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Cell Cycle
Introductory article
Gary S Stein, University of Massachusetts Medical Center, Worcester, Massachusetts, USA
André J van Wijnen, University of Massachusetts Medical Center, Worcester, Massachusetts, USA
Janet L Stein, University of Massachusetts Medical Center, Worcester, Massachusetts, USA
Jane B Lian, University of Massachusetts Medical Center, Worcester, Massachusetts, USA
Thomas A Owen, Pfizer Global Research and Development, Groton, Connecticut, USA
Article Contents
. Introduction
. Principles of Cell Cycle Regulation
. Biochemical and Molecular Parameters of Cell Cycle
Control: Regulatory Strategies
Each period of the cell cycle requires selective expression of genes that encode cell cycle
regulatory proteins. A broad spectrum of signalling mechanisms integrate and amplify
growth-related regulatory cues that mediate fidelity of cell cycle control.
Introduction
Proliferation and cell cycle progression are functionally
linked to the expression of genes associated with growth
control. Both cause and effect relationships between the
factors that modulate the cell division cycle exist, reflecting
multidirectional signalling between segments of regulatory
cascades that operate selectively in specific cells and tissues.
The integration of positive and negative growth regulatory
signals is now appreciated in a broad spectrum of
biological contexts. These include but are not restricted
to: (1) repeated traverse of the cell cycle for cleavage
divisions during the initial stages of embryogenesis and
continued renewal of stem cell populations; (2) stimulation
of quiescent cells to proliferate for tissue remodelling and
wound healing; and (3) exit from the cell cycle with the
option to subsequently proliferate or terminally differentiate. Equally important is an appreciation of the cell
cycle regulatory mechanisms that are compromised in
transformed and tumour cells and in non-malignant
disorders, where there are abnormalities in cell cycle and/
or growth control.
nuclear transplant experiments carried out by David
Prescott. The effects of cytoplasm from various stages of
the cell cycle on transplanted nuclei from other periods
demonstrated the following basic principles of cell cycle
control: (1) that initiation of DNA synthesis is determined
by cytoplasmic factors present throughout S phase but
absent in pre-S phase; (2) that a nuclear mechanism
prevents re-replication of DNA without passage through
mitosis; and (3) that a dominant cytoplasmic factor in
mitotic cells promotes mitosis in interphase cells irrespective of whether DNA replication has occurred. These
important controls are phylogenetically conserved in
organisms as diverse as yeast, protozoans, echinoderms,
amphibian oocytes and mammalian cells.
Requirements for cell cycle stage-specific
modifications in gene expression
The initial indication that modifications in gene expression
are required to support entry into S phase and mitosis was
Cell cycle stages
The cornerstone for investigations into mammalian cell
cycle control is the documentation by A. Howard and S. R.
Pelc, nearly five decades ago, that proliferation of
eukaryotic cells, as in bacteria, requires discrete periods
of DNA synthesis (S phase) and mitotic division (M) with a
postsynthetic, premitotic period designated G2 and a
postmitotic, presynthetic period designated G1 (Figure 1).
The idea that biochemical regulatory mechanisms are
associated with growth control and cell cycle progression
was supported by an elegant series of cell fusion and
Phenotype commitment
G0
Principles of Cell Cycle Regulation
Subdivision of the cell cycle into functional,
biochemically defined stages
Quiescence
Differentiated
M
G2
G1
S
Figure 1 Cell cycle regulation. The four stages of the somatic cell cycle
(G1, S, G2 and M) support duplication of the genome and subsequent
segregation of a diploid set of chromosomes into two progeny cells. Cells
can exit the cell cycle into a quiescent nondividing state (G0) with the
option to re-enter the cell cycle or to differentiate into a committed cell
expressing phenotypic markers characteristic of distinct tissue-specific
lineages.
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1
Cell Cycle
obtained from inhibitor studies. First it was observed that
transcription and protein synthesis are needed for DNA
replication and mitotic division. Restriction points late in
G1 and G2 for competency to initiate S phase and mitosis
were mapped by Arthur Pardee. Subsequently, by the
combined application of gene expression inhibitors and
modulation of growth factor levels in cultured cells, a
mitogen-dependent (growth factor/cytokine responsive)
period was defined early in G1 in which competency for
proliferation is established and a late G1 restriction point
was identified in which competency for cell cycle progression is attained.
Identification of cell cycle checkpoints
Checkpoints have been identified that govern passage
through G1 and G2 (Figure 2), at which competency for cell
cycle traverse is monitored. The first evidence for these
checkpoints was provided by the observation of delayed
entry into S phase or mitosis following exposure to
radiation or carcinogens. Editing functions and decisions
for continued proliferation, growth arrest or apoptotic cell
death occur at these regulatory junctures (Figure 3). The
complexity of the surveillance mechanisms that govern
decisions for cell cycle progression is becoming increasingly apparent. There are multiple checkpoints during S
phase which monitor regulatory events associated with
DNA replication, histone biosynthesis and fidelity of
chromatin assembly. Mitosis is similarly controlled by an
intricate series of checkpoints that are responsive to
Checkpoints
biochemical and structural parameters of chromosome
condensation, mitotic apparatus assembly, chromosome
alignment, chromosome movement and cytokinesis.
Multiple, regulatory cycles operative during
proliferation
Several interdependent cycles are functionally linked to
control of proliferation (Figure 4). The first is a stringently
regulated series of biochemical and molecular parameters
that support genome replication and mitotic division. The
second is a cascade of cyclin-related regulatory factors that
transduces growth factor-mediated signals into discrete
phosphorylation events, controlling expression of genes
responsible for both initiation of proliferation and
competency for cell cycle progression. Other cell cyclerelated regulatory loops involve chromosome condensation, spindle assembly, metabolism and assembly of CDK1
(cdc2) (a key protein kinase in cell cycle regulation) and
assembly/disassembly of DNA replication factor complexes (replicators and potential initiator proteins). It is
becoming increasingly evident that each step in the
regulatory cycles governing proliferation is responsive to
multiple signalling pathways and has multiple regulatory
options. The fact that different cyclin-dependent kinases,
like cdc2, are activated by different cyclin-binding
partners helps to explain the control of proliferation under
multiple biological circumstances and provides functional
redundancy as a compensatory mechanism. The regulatory events associated with the proliferation-related
during cell cycle progression
Multipotent stem cell
Mitotic progression
Self
renewal
Pre-committed
progenitor
Phenotypically
committed cell
G0
Differentiated
Apoptosis
Chromatin fidelity
and DNA repair
M
G2
Cell
cycle
S
DNA synthesis and
chromatin packaging
OFF
DNA synthesis and
chromatin packaging
ON
Expansion
+
G1
–
Growth factors
Cytokines
Adhesion
Cell/cell contact
Senescence
Apoptosis
Apoptosis
Restriction point:
proliferation competency
and DNA repair
Figure 2 Multiple checkpoints control cell cycle progression. The cell cycle is regulated by several critical cell cycle checkpoints (ticks) at which
competency for cell cycle progression is monitored. Entry into and exit from the cell cycle (black lines and lettering) is controlled by growth regulatory
factors (e.g. cytokines, growth factors, cell adhesion and/or cell–cell contact) which determine self-renewal of stem cells and expansion of pre-committed
progenitor cells. The biochemical parameters associated with each cell cycle checkpoint are indicated by red lettering. Options for defaulting to apoptosis
(blue lettering) during G1 and G2 are evaluated by surveillance mechanisms that assess fidelity of structural and regulatory parameters of cell cycle control.
2
ENCYCLOPEDIA OF LIFE SCIENCES / & 2002 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net
Cell Cycle
Checkpoint control
Differentiated
Phenotypically
committed cell
Surveillance:
intracellular levels
architectural integrity
extracellular signals
Stage-specific
preparation:
G1 phase: DNA replication
G2 phase: mitosis
Checkpoint
editing:
DNA repair
Chromatin
Competency for cell
cycle progression:
S phase entry
Mitosis
Cell cycle
block:
DNA damage
Chromatin
Apoptosis
Quiescence
G1
S
Restriction point:
proliferation
competency
Apoptosis
(a)
Apoptosis
G0
G2
Pre-replicative
DNA repair and
chromatin fidelity
M
Pre-replicative
DNA repair and
chromatin fidelity
(b)
CLN
CLN
Intracellular
concentrations
Competency
CDI
CDK
Inactive
Threshold
+
CLN
CLN
CDK
CDK
Active
Inactive
CDI
Growth factors
Cytokines
t
Nucleosome
Chromatin
architecture
DNA
DNA damage/mismatch and
chromatin modifications
DNA repair and
chromatin remodelling
(c)
Figure 3 Surveillance and editing mechanisms mediating checkpoint control. (a) Surveillance mechanisms monitor multiple biochemical and
architectural parameters that control cell cycle progression. These parameters include the intracellular levels of regulatory proteins, structural and
informational integrity of the genome, as well as extracellular signals governing cell cycle progression. The integration of this regulatory input can result in
(i) competency for cell cycle progression (green traffic light and arrows), (ii) cell cycle inhibition and activation of editing mechanisms (yellow traffic light
and arrows), or (iii) the active and regulated destruction of the cell in response to apoptotic signals (red traffic light and red arrow). (b) Traverse of the cell
cycle is regulated by a series of checkpoints at strategic positions within the cell cycle. Several major checkpoints (yellow arrows with ticks and blue lettering)
only allow a cell to commit to a subsequent cell cycle stage upon satisfying essential biochemical and architectural criteria governing competency for cell
cycle progression (green traffic lights). For example, at the ‘restriction point’ surveillance mechanisms (yellow traffic lights) integrate cell growth
stimulatory and inhibitory signals, including growth factors, cell adhesion and nutrient status (blue lettering). Checkpoints in G1 and G2 are necessary to
ensure the integrity of the genome and, if necessary, activate chromatin editing mechanisms (blue lettering). (c) Checkpoint control mechanisms monitor
intracellular levels of cell cycle regulatory factors, as well as parameters of chromatin architecture. For example, the activation of cyclin-dependent kinases
reflects the sensing of intracellular concentrations of the cognate cyclins. CDK activation is attenuated by CDK inhibitor proteins (CDIs) which inactivate
CDK/cyclin complexes. Competency for cell cycle progression requires that cyclin levels reach a threshold (e.g. by exceeding the levels of available CDIs, or
phosphorylation events altering the affinities of cyclins and CDIs for CDKs). As a consequence, activated CDK/cyclin complexes phosphorylate
transcription factors that regulate expression of cell cycle stage-specific genes. Furthermore, key checkpoints in G1 and G2 monitor chromatin integrity and
perform essential editing functions. DNA damage activates DNA-repair mechanisms that fix informational errors in the genome and restore nucleosomal
organization by chromatin remodelling.
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3
Cell Cycle
Ubiquitin
Cytokines
cln D1/2/3
BMP
CAK
pRb
cln D1/2/3
ECM
+
CDK4/6
CDK4/6
CDK
p27
CDK2
P
p107
p15 p16 p18 p19
E2F
+
G1/S start
E2F
DNA replication
Apoptosis
MCM
S
TGFβ
Ubiquitin
cln E/A
–
p57
CDP
CDP
INK-class CDIs
cln E/A
Ubiquitin
pRb
–
CDK2
p21
TGFβ
Adhesion
E2F
+
CIP-class CDIs
Vitamin D
–
Growth
factors
Cyclin
p53
ORC
cln E/A
p107
CDK2
E2F
PCNA
Histone genes
Cell cycle
cln H
CDK7
Ubiquitin
cln A/B1
CDK1
APC/C ubiquitin
ligase complex
G1
CAK
G2
M
+
Apoptosis
wee
–
–
P
cln A/B1
CDK1
+
P
cln A/B1
P
CDK2
P
Ubiquitin
cdc 25
Figure 4 Regulation of the cell cycle by cyclin-dependent kinases and tumour suppressor proteins. Competency for cell cycle progression is determined
by cyclin-dependent kinases (CDKs; yellow rounded boxes) which monitor intracellular levels of cyclins (flat red ovals) and CDK inhibitory proteins (CDIs;
blue circles). CDKs mediate phosphorylation of the pRB class of tumour suppressor proteins (i.e. pRB/p105, p107 and p130), which results in activation of
E2F and CDP/cut-homeodomain transcription factors (red ovals). These E2F-dependent and -independent mechanisms induce expression of genes
required for the G1/S phase transition. The activities of CDKs are also influenced by phosphorylation (e.g. wee1 or CDK-activating kinase (CAK)),
dephosphorylation (CDC25), ubiquitin-dependent proteolysis, and induction of CDIs by the tumour suppressor protein p53. Options for apoptosis are
indicated within the context of cell cycle regulatory factors. Growth factors and cytokines induce the activities of CDKs which mediate the G0/G1 transition
(red arrow). Vitamin D and TGFb-dependent cell signalling pathways upregulate CDIs (e.g. p21 and p27), which blocks cell cycle progression and supports
differentiation in the presence of tissue-specific regulatory factors.
cycles support control within the contexts of: (1) responsiveness to a broad spectrum of positive and negative
mitogenic factors; (2) cell–cell and cell–extracellular
matrix interactions; (3) monitoring sequence integrity of
the genome and invoking editing and/or apoptotic
mechanisms if required; and (4) competency for differentiation.
Cell structure–function interrelationships
mediating control of proliferation
For cells to divide there must be fidelity in the mechanisms
governing DNA metabolism; the organization of chromatin and the production of regulatory factors must also be
tightly controlled. Equally significant is the need to
4
modulate cell cycle-dependent assembly of the mitotic
apparatus, chromosome condensation and decondensation, breakdown and reformation of the nuclear membrane, duplication and functional properties of
centrosomes as well as cytokinesis. Thus, there is a striking
requirement to regulate the biochemical parameters of the
cell cycle and the mechanisms that organize components of
cell cycle regulatory machinery within the three-dimensional context of cellular architecture. Both temporal
modulation of cell cycle regulatory mechanisms and the
cell cycle-dependent placement of regulatory factors at
subcellular sites where activities occur are functionally
linked to growth control. The mechanisms of chromosome
movement and intracellular trafficking of structural and
regulatory proteins are important parameters of cell cycle
control that are operative in vivo.
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Cell Cycle
Biochemical and Molecular Parameters
of Cell Cycle Control: Regulatory
Strategies
Cell cycle regulatory mechanisms
Characterization of the biochemical and molecular components of cell cycle and growth control emerged from
systematic analysis of conditional cell cycle mutants in
yeast by L.H. Hartwell and co-workers. These studies were
the foundation for the concept that cell cycle competency
and progression are controlled by an integrated cascade of
phosphorylation-dependent regulatory signals. Cyclins are
synthesized and activated in a cell cycle-dependent manner
and function as regulatory subunits of cyclin-dependent
kinases (CDKs). The CDKs phosphorylate a broad
spectrum of structural proteins and transcription factors
that control progression through the cell cycle. By
complementation analysis, the genes for mammalian
homologues of the yeast cell cycle regulatory proteins
have been identified. In vivo overexpression, antisense and
antibody analysis have verified conservation of cell cycledependent regulatory activities and have validated functional contributions to control of cell cycle stage-specific
events. The emerging concept is that the cyclins and CDKs
are responsive to regulation by the phosphorylationdependent signalling pathways associated with activities
of the early response genes, which are upregulated
following mitogen stimulation of proliferation (Figure 4).
Cyclin-dependent phosphorylation is functionally linked
to activation and suppression of both p53 and RB-related
tumour suppressor genes, which mediate transcriptional
events involved with passage into S phase. The activities of
the CDKs are downregulated by a series of inhibitors
(designated CDIs) and mediators of ubiquitination, which
signal destabilization and/or destruction of these regulatory complexes in a cell cycle-dependent manner. Particularly significant is the accumulating evidence for functional
interrelationships between activities of cyclin–CDK complexes and growth arrest at G1 and G2 checkpoints, when
editing and repair are monitored following DNA damage
(see Figure 3). It is at these times, and in relation to these
processes, that apoptotic cell death is invoked as a
stringently controlled suicidal mechanism.
During G1, expression of genes associated with deoxynucleotide biosynthesis are upregulated (e.g. thymidine
kinase, thymidylate synthase, dihydrofolate reductase) in
preparation for DNA synthesis. As cells progress through
G1, regulatory factors required for initiation of DNA
replication are sequentially expressed and/or activated.
Following stimulation of quiescent cells to proliferate,
expression of the fos/jun-related early response genes is
induced early in G1, playing a pivotal role in activation of
subsequent cell cycle regulatory events. In S phase, DNA
replication is paralleled by and functionally coupled with
histone gene expression, providing the necessary basic
chromosomal proteins (H1, H4, H3, H2A and H2B) for
packaging newly replicated DNA into chromatin. During
G2, regulatory factors for mitosis are synthesized, and
modifications of chromatin structure to support mitotic
chromosome condensation occur. Mitosis involves a
sequential remodelling of genome architecture from
loosely packaged chromatin to highly condensed chromosomes and back to chromatin; assembly and subsequent
disassembly of the mitotic apparatus; breakdown and reformation of the nuclear membrane; and modifications in
activities of factors required for reinitiation of cell cycle
progression, quiescence or differentiation. As the sophistication of experimental approaches for dissection of
promoter elements and characterization of cognate regulatory factors increases, there is an emerging recognition
of cyclic modifications in occupancy of promoter domains
and protein–protein interactions which control cell cycle
progression.
Cell cycle regulatory factors mediating the G0/
G1, G1/S, S/G2 and G2/M transitions
Cyclin-related proteins are the principal regulators of
proliferation competency and cell cycle progression. In
response to growth factor-mediated signal transduction or
cell intracellular feedback mechanisms, cyclins function as
regulatory subunits of cyclin-dependent kinases (CDKs)
which catalyse the phosphorylation of transcription
factors to control cell cycle-regulated genes and structural
proteins that support proliferation. The CDKs are present
at similar levels throughout the cell cycle. Activities of the
CDKs are controlled by the cell cycle-dependent regulation of specific cyclins and by a series of CDK inhibitors.
Following growth factor induction of proliferation in
quiescent cells or the completion of mitotic division,
complexes of the D-type cyclins with CDK4 and CDK6 are
principal contributors to cell cycle initiation. Insight into
the complexities of regulatory mechanisms controlling the
G1 period of the cell cycle is provided by the presence of
cyclin D in association with CDK4, p21 (a cyclindependent kinase inhibitor also designated WAF1, Cip1,
CAP20, Sdi1, Mda6) and PCNA (proliferating cell nuclear
antigen, which is a subunit of DNA polymerase a and
involved in DNA replication and excision repair). Cyclin D
complexes with CDK4 or CDK6 to phosphorylate pRB
(retinoblastoma tumour suppressor protein). During early
G1 the hypophosphorylated form of pRB binds and
inactivates the E2F transcription factor. Following phosphorylation, E2F is released and transactivates a series of
genes required for DNA replication. Recent results suggest
that the CDKi p16 may be involved in modulating pRB
activity by influencing the association of D cyclins with
CDKs. Progression through the late G1 restriction point is
controlled by a CDK2/cyclin E complex. Although activity
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5
Cell Cycle
of CDK2/cyclin E is required for the G1/S transition, cyclin
E is not requisite for the progression of S phase.
CDK2/cyclin A is required for the initiation of DNA
replication and to support DNA synthesis throughout S
phase. Recent findings suggest that the CDK4/cyclin A
complex phosphorylates a single-stranded DNA-binding
protein designated RPA34, which blocks re-replication of
DNA. CDK2/cyclin A forms a quarternary complex with
the p107 RB-related protein and E2F. In addition, cyclin A
may play a role in cell adhesion and signal transduction
from the plasma membrane.
At the onset of mitosis, chromosome condensation is
mediated by cyclin B/CDK1 complexes. Phosphorylation
of histone H1 by CDK1/cyclin B modifies chromatin
structure through alterations in nucleosome interactions.
CDK1/cyclin B also contributes to chromosome condensation by phosphorylating and consequently activating
caseine kinase which is the topoII activator. CDK1 has
been functionally linked to control of mitosis by phosphorylation-dependent abrogation of lamin phosphorylation, which results in nuclear envelope breakdown.
Additionally, both cyclin A/CDK1 and cyclin B/CDK1
promote microtubule formation from centrosomes.
The complexity of CDK1 regulation in relation to cell
cycle control is illustrated by phosphorylation-dependent
changes at the onset of mitosis. During G2, CDK1 is
inactivated by phosphorylation of Tyr15 by Wee1.
Initiation of mitosis is functionally coupled to inactivation
of Wee1 by phosphorylation which is mediated by a series
of kinases that includes the nim1 kinase. CDC25 phosphatase dephosphorylates CDK1 at the onset of mitosis
and the cyclin B/CDK1 complex phosphorylates and
activates CDC25. Protein phosphatase 1 (pp1) inactivates
CDC25 activity. Yet another component of CDK1
regulation is phosphorylation of Thr161 by CDK-activating kinase (CAK) (MO15 or CDK7), which associates with
cyclin H and is required for maximum activity.
While activity of the CDKs are controlled by complex
formation with cyclins and phosphorylation status,
activity of the cyclins is controlled by cell cycle-dependent
degradation. Cyclins A and B are targeted for proteolysis
by ubiquitination in metaphase. Degradation of other
cyclins is mediated by a PEST sequence (a protein
degredation signal enriched in proline, glutamic acid,
serine and threonine).
Cell cycle stage-specific and ubiquitindependent turnover of cell cycle regulatory
factors
The activation and inactivation of cell cycle regulatory
factors at specific stages of the cell cycle occur at multiple
levels, and are often achieved by a combination of control
mechanisms. CDK-mediated phosphorylation represents
6
an important level of control at the restriction point late in
G1 and at the G1/S phase cell cycle transition.
The inactivation of regulatory factors by ubiquitindependent proteolysis that involves the 22S proteasome
represents an equally important cell cycle control mechanism. The ubiquitin–proteasome system utilizes a large
number of enzymes mediating ubiquitin activation (E1),
ubiquitin conjugation (E2) or ubiquitin ligation (E3) that
modulate turnover of cell cycle regulatory proteins. For
example, degradation of G1 cyclins involves the CDC34
protein. CDC34 is a ubiquitin-conjugating enzyme and is
conserved between yeast and vertebrates. The CDC34 gene
is essential for the G1/S phase transition. Ubiquitindependent degradation is also involved in constitutive
turnover of cyclins throughout G1, reflecting the labile
nature of G1 competency factors such as cyclin E.
Ubiquitin-dependent degradation also performs a key
regulatory function during the G2/M transition. The E2
enzymes encoded by UBC4 and UBC9 are involved in
degradation of specific cyclins prior to the onset of mitosis.
Similarly, completion of mitosis is regulated by the
anaphase-promoting complex (APC). This high-molecular-weight ubiquitination-complex is essential for chromosome segregation, but specific molecular targets have not
been identified. Apart from degradation of cyclins at
specific stages during the cell cycle, ubiquitin-dependent
proteolysis may also be important for regulating the
activities of oncoproteins including c-fos, c-jun and IRF2.
Transcriptional control during the cell cycle
Insight into transcriptional control at strategic points
during the cell cycle has been provided by characterization
of promoters and cognate factors which regulate expression of genes associated with competency for proliferation,
cell cycle progression and mitotic division under diverse
biological circumstances. Transcriptional modulation of
gene expression is required throughout the cell cycle and is
linked to a temporal sequence of events that is necessary for
proliferation. However, for clarity of presentation we will
confine our considerations to examples of transcriptional
control which are operative during G1 and at the onset of S
phase.
Transcriptional control at the restriction point late in G1
and at the G1/S phase transition illustrates the selective
utilization of cell cycle regulatory factors to support gene
expression that controls progression of the proliferation
process. Particularly striking is the temporal discrimination between cyclin E and cyclin A-mediated phosphorylation of transcription factors at the restriction point and G1/
S phase transition. The TK gene provides a paradigm for
restriction during transcriptional control and the histone
genes are indicative of transcriptional regulation that is
functionally linked to initiation of S phase (Figure 5).
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Cell Cycle
G2
G1
E2F dependent
CDK2
Cyclin E
p107
SP1
E2F EGR
TK
M
MT1 MT2 MT3
R
S
CDK1
Cyclin A
pRB CDP
YY1 YY1 YY1 SP1
IRF2
ATF1
CREB
Histone H4
IV
III
I
II
CDP dependent
S
Figure 5 Transcriptional control at the G1/S phase transition. The genes
encoding enzymes involved in nucleotide metabolism (e.g. thymidine
kinase (TK) and histone biosynthesis (e.g. H4) are each controlled by
intricate arrays of promoter regulatory elements (blue and red boxes) that
influence transcriptional initiation by RNA polymerase II (grey oval). E2F
elements in the promoters of the TK gene interact with heterodimeric E2F
factors that associate with CDKs, cyclins and pRb-related proteins. In
contrast, histone genes are controlled by the site II cell cycle regulatory
element, which interacts with CDP-cut and IRF2 proteins. Analogous to
E2F-dependent mechanisms, CDP-cut interacts with CDK1, cyclin A and
pRb, whereas IRF2 performs an activating function similar to ‘free’ E2F. The
presence of SP1 in the promoters of G1/S phase-related genes provides a
shared mechanism for further enhancement of transcription at the onset of
S phase.
Transcriptional activation and suppression of genes
involved in nucleotide metabolism at the restriction
point preceding the G1/S transition
During the G1/S phase transition, three critical events
associated with activities of cell cycle checkpoints occur
which prepare the cell for the duplication of chromatin.
First, genes encoding enzymes involved in nucleotide
metabolism are activated to ensure that cellular deoxynucleoside triphosphate pools are adequate for the onset of
DNA synthesis. Second, multi-protein complexes at DNA
replication origins are assembled that regulate both the
initiation of DNA synthesis and prevent reinitiation at the
same origin. Third, histone proteins are synthesized de
novo to accommodate the packaging of newly replicated
DNA into nucleosomes. Transcriptional activation of gene
expression at the G1/S phase transition represents the
initial rate-limiting step for cell cycle progression into S
phase.
The restriction point prior to the G1/S phase transition
integrates a multiplicity of cell-signalling pathways that
monitor growth factor levels, nutrient status and cell-tocell contact. This integration of positive and negative cell
cycle regulatory cues culminates in the transcriptional
upregulation of genes encoding enzymes and accessory
factors that directly and indirectly control nucleotide
metabolism and DNA synthesis. Analysis of the thymidine
kinase (TK) promoter and cognate promoter factors by
Arthur Pardee (Figure 5) has revealed that maximal TK
gene transcription involves at least three distinct cis-acting
elements that interact with cell cycle-dependent and
constitutive DNA-binding proteins. A principal regulatory complex that interacts with a proximal TK promoter
element includes p107, as well as cyclin- and CDK-related
proteins. The TK regulatory complexes are analogous to or
identical with E2F-related higher order complexes containing cyclins, CDKs and pRB-related proteins. Interestingly, cyclins A and E may represent the labile and ratelimiting restriction point proteins, which were originally
postulated based on results from early studies on cell
growth control.
Each of the G1/S phase genes is controlled by different
arrays of cis-acting promoter elements and cognate
factors. One unifying theme among many promoters of
the R-point genes is the presence of E2F and SP1 consensus
elements. Thus, one mechanism by which the cell achieves
coordinate and temporal regulation of these genes at the
G1/S phase boundary is directly linked to the release of
transcriptionally active E2F from inactive E2F/pRB
complexes. The disruption of E2F/pRB is mediated by
CDK4/CDK6-dependent phosphorylation of pRB in
response to growth factor stimulation and cell cycle entry.
Hence, the E2F-dependent activation of the R-point genes
provides linkage between the onset of S phase and control
of cell growth.
The E2F transcription factor represents a heterogeneous
class of heterodimers formed between one of five different
E2F proteins (i.e. E2F-1 to E2F-5) and one of three distinct
DP factors (DP-1 to DP-3). The various E2F factors may
display preferences in promoter specificity, differ in the
regulation of their DNA-binding activities during the cell
cycle, and bind selectively to distinct pRB proteins. The
mechanism by which this multiplicity of E2F factors
orchestrate transcriptional regulation of diverse sets of
genes at the G1/S phase transition is only beginning to be
understood. Apart from the role of ‘free’ E2F in activating
genes at the G1/S phase transition, promoter-bound
complexes of E2F factors associated with pRB-related
proteins, cyclin A and CDK2 have active roles in the
repression of gene expression during early S phase.
E2F-responsive transcriptional modulation of R-point
genes requires participation of the SP1 family of transcription factors (e.g. SP1 and SP3). For example, the TK
promoter contains one E2F site and one SP1 site and both
are required for maximal transcriptional responsiveness at
the G1/S phase boundary. This synergistic enhancement
involves direct protein–protein interactions between E2F
and SP1. Consistent with the critical role of SP1 in the cell
cycle control of gene expression, protein–protein interactions between SP1 and pRB can also occur, suggesting that
pRB can modulate the activities of E2F and SP1 in concert.
Analogous to the TK promoter, the DHFR promoter is
regulated by four SP1 elements which together with E2F
mediate transcriptional upregulation at the G1/S phase
transition.
ENCYCLOPEDIA OF LIFE SCIENCES / & 2002 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net
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Cell Cycle
Interestingly, SP3 selectively represses SP1 activation of
the DHFR (dihydrofolate reductase) promoter, but not
the TK or histone H4 promoter. It appears that the cellular
ratio of SP1 and SP3 levels may influence specific classes of
cell cycle-regulated genes, but the physiological function of
this regulatory mechanism remains to be elucidated.
Transcriptional control at the G1/S phase transition that
is functionally coupled with initiation of DNA synthesis
Conditions that establish competency for the initiation of
DNA synthesis in vertebrates are monitored in part by the
origin recognition complex (ORC). This complex appears
to contain sequence-specific proteins that mark the
location of DNA replication origins. Prior to S phase,
the labile Cdc6p protein associates with ORC, which stages
the subsequent binding of Mcm proteins (‘licensing
factors’) to form large origin-bound pre-replication complexes. The mechanism by which these complexes facilitate
the onset of template-directed synthesis of DNA remains
to be established. However, activation of S phasedependent CDKs is required for the initiation of DNA
replication. This event is also thought to prevent assembly
of new pre-replication complexes. This hypothesis provides a potential mechanism for stringent control of
chromosomal duplication, which should occur only once
during each somatic cell cycle. Thus, checkpoint controls
at the onset of DNA synthesis serve to signal cellular
competency for S phase entry and maintenance of the
normal diploid genotype upon mitosis.
Once DNA synthesis has been initiated, replicative
activity is confined to specific locations within the nucleus,
referred to as DNA replication foci. DNA replication foci
represent subnuclear domains that are thought to be highly
enriched in multi-subunit complexes (‘DNA replication
factories’) containing enzymes involved in DNA synthesis,
including DNA polymerases a and d, PCNA and DNA
ligase. The concentration of these factors at DNA
replication foci that are associated with the nuclear matrix
provides a solid-phase framework for understanding
catalytic and regulatory components of DNA replication.
Coordinate activation of multiple DNA replicationdependent histone genes at the onset of S phase
The initiation of histone protein synthesis at the G1/S
phase transition is tightly coupled to the start and
progression of DNA synthesis (Figure 5). To prevent
disorganization of nuclear architecture and chromosomal
catastrophe during chromosome segregation at mitosis, it
is critical that newly replicated DNA is packaged
immediately into nucleosomes. Histones permit the precise
packaging of 2 m of DNA into chromatin within each cell
nucleus (diameter approximately 10 mm). This functional
and temporal coupling poses stringent constraints on
multiple parameters of histone gene expression, because
somatic cells do not have storage pools for histone protein
8
or histone mRNAs. The vast number of histone polypeptides that must be synthesized and the limited time of S
phase allotted for this process necessitates a high histone
protein synthesis rate. Mass production of each histone
subtype occurs at an average rate of several thousand
proteins per second throughout S phase. Moreover,
because each 0.2 kb of DNA is packaged by nucleosomal
octamers composed of histones H2A, H2B, H3 and H4, the
stoichiometric synthesis of each of the histone subtypes is
essential for efficient DNA packaging. Consequently,
histone gene regulatory factors integrate a series of cellsignalling pathways that monitor the onset of S phase and
coordinate the expression of 50–100 distinct histone gene
subtypes.
Our laboratory has shown that regulatory protein
interactions with histone gene promoter elements reveal
transcriptional control that is operative at the onset of S
phase. Similar to the R-point genes, the presence of SP1binding sites is critical for maximum activation of histone
genes (Figure 5). However, unlike the R-point genes, the
majority of histone genes does not contain E2F elements.
Rather, a sophisticated and E2F-independent transcriptional mechanism has evolved for coordinate activation of
histone genes.
As with E2F-responsive genes, E2F-independent transcriptional control mechanisms must account for G1/S
phase-dependent enhancement of transcription, as well as
attenuation of gene transcription at later stages of S phase
(Figure 5). The key cell cycle element for histone H4 genes is
a highly conserved promoter domain which encompasses
binding sites for IRF2, the homeodomain-related
‘CCAAT displacement protein’ CDP/cut, and the
TATA-binding complex TFIID. IRF2 is required for
maximal activation of histone gene transcription, and
appears to function at the G1/S phase boundary in a
manner analogous to ‘free’ E2F. Phosphorylation of IRF2
in vivo occurs primarily on serine residues. The CDP/cut
protein is associated with pRB, cyclin A and CDK1/
CDK1. CDP/cut in association with pRB, CDK1/CDK1
and cyclin A may perform a function very similar to that of
the multiplicity of higher order E2F complexes. These
CDP complexes bound to cyclins, CDKs and pRB-related
proteins attenuate the enhanced levels of histone gene
transcription during mid-S phase when physiological
demand for histone mRNAs begins to diminish.
Similar to the R-point genes, histone gene promoters
have auxiliary elements that support transcriptional
activation during the cell cycle. For example, histone H4
genes contain binding sites for YY1 and SP1. The
interaction of SP1 with site I modulates the efficiency of
H4 gene transcription by an order of magnitude. The
binding of YY1 to multiple sites in the histone H4
promoter may facilitate gene–nuclear matrix interactions.
In addition, it has recently been shown that YY1 associates
with the histone deacetylase rpd3. The possibility arises
that posttranslational modifications of histone proteins,
ENCYCLOPEDIA OF LIFE SCIENCES / & 2002 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net
Cell Cycle
when bound as nucleosomes to the H4 promoter, may
parallel the modifications in chromatin structure that
accompany modulations of histone H4 gene expression.
Stoichiometric synthesis of histone mRNAs and proteins requires coordinate control of histone gene expression at several gene regulatory levels. The association of
CDP/cut with PRB/cyclin A and CDK1/CDK1 as
components of multipartite regulatory complexes interacting with DNA replication-dependent histone genes
provides direct functional linkage between transcriptional
coordination of histone gene expression and cyclin/CDK
signalling mechanisms that mediate cell cycle progression.
Accommodation of unique cell cycle
regulatory requirements in specialized cells
and cancer
Consistent with the stringent requirement for fidelity of
DNA replication and DNA repair to execute proliferation,
stage-specific modifications in control of cell cycle regulatory factors have been observed to parallel physiological changes and perturbations in growth control. Some
striking examples of physiological changes are regulatory
mechanisms that support developmental transitions during early embryogenesis, when DNA replication and
mitotic division occur in rapid succession in the absence
of significant G1 or G2 periods. In contrast, proliferation in
somatic cells of the adult requires passage through a cell
cycle with G1, S, G2 and mitotic periods that are operative
and necessary. Often, a prolonged G1 period provides
support for long-term quiescence of cells and tissues while
retaining the competency to reinitiate proliferation for
tissue remodelling and renewal. Stem cells require complex
control of proliferation competency to modulate commitments for cell cycle progression, quiescence or differentiation. Here, responsiveness to cell growth and tissue-specific
regulatory factors must be stringently monitored.
The abrogated components of growth control in
transformed and tumour cells are associated with and
functionally linked to both the regulation and regulatory
activities of cyclin–CDK complexes. Characteristic altera-
tions have been associated with progressive stages of
neoplasia and specific tumours. Frequently, the hallmark
of tumour cells is coexpression of cell growth and tissuespecific genes rather than mutually exclusive expression in
normal diploid cells. Consequently, tumour cells are
providing us with valuable insights into rate-limiting
regulatory steps in cell cycle and cell growth control. In
addition, we are increasing our opportunity to therapeutically rectify proliferative disorders in a targeted manner.
Particularly challenging is the possibility for restoring
fidelity of regulatory mechanisms operative at cell cycle
checkpoints, when responses to apoptotic signals prevent
accumulation and phenotypic expression of mutations
associated with growth control perturbations.
Further Reading
Hartwell LH and Weinert TA (1989) Checkpoints: controls that ensure
the order of cell cycle events. Science 246: 629–634.
King RW, Deshaies RJ, Peters JM and Kirschner MW (1996) How
proteolysis drives the cell cycle. Science 274: 1652–1659.
MacLachlan TK, Sang N and Giordano A (1995) Cyclins, cyclindependent kinases and CDK inhibitors: implications in cell cycle
control and cancer. Critical Reviews in Eukaryotic Gene Expression 5:
127–156.
Nevins JR (1992) E2F: a link between the Rb tumor suppressor protein
and viral oncoproteins. Science 258: 424–429.
Nurse P (1994) Ordering S phase and M phase in the cell cycle. Cell 79:
547–550.
Pardee AB (1989) G1 events and regulation of cell proliferation. Science
246: 603–608.
Sherr CJ (1993) Mammalian G1 cyclins. Cell 73: 1059–1065.
Stein GS, Stein JL, van Wijnen AJ and Lian JB (1994) Histone gene
transcription: a model for responsiveness to an integrated series of
regulatory signals mediating cell cycle control and proliferation/
differentiation interrelationships. Journal of Cellular Biochemistry 54:
393–404.
Stein GS, Montecino M, van Wijnen AJ, Stein JL and Lian JB (2000)
Nuclear structure – gene expression interrelationships: implications
for aberrant gene expression in cancer. Cancer Research 60:
2067–2076.
Weinberg RA (1995) The retinoblastoma protein and cell cycle control.
Cell 81: 323–330.
ENCYCLOPEDIA OF LIFE SCIENCES / & 2002 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net
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