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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. ENCYCLOPEDIA OF LIFE SCIENCES / & 2002 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net 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. ENCYCLOPEDIA OF LIFE SCIENCES / & 2002 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net 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. ENCYCLOPEDIA OF LIFE SCIENCES / & 2002 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net 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 ENCYCLOPEDIA OF LIFE SCIENCES / & 2002 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net 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). ENCYCLOPEDIA OF LIFE SCIENCES / & 2002 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net 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 7 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 9