Review articles

E2F target genes and cell-cycle

checkpoint control
Patrizia Lavia1 and Pidder Jansen-Dürr2*

Summary
In this review, we will focus on the role played by transcription factors of the
E2F/DP family in controlling the expression of genes that carry out important
cell-cycle control functions, thereby ensuring ordered progression through the
mammalian cell division cycle. The emerging picture is that cell-cycle progression
depends on the execution of a regulatory cascade of gene expression, driven by
E2F/DP transcription factors, which are in turn regulated by the products of some
of these genes. That E2F factors are potent regulators of cell-cycle checkpoints in
mammalian cells is supported by experiments demonstrating that ectopic expres-
sion of individual E2F family members is sufficient to modulate cell proliferation
and apoptosis. It is also clear that deregulation of E2F activity will result in the loss
of particular checkpoint controls, thereby predisposing cells to malignant conver-
sion. BioEssays 21:221–230. 1999. r 1999 John Wiley & Sons, Inc.

Introduction alterations in the concentration of key regulatory proteins, the

Ordered progression through the cell cycle is continually kept
on course by regulatory pathways generally referred to as
checkpoint controls. Checkpoint control mechanisms monitor
extracellular and intracellular growth-regulating signals and
ensure the dependency of a cell-cycle phase upon successful
completion of the preceding one. Checkpoint systems essen-
tially act as negative regulatory pathways: ‘‘sensor’’ compo-
nents interact with their monitored substrate in the cell and,
hence, contact an ‘‘effector’’ molecule, which in turn acts on a
key target and prevents its biological activity until the events
required for transition into the next phase are completed.
Typical checkpoint targets are cell-cycle phase-specific cyclin/
kinase complexes whose normal biological activity stimulates
progression into the next phase of the cell cycle. Thus,
progression through the cell cycle is regulated by periodic
1Centro di Genetica Evoluzionistica C.N.R., c/o Universita ‘‘La Sapi-
enza,’’ Rome, Italy.
2Institut f. Biomedizinische Alternsforschung der Österieichischen Akad-
emie der Wissenschaften, Rennweg 10, A-6020 Innsbruck, Austria.
Funding agencies: Deutsche Forschungsgemeinschaft, CNR Progetto
Strategico ‘‘Cell cycle and apoptosis,’’ Associazione Italiana per la
Ricerca sul Cancro, and Biomed 2 program of the European Union.
*Correspondence to: Pidder Jansen-Dürr, Institut f. Biomedizinische
Alternsforschung der Österieichischen Akademie der Wissenschaften,
Rennweg 10, A-6020 Innsbruck, Austria. E-mail: P.Jansen-
Duerr@oeaw.ac.at
level of which is controlled by phase-specific activation of
gene expression and subsequent regulated proteolysis.(1) In
this concept, transcriptional regulation of cell-cycle genes can
be viewed as an essential level of control of cell proliferation
and checkpoint function.
Since the discovery several years ago that the pRb
product of the retinoblastoma susceptibility gene, a major
tumor suppressor, acts as a repressor of transcription of
genes that are activated by the E2F group of transcription
factors, a vast amount of work has been dedicated to clarify
how E2F and pRb-related factors control transcription of
responsive genes. The mechanisms of transcriptional control
by E2F factors have been extensively reviewed recently
(references 2–7); in the present review, instead of re-
examining the well-documented properties of E2F transcrip-
tion factors, we will examine how regulation of specific
E2F-target genes in a particular temporal window of the cell
cycle affects control of further cell-cycle progression. To this
aim, available data on the identity and function of E2F factors
and their partners will be described briefly; we will then
essentially focus on the transcriptional control of several
cyclin genes, which encode G1/S regulatory proteins, and of
the RanBP1 gene, which encodes a regulator of the Ran
signalling network, by the E2F/DP transcription factor family.
The key hypothesis is that the ability of E2F to control
expression of these genes is centrally involved in the modula-
tion of cell-cycle checkpoint controls by E2F.

BioEssays 21:221–230, r 1999 John Wiley & Sons, Inc. BioEssays 21.3 221
Review articles
Levels of regulation of E2F function
The factor originally designated E2F is made up of DNA-
binding heterodimers containing one E2F subunit and one DP
subunit. These subunits are encoded by two gene families:
the E2F family includes five characterized members, E2F-1
through E2F-5, that largely act as transcriptional activators
(references 2–7, see also below); a sixth member, isolated
more recently, E2F-6 or EMA, is instead reported to repress
transcription, because it carries a DNA-binding, but not a
transcriptional activation domain, and may occupy E2F-
binding sequences in promoters, thereby preventing access
to other E2F members.(8,9) The DP family includes two genes,
DP-1 and DP-2, the latter of which gives rise to two functional
products generated by alternative splicing; hence, three
functional proteins actually compose the DP family.(2–4)
The activity of E2F/DP heterodimers is subjected to
several levels of control. The expression of the genes
encoding E2F-1, E2F-2, and E2F-3 is low in G0 and maximal
at the G1/S transition; in the case of the E2F-1 and E2F-2
gene promoters, this regulation is mediated by E2F-binding
sites, which indicates an autoregulatory loop. Conversely,
expression of E2F-4 and E2F-5 is relatively constant during
the cell cycle; therefore, these two factors are the predomi-
nant E2F members to be found in G0 cells, indicating that
they play a role in repression of E2F-mediated transcription in
resting cells. Thus, at least some E2F components are
subjected to cell-cycle control; the DP components show no
significant fluctuations.
E2F/DP heterodimers interact with the pRb, p107, and
p130 members of the pocket protein family. These proteins
antagonize transcriptional activators of the E2F family with a
specificity that is mainly determined by the E2F component.
E2F-1, E2F-2, and E2F-3 preferentially associate with pRb,(10)
whereas E2F-4 predominantly interacts with p107 and
p130(10–12) and E2F-5 with p130 only.(13) However, all pocket
proteins can block the activity of all E2F members in vitro, and
under certain conditions, all members of the E2F family are
complexed with pRb.(10,14) Complexes of E2F/DP het-
erodimers with pocket proteins are destabilized through the
phosphorylation of pRb family members by cyclin-dependent
kinases (reviewed in references 3, 4; see also below).
The biological activity of those E2F members that are
steadily expressed, i.e., E2F-4 and E2F-5, is critically depen-
dent on regulated relocalization during the cell cycle.(15–17)
Although E2F-1 to E2F-3 represent bona fide nuclear pro-
teins, nuclear localization of E2F-4 and E2F-5 depends on
the interaction with an appropriate protein partner, and is
promoted by DP family members that contain a nuclear
localization signal (i.e., the ␣ and ␦ isoforms of DP-3 but not
other DP family members; reference 18), or by association
with a pocket protein (Fig. 1). Association with these different
molecules has opposite effects on transcription of E2F-driven
genes:(19,20) Free E2F complexes that contain E2F-1 (and any
222 BioEssays 21.3
DP family member) have an intrinsic ability to enter the
nucleus and act as transcriptional activators. In contrast,
heterodimers of DP-1, which lacks a nuclear localization
signal, with either E2F-4 or E2F-5 normally only reach the
nucleus in association with a given pocket protein, i.e., when
they function as repressors.(18) This observation marks an
important distinction between the members of the E2F family
(summarized in Fig. 1). These rules are bypassed, however,
in cells transformed by viral oncoproteins: free E2F-5/DP-1
heterodimers are localized to the nucleus in E1A-transformed
cells;(19) similarly, free E2F-4/DP-1 heterodimers have been
observed in the nuclei of HPV-16 E7-transformed cells.(21) A
further level of control of E2F activity is represented by the
proteolytic degradation of some E2F proteins by the ubiquitin/
proteasome pathway. The association with pocket proteins,
e.g., pRb, can greatly increase the stability of the E2F
subunits.(22)
S phase entry and apoptosis: two distinct
functions of E2F genes
Many of the genes that regulate E2F function are subject to
mutation in human cancers,(23) and the phenotype of E2F-1
knockout mice suggests that, in the context of the entire
organism, the E2F-1 gene has properties of both a tumor
promotor and a tumor suppressor gene.(24) This puzzling
finding may be explained by considering that certain E2F
members, including E2F-1, can activate transcription of
target genes in the absence of an associated pocket protein,
yet repress the same genes when complexed with pRb.
Furthermore, E2F proteins can also directly affect cell prolif-
eration in two distinct ways.
First, ectopic expression of E2F-1 overcomes G1 arrest
caused by the inhibition of G1 cyclin-dependent kinases and
drives quiescent human cells into S phase.(25,26) Similarly,
both E2F-2 and E2F-3 can induce S phase in quiescent rat
fibroblasts. The ability of E2F-3 to specifically induce S phase
seems to be directly linked to the ability of E2F-3 to activate
genes encoding origin recognition complex (ORC) and mini-
chromosome maintenance proteins, which are structural
components required for S phase onset.(27,28) Quite differ-
ently, both E2F-4 and E2F-5 require coexpression of a DP
partner to promote S-phase entry.(16) The amino terminus of
E2F-1 is sufficient to enable full-length E2F-4 to induce
S-phase and to transactivate an E2F-responsive promoter;
this reflects the ability of the E2F-1 amino terminus to direct
proteins to the nucleus.(16)
Second, overexpression of E2F-1 induces apoptosis, an
effect that is modulated by p53.(29) The regulated suppression
of E2F-1 DNA-binding activity, mediated through the phosphor-
ylation of DP-1 by cyclin A-kinase, is apparently required to
prevent an apoptotic response.(30) It was concluded that
improper activation of E2F-1 may function as a specific signal
for the initiation of an apoptotic pathway that is normally
 Review articles

Figure 1. Structure and functions of E2F family members. The six known members of the E2F family are shown; the known functional
domains are schematically depicted as boxes (NLS: nuclear localization signal; DBD, DNA binding domain; DD, dimerization domain;
MB, marked box; TAD/PP, transcriptional activation domain, overlapping with the binding site for pocket proteins). For each family
member, the influence of associated heterologous proteins on the subcellular localization and transcriptional capacity is indicated. The
subcellular localization of any complexes involving E2F-6 has not been reported, and the molecular mechanism by which this E2F family
member represses transcription remains to be established.

blocked.(31) Analysis of E2F-1 mutants indicates that DNA
binding, but not necessarily transcriptional activation, is
required for induction of apoptosis.(32) These results indicate
that stimulation of cell-cycle progression and induction of
apoptosis are independent functions of E2F-1.
E2F/DP target genes
The analysis of the promoters of genes that are responsive to
control by E2F might help to understand how different
E2F-containing complexes exert their different biological
effects. Binding sites for E2F factors are characteristic of two
major classes of genes, listed in Table 1: first, a group of
genes encoding essential enzymes in the nucleotide and
DNA biosynthetic pathways that are coordinately up-regu-
lated in late G1; second, genes encoding regulators of
cell-cycle progression. Genes from both groups respond to
ectopic expression of E2F-1.(33)
Cell-cycle regulatory genes are controlled by E2F through
genetically distinct mechanisms: for example, the genes
encoding Cdc2, cyclin E, and ORC1 are up-regulated in an
E2F-dependent manner, whereas the genes encoding
RanBP1, cyclin A, B-myb, and p107 are repressed in G0 and
activated at the G1/S transition; both effects involve E2F. The
genetic effects of E2F-responsive promoters are paralleled
by the protein-binding features of the respective E2F binding
sites in the promoter region. E2F binding sites that have been
identified through mutagenesis or deletion assays to confer
negative control of transcription in G0 cells (e.g., the E2F site
in the cdc2 gene promoter) are footprinted in vivo during G0
and early G1 phases but not upon entrance into the prolifera-
tive cycle.(34) In contrast, genes whose transcription is acti-
vated by E2F factors at the G1/S transition, exemplified by the
DHFR gene, become footprinted in vivo at the G1/S transi-
tion.(35) The best candidates for such a mode of action include
E2F-1, -2, and -3, which are themselves subjected to G1/S
transcriptional induction. Negative control of transcription in
quiescence is essentially attributable to the presence of
E2F-4 and E2F-5 in nuclei, which act to bridge the transcrip-
tional repressors pRb, p107 and p130 to their target DNA
elements in quiescent cells.
There is variation in the nucleotide sequence and binding
affinity of the E2F sites in different gene promoters (some of
which are indicated in reference 36; see also Table 1). The
specificity of E2F complexes for particular target genes,
identified functionally or in DNA/protein interaction assays, is

BioEssays 21.3 223

Review articles

TABLE 1. Features of E2F-Driven Promoters

Regulation by E2F/DP Regulation by E2F/DP
Protein-binding features or pocket proteins or pocket proteins
Genea of E2F sites (transient assays) (endogenous genes)

Regulatory genes
c-myc Two E2F-binding sites complexes contain Transactivation by Ad5 E1A; E2F site Expression not affected in cells lacking
pRb; in vivo protection of E2F sites cor- required for repression by pRb pocket proteinsb
relates with transcriptional activity
cyclin E Multiple E2F-binding sites; a variant site, Repression by pRb; E2F site required for Strong induction by E2F-1c; de-repression
GCGCGA complexes contain p107 and promoter activation in G1; transactiva- in pRb⫺/⫺ cellsb
p130 tion by E2F4/DP-1
B-myb In vivo protection of E2F site correlates Transcriptional repression, corepressors Induction by E2F-1c; de-repression in cells
with transcriptional repression involved? Promoter derepression by E7 lacking p107 and p130b
p107 involved in repression
p107 Two E2F-binding sites Proximal E2F site for basal promoter activ- De-repression in pRb⫺/⫺ cellsb
ity; distal E2F site for repression by pRb
and p107
pRb One E2F site, complexes contain pRb Autorepression of the Rb-1 promoter by Expression not affected in cells lacking
pRb pocket proteinsb
E2F-1 Two E2F-binding sites, each composed of Both sites required for promoter repression De-repression in cells lacking p107 and
two overlapping E2F elements in G0; both sites respond to E2F-1 and p130b
E2F-3
E2F-2 Multiple E2F-binding sites; subtle Proximal E2F sites required for repression Not determined
sequence variations in G0; proximal E2F sites required for
activation by E2F-1, -2, and -3; growth
control also requires E-box elements
cyclin A Variant E2F site AGTCGCG overlaps with Negative control of promoter activity in G0;Induction by E2F-1c; de-repression in cells
CDF-1 binding site; complexes contain E2F site required for promoter activation lacking p107 and p130b
p107; in vivo protection correlates with at G1/S; E2F site required for activation
transcriptional repression by Ad5 E1A; p107 involved in repression
p34cdc2 Nonconsensus E2F site; overlaps with Negative control of promoter activity in G0 Induction by E2F-1c; de-repression in cells
CDE/CHR elements; complexes contain cells lacking p107 and p130b
E2F-4 and p130; in vivo protection cor-
relates with transcriptional repression
RanBP1 Two E2F-binding sites; in vivo protection of Distal site required for activation at G1/S; Induction by E2F-1d
distal site in cycling cells; lack of protec- distal site required for responsiveness to
tion at proximal site in cycling cells E2F-1, E2F-4, pRb, and p107; proximal
site required for G0 repression
Structural genes
DNA pol (␣) Two E2F consensus sites Transactivation by E2F-1 Induction by E2F-1c; expression not
affected in cells lacking pocket proteinsb
HsORC1 Two overlapping E2F sites with reverse E2F sites required for G0 repression; acti- Not determined
orientation vation by E2F-1
DHFR Multiple E2F-binding sites; in vivo occu- E2F sites required for activation at G1/S; Induction by E2F-1c; premature induction
pancy of one E2F site after G1/S transi- activation by E2F-1; cooperation with in cells lacking p107 and p130b
tion; constitutive in vivo occupancy of Sp1
another E2F site
Tk Two overlapping E2F sites; constitutive in E2F sites required for activation at G1/S; Expression not affected in cells lacking
vivo protection during cell cycle cooperation with Sp1 pocket proteinsb
TS One putative E2F site E2F site part of larger element; no activa- Induction by E2F-1c; de-repression in cells
tion by E2F-1 lacking p107 and p130b
PCNA One putative E2F site in first intron Induction by E2F-1/DP-1 Induction by E2F-1c; expression not
affected in cells lacking pocket proteinsb
RRM2 Not determined Not determined Induction by E2F-1c; de-repression in cells
lacking p107 and p130b
Srp20 Two putative E2F binding sites Induction by E2F-1/DP-1 Not determined
Histone H2A One E2F-binding site E2F site required for activation in early S Not determined
activation by E2F-1; repression by pRb
and p107

aMost original references to promoter studies are indicated in the text or are quoted in references 33, 36, and 41.
bData from reference 41.
cData from reference 33.

dData from reference 71.

224 BioEssays 21.3


listed in Table 1. Interestingly, the E2F-binding sites in the
cyclin A,(37) cdc2,(34) and cyclin E(38) gene promoters are
characterized by a variant nucleotide sequence, accompa-
nied by a lower in vitro binding affinity. The reason for the
presence of relatively weak E2F binding sites in the promoter
regions of these key cell-cycle regulatory genes is unclear at
present; it might indicate that the expression of these genes is
controlled by other pathways, in addition to the E2F activation
pathway. In the case of the cyclin A gene, Liu et al.(39)
presented evidence that the variant E2F binding site overlaps
with the binding site for an unrelated transcription factor,
CDF-1; according to these authors, a functional interplay of
E2F and CDF factors brings about the specific timing of
transcription of certain target genes during the cell cycle.(40)
The analysis of cyclin gene expression in mouse fibroblasts in
which specific Rb family members have been deleted indi-
cates that cyclin A gene activity is de-repressed in cells
lacking both p107 and p130; in contrast, expression of the
cyclin E gene is specifically de-repressed in pRb-deficient
cells.(41) These results suggest that the E2F sites in the
promoter regions of these two cyclin genes display significant
functional differences, probably resulting from the coopera-
tion of E2F family members with additional transcription
factors that bind to adjacent or overlapping sites.
E2F factors and phase-specific transcription
of the early cyclin genes
In the following section, we will summarize the current
knowledge of how E2F factors signal from the proliferation
apparatus to the promoters of cell-cycle regulatory genes. In
cells that have entered the division programme, cyclin E and
cyclin A are sequentially synthesized and associate with
cdk2. Although the abundance of cyclin-dependent kinases
(cdk) is relatively constant throughout the cycle in mammalian
cells, the catalytic activity of these enzymes is controlled
through multiple regulatory mechanisms, including regulatory
phosphorylation and the association of specific inhibitors
(CKIs) (for review, see reference 42). However, activation of
cdk enzymes cannot proceed in the absence of an associated
cyclin; hence, the abundance of the various cyclins is a key
determinant for the activity of cyclin-dependent kinases.
There is evidence that S phase entry in mammalian cells is
controlled through a regulatory cascade of cyclin gene expres-
sion (Fig. 2), as described in detail below. In addition to this
domino-like cascade, regulatory feedback loops have been
identified that control the expression of specific cyclin genes,
and it is assumed that these feedback loops are mainly
responsible for the sharp transitions between individual cell-
cycle phases. Furthermore, the regulatory cascade of cyclin
gene expression, as described in Figure 2, can be interrupted
at specific points by external growth-suppressive signals,
which are often mediated through the action of particular cdk
inhibitors.
Review articles
Cyclins D and E
Growth factors induce expression of the cyclin D1 gene
(reviewed in reference 23). Cyclin D1 associates with cdk4 or
cdk6; the kinase activity associated with these complexes
phosphorylates and thereby inactivates the pRb and p107
members of the retinoblastoma protein family; this activity is
subjected to regulation by cdk inhibitors, like p16INK4 or
p21WAF-1 (reviewed in references 5, 43). Through the
inactivation of pRb family members, cyclin D-associated
kinase triggers transcriptional activation of E2F-driven
genes,(44) probably including the cyclin E gene.(45,38) Once
cyclin E is synthesized, it associates with cdk2; the subse-
quent activation of cyclin E-associated kinase is essential for
S phase entry. Cyclin E-associated kinase induces a further
activation of E2F-dependent gene expression, including, in
addition to the autoregulatory activation of the cyclin E gene,
transcriptional activation of the cyclin A gene.(46) Similar to
cyclin E, ectopic expression of cyclin D1 also triggers transcrip-
tion of the cyclin A gene;(37) however, it is clear that the
physiologic functions exerted by D-type cyclins are quite
distinct from those of cyclin E. Cyclin D1 is up-regulated in
response to external conditions and is dispensable in many
tumor cells; on the other hand, cyclin E is an essential part of
the cell-cycle machinery and is required for S phase entry in
all cases studied so far. Furthermore, different molecular
processes are involved in activation of cyclin A transcription
by both cyclins: whereas cyclin D1 induces phosphorylation
of p107 followed by the dissociation of E2F-p107 com-
plexes,(47) cyclin E associates with the cyclin A promoter as
part of a multimeric complex containing E2F, p107, and
cdk2(46) (see Fig. 2).
Cyclin A
The cyclin A protein is required for S phase entry, suggesting
that regulation of cyclin A expression is one of the latest
essential steps before the onset of DNA replication; hence,
this gene is likely to be the target of various cell-cycle
checkpoint controls, summarized as follows: (1) cells grown
to high density or treated by TGF-␤ arrest in G1, which is
accompanied by repression of cyclin A gene expression,
whereas cyclins D1 and E are normally expressed under
these conditions.(48) Regulation of cyclin A gene transcription
also contributes to G1 arrest upon loss of adhesion to the
extracellular matrix,(49,50) (2) when diploid human cells reach
replicative senescence, they arrest in G1, and this effect is
accompanied by repression of cyclin A transcription, whereas
cyclins D1 and E are overexpressed,(51) and (3) cyclin A
transcription is down-regulated in cells in which p53 function
is activated by, for example, DNA damage. Down-regulation
of the cyclin A promoter is not due to direct binding of p53, but
occurs through the p21WAF-1/Cip1 protein, a downstream effec-
tor of p53, which blocks the transactivation potential of E2F
BioEssays 21.3 225
Review articles

Figure 2. The regulatory cascade of early cyclin gene expression. When mammalian fibroblasts exit from G0 to enter S-phase, a
series of cyclin-dependent kinases is consecutively activated. The addition of serum growth factors to quiescent cells triggers
transcription of the cyclin D1 gene, and cyclin D1 molecules associate with preexisting cdk4 molecules. The kinase activity that is
associated with these complexes can phosphorylate specific sites in the retinoblastoma gene product pRb, leading to inactivation of pRb
and a subsequent derepression of the cyclin E gene. Once cyclin E is made, it associates with preexisting cdk2, and the kinase activity
associated with these complexes modulates the function of at least three sets of target proteins: (1) cyclin E/cdk2 complexes associate
with E2F-p107 complexes at the cyclin A promoter and trigger its transcriptional activation, (2) cyclin E/cdk2 complexes cooperate with
cyclin D1 to phosphorylate pRb, leading to its inactivation and the subsequent activation of E2F-driven genes, including the cyclin E gene
itself; thereby, a regulatory feedback loop is established, (3) it is assumed that cyclin E/cdk2 complexes phosphorylate additional
downstream targets that are unrelated to E2F regulation but that may be involved in the initiation of DNA replication. Strong
overexpression of cyclin D1 in vitro, similar to overexpression observed in several human cancers, can trigger activation of the cyclin A
gene by a different pathway that involves the disruption of p107/E2F complexes but may be independent of cyclin E/cdk2. Once cyclin A is
made, it associates with cdk2 and the resulting kinase activity modulates the function of at least two classes of proteins: (1) cyclin A/cdk2
phosphorylates the DP1 subunit of the E2F transcription factor, which leads to a loss of DNA binding capacity and, hence, a shutoff of
E2F-driven transcription; (2) it is assumed that cyclin A/cdk2 complexes phosphorylate additional downstream targets that may be
directly involved in the initiation of DNA replication. The cascade of cyclin gene expression can be interrupted at various points through
the action of cdk inhibitors. Please note that both p16 and p27, as shown here, represent two families of related cdk inhibitors (for details,
see reference 42).

complexes,(52) possibly through direct protein/protein interac-
tion.(53)
In these instances, down-regulation of cyclin A gene
expression is due to a repression of cyclin A promoter activity,
and in most cases, the E2F-binding site in the cyclin A
promoter (see Table 1) is essential for this effect. The E2F site
of the cyclin A gene promoter is also targeted by transforming
oncogenes encoded by DNA tumor viruses such as adenovi-
rus E1A or HPV-16 E7 (for review, see reference 54). These
observations are consistent with the notion that the abroga-
tion of cell-cycle checkpoint controls, monitored here by the
deregulation of cyclin A gene expression, is involved in cell
transformation.
The function of cyclin A/cdk2 complexes in cell-cycle–
regulated transcription is distinct from that exerted by cyclin
E/cdk2 or cyclin D/cdk4. Ectopic expression of cyclin A does
not induce transcription of E2F-dependent genes,(37) on the
contrary, cyclin A/cdk2-mediated phosphorylation of E2F-1/
DP-1 heterodimers results in the loss of E2F binding activ-
ity.(55) These observations suggest that, whereas cyclins D1
and E activate E2F-dependent transcription, cyclin A might be
used to turn off E2F-driven genes, probably as part of its
function as checkpoint protein.
Interestingly, E2F-6, which can antagonize E2F-1-induced
transcription, has been reported to either prolong S phase in
cells growing asynchronously(56) or inhibit the G1/S transition

226 BioEssays 21.3


in cells that have been serum-starved and then restimu-
lated.(57) Although there are some discrepancies in the effects
of E2F-6 in these two studies, possibly owing to the differing
pRb status of the cell lines used, these observations suggest
that this protein acts to antagonize cell-cycle progression, a
function that might apply during cell-cycle checkpoint activa-
tion, which occurs, for example, after DNA damage. Although
entirely hypothetical, two reasons suggest that E2F-6-
containing complexes might have a role in preventing tran-
scriptional activation of E2F target genes in the presence of
DNA-damage. First, E2F-6 lacks a cyclin A-binding domain
and, hence, is not sensitive to cyclin A-mediated phosphoryla-
tion. Second, E2F-6-dependent transcriptional inactivity of
cell-cycle genes may be prolonged in those cases where
cell-cycle arrest or delay is required to sustain the repair
processes and monitor DNA integrity before resumption of
progression through S and G2.
Transcriptional regulation of the RanBP1 gene by
E2F: a novel aspect of checkpoint control?
Among the genes listed in Table 1, the RanBP1 gene
represents a perhaps unexpected target of E2F regulation:
the RanBP1 protein is neither enzymatically involved in DNA
replication nor a direct regulator of cell-cycle progression.
RanBP1 acts as a major regulator of Ran, a member of the
Ras superfamily of GTPases, which controls a puzzling
variety of cell-cycle events, including checkpoint controls at
the G1/S and G2/M transition (for reviews, see references.
58, 59; see also below). In the following two paragraphs, we
first will briefly recapitulate the known functions of the Ran
GTPase and then summarize the available evidence concern-
ing the role of RanBP1 within the Ran regulatory network.
The signalling activity of Ran is dependent on the switch
between the GTP-bound and GDP-bound forms of the mol-
ecule, and the balance between both forms is modulated by
the antagonistic activities of RCC1 (regulator of chromosome
condensation), which acts as the nucleotide exchange factor
for Ran, and of RanGAP (Ran GTPase-activating protein).
Although the molecular targets of Ran are not yet identified,
genetic evidence suggests that the Ran GTPase functions as
a ‘‘timer’’ molecule during cell-cycle progression. Such a role
is supported by the finding that the Ran/RCC1 signalling
activity is absolutely required for: (1) the G2 checkpoint, as
indicated by the appearance of prematurely condensed
chromosomes independent of S-phase completion, which
represents the first recognized alteration associated with loss
of function of RCC1 (reviewed in reference 60); (2) the onset
of S phase (reviewed in references 59, 60; see also reference
61); (3) completion of the mitotic division, as indicated by
fission yeast mutants lacking RCC1 activity that are impaired
in the mitosis-to-interphase transition with hypercondensed
chromosomes (reviewed in reference. 58), a phenotype
Review articles
similar to that of mammalian cells in which RanBP1 expres-
sion is deregulated (see below).
Growing evidence shows that the Ran network also
controls nucleocytoplasmic transport; this function may in fact
underlie the role of Ran and its partners in checkpoint
controls, given that defects in nucleocytoplasmic transport
are often associated with cell-cycle abnormalities. Mutations
in components of the Ran network inhibit both import and
export activities, and the Ran GTPase is itself a shuttling
molecule that can participate in the export of several classes
of RNAs and cytoplasmic proteins, as well as in nuclear
import of proteins that contain a nuclear localization signal
(see reference 62 for review). Intriguingly, the subcellular
localization of several cell-cycle regulatory proteins, such as
E2F-4,(15,16) hCdc18,(63) cdc25C, Nim1, Cdc2, and cyclin B1
(reference 64 and references therein) is regulated stringently
during cell-cycle progression, suggesting that their intracellu-
lar redistribution might play an important role in cell-cycle
control. These lines of evidence converge to suggest that
genes that control nucleocytoplasmic transport, including
Ran, are required to establish orderly cell-cycle progression.
The genes encoding Ran and RCC1 are both induced as
immediate-early genes and are steadily expressed in cycling
cells. In contrast, the RanBP1-encoding gene is transcription-
ally induced at the G1/S boundary.(65) E2F factors control both
growth-dependent repression and S-phase–specific induc-
tion of the RanBP1 gene. An in vivo footprinted E2F-binding
site in the RanBP1 promoter(66) is responsible for G1/S
up-regulation;(67) another E2F-binding site that flanks the
transcription start site controls G0 repression (71). The
finding that the timing of RanBP1 transcriptional induction is
coupled to that of genes required for S phase by the same
family of E2F activators and pocket protein repressors (Fig.
3), suggests that, in mammals at least, RanBP1 might act as
a ‘‘sensor’’ of the cell-cycle phase, connecting the Ran cycle
to the cell cycle: As the RanBP1 protein modulates both
RCC1 and RanGAP activities,(68) E2F-dependent transcrip-
tional activation of RanBP1 at the G1/S transition will modify
the molecular ratio of RCC1 to RanBP1 and, hence, of
RanGTP to RanGDP, during S phase. Regulated expression
of RanBP1 is now emerging as a requirement for orderly
coordination of functions that are controlled by the Ran
network. Replacing the endogenous promoter of the RanBP1
gene with cell-cycle–independent sequences resulted in pro-
duction of the RanBP1 protein throughout the cell cycle and
caused defects in all major cell-cycle transitions, failure of
chromatin decondensation after metaphase and impaired exit
from mitosis to the subsequent interphase.(69) These abnor-
malities suggest that the molecular balance among members
of the Ran network during specific phases is crucial for
orderly cell-cycle progression. Interestingly, a cell line stably
expressing a dominant negative version of E2F-1 displays a
specific phenotype(70) that is similar to the phenotype ob-
BioEssays 21.3 227
Review articles
Figure 3. E2F/DP factors are represented upstream of
some known target genes. Through transcriptional control of
target genes (in ovals), E2F factors control downstream
processes (in squares): G1 progression depends on transcrip-
tional activation of cyclin E and A genes by E2F factors;
E2F-mediated activation of genes encoding DNA replication
components is required for the onset of S phase; concomitant
with this, transcriptional activation of the RanBP1 genes
modifies the activity of the Ran network during S phase, which
in turn controls molecules involved in subsequent progression
through the G2 and M phases; the Ran network also controls
nuclear transport, whose regulated activity is important for at
least certain checkpoint systems. Checkpoint mechanisms
monitor completion of all processes (double arrowheads), or
trigger apoptosis when defective processes are detected.
Apoptosis can also be triggered as a direct response to E2F-1.
served when the RanBP1 gene is expressed throughout the
cell cycle.(69) It is tempting to speculate that the defects
observed in this cell line arise at least in part in consequence
of disruption of cell-cycle regulated RanBP1 expression.
Conclusions
Faulty checkpoint controls have been observed in certain
hereditary cancer syndromes and at early stages of cell
transformation, which suggests that structural mutations,
regulatory mutations, or both, in checkpoint genes might
contribute to the genetic instability associated with neoplastic
growth. Genes that are regulatory targets of E2F/DP transcrip-
tion factors are found in major cell-cycle regulatory pathways,
as schematically diagrammed in Figure 3.
Available data suggest that there is cross-talk between
E2F/DP factors and other regulatory genes, whose products
are themselves implicated in cell-cycle control, and regulated
transcription of particular genes by E2F factors may be
viewed as an additional level of integrated control which
connects the transcriptional apparatus to control of cell
proliferation.
228 BioEssays 21.3
Acknowledgments
We apologize to the many colleagues whose work could not
be cited due to space constraints. Work in the laboratory of
P.J.D. was supported by a grant from the Deutsche Forsch-
ungsgemeinschaft. Work in the laboratory of P.L. was sup-
ported by grants from CNR Progetto Strategico ‘‘Cell cycle
and apoptosis’’ and Associazione Italiana per la Ricerca sul
Cancro.
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