the two E2F sites. Mutation of the E2F1 site or both sites differentially altered PCNA promoter function in transgenic plants. As reported previously for the E2F2 mutation, the E2F1 and E2F1+2 mutations partially relieved the repression of the PCNA promoter in mature leaves. In young tissues, the E2F1 mutation resulted in a threefold reduction in PCNA promoter activity, whereas the E2F1+2 mutation had no detectable effect. The activity of E2F1+2 mutants was indistinguishable from that of E2F2 mutants. These results demonstrate that both E2F elements contribute to the repression of the PCNA promoter in mature leaves, whereas the E2F1 site counters the repression activity of the E2F2 element in young leaves.
[show abstract] [hide abstract]
ABSTRACT: Previous studies have indicated that the presence of an E2F site is not sufficient for G1/S phase transcriptional regulation. For example, the E2F sites in the E2F1 promoter are necessary, but not sufficient, to mediate differential promoter activity in G0 and S phase. We have now utilized the E2F1 minimal promoter to test several hypotheses that could account for these observations. To test the hypothesis that G1/S phase regulation is achieved via E2F-mediated repression of a strong promoter, a variety of transactivation domains were brought to the E2F1 minimal promoter. Although many of these factors caused increased promoter activity, growth regulation was not observed, suggesting that a general repression model is incorrect. However, constructs having CCAAT or YY1 sites or certain GC boxes cloned upstream of the E2F1 minimal promoter displayed E2F site-dependent regulation. Further analysis of the promoter activity suggested that E2F requires cooperation with another factor to activate transcription in S phase. However, we found that the requirement for E2F to cooperate with additional factors to achieve growth regulation could be relieved by bringing the E2F1 activation domain to the promoter via a Gal4 DNA binding domain. Our results suggest a model that explains why some, but not all, promoters that contain E2F sites display growth regulation.Journal of Biological Chemistry 08/1997; 272(29):18367-74. · 4.77 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: The transcription factor E2F regulates the expression of genes involved in the progression of G1/S transition and DNA replication in mammalian cells. We cloned and characterized a cDNA (NtE2F) corresponding to a E2F homolog of tobacco (Nicotiana tabacum). The transcription of NtE2F was induced as cells progressed from G1 to the S phase and expressed much earlier than that of the proliferating cell nuclear antigen (PCNA) gene. We demonstrated that NtE2F can interact with the tobacco retinoblastoma (Rb)-related protein in a yeast two-hybrid assay. To further characterize NtE2F, the trans-activation activity of NtE2F was examined by using a transient assay in the tobacco Bright Yellow-2 (BY-2) cells with NtE2F fused to the DNA-binding domain of the veast transcriptional activator GAL4. NtE2F activated the transcription of the beta-glucuronidase (GUS) reporter gene driven by a cauliflower mosaic virus (CaMV) 35S core promoter containing the GAL4-binding sequence. This is the first report of the identification of a functionally equivalent E2F-like gene in plants.FEBS Letters 11/1999; 460(1):117-22. · 3.54 Impact Factor
Article: Two E2F sites control growth-regulated and cell cycle-regulated transcription of the Htf9-a/RanBP1 gene through functionally distinct mechanisms.[show abstract] [hide abstract]
ABSTRACT: The gene encoding Ran-binding protein 1 (RanBP1) is transcribed in a cell cycle-dependent manner. The RanBP1 promoter contains two binding sites for E2F factors, named E2F-c, located proximal to the transcription start, and E2F-b, falling in a more distal promoter region. We have now induced site-directed mutagenesis in both sites. We have found that the distal E2F-b site, together with a neighboring Sp1 element, actively controls up-regulation of transcription in S phase. The proximal E2F-c site plays no apparent role in cycling cells yet is required for transcriptional repression upon growth arrest. Protein binding studies suggest that each E2F site mediates specific interactions with individual E2F family members. In addition, transient expression assays with mutagenized promoter constructs indicate that the functional role of each site is also dependent on its position relative to other regulatory elements in the promoter context. Thus, the two E2F sites play opposite genetic functions and control RanBP1 transcription through distinct molecular mechanisms.Journal of Biological Chemistry 05/1999; 274(15):10339-48. · 4.77 Impact Factor
The Plant Cell, Vol. 14, 3225–3236, December 2002, www.plantcell.org © 2002 American Society of Plant Biologists
Two E2F Elements Regulate the Proliferating Cell Nuclear
Antigen Promoter Differently during Leaf Development
Erin M. Egelkrout,
and Linda Hanley-Bowdoin
Department of Biochemistry, North Carolina State University, Raleigh, North Carolina 27695
Department of Genetics and Microbiology, University of Pavia, 27100 Pavia, Italy
Department of Botany, North Carolina State University, Raleigh, North Carolina 27695
Sharon B. Settlage,
E2F transcription factors regulate genes expressed at the G1/S boundary of the cell division cycle in higher eukaryotes.
Although animal E2F proteins and their target promoters have been studied extensively, little is known about how these
factors regulate plant promoters. An earlier study identified two E2F consensus binding sites in the promoter of a
gene encoding proliferating cell nuclear antigen (PCNA) and showed that the proximal element
(E2F2) is required for the full repression of PCNA expression in mature leaves. In this study, we examined the distal el-
ement (E2F1) and how it interacts with the E2F2 site to regulate the PCNA promoter. Gel shift assays using plant nu-
clear extracts or purified Arabidopsis E2F and DP proteins showed that different complexes bind to the two E2F sites.
Mutation of the E2F1 site or both sites differentially altered PCNA promoter function in transgenic plants. As reported
previously for the E2F2 mutation, the E2F1 and E2F1
2 mutations partially relieved the repression of the PCNA pro-
moter in mature leaves. In young tissues, the E2F1 mutation resulted in a threefold reduction in PCNA promoter activ-
ity, whereas the E2F1
2 mutation had no detectable effect. The activity of E2F1
that of E2F2 mutants. These results demonstrate that both E2F elements contribute to the repression of the
moter in mature leaves, whereas the E2F1 site counters the repression activity of the E2F2 element in young leaves.
2 mutants was indistinguishable from
In higher eukaryotes, the E2F family of transcription factors
plays an important role in both the positive and negative
regulation of the cell cycle (Black and Azizkhan-Clifford,
1999; Harbour and Dean, 2000; Muller and Helin, 2000;
Trimarchi and Lees, 2002). E2F family members, which het-
erodimerize with DP proteins to form functional transcription
factor complexes, interact with the retinoblastoma gene
product (pRb) during G1. This interaction leads to the tran-
scriptional repression of cell cycle–regulated genes that en-
code proteins required for DNA replication and progression
through S-phase (Lavia and Jansen-Durr, 1999). pRb re-
presses transcription by sequestering free E2F and blocking
its ability to activate transcription (Zacksenhaus et al., 1996)
or by interacting with E2F bound to DNA and recruiting his-
tone deacetylases and SWI/SNF-like enzymes for chromatin
remodeling (Zhang and Dean, 2001). In late G1, phosphory-
lation of pRb by cyclin-dependent kinases disrupts its asso-
ciation with E2F and allows the expression of genes re-
quired for S-phase.
Many components of the pRb-E2F pathway have been
found in plants. pRb homologs (pRBR) have been identified
in maize, tobacco, Arabidopsis, and
al., 1996; Xie et al., 1996; Ach et al., 1997; Fountain et al.,
1999; Nakagami et al., 1999). Like its animal homologs, pRBR
is phosphorylated by cyclin-dependent kinases associated
with G1 and S. pRBR levels are high in differentiated tissues,
consistent with its involvement in the repression of genes
required for cell proliferation (Ach et al., 1997; Nakagami et
al., 1999; Boniotti and Gutierrez, 2001). E2F and DP ho-
mologs have been isolated from wheat, tobacco, carrot,
rice, and Arabidopsis (Ramirez-Parra et al., 1999; Sekine et
al., 1999; Albani et al., 2000; Magyar et al., 2000; Ramirez-
Parra and Gutierrez, 2000; de Jager et al., 2001; Kosugi and
Ohashi, 2002a; Mariconti et al., 2002; Vandepoele et al.,
2002). As in mammals, the six E2F proteins of Arabidopsis
can be separated into two groups that function primarily as
activators or repressors of gene expression (Kosugi and
Ohashi, 2002b; Mariconti et al., 2002).
E2F consensus sites have been found in the promoters of
a number of plant genes, but there are limited functional
data regarding their roles. Purified plant E2F/DP proteins
bchserver.bch.ncsu.edu; fax 919-515-2047.
Article, publication date, and citation information can be found at
To whom correspondence should be addressed. E-mail linda@
3226The Plant Cell
bind in vitro to an E2F site in the Arabidopsis
(Castellano et al., 2001; de Jager et al., 2001). E2F consen-
sus binding sites in the tobacco
gene are required for activation in cultured
cells and intact plants (Chaboute et al., 2000, 2002; Stevens
et al., 2002). E2F sites also have been implicated in de-
velopmental expression of the
nuclear antigen) genes from rice and tobacco (Egelkrout
et al., 2001; Kosugi and Ohashi, 2002a). Overexpression
of Arabidopsis E2Fa and DPa proteins induces mature
leaf cells to reenter S-phase (Rossignol et al., 2002). Ara-
bidopsis plants that ectopically express AtE2Fa also
show sustained cell proliferation in cotyledons and hypo-
cotyls and extra DNA replication in endoreduplicating
cells (De Veylder et al., 2002).
There are several important differences between the pRb/
E2F networks of plants and animals. pRb is a member of a
multigene family in mammals but is represented by a single
gene in Arabidopsis (Durfee et al., 2000). Naturally oc-
curring mutations in the
gene have not been identified,
and there is no known link between pRBR and tumor forma-
tion in plants. The DNA binding domains of plant E2F ho-
mologs show the highest conservation with animal E2F family
members. However, in some Arabidopsis E2F homologs, this
binding domain is duplicated and these proteins can bind to
DNA in the absence of a DP partner (Kosugi and Ohashi,
2002b; Mariconti et al., 2002). Arabidopsis E2F family mem-
bers associated with transcriptional activation are dependent
on their DP partners for nuclear localization, whereas their hu-
man counterparts can translocate into the nucleus indepen-
dent of their DP partners (Kosugi and Ohashi, 2002c).
Many mammalian DNA tumor viruses disrupt the pRb/E2F
pathway (Nevins, 1992; Weinberg, 1995) and activate genes
required for DNA replication, leading to untimely entry into
S-phase and cell transformation. Geminiviruses are a di-
verse family of single-stranded DNA viruses that infect
plants and replicate in nuclei using host replication machin-
ery (Gutierrez, 2000; Hanley-Bowdoin et al., 2000). The
Tomato golden mosaic virus
cumulation of the DNA polymerase
PCNA, in mature plant tissues by altering transcription of
gene (Nagar et al., 1995; Egelkrout et al., 2001).
The PCNA promoter of
E2F consensus binding sites. Mutation of the proximal E2F2
site in the
N. benthamiana PCNA
duction of the PCNA promoter in infected leaves (Egelkrout
et al., 2001), and induction of PCNA transcription depends
on interactions between pRBR and the TGMV replication
protein, AL1 (Ach et al., 1997; Kong et al., 2000). Together,
these results suggest that geminiviruses also disrupt the
pRBR/E2F pathway in plants.
Many E2F-responsive genes, including those that encode
human Htf9-a/RanBP1, human p107, and
(Yamaguchi et al., 1995; Zhu et al., 1995; Di Fiore et al.,
1999), contain multiple E2F sites that cooperate to regulate
their expression. The tobacco
genes and the Arabi-
(TGMV) causes ac-
gene abrogates TGMV in-
gene has two E2F sites
(Chaboute et al., 2000), both of which are required for acti-
vation during S-phase and one that negatively regulates
RNR2 expression outside of S-phase. One of the E2F sites
in the Arabidopsis
gene is required for meristematic
expression in seedlings, whereas the other site negatively
regulates the promoter during G2 in cultured cells (Stevens
et al., 2002). The
and rice PCNA promoters also
contain two E2F sites, but their relative contributions to
gene regulation are not known (Egelkrout et al., 2001;
Kosugi and Ohashi, 2002a). In a previous study, we
showed that the proximal E2F site (E2F2) in the
PCNA promoter is required for repression in ma-
ture leaves (Egelkrout et al., 2001). Here, we examined the
role of the distal E2F site (E2F1) and its interaction with the
E2F2 site in the regulation of PCNA expression. Our data
are consistent with a model in which the E2F sites interact
in both young and mature tissues to regulate PCNA ex-
pression during plant development.
Two E2F Sites Display Different Binding Properties
The PCNA promoter from
ments that have strong homology with the consensus bind-
ing site for the E2F family of transcription factors (Figure 1A)
(Egelkrout et al., 2001). The upstream site (E2F1) consists of
a single copy of the consensus sequence (Herwig and Strauss,
1997; Black and Azizkhan-Clifford, 1999). The downstream
element (E2F2) is an inverted repeat of the consensus
(Wade et al., 1995) and also shows homology with the CDE/
CHR bipartite repressor element (Zwicker et al., 1995;
Zwicker and Muller, 1997). A previous study showed that the
E2F2 site is bound specifically by a factor(s) in nuclear ex-
suspension cultures (Egelkrout et
al., 2001). In Figure 1B, we used electrophoretic mobility
shift assays to determine if the E2F1 motif also binds to a
nuclear protein(s) in vitro. A single shifted product was de-
tected in assays containing a radiolabeled, double-stranded
DNA corresponding to the E2F1 site and a
nuclear extract from asynchronous cycling cells (Figure 1B,
lane 2). Formation of the E2F1 protein complex was com-
peted by excess unlabeled E2F1 DNA (Figure 1B, lanes 3 to
5) but not by an oligonucleotide carrying a 2-bp mutation in
the consensus site (lanes 6 to 8). Interestingly, the E2F1
binding activity also was not competed by excess unlabeled
E2F2 DNA (Figure 1B, lanes 9 to 11). Binding to radiolabeled
E2F2 DNA also was not competed by excess unlabeled E2F1
DNA (Figure 1C, lanes 9 to 11), even though competition
was observed in the presence of unlabeled E2F2 DNA (lanes
3 to 5). Together, these results suggest that the two E2F ele-
ments in the
PCNA promoter bind to differ-
contains two ele-
Regulation of PCNA Transcription 3227
ent proteins in the nuclear extract even though they form
complexes of similar electrophoretic mobilities (data not
The Arabidopsis genome encodes six members of the
E2F family (E2Fa, E2Fb, E2Fc, E2Fd, E2Fe, and E2Ff) and
the two DP homologs (DPa and DPb) (Magyar et al., 2000).
Recently, purified recombinant proteins corresponding to
Arabidopsis E2F and DP family members were produced
and shown to bind to the canonical E2F consensus se-
quence in vitro (Mariconti et al., 2002). Because the homol-
proteins have not been isolated or
expressed in vitro, we examined the binding of the recombi-
nant Arabidopsis proteins to the
moter E2F elements. E2F1 and E2F2 DNAs displayed similar
binding patterns with E2Fa (Figure 2A), E2Fd, and E2Fe (Fig-
ure 2D), and neither site bound to E2Ff (data not shown). Both
DNAs bound to E2Fa in combination with either DPa or DPb
(Figure 2A, lanes 2 and 5) but not to E2Fa alone (lanes 1 and
4). By contrast, the E2F1 and E2F2 probes bound to E2Fd
and E2Fe independent of the presence of a DP partner (Fig-
ure 2D, lanes 2, 4, 6, and 8). However, the presence of DPb
may have enhanced the binding of E2Fd and E2Fe to the
E2F2 site (Figure 2D, cf. lanes 6 and 8). DNAs corresponding
to mutant E2F1 and E2F2 sites did not bind to E2Fa/DP (Fig-
ure 2A, lanes 3 and 6), E2Fd, and E2Fe (Figure 2D, lanes 3
and 7), thereby establishing the specificity of the interactions.
The PCNA E2F sites showed distinct binding patterns
with Arabidopsis E2Fb and E2Fc (Figures 2B and 2C). The
E2F1 DNA was unable to recruit E2Fb in the presence of
DPa (Figure 2B, top gel, lane 1), whereas E2F2 formed a sta-
ble complex with E2Fb/DPa (lane 3). Neither DNA bound to
E2Fb alone (Figure 2B, lane 2) or in combination with DPb
(Figure 2C, lane 1). E2F1 formed a faint complex with E2Fc
in the presence of DPa (Figure 2B, bottom gel, lane 1),
whereas E2F2 gave a strong band (lane 3). Both E2F1 and
E2F2 bound weakly to E2Fc in combination with DPb (Fig-
ure 2C, lane 3) but not to E2Fc alone (lane 2). As summa-
rized in Figure 2E, the binding characteristics of the E2F1
and E2F2 sites showed differences, consistent with the data
presented in Figure 1, indicating that they interact with dif-
ferent proteins from nuclear extracts.
E2F Elements Differentially Regulate the
Because multiple E2F sites within a promoter can interact
to regulate gene expression (Lavia and Jansen-Durr, 1999),
we assessed the relative roles of the PCNA promoter E2F
PCNA E2F Sites Show Different Binding Properties with
the transcription start site (
shown in boldface italic type. The TATA box is shown in boldface
type. The promoter sequences included in the probes used for gel
shift analysis (
and Figure 2) are underlined. Mutations in E2F
sites are shown with lowercase letters beneath the probes.
Oligonucleotides corresponding to the E2F1 or E2F2
sites were annealed, labeled with
nuclear proteins from
quences of the probe and competitor DNAs were as follows: E2F1
; and mE2F2 (m2), 5
. Lowercase letters represent mutations in the E2F con-
sensus sequences or unrelated flanking sequence. The reactions in
contained the E2F1 (wt1) probe, whereas those in
the E2F2 (wt2) probe. Lanes 1 show no shift in the absence of nu-
The sequence of the PCNA promoter from nucleotide
1). The two E2F consensus sites are
P, and assayed for binding to
suspension cultures. The se-
; mE2F1 (m1), 5
; E2F2 (wt2), 5
clear extract. Lanes 2 show a shifted complex in the presence of nu-
clear extract. In lanes 3 to 11, increasing amounts (10-, 100-, and
250-fold excess) of unlabeled competitor DNAs were added as indi-
cated at top. The bound complexes are identified at left.
3228The Plant Cell
Figure 2. Electrophoretic Mobility Shift Analysis with Recombinant E2F and DP Proteins.
(A) to (D) Representative electrophoretic mobility shift assays (EMSAs) are shown with the probes indicated at top. The probe designations (see
Figure 1A) are E2F1 (wt1), mE2F1 (m1), E2F2 (wt2), and mE2F2 (m2). E2F proteins were included in all of the reactions except where indicated in
(D). The presence (?) or absence (?) of DP is indicated at the top of each panel. The bound complexes are marked.
(A) EMSA with E2Fa and DPa (top, lanes 2, 3, 5, and 6) or DPb (bottom, lanes 2, 3, 5, and 6) and the indicated probes. Lanes 1 and 4 show E2Fa
and probe without DP.
(B) EMSA with E2Fb and DPa (top, lanes 1, 3, and 4) or E2Fc and DPa (bottom, lanes 1, 3, and 4) and the indicated probes. Lane 2 shows E2F
and probe without DP.
(C) EMSA with E2Fb (lane 1) and E2Fc (lanes 3 and 4) with DPb and the indicated probes. Lane 2 shows E2Fc and probe without DP.
(D) EMSA with E2Fd (top) or E2Fe (bottom) and DPb with the indicated probes (lanes 4 and 8). Lanes 2, 3, 6, and 7 show E2F and probe without
DP; lanes 1 and 5 show DP and probe without E2F. Binding of E2Fd and E2Fe in the presence of DPa was not tested.
(E) Summary of the binding properties of combinations of recombinant Arabidopsis E2F and DP proteins for the E2F1 and E2F2 sites of the N.
benthamiana PCNA promoter. The data for E2Ff are not shown. ?, strong interactions; (?), weak interactions; ?, no detectable interaction.
Regulation of PCNA Transcription3229
elements in transgenic plants. A series of promoter con-
structs with mutations (Figure 1B) in E2F1, E2F2, or both
sites were generated in the background of the
633 PCNA promoter fragment shown previously to be
regulated developmentally in plants (Egelkrout et al., 2001).
The promoter constructs were fused to the luciferase re-
porter gene and introduced into
We compared the impact of the individual and double
mutations on PCNA promoter activity in T1 plants selected
on kanamycin. The average reporter activities in young
(leaves 1 and 2) and mature (leaf 9) leaves for the PCNA
promoter constructs are shown in Figure 3A. Figure 3B
shows a scatterplot of average reporter activities for indi-
vidual transgenic lines for each construct. Our previous re-
sults with the wild-type promoter and the E2F2 site mutant
are included for comparison (Egelkrout et al., 2001). The
results of two-tailed Student’s
porter activities for each of the four constructs is shown in
Figure 3C. In mature tissue, there was a statistically signifi-
cant increase in reporter activity for each of the three E2F
mutant PCNA promoter constructs relative to the wild-type
promoter, indicative of a partial loss of repression. The ac-
tivities of E2F2 and E2F1
2 promoters both increased
sevenfold to eightfold relative to that of the wild-type
PCNA promoter, whereas the activity of the E2F1 mutant
was threefold greater than that of the wild type. In young
leaves, the activities of the E2F2 and E2F1
moters were similar to those of the wild type, whereas the
E2F1 promoter showed a threefold decrease in activity
compared with the wild-type PCNA promoter. These changes
resulted in a decrease in the ratio of reporter activity in
young versus mature leaves. For the E2F1, E2F2, and
2 mutant constructs, the ratios were 2.2, 2.7, and
2.4, respectively, whereas the ratio was 19 for the wild-
type promoter (Figure 3D).
We also analyzed the expression of the PCNA promoter–
luciferase constructs in every leaf of transgenic plants. Our
previous study showed a sharp decrease in PCNA expres-
sion during development from high activity in the youngest
leaves (leaves 1 to 3) to low activity by leaf 5 (Egelkrout et
al., 2001). Figure 4 shows a similar developmental analysis
for two plant lines carrying either the E2F1 (A) or the
2 (B) PCNA promoter construct and displaying re-
porter gene activities near the overall mean for each con-
struct. Our previous results for the wild-type and E2F2 mu-
tant promoters are included for comparison. As observed
with the E2F2 mutant, the activities of both the E2F1 and
2 constructs declined more gradually over the
course of development than those of the wild type. How-
ever, expression levels for the E2F1 mutant construct were
lower than those for the other two mutant constructs
throughout development. Surprisingly, the expression pat-
tern for the E2F1
2 double-mutant construct was similar
to that for the E2F2 construct and not a composite of the
patterns seen for the individual E2F1 and E2F2 mutants.
tests comparing mean re-
2 mutant pro-
These results suggested that both E2F sites contribute to
the repression of PCNA activity in mature tissue, with the
E2F2 site playing the major role. Analysis of the single-site
mutants indicated that the E2F1 site is required for full activity
of the PCNA promoter in young tissue, whereas the E2F2 site
has no apparent involvement in transcription in young tissue.
However, our results do not exclude a role for the E2F2 site at
a specific stage of the cell cycle that cannot be detected in an
asynchronous cell population, as occurs in young leaves.
Analysis of E2F1
2 mutant lines revealed that the E2F1 site
is required in young tissues only when the E2F2 site is intact.
These data are consistent with the idea that the E2F2 site
also functions as a negative regulatory element in young tis-
sues but that this activity is countered by the E2F1 element in
the wild-type promoter. The E2F1 mutant lines uncovered the
negative regulatory potential of the E2F2 element, whereas
2 mutants revealed that E2F1 acts as an antire-
pressor or a strong activator to overcome repression through
the E2F2 element.
We also examined the impact of the E2F mutations on
promoter activity in transient assays using
periments failed to detect any statistically significant impact
of the mutations on PCNA promoter activity in asynchro-
nous cultured cells (data not shown).
suspension cells. These ex-
Geminivirus Infection Has No Effect on the E2F
Because E2F mutations interfere with the correct develop-
mental regulation of the PCNA promoter, we hypothesized
that the same mutations might affect geminivirus-mediated
activation of the promoter in mature tissues (Nagar et al.,
1995; Egelkrout et al., 2001). We monitored luciferase activ-
ity in consecutive leaves starting at the apex during infection
to determine if PCNA promoter constructs carrying the E2F1
or the E2F1
2 mutation could be activated by TGMV. The
activity of the E2F1 mutant construct was very similar for
healthy untreated, mock-inoculated, and TGMV-infected
leaves at different stages of development (Figure 5A). Pro-
moter activity also was comparable in untreated, mock-inoc-
ulated, and infected plants carrying the E2F1
construct (Figure 5B). We previously found that TGMV infec-
tion has no detectable effect on the activity of the E2F2 mu-
tant promoter (Egelkrout et al., 2001). Thus, the presence of
intact E2F sites is necessary for the induction of PCNA ex-
pression over basal levels in infected mature tissue. However,
the basal levels of reporter activity in healthy mature tissues
of the various E2F mutant plants were threefold to eightfold
higher than those of uninfected wild-type plants. Given that
TGMV induction of the wild-type PCNA promoter ranged
from twofold to sevenfold (Figure 5B, bottom), we cannot
exclude the possibility that there is a response to geminivirus
infection that is masked by the high basal levels of reporter
gene expression in mature leaves of the mutant lines.
3230 The Plant Cell
PCNA Promoter Induction Is Characteristic of
We asked if other geminiviruses in addition to TGMV acti-
vate the PCNA promoter during infection. For these experi-
ments, we used
Cabbage leaf curl virus
geminivirus of the begomovirus genus. CbLCV and TGMV
have similarly arranged bipartite genomes and share a com-
mon host in
CbLCV is representative of a small group of viruses whose
replication protein has diverged significantly from that of the
(Hill et al., 1998). However,
majority of begomoviruses, including TGMV (our unpub-
lished observation). Transgenic plants with the wild-type
PCNA promoter fused to luciferase were infected with
CbLCV, and PCNA promoter activity was monitored 11
days after infection (Figure 6). Infected plants showed a
sixfold increase in PCNA promoter activity in leaf 8 rela-
tive to the equivalent mock-inoculated leaf. The CbLCV
induction profile was similar to that observed for TGMV
infection of the same transgenic line (Figure 5B, bottom)
(Egelkrout et al., 2001). We also detected PCNA mRNA in
mature leaves of CbLCV-infected plants (J.T. Ascencio-
Figure 3. Mutation of the E2F Sites Alters the Developmental Regulation of the PCNA Promoter.
(A) The average luciferase specific activity in young (leaves 1 and 2; black bars) and mature (leaf 9; white bars) leaves of N. benthamiana plants
carrying the wild-type, E2F1 mutant, E2F2 mutant, or E2F1?2 mutant PCNA:luciferase construct. The transgene is shown at left, and the num-
ber of lines and plants examined for each construct is given at right. RLU, relative light units. Error bars correspond to 2 SD.
(B) Scatterplot showing luciferase specific activity in young (left) and mature (right) leaves for individual transgenic lines carrying the constructs
(bottom) described in (A). Each symbol represents the average activity of a minimum of three plants for each transgenic line. The arrows indicate
the transgenic lines analyzed in Figure 4.
(C) Statistical analysis of the data shown in (B) using a two-tailed Student’s t-test. Boldface type indicates significant comparisons (P ? 0.05).
(D) Summary of significant fold changes in reporter activities for young and mature leaves of E2F1 mutant, E2F2, and E2F1?2 mutant lines rela-
tive to the wild-type PCNA promoter. Wild-type levels of reporter activities are designated as wt. The average ratios of luciferase specific activity
in young versus mature leaves also are given.
Regulation of PCNA Transcription3231
Ibañez and L. Hanley-Bowdoin, unpublished data), thereby
providing independent verification of PCNA promoter ac-
tivation by CbLCV.
E2F transcription factors interact with each other and with
other components of the transcription apparatus to regu-
late gene expression during the cell cycle and develop-
ment. In animal systems, it is not uncommon for promoters
to contain multiple E2F binding sites that act synergisti-
cally or antagonistically to regulate transcription (Yamaguchi
et al., 1995; Zhu et al., 1995; Di Fiore et al., 1999). The
PCNA promoter contains two E2F consensus
sequences (Egelkrout et al., 2001). We showed that both
sequences, albeit to different degrees, contribute to the re-
pression of the PCNA promoter in mature leaves. In young
plant tissues, one of the elements can function as a nega-
tive regulator, but its activity is masked by the other ele-
ment, which acts either as an antirepressor or as a strong
activator to ensure high levels of PCNA expression in cy-
cling cells. Our results and the results of others demon-
strated that the regulation of PCNA transcription in plants
is mediated by an intricate set of interactions involving dif-
ferent E2F complexes as well as other transcription factors
(Kosugi et al., 1995; Kosugi and Ohashi, 1997; Egelkrout et
al., 2001). Our studies also established the efficacy of us-
ing intact plants to study the mechanisms underlying the
developmental regulation of cell cycle–associated genes in
Our results differ significantly from the expression studies
of the tobacco
gene by Kosugi and Ohashi (2002a),
who reported that mutations in either E2F site or in both
sites together reduced the activity of the tobacco PCNA
promoter in both young and mature tissues of transgenic to-
bacco. This finding was surprising given that
PCNA genes are 92% identical over a
321-bp region encompassing both the proximal promoter
and the leader, and because their E2F sites are identical in
sequence and position. However, there are several key dif-
ferences between the transgenic plants analyzed by Kosugi
and Ohashi (2002a) and in the experiments reported here.
species and reporter genes were used.
In addition, different lengths of the PCNA promoters were
fused to the reporters. We examined the E2F mutations in
the context of a 743-bp fragment (a 677-bp promoter plus a
66-bp transcribed sequence) of the
whereas Kosugi and Ohashi tested their mutations in a 321-bp
fragment (a 208-bp promoter plus a 113-bp transcribed se-
quence) of the tobacco gene. The tobacco
construct displayed low activity in transgenic plants (Kosugi
and Ohashi, 2002a), and a
construct was 60% less active than the longer
633 constructs in cultured cells (Egelkrout et al., 2001).
Together, these results showed that upstream sequences
lacking in the
?275 constructs are necessary for
full PCNA promoter activity. There is evidence in animals
that the function of E2F sites is dependent on the promoter
context and interactions with other transcription factors (Fry
Figure 4. Developmental Profiles of the PCNA Promoter Carrying
E2F1 or E2F1?2 Mutations.
Luciferase specific activity (left) was measured in each leaf of trans-
genic plants carrying the E2F1 mutant and E2F1?2 mutant
PCNA:luciferase constructs. The lines used for these studies are
marked with arrows in Figure 3B. A minimum of four plants was ana-
lyzed for each line. RLU, relative light units. The vertical lines repre-
sent 2 SE.
(A) Average luciferase specific activities for E2F1 mutant plant
lines 46 (open circles) and 43 (closed circles) are shown com-
pared with the average activity supported by the wild-type pro-
moter (solid line).
(B) Average luciferase specific activity is shown for E2F1?2 lines 7
(open triangles) and 49 (closed triangles). The activity supported by
the wild-type (solid line) and the E2F2 mutant (dashed line) promot-
ers are shown for comparison.
3232 The Plant Cell
et al., 1997; van Ginkel et al., 1997) whose recognition sites
may be absent in the shorter PCNA promoter constructs.
Alternatively, the choice of mutations may have influ-
enced the results. Both studies mutated the same two nu-
cleotides in the E2F1 site and obtained similar results
when differences in promoter strength are taken into con-
sideration. By contrast, the E2F2 site was modified at only
two positions in the tobacco promoter, whereas four posi-
tions were changed in the N. benthamiana promoter. In
vitro binding studies verified that the 4-bp mutation pre-
vents E2F binding, but there is no information regarding
the impact of the 2-bp mutation on binding. Both types of
mutations had minimal impact in young leaves when ex-
perimental scatter is considered, but only the 4-bp muta-
tion resulted in the relief of repression in mature leaves.
Because the E2F2 site consists of two overlapping con-
sensus sequences (Wade et al., 1995), it is possible that
the 2-bp mutation was not sufficient to block E2F binding.
In this case, the prediction would be that the tobacco
E2F2 mutant would resemble the wild type, whereas the
double mutant would resemble the E2F1 mutant, as re-
ported previously (Kosugi and Ohashi, 2002a).
Many eukaryotic promoters contain multiple E2F sites,
but whether the sites have synergistic or opposing activi-
ties varies among promoters (Yamaguchi et al., 1995; Zhu
et al., 1995; Di Fiore et al., 1999). Two lines of evidence
suggest that the two E2F elements in the N. benthamiana
PCNA promoter perform different functions. First, the two
elements have distinct sequences, with E2F1 consisting of
a single copy of the consensus motif and E2F2 containing
inverted, overlapping motifs. In animal systems, the bind-
Figure 5. E2F Mutant Promoters Are Not Activated by Geminivirus
Plants carrying the E2F1 mutant or E2F1?2 mutant PCNA:luciferase
constructs were infected with TGMV by agroinoculation, and lu-
ciferase activity was measured in soluble protein extracts from each
leaf of mature plants. Average luciferase specific activity in each leaf
of healthy (dotted line), mock-inoculated (open circles or triangles),
or TGMV-infected (closed circles or triangles) plants is shown for
E2F1 lines 43 and 46 (A) and E2F1?2 lines 7 and 49 (B). A minimum
of four plants were analyzed for each line and treatment. The vertical
lines represent 2 SE. The gray lines in the bottom graph in (B) corre-
spond to healthy (dotted) and TGMV-infected (solid) profiles of
plants carrying the wild-type PCNA:luciferase construct. RLU, rela-
tive light units.
Figure 6. CbLCV Infection Activates the Wild-Type PCNA Promoter
in Mature Leaves.
Transgenic plants carrying the wild type ?633 PCNA:luciferase con-
struct were infected with CbLCV by agroinoculation, and luciferase
activity was measured in soluble protein extracts from each leaf of
three infected (closed circles, diamonds, and triangles) and three
mock-inoculated (open circles, diamonds, and triangles) plants. RLU,
relative light units.
Regulation of PCNA Transcription 3233
ing affinities of these types of elements differ, with the
overlapping motif binding to E2F/DP heterodimers more
strongly (Wade et al., 1995). Second, in vitro binding as-
says using nuclear extracts or recombinant E2F and DP
proteins showed that the two sites can interact with differ-
ent protein complexes. In particular, the E2F1 and E2F2
sites did not compete with each other in assays containing
nuclear extracts. In addition, only the E2F2 element bound
to recombinant E2Fb/DPa, and the E2F2 site bound E2Fc/
DPa more strongly than the E2F1 site. There is precedent
in animal promoters for different E2F sites to interact with
different E2F/DP complexes (Zhu et al., 1995; Di Fiore et
al., 1999), but this type of selectivity has not been de-
scribed for plant E2F elements.
Mutations in the E2F sites affected PCNA promoter activ-
ity differently in transgenic plants. In young tissues, the
E2F1 mutation resulted in a threefold decrease in promoter
activity, whereas the E2F2 mutation had no detectable effect.
Mutations in either element derepressed the PCNA promoter
in mature leaves, but the E2F2 mutation was significantly
stronger than the E2F1 mutation (eightfold versus threefold
increase). The different results in young versus mature tis-
sues may reflect developmental regulation of the factors
that bind to the E2F1 and E2F2 sites. This idea, which is
supported by RNA data indicating that the various E2F and
DP family members have distinct developmental and tis-
sue-specific expression patterns (Magyar et al., 2000;
Kosugi and Ohashi, 2002b), provides an explanation for
why the E2F1 element activates transcription in young tis-
sues and represses it in mature tissues. In addition, the se-
quence and context of an E2F element can influence it
binding properties (Fry et al., 1997; van Ginkel et al., 1997),
thereby providing a basis for why the E2F2 site recruits a
strong repressor and the E2F1 site binds to a weak repres-
sor in mature tissues.
Analysis of the E2F1?2 double mutant revealed that
PCNA transcriptional repression is complex and that the
two E2F sites do not act independently. Failure of the
E2F1?2 mutation to fully derepress the PCNA promoter in
mature leaves indicated that other cis elements or irrevers-
ible changes in chromatin structure also contribute to re-
pression. The role for chromatin structure is supported by
the observation that the activities of the E2F mutant promot-
ers were not altered significantly in transient assays. These
assays used plasmid-based reporter cassettes, and it is un-
likely that the chromatin remodeling factors recruited by
E2F/pRb complexes affect episomal and chromosomal DNA
similarly. Another possibility is that transcriptional activators
in mature tissues are not as effective as those in young tis-
sues at activating the PCNA promoter, even in the absence
Comparison of the E2F1?2 double mutant with individual
mutants showed that the E2F1 site is required in young
leaves only when a functional E2F2 element is present. This
result is consistent with a model in which the E2F1 site over-
comes the repressive activity of the E2F2 site to ensure high
Figure 7. Model for the Regulation of a Plant PCNA Promoter.
A model for PCNA promoter regulation is shown, with activators in-
dicated in blue, repressors indicated in red, and antirepressors indi-
cated in yellow. E2F1 and E2F2 may be occupied by different family
members at different stages, as reflected by the different shapes.
The sizes of the shapes reflect the relative strengths of the factors
occupying the sites.
(A) In young tissue, E2F1 may recruit a strong activator that masks
the activity of a weak repressor bound to the E2F site. Other tran-
scription factors also contribute to the full activation of the promoter.
(B) Alternatively, in young tissue, binding of an antirepressor to the
E2F1 site prevents the binding of a repression complex to the E2F2
site or interferes with protein interactions required for repression.
Other transcription factors are responsible for the activation of the
(C) As plant tissues mature, changes in the E2F1 binding complex
allow recruitment of a pRBR/E2F complex to the E2F2 site, leading
to the repression of the PCNA promoter. Factors binding to the
E2F1 site also contribute to repression, albeit to a lesser extent.
(D) In geminivirus-infected cells, AL1 interacts with pRBR to induce
the release of the repressive complex bound to the E2F sites and the
reassembly of an active promoter. It is not known if factors bound to
the E2F1 and E2F2 sites function as activators during infection.
3234 The Plant Cell
PCNA promoter activity in young tissues. One possibility is
that E2F1 recruits a strong transcriptional activator that
counters the activity of a repressor bound to E2F2. Alterna-
tively, E2F1 may recruit an antirepressor that interferes di-
rectly with the activity of the repressor but is not itself a tran-
scriptional activator. The best-characterized example of an
antirepressor element associated with the cell division cycle
is the overlapping CDE/CHR motif in the genes that encode
cdc25C, cdc2, cyclin A, and B-myb (Liu et al., 1998). Inter-
estingly, the E2F2 element in the N. benthamiana PCNA pro-
moter shows homology with the CHR. There is evidence
that competitive binding of E2F and an E2F-unrelated tran-
scriptional repressor to the CDE/CHR site regulates pro-
moter activity (Liu et al., 1997, 1998). However, the separa-
tion of the E2F1 and E2F2 sites in the N. benthamiana PCNA
promoter suggests that the two sites function via a different
Plant PCNA promoters are likely to be controlled by a
combination of transcriptional activation, repression, and
antirepression (Figure 7). Our data showed that PCNA ex-
pression is repressed via the conserved E2F1 and E2F2
sites in the proximal promoter region in mature tissues. By
analogy to animal E2F-regulated promoters, these sites act
by recruiting E2F/pRBR complexes that alter chromatin
structure and block access to the promoter (Figure 7C). Like
mammalian DNA tumor antigen proteins, the geminivirus
AL1 protein relieves repression through its interaction with
pRBR, leading to the disruption of repressive E2F/pRBR
complexes (Figure 7D). In young plant tissues, high levels of
PCNA expression are achieved by the combined action of
transcriptional activators and the antirepression activity of fac-
tors recruited to the E2F1 site (Figures 7A and 7B). Antire-
pression activity may reflect factors bound to the E2F1 site
that block the binding of the repressor to the E2F2 site, in-
terfering with the ability of a bound repressor to contact other
components of the transcription apparatus or masking re-
pression through the recruitment of a strong activator. The
different activities of the E2F1 and E2F2 elements at the
same developmental stages and their changing activities dur-
ing leaf development may reflect the binding of different E2Fs
and their partners, which also are likely to be subject to de-
velopmental regulation. Future experiments to characterize
the protein complexes recruited to the N. benthamiana PCNA
promoter as well as their interactions and occupancy in
young and mature leaves will address these possibilities and
provide insight into the functions of pRBR and different E2F
family members during plant development.
E2F Mutant Constructs and Transient Transfection Assays
The proliferating cell nuclear antigen (PCNA) E2F1 and E2F1?2 mu-
tant constructs were generated by two-step overlap extension PCR,
as described previously (Egelkrout et al., 2001). The gene-specific
primers for the E2F1 construct were E2F1 #1 (5?-CCAAAATAGAGG-
taGGAAAAATATTTTTTCCAC-3?) and E2F1 #2 (5?-ATATTTTTCCta-
CCTCTATTTTGGGC-3?), with mutations shown in lowercase letters.
The PCR product was gel purified, repaired with Klenow, and cloned
into the SmaI site of pUC119 to give pNSB925. To prepare the
E2F1?2 mutant, pNSB925 was used as a template for PCR mu-
tagenesis of the E2F2 site using the gene-specific primers E2F2 #1
and E2F2 #2 (Egelkrout et al., 2001). The PCR product was cloned
into pUC119 to produce pNSB927. Mutant promoter fragments
from pNSB925 and pNSB927 with BamHI and trimmed SacI ends
were fused to the luciferase coding sequence in pMON8796 (Eagle
et al., 1994) with BglII and trimmed PstI ends to produce pNSB939
and pNSB940, respectively. Preparation of protoplasts from Nicoti-
ana benthamiana and N. tabacum BY2 cells, DNA transfections,
and enzyme assays were as described previously (Eagle et al.,
Generation of Transgenic Plants and Reporter Assays
The PCNA promoter–luciferase fusions from pNSB939 and pNSB940
were cloned as NotI expression cassettes into NotI-digested pMON721
(Lanahan et al., 1994) to generate pNSB936 and pNSB938, respec-
tively. The constructs were introduced stably into N. benthamiana
plants via Agrobacterium tumefaciens–mediated transformation us-
ing standard protocols (Horsch et al., 1985). The transgene promot-
ers were verified in planta by PCR amplification of chromosomal
DNA using the primers pMON721 (5?-TCGAAGCCGTGTGCGAGA-
GACACC-3?) and LUCSEQ (5?-GGCGTATCTCTTCATAGCCTTATG-
C-3?) followed by DNA sequencing. Plants were grown in a con-
trolled-environment chamber at 25?C with a 16-h/8-h light/dark cycle
and 65% RH. Preparation of crude extracts, infection of plants by
agroinoculation, luciferase reporter assays, and statistical analysis
were as described previously (Egelkrout et al., 2001). In all cases,
plants were of the T1 generation and selected on kanamycin for the
presence of the transgene.
Electrophoretic Mobility Shift Assays
N. benthamiana nuclear extracts were prepared from log-phase cul-
tured cells as described previously (Albani et al., 2000; Egelkrout et
al., 2001) except that pelleted nuclei were frozen at ?80?C overnight
before lysis in some cases. Electrophoretic mobility shift assays with
nuclear extracts (1.9 ?g per reaction) were as described previously
(Egelkrout et al., 2001) except that 2.5 ? 104 or 2.5 ? 105 cpm/probe
was used for the E2F2 and E2F1 experiments, respectively. Produc-
tion of recombinant Arabidopsis E2F and DP proteins and electro-
phoretic mobility shift assays with purified proteins were as de-
scribed (Albani et al., 2000; Mariconti et al., 2002) except that the
probes indicated in Figure 1A were used. Each assay contained 100
ng of each recombinant protein and 5 ? 104 cpm/probe.
Cabbage leaf curl virus Clones and Infectivity Assays
The plasmids pCpCLCVA.003.2 and pCpCLCVB.002.2, carrying sin-
gle copies of the Cabbage leaf curl virus (CbLCV) A and B compo-
nents, respectively, have been described (Turnage et al., 2002). Sin-
Regulation of PCNA Transcription3235
gle copies of the A and B components were isolated from these
clones as KpnI-EcoRI and XbaI-EcoRI fragments. The resulting frag-
ments were ligated into the Agrobacterium transformation vector
pMON721 (Lanahan et al., 1994) and digested with the same en-
zymes to produce pNSB1087 and pNSB1088. pNSB1087 was di-
gested with EcoRI and BamHI and ligated to the EcoRI-BamHI frag-
ment from pCpCLCVA.003.2 to generate a partial tandem copy of
the CbLCV A component in pNSB1090. pNSB1088 digested with
EcoRI was ligated to the EcoRI fragment from pCpCLCVB.002.2 to
generate a partial tandem copy of the CbLCV B component in
pNSB1091. Transformation into Agrobacterium, plant infection by sy-
ringe inoculation, preparation of protein extracts, and reporter assays
all were as described (Egelkrout et al., 2001) except that tissue was
harvested 11 days after inoculation. The ?633 PCNA:luciferase line L-39
(Egelkrout et al., 2001) was used in the CbLCV infectivity assays.
Upon request, all novel materials described in this article will be made
available in a timely manner for noncommercial research purposes.
We thank Tara Nash for her valuable technical support. This research
was supported by grants from the National Research Initiative Com-
petitive Grants Program of the U.S. Department of Agriculture
(98-01392 to L.H.-B. and D.R.), the National Science Foundation
(MCB-9809953 and MCB-0110536 to L.H.-B.), and the Ministero
dell’Istruzione, dell’Università e della Ricerca (to L.M. and R.C).
Received July 15, 2002; accepted September 20, 2002.
Ach, R.A., Durfee, T., Miller, A.B., Taranto, P., Hanley-Bowdoin,
L., Zambryski, P.C., and Gruissem, W. (1997). RRB1 and RRB2
encode maize retinoblastoma-related proteins that interact with a
plant D-type cyclin and geminivirus replication protein. Mol. Cell.
Biol. 17, 5077–5086.
Albani, D., Mariconti, L., Ricagno, S., Pitto, L., Moroni, C., Helin,
K., and Cella, R. (2000). DcE2F, a functional plant E2F-like tran-
scriptional activator from Daucus carota. J. Biol. Chem. 275,
Black, A.R., and Azizkhan-Clifford, J. (1999). Regulation of E2F: A
family of transcription factors involved in proliferation control.
Gene 237, 281–302.
Boniotti, M.B., and Gutierrez, C. (2001). A cell-cycle-regulated kinase
activity phosphorylates plant retinoblastoma protein and contains, in
Arabidopsis, a CDKA/cyclin D complex. Plant J. 28, 341–350.
Castellano, M.M., del Pozo, J.C., Ramirez-Parra, E., Brown, S., and
Gutierrez, C. (2001). Expression and stability of Arabidopsis CDC6
are associated with endoreplication. Plant Cell 13, 2671–2686.
Chaboute, M.E., Clement, B., and Philipps, G. (2002). S phase and
meristem-specific expression of the tobacco RNR1b gene is
mediated by an E2F element located in the 5? leader sequence. J.
Biol. Chem. 277, 17845–17851.
Chaboute, M.E., Clement, B., Sekine, M., Philipps, G., and
Chaubet-Gigot, N. (2000). Cell cycle regulation of the tobacco
ribonucleotide reductase small subunit gene is mediated by E2F-
like elements. Plant Cell 12, 1987–2000.
de Jager, S.M., Menges, M., Bauer, U.M., and Murray, J.A. (2001).
Arabidopsis E2F1 binds a sequence present in the promoter of
S-phase-regulated gene AtCDC6 and is a member of a multigene
family with differential activities. Plant Mol. Biol. 47, 555–568.
De Veylder, L., Beeckman, T., Beemster, G.T., de Almeida Engler,
J., Ormenese, S., Maes, S., Naudts, M., Van Der Schueren, E.,
Jacqmard, A., Engler, G., and Inze, D. (2002). Control of prolifer-
ation, endoreduplication and differentiation by the Arabidopsis
E2Fa-DPa transcription factor. EMBO J. 21, 1360–1368.
Di Fiore, B., Guarguaglini, G., Palena, A., Kerkhoven, R.M.,
Bernards, R., and Lavia, P. (1999). Two E2F sites control growth-
regulated and cell cycle-regulated transcription of the Htf9-a/
RanBP1 gene through functionally distinct mechanisms. J. Biol.
Chem. 274, 10339–10348.
Durfee, T., Feiler, H.S., and Gruissem, W. (2000). Retinoblastoma-
related proteins in plants: Homologues or orthologues of their
metazoan counterparts? Plant Mol. Biol. 43, 635–642.
Eagle, P.A., Orozco, B.M., and Hanley-Bowdoin, L. (1994). A DNA
sequence required for geminivirus replication also mediates tran-
scriptional regulation. Plant Cell 6, 1157–1170.
Egelkrout, E.M., Robertson, D., and Hanley-Bowdoin, L. (2001).
Proliferating cell nuclear antigen transcription is repressed
through an E2F consensus element and activated by geminivirus
infection in mature leaves. Plant Cell 13, 1437–1452.
Fountain, M.D., Murray, J.A.H., and Beck, E. (1999). Isolation of a
full-length cDNA encoding a retinoblastoma (accession No.
AJ011681) protein from suspension cultured photoautotrophic
Chenopodium rubrum L. cells. Plant Physiol. 119, 363.
Fry, C.J., Slansky, J.E., and Farnham, P.J. (1997). Position-dependent
transcriptional regulation of the murine dihydrofolate reductase promo-
ter by the E2F transactivation domain. Mol. Cell. Biol. 17, 1966–1976.
Grafi, G., Burnett, R.J., Helentjaris, T., Larkins, B.A., DeCaprio, J.A.,
Sellers, W.R., and Kaelin, W.G., Jr. (1996). A maize cDNA encoding
a member of the retinoblastoma protein family: Involvement in
endoreduplication. Proc. Natl. Acad. Sci. USA 93, 8962–8967.
Gutierrez, C. (2000). DNA replication and cell cycle in plants: Learn-
ing from geminiviruses. EMBO J. 19, 792–799.
Hanley-Bowdoin, L., Settlage, S.B., Orozco, B.M., Nagar, S., and
Robertson, D. (2000). Geminiviruses: Models for plant DNA repli-
cation, transcription, and cell cycle regulation. Crit. Rev. Biochem.
Mol. Biol. 35, 105–140.
Harbour, J.W., and Dean, D.C. (2000). The Rb/E2F pathway: Expand-
ing roles and emerging paradigms. Genes Dev. 14, 2393–2409.
Herwig, S., and Strauss, M. (1997). The retinoblastoma protein: A
master regulator of cell cycle, differentiation and apoptosis. Eur.
J. Biochem. 246, 581–601.
Hill, J.E., Strandberg, J.O., Hiebert, E., and Lazarowitz, S.G.
(1998). Asymmetric infectivity of pseudorecombinants of cabbage
leaf curl virus and squash leaf curl virus: Implications for bipartite
geminivirus evolution and movement. Virology 250, 283–292.
Horsch, R.B., Fry, J.E., Hoffman, N.L., Wallroth, M., Eicholtz, D.,
Rogers, S.G., and Fraley, R.T. (1985). A simple and general method
for transferring cloned genes into plants. Science 227, 1229–1231.
Kong, L.J., Orozco, B.M., Roe, J.L., Nagar, S., Ou, S., Feiler, H.S.,
Durfee, T., Miller, A.B., Gruissem, W., Robertson, D., and
Hanley-Bowdoin, L. (2000). A geminivirus replication protein
interacts with the retinoblastoma protein through a novel domain
to determine symptoms and tissue specificity of infection in
plants. EMBO J. 19, 3485–3495.
3236 The Plant Cell
Kosugi, S., and Ohashi, Y. (1997). PCF1 and PCF2 specifically bind
to cis elements in the rice proliferating cell nuclear antigen gene.
Plant Cell 9, 1607–1619.
Kosugi, S., and Ohashi, Y. (2002a). E2F sites that can interact with
E2F proteins cloned from rice are required for meristematic tis-
sue-specific expression of rice and tobacco proliferating cell
nuclear antigen promoters. Plant J. 29, 45–59.
Kosugi, S., and Ohashi, Y. (2002b). E2Ls, E2F-like repressors of
Arabidopsis that bind to E2F-sites in a monomeric form. J. Biol.
Chem. 277, 16553–16558.
Kosugi, S., and Ohashi, Y. (2002c). Interaction of the Arabidopsis
E2F and DP proteins confers their concomitant nuclear transloca-
tion and transactivation. Plant Physiol. 128, 833–843.
Kosugi, S., Suzuka, I., and Ohashi, Y. (1995). Two of three pro-
moter elements identified in a rice gene for proliferating cell
nuclear antigen are essential for meristematic tissue-specific
expression. Plant J. 7, 877–886.
Lanahan, M.B., Yen, H.-C., Giovannoni, J.J., and Klee, H.J.
(1994). The never ripe mutation blocks ethylene perception in
tomato. Plant Cell 6, 521–530.
Lavia, P., and Jansen-Durr, P. (1999). E2F target genes and cell-
cycle checkpoint control. Bioessays 21, 221–230.
Liu, N., Lucibello, F.C., Engeland, K., and Muller, R. (1998). A new
model of cell cycle-regulated transcription: Repression of the
cyclin A promoter by CDF-1 and anti-repression by E2F. Onco-
gene 16, 2957–2963.
Liu, N., Lucibello, F.C., Korner, K., Wolfraim, L.A., Zwicker, J.,
and Muller, R. (1997). CDF-1, a novel E2F-unrelated factor, inter-
acts with cell cycle-regulated repressor elements in multiple pro-
moters. Nucleic Acids Res. 25, 4915–4920.
Magyar, Z., Atanassova, A., De Veylder, L., Rombauts, S., and
Inze, D. (2000). Characterization of two distinct DP-related genes
from Arabidopsis thaliana. FEBS Lett. 486, 79–87.
Mariconti, L., Pellegrini, B., Cantoni, R., Stevens, R., Bergounioux,
C., Cella, R., and Albani, D. (2002). The E2F family of transcrip-
tion factors from Arabidopsis thaliana: Novel and conserved com-
ponents of the retinoblastoma/E2F pathway in plants. J. Biol.
Chem. 277, 9911–9919.
Muller, H., and Helin, K. (2000). The E2F transcription factors: Key reg-
ulators of cell proliferation. Biochim. Biophys. Acta 1470, M1–M12.
Nagar, S., Pedersen, T.J., Carrick, K.M., Hanley-Bowdoin, L.,
and Robertson, D. (1995). A geminivirus induces expression of a
host DNA synthesis protein in terminally differentiated plant cells.
Plant Cell 7, 705–719.
Nakagami, H., Sekine, M., Murakami, H., and Shinmyo, A. (1999).
Tobacco retinoblastoma-related protein phosphorylated by a dis-
tinct cyclin-dependent kinase complex with Cdc2/cyclin D in vitro.
Plant J. 18, 243–252.
Nevins, J.R. (1992). E2F: A link between the Rb tumor suppressor
protein and viral oncoproteins. Science 258, 424–429.
Ramirez-Parra, E., and Gutierrez, C. (2000). Characterization of
wheat DP, a heterodimerization partner of the plant E2F transcription
factor which stimulates E2F-DNA binding. FEBS Lett. 486, 73–78.
Ramirez-Parra, E., Xie, Q., Boniotti, M.B., and Gutierrez, C.
(1999). The cloning of plant E2F, a retinoblastoma-binding pro-
tein, reveals unique and conserved features with animal G(1)/S
regulators. Nucleic Acids Res. 27, 3527–3533.
Rossignol, P., Stevens, R., Perennes, C., Jasinski, S., Cella, R.,
Tremousaygue, D., and Bergounioux, C. (2002). AtE2F-a and
AtDP-a, members of the E2F family of transcription factors,
induce Arabidopsis leaf cells to re-enter S phase. Mol. Genet.
Genomics 266, 995–1003.
Sekine, M., Ito, M., Uemukai, K., Maeda, Y., Nakagami, H., and
Shinmyo, A. (1999). Isolation and characterization of the E2F-like
gene in plants. FEBS Lett. 460, 117–122.
Stevens, R., Mariconti, L., Rossignol, R., Perennes, C., Cella, R.,
and Bergounioux, C. (2002). Two E2F sites in the Arabidopsis
MCM3 promoter have different roles in cell cycle activation and
meristematic expression. J. Biol. Chem. 277, 32978–32984.
Trimarchi, J.M., and Lees, J.A. (2002). Sibling rivalry in the E2F
family. Nat. Rev. Mol. Cell. Biol. 3, 11–20.
Turnage, M.A., Muangsan, N., Peele, C.G., and Robertson, D.
(2002). Geminivirus-based vectors for gene silencing in Arabidop-
sis. Plant J., 29, 1–9.
Vandepoele, K., Raes, J., De Veylder, L., Rouze, P., Rombauts,
S., and Inze, D. (2002). Genome-wide analysis of core cell cycle
genes in Arabidopsis. Plant Cell 14, 903–916.
van Ginkel, P.R., Hsiao, K.M., Schjerven, H., and Farnham, P.J.
(1997). E2F-mediated growth regulation requires transcription fac-
tor cooperation. J. Biol. Chem. 272, 18367–18374.
Wade, M., Blake, M.C., Jambou, R.C., Helin, K., Harlow, E., and
Azizkhan, J.C. (1995). An inverted repeat motif stabilizes binding
of E2F and enhances transcription of the dihydrofolate reductase
gene. J. Biol. Chem. 270, 9783–9791.
Weinberg, R.A. (1995). The retinoblastoma protein and cell cycle
control. Cell 81, 323–330.
Xie, Q., Sanz-Burgos, A.P., Hannon, G.J., and Gutierrez, C.
(1996). Plant cells contain a novel member of the retinoblastoma
family of growth regulatory proteins. EMBO J. 15, 4900–4908.
Yamaguchi, M., Hayashi, Y., and Matsukage, A. (1995). Essential
role of E2F recognition sites in regulation of the proliferating cell
nuclear antigen gene promoter during Drosophila development. J.
Biol. Chem. 270, 25159–25165.
Zacksenhaus, E., Jiang, Z., Phillips, R.A., and Gallie, B.L. (1996).
Dual mechanisms of repression of E2F1 activity by the retinoblas-
toma gene product. EMBO J. 15, 5917–5927.
Zhang, H.S., and Dean, D.C. (2001). Rb-mediated chromatin structure
regulation and transcriptional repression. Oncogene 20, 3134–3138.
Zhu, L., Zhu, L., Xie, E., and Chang, L.S. (1995). Differential roles of
two tandem E2F sites in repression of the human p107 promoter
by retinoblastoma and p107 proteins. Mol. Cell. Biol. 15, 3552–
Zwicker, J., Lucibello, F.C., Wolfraim, L.A., Gross, C., Truss, M.,
Engeland, K., and Muller, R. (1995). Cell cycle regulation of the
cyclin A, cdc25C and cdc2 genes is based on a common mecha-
nism of transcriptional repression. EMBO J. 14, 4514–4522.
Zwicker, J., and Muller, R. (1997). Cell-cycle regulation of gene
expression by transcriptional repression. Trends Genet. 13, 3–6.