Table of contents

Transcriptional regulation of CDK inhibitors

Although insights have emerged regarding genes controlling the early stages of eye formation, little is known about lens-fiber differentiation and elongation. The expression pattern of Prox1, a prospero related homeobox gene, suggests it has a role in a variety of embryonic tissues, including lens. To analyse the requirement for Prox1 during mammalian development, the locus was inactivated in mice. Homozygous Prox1-null mice die at mid-gestation from multiple developmental defects; the specific effect on lens development is described. Prox1 inactivation causes abnormal cellular proliferation, downregulated expression of the cell-cycle inhibitors Cdkn1b (also known as p27KIP1) and Cdkn1c (also known as p57KIP2), misexpression of E-cadherin, and inappropriate apoptosis. Consequently, mutant lens cells fail to polarize and elongate properly, resulting in a hollow lens. These data provide evidence that the progression of terminal fiber differentiation and elongation is dependent on Prox1 activity during lens development (Wigle, 1999).

The cell cycle inhibitor p21/WAF1/Cip1 is expressed in many cell types and is regulated by p53-dependent and p53-independent mechanisms. p21 is an important regulator of hepatocyte cell cycle, differentiation, and liver development, but little is known about the regulation of its synthesis in hepatocytes. The p21 gene is shown to be constitutively expressed in human hepatoma HepG2 cells. Deletion analysis of the p21 promoter shows that it contains a distal (positions -2,300/-210) and a proximal (positions -124 to -61) region that act synergistically to achieve high levels of constitutive expression. The proximal region that consists of multiple Sp1 binding sites is essential for constitutive p21 promoter activity in hepatocytes. This region also mediates the transcriptional activation of the p21 promoter by members of the Smad family of proteins (See Drosophila Mad), which play important roles in the transduction of extracellular signals, such as transforming growth factor beta, activin, etc. Constitutive expression of p21 is severely reduced by a C-terminally truncated form of Smad4 that has been shown previously to block signaling through Smads. Smad3/4, and to a much lesser extent Smad2/4, causes high levels of transcriptional activation of the p21 promoter. Transactivation is compromised by N- or C-terminally truncated forms of Smad3. By using Gal4-Sp1 fusion proteins, it has been shown that Smad proteins can activate gene transcription via functional interactions with the ubiquitous factor Sp1. These data demonstrate that Smad proteins and Sp1 participate in the constitutive or inducible expression of the p21 gene in hepatic cells (Moustakas, 1998).

The p21WAF1/CIP1 gene, which encodes a cyclin-dependent kinase inhibitor, may be critical for tumor suppressor gene p53-induced cell cycle arrest. The p53 gene is known to regulate the G1 checkpoint, which can either induce G1 arrest or initiate apoptosis. To directly examine the role of p21WAF1/CIP1 in the control of p53 function, human p21WAF1/CIP1 gene was introduced into a p53-deficient human non-small cell lung cancer cell line (H1299) using a p21WAF1/CIP1-expressing adenoviral vector (AdCMVp21). Infection with AdCMVp21 results in high levels of p21WAF1/CIP1 expression and significantly suppresses the growth of H1299 cells through the G1 arrest of the cell cycle. In contrast, transient expression of the wild-type p53 gene by a recombinant adenoviral vector (AdCMVp53) in H1299 cells induces apoptotic cell death and results in a rapid loss of cell viability. Combined infection with AdCMVp21 and AdCMVp53 overcomes p21WAF1/CIP1-mediated cell cycle arrest at G1 and induces apoptosis, although viral-transduced p21WAF1/CIP1 expression level is unaffected. These observations suggest that p53 expression converts a p21WAF1/CIP1-mediated growth arrest into apoptosis. The result was repeated with two additional human colon adenocarcinoma cell lines with the different p53 status (mutant p53-expressing DLD-1 and wild-type p53-expressing LoVo), suggesting that this phemonenon is a general event among human cancer cells. Thus, the p53-mediated apoptotic pathway is dominant over the growth arrest pathway, indicating that p53 may be an essential upstream mediator of p21WAF1/CIP1 in the regulation of a cell process leading either to growth arrest or to apoptotic suicide (Kagawa, 1997).

Butyrate is a colonic luminal short chain fatty acid; it arrests cell growth and induces differentiation in various cell types. The effect of butyrate was examined on the expression of WAF1/Cip1, a potent inhibitor of cyclin-dependent kinases, and its relation to growth arrest in a p53-mutated human colon cancer cell line WiDr. Butyrate completely inhibits the growth of WiDr and causes G1-phase arrest. WAF1/Cip1 mRNA is rapidly induced within 3 h by treatment with butyrate, and drastic WAF1/Cip1 protein induction is detected. The butyrate-responsive elements are two Sp1 sites at -82 and -69 relative to the transcription start site. A TATA element at -46 and two overlapping consensus Sp1 sites at -60 and -55 are essential for the basal promoter activity of WAF1/Cip1. These findings suggest that butyrate arrests the growth of WiDr by activating the WAF1/Cip1 promoter through specific Sp1 sites in a p53-independent fashion (Nakano, 1997).

Terminal differentiation is associated with withdrawal from the cell cycle. The cyclin-dependent kinase inhibitor (CKI) p21Cip1 is transcriptionally regulated by p53 and can induce growth arrest. CKIs are therefore potential mediators of developmental control of cell proliferation. The expression pattern of mouse p21 correlates with terminal differentiation of multiple cell lineages, including skeletal muscle, cartilage, skin, and nasal epithelium. Although the muscle-specific transcription factor MyoD is sufficient to activate p21 expression in cultured cells, p21 is expressed in myogenic cells of mice lacking the genes encoding MyoD and myogenin, demonstrating that p21 expression does not require these transcription factors. The p21 protein may function during development as an inducible growth inhibitor that contributes to cell cycle exit and differentiation (Parker, 1995).

The mammalian UV response results in rapid and dramatic induction of c-jun. Induction of a protooncogene, normally involved in mitogenic responses, by a genotoxic agent that causes growth arrest seems paradoxical. An explanation for the role of c-Jun in the UV response of mouse fibroblasts is provided by this study. c-Jun is necessary for cell-cycle reentry of UV-irradiated cells, but does not participate in the response to ionizing radiation. Cells lacking c-Jun undergo prolonged cell-cycle arrest, but resist apoptosis, whereas cells that express c-Jun constitutively do not arrest and undergo apoptosis. This function of c-Jun is exerted through negative regulation of p53 association with the p21 promoter. Cells lacking c-Jun exhibit prolonged p21 induction, whereas constitutive c-Jun inhibits UV-mediated p21 induction (Shaulian, 2000).

Cut is a homeodomain transcription factor which has the unusual property of containing several DNA-binding domains: three regions (termed Cut repeats) and the Cut homeodomain. Genetic studies in Drosophila indicate that cut plays important roles in the determination and maintenance of cell-type specificity. Mammalian Cut proteins may yet play another biological role, specifically in proliferating cells. The binding of Cut to a consensus binding site is found to vary during the cell cycle. Binding is virtually undetectable in G0 and early G1, but becomes very strong as cells reach S phase. This results both from an increase in Cut expression and dephosphorylation of the Cut homeodomain by the Cdc25A phosphatase. Cdc25A has been found to play a role in the G1-S transition. It becomes active in late G1, activated by the cdk2-cyclin E complex and blocks the inhibitory effect of p21 (WAF1) on cyclin-cdk complexes. Microinjection of anti-Cdc25A antibodies in G1 cells is known to prevent progression into S phase, and Cdc25A expression is upregulated by c-Myc. Thus, Cdc25A plays an important role in the control of cell-cycle progression; however, apart from cdk2 and cdk4, its physiological targets have remained unknown. The increase in Cut activity coincides with a decrease in p21 mRNAs. In co-transfection experiments, Cut proteins repress p21 gene expression through binding to a sequence that overlaps the TATA box. Moreover, p21 expression is repressed equally well by either Cdc25A or Cut. Together, these results suggest a model by which Cdc25A activates the Cut repressor, which then downregulates transcription of p21 in S phase. Thus, in addition to their role during cellular differentiation, Cut proteins also serve as cell-cycle-dependent transcriptional factors in proliferating cells (Coqueret, 1998).

Cux-1, coding for a Cut family protein, is a murine homeobox gene that is highly expressed in the developing kidney with expression restricted to the nephrogenic zone. Cux-1 is highly expressed in cyst epithelium of polycystic kidneys from C57BL/6J-cpk/cpk mice, but not in kidneys isolated from age-matched phenotypically normal littermates. To further elucidate the role of Cux-1 in renal development, transgenic mice were generated expressing Cux-1 under the control of the CMV immediate early gene promoter. Mice constitutively expressing Cux-1 develop multiorgan hyperplasia and organomegaly, but not an overall increase in body size. Transgenic kidneys were enlarged 50% by 6 weeks of age, with the increased growth primarily restricted to the cortex. Proliferating cells were found in proximal and distal tubule epithelium throughout the cortex, and the squamous epithelium that normally lines Bowman's capsule was replaced with proximal tubule epithelium. However, the total number of nephrons was not increased. In the developing kidneys of transgenic mice, Cux-1 is ectopically expressed in more highly differentiated tubules and glomeruli, and this is associated with reduced expression of the cyclin kinase inhibitor, p27. Transient transfection experiments have revealed that Cux-1 is an inhibitor of p27 promoter activity. These results suggest that Cux-1 regulates cell proliferation during early nephrogenesis by inhibiting expression of p27 (Ledford, 2002).

The cell cycle inhibitor protein p21WAF1/Cip1 (p21) is a critical downstream effector in p53-dependent mechanisms of growth control and p53-independent pathways of terminal differentiation. The transforming growth factor-beta pathway-specific Smad3 and Smad4 proteins transactivate the human p21 promoter via a short proximal region, which contains multiple binding sites for the ubiquitous transcription factor Sp1. The Sp1-occupied promoter region mediates transactivation of the p21 promoter by c-Jun and the related proteins JunB, JunD, and ATF-2. Gel electrophoretic mobility shift assays show that this region does not contain a binding site for c-Jun. In accordance with the DNA binding data, c-Jun is unable to transactivate the p21 promoter when overexpressed in the Sp1-deficient Drosophila-derived SL2 cells. Coexpression of c-Jun and Sp1 in these cells results in a strong synergistic transactivation of this promoter. In addition, a chimeric promoter consisting of six tandem high affinity Sp1-binding sites fused with the CAT gene is transactivated by overexpressed c-Jun in HepG2 cells. The above data suggest functional cooperation between c-Jun and Sp1. Physical interactions between the two factors have been demonstrated in vitro by using GST-Sp1 hybrid proteins expressed in bacteria and in vitro transcribed-translated c-Jun. The region of c-Jun mediating interaction with Sp1 maps within the basic region leucine zipper domain. In vivo, functional interactions between c-Jun and Sp1 have been demonstrated using a GAL4-based transactivation assay. Overexpressed c-Jun transactivates only a chimeric promoter consisting of five tandem GAL4-binding sites when coexpressed with GAL4-Sp1-(83-778) fusion proteins in HepG2 cells. By utilizing the same assay, it was found that the glutamine-rich segment of the B domain of Sp1 (Bc, amino acids 424-542) is sufficient for c-Jun-induced transactivation of the p21 promoter. In conclusion, these data support a mechanism for superactivation of Sp1 by c-Jun that is based on physical and functional interactions between these two transcription factors on the human p21 and possibly other Sp1-dependent promoters (Kardassis, 1999).

XBF-1 (Drosophila homolog: Sloppy paired) is an anterior neural plate-specific, winged helix transcription factor that affects neural development in a concentration-dependent manner. A high concentration of XBF-1 results in suppression of endogenous neuronal differentiation and an expansion of undifferentiated neuroectoderm. The mechanism by which this expansion is achieved has been investigated. The findings suggest that XBF-1 converts ectoderm to a neural fate and it does so independently of any effects on the mesoderm. In addition, a high dose of XBF-1 promotes the proliferation of neuroectodermal cells while a low dose inhibits ectodermal proliferation. Thus, the neural expansion observed after high dose XBF-1 misexpression is due both to an increase in the number of ectodermal cells devoted to a neural fate and an increase in their proliferation. The effect on cell proliferation is likely to be mediated by p27XIC1, a cyclin-dependent kinase (cdk) inhibitor. p27XIC1 is expressed in a spatially restricted pattern in the embryo, including the anterior neural plate, and when misexpressed it is sufficient to block the cell cycle in vivo. p27XIC1 is transcriptionally regulated by XBF-1 in a dose-dependent manner such that it is suppressed or ectopically induced by a high or low dose of XBF-1, respectively. However, while a low dose of XBF-1 induces ectopic p27XIC1 and ectopic neurons, misexpression of p27XIC1 does not induce ectopic neurons, suggesting that the effects of XBF-1 on cell fate and cell proliferation are distinct. p27XIC1 is suppressed by XBF-1 in the absence of protein synthesis, suggesting that at least one component of p27XIC1 regulation by XBF-1 may be direct. Thus, XBF-1 is a neural-specific transcription factor that can independently affect both the cell fate choice and the proliferative status of the cells in which it is expressed (Hardcastle, 2000).

The enamel knot, a transient epithelial structure, appears at the onset of mammalian tooth shape development. Until now, the morphological, cellular and molecular events leading to the formation and disappearance of the enamel knot have not been described. The cessation of cell proliferation in the enamel knot in mouse molar teeth is linked with the expression of the cyclin-dependent kinase inhibitor p21. p21 expression is induced by bone morphogenetic protein 4 (BMP-4) in isolated dental epithelia. Since BMP-4 (Drosophila homolog: Dpp) is expressed only in the underlying dental mesenchyme at the onset of the enamel knot formation, these results support the role of the cyclin-dependent kinase inhibitors as inducible cell differentiation factors in epithelial-mesenchymal interactions. The expression of p21 in the enamel knot is followed by BMP-4 expression, and subsequently by apoptosis of the differentiated enamel knot cells. Three-dimensional reconstructions of serial sections after in situ hybridization and Tunel-staining indicate an exact codistribution of BMP-4 transcripts and apoptotic cells. Apoptosis is stimulated by BMP-4 in isolated dental epithelia, but only in one third of the explants. It is concluded that BMP-4 may be involved both in the induction of the epithelial enamel knot, as a mesenchymal inducer of epithelial cyclin-dependent kinase inhibitors, and later in the termination of the enamel knot signaling functions by participating in the regulation of programmed cell death. These results show that the life history of the enamel knot is intimately linked to the initiation of tooth shape development and support the role of the enamel knot as an embryonic signaling center (Jernvall, 1998).

Ras proteins play a key role in integrating mitogenic signals with cell cycle progression through G1. Ras is required for cell cycle progression and activation of both Cdk2 and Cdk4 until approximately 2 h before the G1/S transition, corresponding to the restriction point. Analysis of Cdk-cyclin complexes indicates that Ras signaling is required both for induction of cyclin D1 and for downregulation of the Cdk inhibitor p27KIP1. Constitutive expression of cyclin D1 circumvents the requirement for Ras signaling in cell proliferation, indicating that regulation of cyclin D1 is a critical target of the Ras signaling cascade (Aktas, 1997).

It is well documented that Ras functions as a molecular switch for reentry into the cell cycle at the border between G0 and G1 by transducing extracellular growth stimuli into early G1 mitogenic signals. The role of Ras was investigated during the late stage of the G1 phase by using NIH 3T3 (M17) fibroblasts in which the expression of a dominant negative Ras mutant [Ha-Ras(Asn17)] was induced in response to dexamethasone treatment. Delaying the expression of Ras(Asn17) until late in the G1 phase by introducing dexamethasone 3 h after the addition of epidermal growth factor (EGF) abolishes the downregulation of the p27kip1 cyclin-dependent kinase (CDK) inhibitor that normally occurs during this period, with resultant suppression of cyclin Ds/CDK4 and cyclin E/CDK2 and G1 arrest. The immunodepletion of p27kip1 completely eliminates the CDK inhibitor activity from EGF-stimulated, dexamethasone-treated cell lysate. The failure of p27kip1 downregulation and G1 arrest is also observed in cells in which Ras(Asn17) is induced after growth stimulation with either a phorbol ester or alpha-thrombin and is mimicked by the addition of inhibitors for phosphatidylinositol-3-kinase late in the G1 phase. Ras-mediated downregulation of p27kip1 involves both the suppression of synthesis and the stimulation of the degradation of the protein. Unlike the earlier expression of Ras(Asn17) at the border between G0 and G1, its delayed expression does not compromise the EGF-stimulated transient activation of extracellular signal-regulated kinases or inhibit the stimulated expression of a principal D-type cyclin, cyclin D1, until close to the border between G1 and S. It is concluded that Ras plays temporally distinct, phase-specific roles throughout the G1 phase and that Ras function late in G1 is required for p27kip1 downregulation and passage through the restriction point, a prerequisite for entry into the S phase (Takuwa, 1997).

During development, neuronal differentiation is closely coupled with cessation of proliferation. Studying nerve growth factor (NGF)-induced differentiation of PC12 pheochromocytoma cells, a novel signal transduction pathway was found that blocks cell proliferation. Treatment of PC12 cells with NGF leads to induction of nitric oxide synthase (See Drosophila NOS) . The resulting nitric oxide (NO) acts as a second messenger, activating the p21(WAF1) promoter and inducing expression of p21(WAF1) cyclin-dependent kinase inhibitor. NO activates the p21(WAF1) promoter by p53-dependent and p53-independent mechanisms. Blocking production of NO with an inhibitor of NOS reduces accumulation of p53, activation of the p21(WAF1) promoter, expression of neuronal markers, and neurite extension. To determine whether p21(WAF1) is required for neurite extension, PC12 line was prepared with an inducible p21(WAF1) expression vector. Blocking NOS with an inhibitor decreases neurite extension, but induction of p21(WAF1) with isopropyl-1-thio-beta-D-galactopyranoside restores this response. Levels of p21(WAF1) induced by isopropyl-1-thio-beta-D-galactopyranoside were similar to those induced by NGF. Therefore, a signal transduction pathway has been identified that is activated by NGF; proceeds through NOS, p53, and p21(WAF1) to block cell proliferation; and is required for neuronal differentiation by PC12 cells (Poluha, 1997).

Telomere loss has been proposed as a mechanism for counting cell divisions during aging in normal somatic cells. It is not known how such a mitotic clock initiates the intracellular signalling events that culminate in G1 cell cycle arrest and senescence to restrict the lifespan of normal human cells. In aging cells, critically short telomere length activates a DNA damage response pathway involving p53 and p21(WAF1). The DNA binding and transcriptional activity of p53 protein increases with cell age, in the absence of any marked increase in the level of p53 protein; p21(WAF1) promoter activity in senescent cells is dependent on both p53 and the transcriptional co-activator p300. Increased specific activity of p53 protein is detected in AT fibroblasts, which exhibit accelerated telomere loss and undergo premature senescence, as compared with normal fibroblasts. Poly(ADP-ribose) polymerase is involved in the post-translational activation of p53 protein in aging cells. p53 protein can associate with PARP; inhibition of PARP activity leads to abrogation of p21 and mdm2 expression in response to DNA damage. Inhibition of PARP activity leads to extension of cellular lifespan. In contrast, hyperoxia, an activator of PARP, is associated with accelerated telomere loss, activation of p53 and premature senescence. It is proposed that p53 is post-translationally activated, not only in response to DNA damage but also in response to the critical shortening of telomeres that occurs during cellular aging (Vaziri, 1997).

The helix-loop-helix transcription factor E2A plays several important developmental roles: not only does it promote cellular differentiation, but it also suppresses cell growth. Id proteins, inhibitors of E2A, have the opposite effects on cell differentiation and growth. To understand the mechanisms by which E2A suppresses cell growth, the role of E2A was examined in regulating the expression of the cyclin-dependent kinase inhibitor p21CIP1/WAF1/SD11, which when overexpressed, prevents cell cycle progression. Overexpression of E2A can transcriptionally activate the p21 gene. Out of the eight putative E2A-binding sequences (E1 to E8) in the promoter, the E1 to E3 sequences located close to the transcription start site have been found to be essential. Loss of the E boxes in the promoter also reduces p21 expression without cotransfection with E2A in HIT pancreatic cells, where the endogenous E2A-like activity is high. Overexpression of E2A in 293T cells activates expression of the endogenous p21 gene at both the mRNA and protein levels. In correlation with the finding that E47 overexpression leads to growth arrest in NIH 3T3 cells, it has been shown that Id1 (Drosophila homolog: Extramachrochaetae) overexpression T3 cells accelerates cell growth and inhibits p21 expression. Taken together, these results provide insight into the mechanisms by which E2A and Id proteins control cell growth (Prabhu, 1997).

The proliferation of lymphocytes in response to cytokine stimulation is essential for a variety of immune responses. Studies of Stat6-deficient mice have demonstrated that this protein is required for the normal proliferation of lymphocytes in response to interleukin-4 (IL-4). The impaired IL-4-induced proliferative response of Stat6-deficient lymphocytes is not due to an inability to activate alternate signaling pathways, such as those involving insulin receptor substrates, or to a failure to upregulate IL-4 receptor levels. Cell cycle analysis shows that the percentage of Stat6-deficient lymphocytes that transit from the G1 to the S phase of the cell cycle following IL-4 stimulation is lower than that of control lymphocytes. Although the regulation of many genes involved in the control of cytokine-induced proliferation is normal in Stat6-deficient lymphocytes, protein levels of the cdk inhibitor p27Kip1 are markedly dysregulated. p27Kip1 is expressed at significantly higher levels in Stat6-deficient lymphocytes than in control cells following IL-4 stimulation. The higher level of p27Kip1 expression seen in IL-4-stimulated Stat6-deficient lymphocytes correlates with decreased cdk2-associated kinase activity and is the result of the increased accumulation of protein rather than altered mRNA expression. Similarly, higher levels of p27Kip1 protein expression are also seen following IL-12 stimulation of Stat4-deficient lymphocytes than are seen following stimulation of control cells. These data suggest that Stat proteins may control the cytokine-induced proliferative response of activated T cells by regulating the expression of cell cycle inhibitors so that cyclin-cdk complexes may function to promote transition from the G1 to the S phase of the cell cycle (Kaplan, 1998).

Cyclin-dependent kinase inhibitor p21(Waf1/Cip1/Sdi1) has been suggested to be involved in the antiproliferative effect of nitric oxide (NO) in vascular smooth muscle cells (VSMCs). To elucidate the mechanism underlying NO-induced p21 expression, the roles of tumor suppressor p53 and the guanylate cyclase-cGMP pathway were investigated. The induction of p21 by the NO donor S-nitroso-N-acetylpenicillamine (SNAP) seems to be due to p21 transactivation because SNAP elevates the activity of the p21 promoter but does not stabilize p21 mRNA and protein. Because SNAP does not stimulate the deletion mutant of the p21 promoter that lacks p53 binding sites, the involvement of p53 was investigated. The expression level of p53 is down-regulated after mitogenic stimulation, whereas it is sustained in the presence of SNAP. SNAP markedly stimulates DNA binding activity of p53. Furthermore, SNAP fails to induce p21 in VSMCs of p53-knock out mice and in A431 cells that contain mutated p53. The antiproliferative effect of SNAP also is attenuated in these cells. NO stimulates guanylate cyclase and its product cGMP has been shown to inhibit VSMC proliferation. However a guanylate cyclase inhibitor does not prevent SNAP-induced p21 expression. 8-bromo-cGMP, 3-isobutyl-1-methylxanthine, and their combination does not induce p21. Although 8-bromo-cGMP has a small antiproliferative effect, the elevation of cGMP concentration induced by SNAP is little throughout the G(1) phase. The antiproliferative effect of SNAP is not attenuated by an inhibitor of cGMP-dependent protein kinase. These results suggest that NO induces p21 through a p53-dependent but cGMP-independent pathway (Ishida, 1999).

Nitric oxide (NO) regulates the expression of p21(Waf1/Cip1) in several cell types. The present study examined the role of both the extracellular signal-regulated kinase (ERK) and p70 S6 kinase [p70(S6k): see Drosophila RPS6-p70-protein kinase] in the NO-induced increase in p21 expression that occurs in adventitial fibroblasts during the cell cycle. Both ERK and p70(S6k) are phosphorylated in response to the NO donor S-nitroso-N-acetylpenicillamine (SNAP) and the activation is rapid, transient, and precedes increased p21 expression under defined conditions where serum is present. Addition of a selective inhibitor of ERK phosphorylation (PD98059) prevents the subsequent phosphorylation of p70(S6k) and the increase in p21 protein. Both cGMP and cAMP activate both ERK and p70(S6k), whereas only selective inhibitors of protein kinase G prevent the activation of the kinases by SNAP. A complex between ERK and p70(S6k) was documented by immunoprecipitation procedures. Rapamycin blocks p70(S6k) phosphorylation induced by NO and also inhibits p53 phosphorylation and p21 expression whereas PD98059 only prevents the NO-induced increase in p21 protein without influencing either p53 activation or p21 mRNA expression. The studies show a unique relationship between NO, ERK, and p70(S6k) and also provide evidence for a novel role of p70(S6k) in the activation of p53 (Gu, 2000).

The myeolomonocytic cell line U937 differentiates into macrophages in response to a variety of agents. Several genes including the cyclin-dependent kinase inhibitor p21waf1/cip1 and the homeobox gene transcription factor HOXA10 are induced at the onset of differentiation. Ectopic expression of either gene results in U937 differentiation. In this paper, a mechanism is described by which p21 and HOXA10 may act in concert, where HOXA10 can bind directly to the p21 promoter and, together with its trimeric partners PBX1 and MEIS1, activate p21 transcription, resulting in cell cycle arrest and differentiation. These experiments for the first time identify p21 as a selective target for a HOX protein and link the differentiative properties of a transcription factor and a cell cycle inhibitor. Interestingly, various studies indicate that both p21 and HOXA10 have opposing effects on myeloid cells at different stages. In the earliest progenitors, p21 slows growth in order to maintain the stem cell pool, whereas a proliferative effect has been described for more mature myeloid progenitors. In U937 cells, p21 expression leads to G1 arrest and differentiation. HOXA10 expression is also associated with cell growth and some leukemias and has an antidifferentiative effect in early progenitors, whereas a differentiative and anticycling effect has been demonstrated in the intermediate-staged U937 cells. It is tempting to conclude that the various roles of p21 at different stages of myeloid differentiation account for the apparent paradoxical effects of HOXA10. It is becoming clear, however, that HOX proteins have their own mechanism for generating specificity, by making use of heterodimers and trimers and varying composite cis-acting elements for function. How HOXA10 exerts its various cellular effects will only be understood when its other stage- and tissue-specific target genes are identified (Bromleigh, 2000).

Calcium functions as a trigger for the switch between epithelial cell growth and differentiation. The calcium/calmodulin-dependent phosphatase calcineurin is involved in this process. Treatment of primary mouse keratinocytes with cyclosporin A, an inhibitor of calcineurin activity, suppresses the expression of terminal differentiation markers and of p21WAF1/Cip1 and p27KIP1, two cyclin-dependent kinase inhibitors that are usually induced with differentiation. In parallel with down-modulation of the endogenous genes, suppression of calcineurin function blocks induction of the promoters for the p21WAF1/Cip1 and loricrin differentiation marker genes, whereas activity of these promoters is enhanced by calcineurin overexpression. The calcineurin-responsive region of the p21 promoter maps to a 78-bp Sp1/Sp3-binding sequence next to the TATA box, and calcineurin induces activity of the p21 promoter through Sp1/Sp3-dependent transcription. The endogenous NFAT-1 and -2 transcription factors, major downstream targets of calcineurin, associate with Sp1 in keratinocytes in a calcineurin-dependent manner, and calcineurin up-regulates Sp1/Sp3-dependent transcription and p21 promoter activity in synergism with NFAT1/2. Thus, this study reveals an important role for calcineurin in control of keratinocyte differentiation and p21 expression, and points to a so-far-unsuspected interconnection among this phosphatase, NFATs, and Sp1/Sp3-dependent transcription (Santini, 2001).

The differentiation, survival, and proliferation of developing sympathetic neuroblasts are all coordinately promoted by neurotrophins. Bone morphogenetic protein 4 (BMP4), a factor known to be necessary for the differentiation of sympathetic neurons, conversely reduces both survival and proliferation of cultured E14 sympathetic neuroblasts. The anti-proliferative effects of BMP4 occur more rapidly than the pro-apoptotic actions and appear to involve different intracellular mechanisms. BMP4 treatment induces expression of the transcription factor Msx-2 and the cyclin-dependent kinase inhibitor p21CIP1/WAF1 (p21). Treatment of cells with oligonucleotides antisense to either of these genes prevents cell death after BMP4 treatment but does not significantly alter the anti-proliferative effects. Thus Msx-2 and p21 are necessary for BMP4-mediated cell death but not for promotion of exit from cell cycle. Although treatment of cultured E14 sympathetic neuroblasts with neurotrophins alone does not alter cell numbers, BMP4-induced cell death was prevented by co-treatment with either neurotrophin-3 (NT-3) or nerve growth factor (NGF). This suggests that BMP4 may also induce dependence of the cells on neurotrophins for survival. Thus, sympathetic neuron numbers may be determined in part by factors that inhibit the proliferation and survival of neuroblasts and make them dependent upon exogenous factors for survival (Gomes, 2001).

Histone deacetylases (HDACs) modulate chromatin structure and transcription, but little is known about their function in mammalian development. Previously, HDACs have been shown to be required for embryonic development of invertebrates. In addition, loss of specific components of the Sin3 and the NuRD complexes such as RbAp46/48 (lin-53, rba-1), Sin3 (dSin3A: see Drosophila Sin3A), Mi-2 (dMi-2, chd-3, chd-4) and MTA1/MTA2 (egl-27, egr1) affect embryonic viability and development of Drosophila melanogaster and Caenorhabditis elegans. Mammalian HDAC1 (Drosophila homolog: Rpd3) has been implicated in the repression of genes required for cell proliferation and differentiation. Targeted disruption of both HDAC1 alleles results in embryonic lethality before E10.5 due to severe proliferation defects and retardation in development. HDAC1-deficient embryonic stem cells show reduced proliferation rates, which correlate with decreased cyclin-associated kinase activities and elevated levels of the cyclin-dependent kinase inhibitors p21WAF1/CIP1 and p27KIP1. Similarly, expression of p21 and p27 is up-regulated in HDAC1-null embryos. In addition, loss of HDAC1 leads to significantly reduced overall deacetylase activity, hyperacetylation of a subset of histones H3 and H4 and concomitant changes in other histone modifications. The expression of HDAC2 and HDAC3 is induced in HDAC1-deficient cells, but cannot compensate for loss of the enzyme, suggesting a unique function for HDAC1. This study provides the first evidence that a histone deacetylase is essential for unrestricted cell proliferation by repressing the expression of selective cell cycle inhibitors (Lagger, 2002).

HDAC1-deficient embryos and HDAC1-null cells show proliferation defects. Together with previous data showing high expression levels of HDAC1 in proliferating cells, these results are suggestive of a proliferation-linked function for the enzyme. Paradoxically, the recruitment of class I HDACs by Rb seems to be important for the repression of proliferation-associated genes, and HDAC1 should therefore act rather as a growth inhibitor. However, the data shown in this report demonstrate that one of the key functions of HDAC1 is to prevent the expression of antiproliferative genes in cycling cells. These findings indicate that deacetylases other than HDAC1 as well as deacetylase-independent mechanisms ensure the proper regulation of Rb target genes in HDAC1-null cells. Evidence has been presented that HDAC1 controls the expression of a specific subset of CDK inhibitors. The induction of p21 and p27 in HDAC1-null cells correlates with the hyperacetylation of the corresponding promoters. The specificity of this response is underlined by the fact that only the proximal but not the distal portion of the p21 promoter was found to be associated with hyperacetylated histone H3. The proximal p21 promoter contains Sp1-binding sites that are required for the induction of the p21 gene by deacetylase inhibitors. Activation of tumor suppressors has been shown to be a crucial function of HDAC inhibitors as anti-cancer drugs in human cells. These results strongly support the idea that HDAC1 might be a relevant target in tumor treatment (Lagger, 2002).

The Myc oncoprotein represses initiator-dependent transcription through the POZ domain transcription factor Miz-1. Transactivation by Miz-1 is negatively regulated by association with topoisomerase II binding protein (TopBP1); UV irradiation downregulates expression of TopBP1 and releases Miz-1. Miz-1 binds to the p21Cip1 core promoter in vivo and is required for upregulation of p21Cip1 upon UV irradiation. Using both c-myc-/- cells and a point mutant of Myc that is deficient in Miz-1 dependent repression, it has been shown that Myc negatively regulates transcription of p21Cip1 upon UV irradiation and facilitates recovery from UV-induced cell cycle arrest through binding to Miz-1. These data implicate Miz-1 in a pathway that regulates cell proliferation in response to UV irradiation (Herold, 2002).

The transactivation of TCF target genes induced by Wnt pathway mutations constitutes the primary transforming event in colorectal cancer (CRC). Disruption of ß-catenin/TCF-4 activity in CRC cells induces a rapid G1 arrest and blocks a genetic program that is physiologically active in the proliferative compartment of colon crypts. Coincidently, an intestinal differentiation program is induced. The TCF-4 target gene c-MYC plays a central role in this switch by direct repression of the p21CIP1/WAF1 promoter. Following disruption of ß-catenin/TCF-4 activity, the decreased expression of c-MYC releases p21CIP1/WAF1 transcription, which in turn mediates G1 arrest and differentiation. Thus, the ß-catenin/TCF-4 complex constitutes the master switch that controls proliferation versus differentiation in healthy and malignant intestinal epithelial cells (van de Wetering, 2002).

c-MYC plays a central role in the proliferative capacity of many cancers, including CRC. tHE data imply that c-MYC blocks the expression of the cell cycle inhibitor p21CIP1/WAF1. The region responsible for p21CIP1/WAF1 regulation has been mapped to a 200 bp fragment of the proximal promoter. The presence of MIZ-1 and c-MYC on this promoter suggests that c-MYC-mediated repression of p21CIP1/WAF1 occurs by a mechanism resembling c-MYC control of p15INK4b, i.e., through preventing promoter activation by the transcription factor MIZ-1. Decreased expression of c-MYC would allow MIZ-1 to activate p21CIP1/WAF1 transcription. The complementarity in the expression of c-MYC and p21CIP1/WAF1 in the intestine supports this mechanism (van de Wetering, 2002).

Activation of the tumor suppressor p53 by DNA damage induces either cell cycle arrest or apoptotic cell death. The cytostatic effect of p53 is mediated by transcriptional activation of the cyclin-dependent kinase (CDK) inhibitor p21Cip1, whereas the apoptotic effect is mediated by transcriptional activation of mediators including PUMA and PIG3. What determines the choice between cytostasis and apoptosis is not clear. The transcription factor Myc is shown to be a principal determinant of this choice. Myc is directly recruited to the p21Cip1 promoter by the DNA-binding protein Miz-1. This interaction blocks p21Cip1 induction by p53 and other activators. As a result Myc switches, from cytostatic to apoptotic, the p53-dependent response of colon cancer cells to DNA damage. Myc does not modify the ability of p53 to bind to the p21Cip1 or PUMA promoters, but selectively inhibits bound p53 from activating p21Cip1 transcription. By inhibiting p21Cip1 expression Myc favors the initiation of apoptosis, thereby influencing the outcome of a p53 response in favor of cell death (Seoane, 2002).

Several conclusions can be drawn from these results. Myc selectively targets p21Cip1 in the p53 transcriptional program, sparing the ability of p53 to induce the expression of PUMA or PIG3. Myc does not alter the ability of p53 to bind to the p21Cip1 promoter but inhibits p21Cip1 transcriptional activation by promoter-bound p53. In the presence of p21, p53 can still bind to the PUMA promoter and induce the accumulation of its product, but apoptosis is not achieved. Thus, the p21-dependent block in apoptosis maps to a step downstream of the DNA damage-p53-PUMA pathway. The mechanism for this provocative observation is not obvious. These results suggest a model in which Myc selectively prevents p53-dependent transcriptional activation of p21Cip1, enabling pro-apoptotic factors such as PUMA to execute a cell death program. Thus, these results define, in mechanistic terms, how one element of the cellular context, that is, the level of Myc activity, can determine the outcome of the p53 response. Although it remains to be seen whether repression of p21Cip1 would be beneficial in cancer treatment, the mechanism proposed here suggests ways to influence the cell's response to stresses that result in activation of p53 (Seoane, 2002).

Myc and E2f1 promote cell cycle progression, but overexpression of either can trigger p53-dependent apoptosis. Mice expressing an Eμ-Myc transgene in B lymphocytes develop lymphomas, the majority of which sustain mutations of either Arf (a tumor suppressor whose product inhibits Mdm2, thereby stabilizing p53) or p53. Eμ-Myc transgenic mice lacking one or both E2f1 alleles exhibit a slower onset of lymphoma development associated with increased expression of the cyclin-dependent kinase inhibitor p27Kip1 and a reduced S phase fraction in precancerous B cells. In contrast, Myc-induced apoptosis and the frequency of Arf and p53 mutations in lymphomas were unaffected by E2f1 loss. Therefore, Myc does not require E2f1 to induce Arf, p53, or apoptosis in B cells, but depends upon E2f1 to accelerate cell cycle progression and downregulate p27Kip1 (Baudino, 2003).

The Forkhead transcription factors AFX, FKHR and FKHR-L1 (Drosophila homolog: Foxo) are orthologs of DAF-16, a Forkhead factor that regulates longevity in Caenorhabditis elegans. Overexpression of these Forkhead transcription factors causes growth suppression in a variety of cell lines, including a Ras-transformed cell line and a cell line lacking the tumor suppressor PTEN. Expression of AFX blocks cell-cycle progression at phase G1, independent of functional retinoblastoma protein (pRb) but dependent on the cell-cycle inhibitor p27kip1. Indeed, AFX transcriptionally activates p27kip1, resulting in increased protein levels. It is concluded that AFX-like proteins are involved in cell-cycle regulation and that inactivation of these proteins is an important step in oncogenic transformation (Medema, 2000).

FoxO Forkhead transcription factors have been shown to act as signal transducers at the confluence of Smad, PI3K, and FoxG1 pathways. Smad proteins activated by TGF-ß form a complex with FoxO proteins to turn on the growth inhibitory gene p21Cip1. This process is negatively controlled by the PI3K pathway, a known inhibitor of FoxO localization in the nucleus, and by the telencephalic development factor FoxG1, which binds to FoxO-Smad complexes and blocks p21Cip1 expression. It is suggested that the activity of this network confers resistance to TGF-ß-mediated cytostasis during the development of the telencephalic neuroepithelium and in glioblastoma brain tumor cells (Seoane, 2004).

The insulin-like growth factor I (IGF-I) stimulates muscle satellite cell proliferation. IGF-I-stimulated proliferation of primary satellite cells is associated with the activation of phosphatidylinositol 3'-kinase (PI3K)/Akt and the downregulation of a cell-cycle inhibitor p27Kip1. To understand mechanisms by which IGF-I signals the downregulation of p27Kip1 in rat skeletal satellite cells, the role of Forkhead transcription factor FoxO1 in transcriptional activity of p27Kip1 was examined. When primary rat satellite cells are transfected with a p27Kip1 promoter-reporter gene construct, IGF-I inhibits specific p27Kip1 promoter activity. Addition of LY294002, an inhibitor of PI3K, reverses the IGF-I-mediated downregulation of p27Kip1 promoter activity. Co-transfection of wild type (WT) FoxO1 into satellite cells increases p27Kip1 promoter activity in the absence of IGF-I supplementation. Addition of IGF-I reverses the induction of p27Kip1 promoter activity by WT FoxO1. When a mutated FoxO1 (without Thr24, Ser256, and Ser316 Akt phosphorylation sites) is used, IGF-I is no longer able to reverse the FoxO1 induced stimulation of p27Kip1 promoter activity that is seen when WT FoxO1 is present. When the satellite cells are treated with IGF-I, phosphorylation of Akt-Ser473 and FoxO1-Ser256 is increased. In addition, when the cells are pre-incubated with LY294002 before IGF-I stimulation, the phosphorylation of Akt-Ser473 and FoxO1-Ser256 is inhibited, implying that phosphorylation of Akt and FoxO1 is downstream of IGF-I-induced PI3K signaling. However, IGF-I does not induce phosphorylation of FoxO1 on residues Thr24 and Ser316. These results suggest that IGF-I induces the phosphorylation of Ser256 and inactivates FoxO1, thereby downregulating the activation of the p27Kip1 promoter. Thus, inactivation of FoxO1 by IGF-I plays a critical role in rat skeletal satellite cell proliferation through regulation of p27Kip1 expression (Machida, 2003).

Cytochrome P450-derived epoxyeicosatrienoic acids (EETs) stimulate endothelial cell proliferation and angiogenesis. The involvement of the FOXO family of transcription factors and their downstream target p27Kip1 has been investigated in EET-induced endothelial cell proliferation. Incubation of human umbilical vein endothelial cells with 11,12-EET induces a time- and dose-dependent decrease in p27Kip1 protein expression, whereas p21Cip1 is not significantly affected. This effect on p27Kip1 protein is associated with decreased mRNA levels as well as p27Kip1 promoter activity. 11,12-EET also stimulates the time-dependent phosphorylation of Akt and of the forkhead factors FOXO1 and FOXO3a, effects prevented by the phosphatidylinositol 3-kinase inhibitor LY 294002. Transfection of endothelial cells with either a dominant-negative or an 'Akt-resistant'/constitutively active FOXO3a mutant reverses the 11,12-EET-induced down-regulation of p27Kip1, whereas transfection of a constitutive active Akt decreases p27Kip1 expression independent of the presence or absence of 11,12-EET. To determine whether these effects are involved in EET-induced proliferation, endothelial cells were transfected with the 11,12-EET-generating epoxygenase CYP2C9. Transfection of CYP2C9 elicits endothelial cell proliferation and this effect is inhibited in cells co-transfected with CYP2C9 and either a dominant-negative Akt or constitutively active FOXO3a. However, reducing FOXO expression using RNA interference attenuates p27Kip1 expression and stimulates endothelial cell proliferation. These results indicate that EET-induced endothelial cell proliferation is associated with the phosphatidylinositol 3-kinase/Akt-dependent phosphorylation and inactivation of FOXO factors and the subsequent decrease in expression of the cyclin-dependent kinase inhibitor p27Kip1 (Potente, 2003).

The tumor suppressor protein p53 regulates transcriptional programs that control the response to cellular stress. Distinct mechanisms exist to activate p53 target genes as revealed by marked differences in affinities and damage-specific recruitment of transcription initiation components. p53 functions in a temporal manner to regulate promoter activity both before and after stress. Before DNA damage, basal levels of p53 are required to assemble a poised RNA polymerase II initiation complex on the p21 promoter. RNA pol II is converted into an elongating form shortly after stress but before p53 stabilization. Proapoptotic promoters, such as Fas/APO1, have low levels of bound RNA pol II but undergo damage-induced activation through efficient reinitiation. Surprisingly, in a p53-dependent process key basal factors TAFII250 and TFIIB assemble into the transcription machinery in a stress- and promoter-specific manner, behaving as differential cofactors for p53 action after distinct types of DNA damage (Espinosa, 2003).

In hypoxic cells, HIF-1alpha escapes from oxygen-dependent proteolysis and binds to the hypoxia-responsive element (HRE) for transcriptional activation of target genes involved in angiogenesis and glycolysis. The G1 checkpoint gene p21(cip1)is activated by HIF-1alpha with a novel mechanism that involves the HIF-1alpha PAS domains to displace Myc binding from p21(cip1) promoter. This HIF-1alpha-Myc pathway may account for up- and down-regulation of other hypoxia-responsive genes that lack the HRE. Moreover, the role of HIF-1alpha in cell cycle control indicates a dual, yet seemingly conflicting, nature of HIF-1alpha: promoting cell growth and arrest in concomitance. It is speculated that a dynamic balance between the two processes is achieved by a 'stop-and-go' strategy to maintain cell growth and survival. Tumor cells may adopt such a scheme to evade the killing by chemotherapeutic agents (Koshiji, 2004).

Mammalian organogenesis requires the expansion of pluripotent precursor cells before the subsequent determination of specific cell types, but the tissue-specific molecular mechanisms that regulate the initial expansion of primordial cells remain poorly defined. It has been genetically established that Six6 homeodomain factor, acting as a strong tissue-specific repressor, regulates early progenitor cell proliferation during mammalian retinogenesis and pituitary development. Six6, in association with Dach corepressors, regulates proliferation by directly repressing cyclin-dependent kinase inhibitors, including the p27Kip1 promoter. These data reveal a molecular mechanism by which a tissue-specific transcriptional repressor-corepressor complex can provide an organ-specific strategy for physiological expansion of precursor populations (Li, 2002).

p27Kip1 restrains cell proliferation by binding to and inhibiting cyclin-dependent kinases. To investigate the mechanisms of p27 translational regulation, a complete p27 cDNA was isolated and an internal ribosomal entry site (IRES) was identified, located in the p27 5'UTR. The IRES allows for efficient p27 translation under conditions where cap-dependent translation is reduced. Searching for possible regulators of IRES activity the neuronal ELAV protein HuD was identified as a specific binding factor of the p27 5'UTR. Increased expression of HuD or the ubiquitously expressed HuR protein specifically inhibits p27 translation and p27 IRES activity. Consistent with an inhibitory role of Hu proteins in p27 translation, siRNA mediated knockdown of HuR induces endogenous p27 protein levels as well as IRES-mediated reporter translation and leads to cell cycle arrest in G1 (Kullmann, 2002).

What is the physiologic role of the IRES? Analysis of the effect of PI3 kinase inhibition on p27 translation suggests that p27 translation can be mediated in large part via its IRES element. It is speculated that the IRES serves to sustain p27 synthesis under unfavorable growth conditions, when overall cap-dependent protein synthesis is reduced and persisting p27 expression is required, for example, in the case of some viral infections. In addition, maintenance of translation under stress conditions or during quiescence may relay on IRES elements. For example, VEGF is thought to be translated by internal ribosomal entry under hypoxia, when overall protein synthesis is compromised. Interestingly hypoxia has also been shown to induce p27 protein levels. Efficient translation of the Cdk inhibitor under these conditions may therefore depend on its IRES element (Kullmann, 2002).

Cyclin E, in conjunction with its catalytic partner cdk2, is rate limiting for entry into the S phase of the cell cycle. Cancer cells frequently contain mutations within the cyclin D-Retinoblastoma protein pathway that lead to inappropriate cyclin E-cdk2 activation. Although deregulated cyclin E-cdk2 activity is believed to directly contribute to the neoplastic progression of these cancers, the mechanism of cyclin E-induced neoplasia is unknown. The consequences of deregulated cyclin E expression have been studied in primary cells; cyclin E was found to initiate a p53-dependent response that prevents excess cdk2 activity by inducing expression of the p21Cip1 cdk inhibitor. The increased p53 activity is not associated with increased expression of the p14ARF tumor suppressor. Instead, cyclin E leads to increased p53 serine15 phosphorylation that is sensitive to inhibitors of the ATM/ATR family. When either p53 or p21cip1 is rendered nonfunctional, then the excess cyclin E becomes catalytically active and causes defects in S phase progression, increased ploidy, and genetic instability. It is concluded that p53 and p21 form an inducible barrier that protects cells against the deleterious consequences of cyclin E-cdk2 deregulation. A response that restrains cyclin E deregulation is likely to be a general protective mechanism against neoplastic transformation. Loss of this response may thus be required before deregulated cyclin E can become fully oncogenic in cancer cells. Furthermore, the combination of excess cyclin E and p53 loss may be particularly genotoxic, because cells cannot appropriately respond to the cell cycle anomalies caused by excess cyclin E-cdk2 activity (Minella, 2002).

How might deregulated cyclin E cause S phase abnormalities that activate an S phase checkpoint? In yeast, S phase-promoting cyclins inhibit the transition of replication origins to the prereplicative state. Furthermore, when early-firing origins are inhibited by hydroxyurea, then the stalled early origins inhibit late origins through a checkpoint that requires the Mec1 protein (the budding yeast ATM/ATR homolog. Similarly, inhibition of ATR function in a human cell line by a kinase-inactive ATR mutant renders these cells hypersensitive to treatments that prolong DNA synthesis, and cyclin E overexpression is synthetically lethal with ATR inhibition. Thus, perhaps cyclin E deregulation leads to aberrant licensing of replication origins, and the resultant S phase progression defect may be sensed by a protein such as ATR, which then enforces an S phase checkpoint. Furthermore, the stalled replication origins associated with this prolonged S phase may be fragile and constitute the precursors to genetic instability. Another mechanism through which enforced cyclin E expression might impair normal cell cycle progression is by cyclin A-cdk2 inhibition, since cyclin A-cdk2 activity (and cyclin A expression) drops substantially in cells with ectopic cyclin E expression. However, cyclin E-induced cell cycle anomalies persist in E6-expressing cells with high levels of cyclin A-cdk2 kinase activity, so cyclin A-cdk2 activity cannot be the principle cause of the cyclin E-associated S phase phenotype (Minella, 2002).

CCAAT displacement protein/cut homolog (CDP/cut) is a highly conserved homeodomain protein that contains three cut repeat sequences. CDP/cut interacts with a histone lysine methyltransferase (HKMT), G9a, in vivo and in vitro. The deletion of the cut repeats within CDP/cut abrogates the interaction with G9a. The transcriptional repressor function of CDP/cut is mediated through HKMT activity of G9a associated with CDP/cut. The recruitment of G9a to the human p21(waf1/cdi1) promoter is contingent on the interaction with CDP/cut, and CDP/cut is directly associated with an increase in the methylation in vivo of Lys-9 in histone H3 within the CDP/cut-regulatory region of the p21(waf1/cdi1) promoter. The endogenous level of p21(waf1/cdi1) expression is repressed through CDP/cut and mediated by HKMT activity of G9a. This study identifies G9a as a component of CDP/cut complex. G9a colocalizes with CDP/cut in the nucleus. These results indicate that G9a functions as a transcriptional corepressor in association with a CDP/cut complex. These studies now reveal the interaction of G9a with a sequence-specific transcription factor that regulates gene repression through CDP/cut (Nishio, 2004).

The Myc transcription factor is an essential mediator of cell growth and proliferation through its ability to both positively and negatively regulate transcription. The mechanisms by which Myc silences gene expression are not well understood. The current model is that Myc represses transcription through functional interference with transcriptional activators. Myc is shown to bind the corepressor DNA CpG methyltransferase Dnmt3a and associate with DNA methyltransferase activity in vivo. In cells with reduced Dnmt3a levels, specific reactivation of the Myc-repressed p21Cip1 gene is seen, whereas the expression of Myc-activated E-boxes genes is unchanged. In addition, it was found that Myc can target Dnmt3a selectively to the promoter of p21Cip1. Myc is known to be recruited to the p21Cip1 promoter by the DNA-binding factor Miz-1. Consistent with this, Myc and Dnmt3a form a ternary complex with Miz-1 and this complex can corepress the p21Cip1 promoter. Finally, it is shown that DNA methylation is required for Myc-mediated repression of p21Cip1. These data identify a new mechanism by which Myc can silence gene expression not only by passive functional interference but also by active recruitment of corepressor proteins. Furthermore, these findings suggest that targeting of DNA methyltransferases by transcription factors is a wide and general mechanism for the generation of specific DNA methylation patterns within a cell (Brenner, 2005).

The Notch and Calcineurin/NFAT pathways have both been implicated in control of keratinocyte differentiation. Induction of the p21WAF1/Cip1 gene by Notch 1 activation in differentiating keratinocytes is associated with direct targeting of the RBP-Jκ protein to the p21 promoter. Notch 1 activation functions also through a second Calcineurin-dependent mechanism acting on the p21 TATA box-proximal region. Increased Calcineurin/NFAT activity by Notch signaling involves downregulation of Calcipressin, an endogenous Calcineurin inhibitor, through a HES-1-dependent mechanism. Besides control of the p21 gene, Calcineurin contributes significantly to the transcriptional response of keratinocytes to Notch 1 activation, both in vitro and in vivo. In fact, deletion of the Calcineurin B1 gene in the skin results in a cyclic alopecia phenotype, associated with altered expression of Notch-responsive genes involved in hair follicle structure and/or adhesion to the surrounding mesenchyme. Thus, an important interconnection exists between Notch 1 and Calcineurin-NFAT pathways in keratinocyte growth/differentiation control (Mammucari, 2005).

Levels of extra- and intra-cellular calcium play a major role in keratinocyte growth/differentiation control, and the calcium/Calmodulin-dependent phosphatase Calcineurin has been implicated in this process. Calcineurin is the only known serine/threonine phosphatase under calcium/calmodulin control. Among the proteins that are dephosphorylated as a consequence of Calcineurin activation are the nuclear factors of activated T cells (NFATs). Increased Calcineurin activity promotes the localization of NFATs to the nucleus, and its effect is counteracted by the phosphorylation of these factors by a number of both constitutive and inducible kinases such as GSK3, CK1, p38, and JNK1. Such a complexity of regulation is reflected by the fact that induction of NFAT-dependent transcription by Calcineurin activation is not immediately associated with increases in intracellular calcium levels, but requires a prolonged stimulus consistent with an oscillatory and accumulative mechanism of NFAT dephosphorylation and nuclear translocation (Mammucari, 2005).

Studies on the biological function of Calcineurin have been greatly facilitated by the use of the inhibitory drugs Cyclosporin A (CsA) and FK506. Several endogenous Calcineurin inhibitors have also been reported. Among these is Calcipressin (CALP1), also known as the DSCR1 gene product, located in the Down Syndrome Critical Region of human chromosome 21 and mouse chromosome 16. This protein binds directly to the CnA subunit and inhibits its activity. Importantly, Calcipressin gene expression is under direct positive control of Calcineurin/NFAT activity, so that this protein is thought to function as a feedback inhibitor of Calcineurin signaling, with an impact on T cell activation as well as the response to different stress stimuli in cardiac hypertrophy (Mammucari, 2005).

The function of Calcineurin has been elucidated in great detail in T cells, but has also been studied in the hematopoietic, neuronal, myogenic, and vascular systems. Calcineurin/NFAT activity has also been directly implicated in keratinocyte growth/differentiation control and, in vivo, in control of the hair cycle. Molecular analysis of the role of this pathway in keratinocytes has focused on control of p21 gene transcription. Induction of p21(WAF1/Cip1) is one of the earliest regulatory events associated with keratinocyte differentiation, contributing to withdrawal from the cell cycle. In mouse primary keratinocytes, p21 expression is induced by increased extracellular calcium, and the responsive region of the p21 promoter maps to a 78 bp GC-rich region close to the TATA box, containing six Sp1/Sp3 binding sites. Calcineurin induces activation of this promoter through the Calcineurin-dependent association of NFAT with the transcription factors Sp1/Sp3 (Mammucari, 2005).

Notch 1 activation induces p21 transcription not only through direct binding of the RBP-Jκ protein to the p21 promoter, but also through the calcium/Calcineurin-responsive TATA box-proximal region. Underlying this effect, induction of Calcineurin/NFAT activity by Notch signaling involves downregulation of Calcipressin, in opposition to positive control of this gene by Calcineurin/NFAT itself. Besides control of p21 expression, Calcineurin signaling plays a significantly broader role in the transcriptional response of keratinocytes to Notch 1 activation. In particular, inducible deletion of the CnB1 gene in the skin causes a cyclic alopecia phenotype that is linked to altered expression of several Notch-responsive genes involved in hair follicle structure and adhesion to the surrounding mesenchyme (Mammucari, 2005).

Development is typically studied as a continuous process under laboratory conditions, but wild animals often develop in variable and stressful environments. C. elegans larvae hatch in a developmentally arrested state (L1 arrest) and initiate post-embryonic development only in the presence of food (E. coli in lab). In contrast to the well-studied dauer arrest, L1 arrest occurs without morphological modification, although larvae in L1 arrest are more resistant to environmental stress than developing larvae. Consistent with its role in dauer formation and aging, insulin/insulin-like growth factor (IGF) signaling is shown to regulate L1 arrest. daf-2 insulin/IGF receptor mutants have a constitutive-L1-arrest phenotype when fed and extended survival of L1 arrest when starved. Conversely, daf-16/FOXO mutants (see Drosophila Foxo) have a defective-arrest phenotype, failing to arrest development and dying rapidly when starved. DAF-16 is required for transcription of the cyclin-dependent kinase inhibitor cki-1 in stem cells in response to starvation, accounting for the failure of daf-16/FOXO mutants to arrest cell division during L1 arrest. Other developmental events such as cell migration, cell fusion, and expression of the microRNA lin-4, a temporal regulator of post-embryonic development, are also observed in starved daf-16/FOXO mutants. These results suggest that DAF-16/FOXO promotes developmental arrest via transcriptional regulation of numerous target genes that control various aspects of development (Baugh, 2006).

Smad3, a transforming growth factor β/activin signaling effector, is expressed in discrete progenitor domains along the dorsoventral axis of the developing chick spinal cord. Restriction of Smad3 expression to the dP6-p2 and p3 domains together with exclusion from the motoneuron progenitor domain, are the result of the activity of key transcription factors responsible for patterning the neural tube. Smad3-mediated TGFβ activity promotes cell-cycle exit and neurogenesis by inhibiting the expression of Id proteins, and activating the expression of neurogenic factors and the cyclin-dependent-kinase-inhibitor p27kip1. Furthermore, Smad3 activity induces differentiation of selected neuronal subtypes at the expense of other subtypes. Within the intermediate and ventral domains, Smad3 promotes differentiation of ventral interneurons at the expense of motoneuron generation. Consequently, the absence of Smad3 expression from the motoneuron progenitor domain during pattern formation of the neural tube is a prerequisite for the correct generation of spinal motoneurons (Garcia-Campmany, 2007).

Recent studies have identified stem cells in brain cancer. However, their relationship to normal CNS progenitors, including dependence on common lineage-restricted pathways, is unclear. Expression of the CNS-restricted transcription factor, OLIG2, has been observed in human glioma stem and progenitor cells reminiscent of type C transit-amplifying cells in germinal zones of the adult brain. Olig2 function is required for proliferation of neural progenitors and for glioma formation in a genetically relevant murine model. Moreover, this study shows that p21WAF1/CIP1, a tumor suppressor and inhibitor of stem cell proliferation, is directly repressed by OLIG2 in neural progenitors and gliomas. These findings identify an Olig2-regulated lineage-restricted pathway critical for proliferation of normal and tumorigenic CNS stem cells (Ligon, 2007).

Both normal and tumorigenic progenitor cells share an Olig2 requirement for growth. At what developmental time point might such effects be relevant? Loss of Olig2 function in the embryonic forebrain ganglionic eminence does not result in global changes to ventricular zone proliferation or cell death. Rather, one observes a failure to develop oligodendrocytes, suggesting primarily an early role in cell fate specification. However, sustained expression of Olig2 at adult stages, in a subset of type B stem cells and transit-amplifying (type C) progenitors suggests additional roles for this transcription factor in late-stage proliferating progenitor cells found in the adult SVZ. It is further speculated that Olig2 could also serve cell cycle regulatory roles in slowly replicating NG2 progenitor cells that are abundant in the adult cerebral cortex (Ligon, 2007).

To better understand the mechanisms critical for growth of glioma, focus was placed on the proliferating subset of cells in human glioblastoma multiforme (GBM). Approximately 10%-15% of cells in these tumors are actively dividing, and of these, 85% expressed OLIG2. Both OLIG1 and OLIG2 are expressed in human glioma. However, the current findings indicate that Olig2 function in particular is necessary for tumorigenesis in a robust murine model of primary glioma that incorporates activation of EGFR and mutation of the Ink4a/Arf locus. Interestingly, similar findings are obtained when Olig2 is conditionally deleted from an independent glioma model based on mutations of the Nf1 and p53 loci. Thus, the requirement for Olig2 function may apply to glioma formation in general (Ligon, 2007).

Olig2 fulfills criteria of a lineage-restricted competence factor for brain cancer, and in this respect it bears striking analogy to the bHLH-LZ transcription factor, MITF, in melanoma. First, Olig2 function is crucial for the development of neural progenitors and progeny cells specifically in the CNS. Second, its expression is deregulated in brain cancer. Finally, its function is required for tumor formation. However, the notion of Olig2 as a critical lineage-restricted competence factor for brain cancer does not depend on functions as an oncogene per se. Although OLIG2 ectopic expression in the T-cell lineage is associated with T-cell leukemia, gain-of-function studies in mice suggest it is unlikely to be sufficient for brain cancer formation (Ligon, 2007).

The data also argue against critical functions for Olig2 in specification of stem cells. Olig1/2-null neurospheres are capable of self-renewal and differentiation into two of the three principal neural cell types. Expression of stem cell marker genes is either unaffected in Olig2 null neurospheres compared to controls or actually upregulated in the Olig2 null cells. Olig2 is expressed in type B cells of the SVZ and CD133-positive cells in human glioma, and so it might regulate the transition from quiescent stem cell to transit-amplifying progenitor cell in the context of normal development and tumorigenesis, respectively (Ligon, 2007).

The p21 cell cycle inhibitor gene is notable for its important role in maintaining the relatively quiescent state of stem cells for both blood and brain. In addition, p53 regulation of neural stem cell growth is also mediated in part through its effects on p21. Targeted disruption of p21 enhances the proliferation rate of neural stem cells in the adult mammalian forebrain. The current data show that the p21 locus is subject to direct Olig2 transcriptional repression, thus providing a direct link between Olig2 and the cell cycle regulatory apparatus in normal and gliomagenic progenitors. Collectively, these observations raise the interesting possibility that OLIG2 function might be dispensable for tumor formation in a p21 null context. However, an alternative, more complex scenario is suggested by a computational screen, which reveals three additional p53-inducible genes that may serve as direct genetic targets for Olig2. The p21 gene product does function as a tumor suppressor in knockout mice but its activity as such is rather weak. Moreover, p21 loss-of-function mutations are infrequent in human cancers. Laboratory and clinical data suggest that loss of p53 gene function upstream of p21 is a far more effective route to cancer. Thus, OLIG2 might suppress multiple p53 targets and/or other non-p53 target genes in its role as a 'gateway' gene for brain cancer (Ligon, 2007).

Interestingly, primary glioblastomas are highly resistant to radiation and cytotoxic drugs despite the fact that p53 is generally intact within these tumors. Although there are multiple routes to suppression of p53 function, the data suggest a novel, OLIG2-dependent avenue to p53 pathway antagonism which is potentially active even at early stages of gliomagenesis. One might imagine that increased OLIG2 and decreased p21 expression would predict more aggressive tumor and shorter survival times. However, the literature on p21 and glioma survival is ambivalent. Some studies show that p21 expression is higher in low-grade than in high-grade astrocytomas and that the presence of p21 is associated with prolonged survival. Other studies indicate that higher p21 staining is associated strongly with higher histological grade, a higher rate of proliferation, and worse survival. Still other studies report that p21 levels have no impact at all on survival in high-grade gliomas. There are many pitfalls to interpreting the impact of p21 (or any gene, for that matter) on survival in gliomas. However, one obvious problem is that previous studies have monitored p21 expression in the whole tumor. It is conceivable that important prognostic insights have been hitherto obscured for this reason. It is possible that useful prognostic insights could be derived from an analysis of p21 expression in CD133/OLIG2-positive glioma cells (Ligon, 2007).

Neurogenesis requires the coordination of neural progenitor proliferation and differentiation with cell-cycle regulation. However, the mechanisms coordinating these distinct cellular activities are poorly understood. This study demonstrates that a Cut-like homeodomain transcription factor family member, Cux2 (Cutl2), regulates cell-cycle progression and development of neural progenitors. Cux2 loss-of-function mouse mutants exhibit smaller spinal cords with deficits in neural progenitor development as well as in neuroblast and interneuron differentiation. These defects correlate with reduced cell-cycle progression of neural progenitors coupled with diminished NeuroD and p27Kip1 activity. Conversely, in Cux2 gain-of-function transgenic mice, the spinal cord is enlarged in association with enhanced neuroblast formation and neuronal differentiation, particularly with respect to interneurons. Furthermore, Cux2 overexpression induces high levels of NeuroD and p27Kip1. Mechanistically, it was discovered through chromatin immunoprecipitation assays that Cux2 binds both the NeuroD and p27Kip1 promoters in vivo, indicating that these interactions are direct. These results therefore show that Cux2 functions at multiple levels during spinal cord neurogenesis. Cux2 initially influences cell-cycle progression in neural progenitors but subsequently makes additional inputs through NeuroD and p27Kip1 to regulate neuroblast formation, cell-cycle exit and cell-fate determination. Thus this work defines novel roles for Cux2 as a transcription factor that integrates cell-cycle progression with neural progenitor development during spinal cord neurogenesis (Iulianella, 2008).

Histone deacetylases (HDACs) regulate gene expression by deacetylating histones and also modulate the acetylation of a number of nonhistone proteins, thus impinging on various cellular processes. This study analyzed the major class I enzymes HDAC1 and HDAC2 in primary mouse fibroblasts and in the B-cell lineage. Fibroblasts lacking both enzymes fail to proliferate in culture and exhibit a strong cell cycle block in the G1 phase that is associated with up-regulation of the CDK inhibitors p21(WAF1/CIP1) and p57(Kip2) and of the corresponding mRNAs. This regulation is direct, as in wild-type cells HDAC1 and HDAC2 are bound to the promoter regions of the p21 and p57 genes. Furthermore, analysis of the transcriptome and of histone modifications in mutant cells demonstrated that HDAC1 and HDAC2 have only partly overlapping roles. Next, HDAC1 and HDAC2 were eliminated in the B cells of conditionally targeted mice. It was found that B-cell development strictly requires the presence of at least one of these enzymes: When both enzymes are ablated, B-cell development is blocked at an early stage, and the rare remaining pre-B cells show a block in G1 accompanied by the induction of apoptosis. In contrast, elimination of HDAC1 and HDAC2 in mature resting B cells has no negative impact, unless these cells are induced to proliferate. These results indicate that HDAC1 and HDAC2, by normally repressing the expression of p21 and p57, regulate the G1-to-S-phase transition of the cell cycle (Yamaguchi, 2010).

p53 target promoters are structurally diverse and display pronounced differences in RNA polymerase II (RNAP II) occupancy even in unstressed cells, with higher levels observed on cell cycle arrest genes (p21) compared with apoptotic genes (Fas/APO1). This occupancy correlates well with their ability to undergo rapid or delayed stress induction. To understand the basis for such distinct temporal assembly of transcription complexes, the role of core promoter structures in this process was examined. It was found that the p21 core promoter directs rapid, TATA box-dependent assembly of RNAP II preinitiation complexes (PICs), but permits few rounds of RNAP II reinitiation. In contrast, PIC formation at the Fas/APO1 core promoter is very inefficient but supports multiple rounds of transcription. A downstream element was defined within the Fas/APO1 core promoter that is essential for its activation, and nuclear transcription factor Y (NF-Y) was identified as the downstream element binding partner. NF-Y acts as a bifunctional transcription factor that regulates basal expression of Fas/APO1 in vivo. Thus, two critical parameters of the stress-induced p53 transcriptional response are the kinetics of gene induction and duration of expression through frequent reinitiation. These features are intrinsic, DNA-encoded properties of diverse core promoters that may be fundamental to anticipatory programming of p53 response genes upon stress (Morachis, 2010).

In unstressed cells, certain p53 target promoters, like p21, are 'preloaded' with paused RNAP II, whereas proapoptotic promoters, among others, have negligible RNAP II association. Such striking variation in levels of promoter-bound RNAP II may have direct bearing on the differential activation kinetics observed after stress induction of p53-responsive genes. The existence of such regulatory mechanisms acting before DNA damage to establish a default programmatic transcriptional response to stress is very intriguing. These data suggest that the intrinsic properties of diverse p53 core promoters play a key role in regulating RNAP II affinity and dynamics to coordinate appropriate responses to different stress conditions. An unexpected level was found of transcriptional regulation governing RNAP II dynamics that is encoded within the DNA sequence of diverse core promoters that drives expression of p53-responsive genes. The TATA box within the p21 promoter has a critical role in recruiting the transcriptional machinery by promoting rapid formation of a functional PIC that is poised for initiation. However, the p21 core promoter is intrinsically inefficient for reinitiation, which may be enhanced by signal-dependent components acting at other levels of regulation to facilitate PIC reformation and prolonged RNA synthesis. In contrast to p21, the Fas/APO1 promoter does not contain a TATA box or other well-characterized core motifs, and the rate of PIC formation is very slow. A Fas downstream element that binds to NF-Y is essential for core promoter activity in vitro, and may nucleate PIC assembly by direct interaction with the general transcription machinery. Surprisingly, once transcription is engaged, the Fas/APO1 promoter is capable of efficient RNAP II reinitiation events. Published reports have demonstrated that initiation and reinitiation can be experimentally uncoupled and, in one example, reinitiation is faster than initiation, which was also observe with Fas/APO1 (Morachis, 2010).

Thus, two critical parameters of p53-dependent gene activation -- the kinetics of induction and duration of expression through frequent reinitiation -- are intrinsic, DNA-encoded features of diverse core promoters that may be fundamental to anticipatory programming of p53 response genes. The default mode, as seen at the p21 promoter, is to rapidly form a PIC but undergo few rounds of reinitiation, whereas that of Fas/APO1 is the opposite. Of course, sustained p21 expression requiring multiple rounds of RNAP II reinitiation and reduced Fas/APO1 expression by infrequent reinitiation can be achieved by overriding the default programming through sophisticated epigenetic processes that are tailored to specific stress environments. Considering the advantage of preserving flexibility to fine-tune cell fate decisions, having a default, genetic program embedded in core promoter DNA would safeguard against misregulation, particularly of apoptotic genes. This may reflect an evolutionary need to balance cell growth while limiting the ability to self-destruct. It is difficult to envision a cellular system that would evolve to activate cell cycle arrest and apoptotic genes identically. If this were true, apoptosis would likely override the cell cycle arrest program without allowing the cell to recover from stress or DNA damage. Further investigation into the default mechanisms used by structurally diverse p53 target genes may provide insight into how the programmatic response to stress is regulated and how it can be manipulated for targeted therapies (Morachis, 2010).

The decision of a neural precursor to stop dividing and begin its terminal differentiation at the correct place, and at the right time, is a crucial step in the generation of cell diversity in the nervous system. This study shows that the Down's syndrome candidate gene (Mnb/Dyrk1a) is transiently expressed in prospective neurons of vertebrate CNS neuroepithelia. The gain of function (GoF) of Mnb/Dyrk1a induces proliferation arrest. Conversely, its loss of function (LoF) caused over proliferation and cell death. It was found that MNB/DYRK1A is both necessary and sufficient to upregulate, at transcriptional level, the expression of the cyclin-dependent kinase inhibitor p27KIP1 in the embryonic chick spinal cord and mouse telencephalon, supporting a regulatory role for MNB/DYRK1A in cell cycle exit of vertebrate CNS neurons. All these actions required the kinase activity of MNB/DYRK1A. It was also observed that MNB/DYRK1A is co-expressed with the Notch ligand Delta1 in single neuronal precursors. Furthermore, MNB/DYRK1A was found to suppress Notch signaling, counteracting the pro-proliferative action of the Notch intracellular domain (NICD), stimulatiing Delta1 expression, and is required for the neuronal differentiation induced by the decrease in Notch signaling. Nevertheless, although Mnb/Dyrk1a GoF leads to extensive withdrawal of neuronal precursors from the cell cycle, it is insufficient to elicit their differentiation. Remarkably, a transient (ON/OFF) Mnb/Dyrk1a GoF efficiently induces neuronal differentiation. It is proposed that the transient expression of MNB/DYRK1A in neuronal precursors acts as a binary switch, coupling the end of proliferation and the initiation of neuronal differentiation by upregulating p27KIP1 expression and suppressing Notch signaling (Hämmerle, 2011).

A role for Chk1 in blocking transcriptional elongation of p21 RNA

When cells are arrested in S phase, a subset of p53 target genes fails to be strongly induced despite the presence of high levels of p53. When DNA replication is inhibited, reduced p21 mRNA accumulation is correlated with a marked reduction in transcription elongation. This study shows that ablation of the protein kinase Chk1 rescues the p21 transcription elongation defect when cells are blocked in S phase, as measured by increases in both p21 mRNA levels and the presence of the elongating form of RNA polymerase II (RNAPII) toward the 3' end of the p21 gene. Recruitment of specific elongation and 3' processing factors (DSIF, CstF-64, and CPSF-100) is also restored. While additional components of the RNAPII transcriptional machinery, such as TFIIB and CDK7, are recruited more extensively to the p21 locus after DNA damage than after replication stress, their recruitment is not enhanced by ablation of Chk1. Significantly, ablating Chk2, a kinase closely related in substrate specificity to Chk1, does not rescue p21 mRNA levels during S-phase arrest. Thus, Chk1 has a direct and selective role in the elongation block to p21 observed during S-phase arrest. These findings demonstrate for the first time a link between the replication checkpoint mediated by ATR/Chk1 and the transcription elongation/3' processing machinery (Beckerman, 2009).

This study establishes a previously unknown link between the polyadenylation machinery and a DNA damage effector kinase. What is perhaps most striking about the results is that several proteins (DSIF, CPSF-100, and CstF-64) show no difference in recruitment to the TATA region of the p21 promoter after HU, but show a progressive loss in occupancy as the p21 gene is traversed by RNAPII. This is most pronounced at or downstream from the poly(A) site. DSIF, CPSF-100, and CstF-64 have all been shown to associate with RNAPII along the gene, and treatment of cells with DRB, which inhibits RNAPII phosphorylation at Ser2, decreases the association of CstF and CPSF subunits with distal regions of p21. Therefore, it cannot be ruled out, at this point, that the decreased recruitment of these processing factors at the 3' end of p21, and their subsequent rescue by Chk1 inhibition, does not simply reflect the decrease in Ser2-phosphorylated RNAPII under these conditions. However, a more interesting hypothesis is that these polyadenylation factors are regulated by Chk1 in such a way as to affect the efficiency of transcription elongation. Several reports have shown that polyadenylation factors impact transcription, especially elongation and termination, and several SR proteins have been shown to stimulate transcription elongation. Intriguingly, preliminary data indicate that Chk1 is recruited to the TATA region of p21 after HU treatment. This provides further support for a direct role for Chk1 in regulating transcription elongation at this locus. Future studies will investigated whether Chk1 directly phosphorylates CstF and CPSF subunits and if so, whether this affects transcription elongation (Beckerman, 2009).

p21 transcription is regulated by differential localization of histone H2A.Z

In yeast cells, H2A.Z regulates transcription and is globally associated within a few nucleosomes of the initiator regions of numerous promoters. H2A.Z is deposited at these loci by an ATP-dependent complex, Swr1.com. H2A.Z suppresses the p53 --> p21 transcription and senescence responses. Upon DNA damage, H2A.Z is first evicted from the p21 promoter, followed by the recruitment of the Tip60 histone acetyltransferase to activate p21 transcription. p400, a human Swr1 homolog, is required for the localization of H2A.Z, and largely colocalizes with H2A.Z at multiple promoters investigated. Notably, the presence of sequence-specific transcription factors, such as p53 and Myc, provides positioning cues that direct the location of H2A.Z-containing nucleosomes within these promoters. Collectively, this study strongly suggests that certain sequence-specific transcription factors regulate transcription, in part, by preferentially positioning histone variant H2A.Z within chromatin. This H2A.Z-centered process is part of an epigenetic process for modulating gene expression (Gévry, 2007).

Eukaryotic DNA is condensed many fold (e.g., 10,000) into chromatin, the basic unit of which contains 146 base pairs (bp) of DNA and an octamer of histone proteins (H2A, H2B, H3, and H4). Due to the high level of compaction, chromatin typically represses certain cellular DNA transactions, including transcription. For successful transcription, it is argued that nucleosomes need to be remodeled or evicted from promoter regions for the transcriptional machinery to be efficiently recruited to a target gene (Gévry, 2007).

The incorporation of histone variants into specific nucleosomes within a promoter region constitutes a mechanism by which promoter region chromatin can become more permissive to transcription initiation and elongation following receipt of a proper physiological cue. One such histone variant is H2A.Z. In Saccharomyces cerevisiae, it can elicit positive effects on gene expression. In addition, H2A.Z regulates genes that are proximal to telomeres and acts as a 'buffer' to antagonize the spread of heterochromatin into euchromatic regions (Meneghini, 2003). Furthermore, recent reports (Guillemette, 2005; Li, 2005; Raisner, 2005; Zhang, 2005) have shown that H2A.Z is preferentially localized within a few nucleosomes of the initiator regions of multiple promoters in the yeast genome. Interestingly, these H2A.Z-rich loci are largely devoid of transcriptional activity, which suggests that the variant histone prepares genes for activation (Guillemette, 2005) and/or operates as a transcriptional repressor. Finally, yeast H2A.Z has been shown to regulate nucleosome positioning, which provides mechanistic insight into how its presence can alter promoter transcriptional state (Gévry, 2007).

An ATP-dependent chromatin remodeling complex that specifically loads H2A.Z onto chromatin and exchanges it with H2A exists in yeast (Krogan, 2003; Kobor, 2004; Mizuguchi, 2004). This complex, in which the catalytic subunit is Swr1, also shares essential subunits with the NuA4 histone acetyltransferase complex (Krogan, 2003; Kobor, 2004). In addition to their importance in gene regulation, the Swr1 complex, H2A.Z, and NuA4 are all involved in the regulation of yeast chromosome stability (Krogan, 2004). This is noteworthy because, in mammalian cells, depletion of H2A.Z causes major nuclear and chromosomal abnormalities (Rangasamy, 2004) as witnessed by a high incidence of lagging chromosomes and chromatin bridges (Gevry, 2007).

There are two homologs of Swr1 in human cells: p400/Domino (referred to as p400), and SRCAP. There are also three uncharacterized p400-type SWI2-SNF2 molecules, including hIno80. Members of this family of SWI2/SNF2 chromatin remodeling enzymes each contain a spacer region inserted into the SWI2/SNF2 homology region (Gevry, 2007).

p400 was originally isolated as an E1A-associated protein, and it was also shown to interact with p53, Myc, and SV40 large T antigen. It is also required for E1A to induce p53-mediated apoptosis. SRCAP has been isolated as a CREB-binding protein. While one report shows that both p400 and SRCAP constitute part of the same complex, a recent study shows that SRCAP and p400 exist in distinct complexes with H2A.Z (Jin, 2005; Ruhl, 2006). Recently an SRCAP-containing complex was purified, and it was shown to have the ability to exchange H2A-H2B for H2A.Z-H2B in reconstituted mononucleosomes (Ruhl, 2006). It remains to be determined whether mammalian homolog(s) of Swr1, such as p400 and SRCAP, also catalyze H2A.Z deposition in vivo (Gevry, 2007).

Depletion of p400 elevates p21 synthesis to initiate premature senescence in primary human fibroblasts (Chan, 2005). Senescence has been observed in tissue culture cells as a stable form of cell growth arrest provoked by diverse stresses. Recently, oncogene-induced senescence was shown to occur in various precancerous lesions both in humans and mice, further suggesting that senescence acts as a defense mechanism against malignant cell development. Importantly, the action of p400 at p21 depends on the function of p53, a key regulator of p21 transcription (Gevry, 2007).

Given the possibility of a link between p400 and H2A.Z, it was asked whether H2A.Z is also an important regulator of p21 expression. The results of this effort show that H2A.Z depletion induces p21 expression in a p53-dependent fashion, as well as the premature senescence of primary diploid fibroblasts. Similar to senescence induced by p400 depletion, inactivating p53 or p21 blocked the emergence of certain senescent phenotypes following H2A.Z depletion. In a normal setting, H2A.Z is highly enriched at discrete p53-binding sites that lie within the p21 promoter. This distinctive localization pattern depends on the presence of p53, and was detected at other p53 target gene promoters as well. The presence of p400 is required to localize H2A.Z at those loci, and purified recombinant p400 from insect cells can carry out in vitro exchange of H2A.Z-H2B dimers into chromatin. H2A.Z and p400 localization at the p53-binding sites in p21 is severely diminished following p21 induction, and this process is not dependent on active p21 transcription per se. After H2A.Z and p400 eviction from the p53-binding sites in p21, it was observed that the Tip60 histone acetyltransferase isrecruited to the distal p53-binding site in the promoter to positively regulate p21 expression. Finally, overexpression of Myc, a known suppressor of p21 synthesis, significantly increases H2A.Z localization at the Myc-binding site in the TATA initiator region of the p21 promoter. This observation is consistent with the view that Myc represses p21 expression by preferentially recruiting H2A.Z-containing nucleosome(s) to this element (Gevry, 2007).

Post-transcriptional regulation of CDK inhibitors

Key regulators of 3' untranslated regions (3' UTRs) are microRNAs and RNA-binding proteins (RBPs). The p27 tumour suppressor is highly expressed in quiescent cells, and its downregulation is required for cell cycle entry after growth factor stimulation. Intriguingly, p27 accumulates in quiescent cells despite high levels of its inhibitors miR-221 and miR-222. This study shows that miR-221 and miR-222 are underactive towards p27-3' UTR in quiescent cells, as a result of target site hindrance. Pumilio-1 (PUM1) is a ubiquitously expressed RBP that was shown to interact with p27-3' UTR. In response to growth factor stimulation, PUM1 is upregulated and phosphorylated for optimal induction of its RNA-binding activity towards the p27-3' UTR. PUM1 binding induces a local change in RNA structure that favours association with miR-221 and miR-222, efficient suppression of p27 expression, and rapid entry to the cell cycle. This study has therefore uncovered a novel RBP-induced structural switch modulating microRNA-mediated gene expression regulation (Kedde, 2010).

The Ski-interacting protein SKIP/SNW1 functions as both a splicing factor and a transcriptional coactivator for induced genes. Transcription elongation factors such as SKIP have been shown to be dispensable in cells subjected to DNA damage stress. However, this study reporta that SKIP is critical for both basal and stress-induced expression of the cell cycle arrest factor p21Cip1. RNAi chromatin immunoprecipitation (RNAi-ChIP) and RNA immunoprecipitation (RNA-IP) experiments indicate that SKIP is not required for transcription elongation of the gene under stress, but instead is critical for splicing and p21Cip1 protein expression. SKIP interacts with the 3' splice site recognition factor U2AF65 and recruits it to the p21Cip1 gene and mRNA. Remarkably, SKIP is not required for splicing or loading of U2AF65 at other investigated p53-induced targets, including the proapoptotic gene PUMA. Consequently, depletion of SKIP induces a rapid down-regulation of p21Cip1 and predisposes cells to undergo p53-mediated apoptosis, which is greatly enhanced by chemotherapeutic DNA damage agents. ChIP experiments reveal that SKIP is recruited to the p21Cip1, and not PUMA, gene promoters, indicating that p21Cip1 gene-specific splicing is predominantly cotranscriptional. The SKIP-associated factors DHX8 and Prp19 are also selectively required for p21Cip1 expression under stress. Together, these studies define a new step that controls cancer cell apoptosis (Chen, 2011).

p21 is a negative transcriptional regulator of Wnt4 expression downstream of Notch1 activation

In keratinocytes, the cyclin/CDK inhibitor p21WAF1/Cip1 is a direct transcriptional target of Notch1 activation; loss of either the p21 or Notch1 genes expands stem cell populations and facilitates tumor development. The Notch1 tumor-suppressor function has been associated with down-regulation of Wnt signaling. This study shows that suppression of Wnt signaling by Notch1 activation is mediated, at least in part, by down-modulation of Wnts gene expression. p21 is a negative regulator of Wnts transcription downstream of Notch1 activation, independent of effects on the cell cycle. More specifically, expression of the Wnt4 gene is under negative control of endogenous p21 both in vitro and in vivo. p21 associates with the E2F-1 transcription factor at the Wnt4 promoter and causes curtailed recruitment of c-Myc and p300, and histone hypoacetylation at this promoter. Thus, p21 acts as a selective negative regulator of transcription and links the Notch and Wnt signaling pathways in keratinocyte growth control (Devgan, 2005).

Thus in keratinocytes, p21 functions as a transcriptional regulator that associates physically to the promoter of the Wnt4 gene. While increased p21 expression suppresses both Wnt3 and Wnt4 expression and endogenous p21 is required for the effective down-modulation of both genes by Notch1, in the skin of p21-/- mice and in primary p21-/- keratinocytes under basal conditions, only Wnt4 is up-regulated. This is likely a reflection of the fact that, biochemically, association of the endogenous as well as overexpressed p21 protein to the Wnt4 promoter is readily observed, while association to the Wnt3 promoter, if it occurs, is much weaker and harder to demonstrate. By ChIP assays, it was found that E2F-1 binds the same region of the Wnt4 promoter as p21, and that the two proteins can be recovered in association at this promoter. These findings are consistent with an elegant model, whereby E2F-1-p21 association provides a bridging mechanism for bringing p21 to target promoters. Importantly, however, in the cells used, p21 binding is specific for the Wnt4 promoter and does not occur at the promoter of another 'classical' E2F-1 target gene such as PCNA, the expression of which is unaffected by increased p21 expression. Concomitantly, p21 binding at the Wnt4 promoter is linked to curtailed recruitment of c-Myc and p300. By exogenous expression and promoter activity studies, p21 was previously reported to associate with the c-Myc protein suppressing its activity. The data are consistent with such a mechanism taking place at the Wnt4 promoter. Even in this case, however, there is an important element of selectivity, in that expression of other classical c-Myc target genes (such as that for ornithine decarboxylase), remains unaffected by p21 expression in keratinocytes (our unpublished observations). Thus, the findings are overall consistent with the emerging crucial role of chromatin configuration and promoter context in control of gene expression, with a physical and functional interplay between p21 and the specific transcription regulatory apparatus of individual genes such as that for Wnt4 (Devgan, 2005).

In summary, the common biological function of Notch1 and p21 as negative regulators of keratinocyte self renewal and tumorigenesis can be explained, in part, by one being a mediator of the other in transcriptional suppression of Wnt family members with consequent down-modulation of ß-catenin signaling. More specifically, p21 is directly involved in transcription regulation of the Wnt4 target gene, the control of which at the integrated chromatin level, remains an exciting topic for future studies (Devgan, 2005).

Degradation of CDK inhibitors

Deregulation of cell proliferation is a hallmark of cancer. In many transformed cells, the cyclin A/CDK2 complex that contains S-phase kinase associated proteins 1 and 2 (SKP1 and SKP2) is highly induced. To determine the roles of this complex in the cell cycle regulation and transformation, the composition of this complex was analyzed. This complex contains an additional protein, human CUL-1, a member of the cullin/CDC53 family. The identification of CUL-1 as a member of the complex raises the possibility that the p19(SKP1)/p45(SKP2)/CUL-1 complex may function as the yeast SKP1-CDC53-F-box (SCF) protein complex that acts as a ubiquitin E3 ligase to regulate the G1/S transition. In mammalian cells, cyclin D, p21(CIP1/WAF1), and p27(KIP1) are short-lived proteins that are controlled by ubiquitin-dependent proteolysis. To determine the potential in vivo targets of the p19(SKP1)/p45(SKP2)/CUL-1 complex, the specific antisense oligodeoxynucleotides were used against either SKP1, SKP2, or CUL-1 RNA to inhibit their expression. Treatment of cells with these oligonucleotides causes the selective accumulation of p21 and cyclin D proteins. The protein level of p27 is not affected. These data suggest that the human p19(SKP1)/p45(SKP2)/CUL-1 complex is likely to function as an E3 ligase to selectively target cyclin D and p21 for the ubiquitin-dependent protein degradation. Aberrant expression of human p19(SKP1)/p45(SKP2)/CUL-1 complex thus may contribute to tumorigenesis by regulating the protein levels of G1 cell cycle regulators (Yu, 1998).

Entry into S phase is dependent on the coordinated activation of CDK4,6 and CDK2 kinases. Once a cell commits to S phase, there must be a mechanism to ensure the irreversibility of this decision. The activity of these kinases is inhibited by their association with p27. In many cells, p27 plays a major role in the withdrawal from the cell cycle in response to environmental cues. Thus, it is likely that p27 is a target of the machinery required to ensure the irreversibility of S-phase entry. A cell-free degradation system is described that faithfully recapitulates the cell cycle phase-specific degradation of p27. This reaction is dependent on active CDK2 activity, suggesting that CDK2 activity is directly required for p27 degradation. In addition to CDK2, other S-phase-specific factors are required for p27 degradation. At least some of these factors are ubiquitin and proteasome dependent. Also discussed are the relationships between CDK2 activity and ubiquitin-dependent (and possibly ubiquitin-independent) proteasomal activities in S-phase extracts as related to p27 (Nguyen, 1999).

The cellular abundance of the cyclin-dependent kinase (Cdk) inhibitor p27 is regulated by the ubiquitin-proteasome system. Activation of p27 degradation is seen in proliferating cells and in many types of aggressive human carcinomas. p27 can be phosphorylated on threonine 187 by Cdks, and cyclin E/Cdk2 overexpression can stimulate the degradation of wild-type p27, but not of a threonine 187-to-alanine p27 mutant [p27(T187A)]. However, it remains unknown whether threonine 187 phosphorylation stimulates p27 degradation through the ubiquitin-proteasome system or some alternative pathway. In this study, it is demonstrated that p27 ubiquitination (as assayed in vivo and in an in vitro reconstituted system) is cell-cycle regulated and that Cdk activity is required for the in vitro ubiquitination of p27. Furthermore, ubiquitination of wild-type p27, but not of p27(T187A), can occur in G1-enriched extracts only on addition of cyclin E/Cdk2 or cyclin A/Cdk2. Using a phosphothreonine 187 site-specific antibody for p27, it has been shown that threonine 187 phosphorylation of p27 is also cell-cycle dependent, being present in proliferating cells but undetectable in G1 cells. In addition to threonine 187 phosphorylation, efficient p27 ubiquitination requires formation of a trimeric complex with the cyclin and Cdk subunits. In fact, cyclin B/Cdk1, which can phosphorylate p27 efficiently, but cannot form a stable complex with it, is unable to stimulate p27 ubiquitination by G1 extracts. Furthermore, another p27 mutant [p27(CK-)] that can be phosphorylated by cyclin E/Cdk2 but cannot bind this kinase complex, is refractory to ubiquitination. Thus throughout the cell cycle, both phosphorylation and trimeric complex formation act as signals for the ubiquitination of a Cdk inhibitor (Montagnoli, 1999).

The proliferation of mammalian cells is under strict control, and the cyclin-dependent-kinase inhibitory protein p27Kip1 is an essential participant in this regulation both in vitro and in vivo. Although mutations in p27Kip1 are rarely found in human tumours, reduced expression of the protein correlates well with poor survival among patients with breast or colorectal carcinomas, suggesting that disruption of the p27Kip1 regulatory mechanisms contributes to neoplasia. The abundance of p27Kip1 in the cell is determined either at or after translation, for example as a result of phosphorylation by cyclinE/Cdk2 complexes, degradation by the ubiquitin/proteasome pathway, sequestration by unknown Myc-inducible proteins, binding to cyclinD/Cdk4 complexes, or inactivation by the viral E1A oncoprotein. A mouse 38K protein (p38) encoded by the Jab1 gene interacts specifically with p27Kip1 and overexpression of p38 in mammalian cells causes the translocation of p27Kip1 from the nucleus to the cytoplasm, decreasing the amount of p27Kip1 in the cell by accelerating its degradation. Ectopic expression of p38 in mouse fibroblasts partially overcomes p27Kip1-mediated arrest in the G1 phase of the cell cycle and markedly reduces their dependence on serum. These findings indicate that p38 functions as a negative regulator of p27Kip1 by promoting its degradation (Tomoda, 1999).

The Cdk inhibitor p21Cip1 is an unstable protein. Pharmacologic inhibition of the proteasome increases the half-life of p21 from less than 30 min to more than 2 hr and results in the accumulation of p21-ubiquitin conjugates. To determine whether ubiquitination is required for proteasomal degradation of p21, mutant versions of p21 have been constructed that are not ubiquitinated in vivo. Remarkably, these mutants remain unstable and increase in abundance upon proteasome inhibition, indicating that direct ubiquitination of p21 is not necessary for its turnover by the proteasome. The frequently observed correlation between protein ubiquitination and proteasomal degradation is insufficient to conclude that ubiquitination is a prerequisite for degradation (Sheaff, 2000).

How might p21 be degraded by the proteasome, independent of ubiquitin attachment? The clearest example of a protein whose turnover by the proteasome is ubiquitin independent is ornithine decarboxylase (ODC), and additional examples have been proposed. ODC is directed to the proteasome by its specific binding partner, antizyme. Similarly, the interaction of p21 with its known binding partners affects its turnover, although these relationships are complex and poorly understood. Perhaps the simplest explanation is that nonubiquitinated p21 is directly recognized by the proteasome. Unstructured proteins can be directly recognized and degraded by proteasomes in vitro and in vivo without ubiquitination. Moreover, free p21 does not have a well-defined tertiary structure. Thus, p21 and perhaps other small Cdk inhibitors might be directly recognized by the proteasome when they are free of cyclin/Cdk complexes (Sheaff, 2000).

The Cdk2 inhibitor, p27Kip1, is degraded in a phosphorylation- and ubiquitylation-dependent manner at the G1 -S transition of the cell cycle. Degradation of p27Kip1 requires import into the nucleus for phosphorylation by Cdk2. Phosphorylated p27Kip1 is thought to be subsequently re-exported and degraded in the cytosol. Using two-hybrid screens, p27Kip1 has been shown to interact with a nuclear pore-associated protein, mNPAP60: the interaction maps to the 310  helix of p27 and a point mutant in p27Kip1 has been identified that is deficient for interaction (R90G). Whether in vivo or in vitro, the loss-of-interaction mutant is poorly transported into the nucleus, while ubiquitination of p27R90G occurs normally. Co-expression of cyclin E and Cdk2 in vivo rescues the import defect. However, mutant p27Kip1 accumulates in a phosphorylated form in the nucleus and is not efficiently degraded, arguing that at least one step in the degradation of phosphorylated p27Kip1 requires an interaction with the nuclear pore. These results identify a novel component involved in p27Kip1 degradation and suggest that degradation of p27Kip1 is tightly linked to its intracellular transport (Muller, 2000).

The c-Myc oncoprotein plays an important role in the growth and proliferation of normal and neoplastic cells. To execute these actions, c-Myc is thought to regulate functionally diverse sets of genes that directly govern cellular mass and progression through critical cell cycle transitions. Several lines of evidence are provided that c-Myc promotes ubiquitin-dependent proteolysis by directly activating expression of the Cul1 gene, encoding a critical component of the ubiquitin ligase SCFSKP2. The cell cycle inhibitor p27kip1 is a known target of the SCFSKP2 complex, and Myc-induced Cul1 expression matches well with the kinetics of declining p27kip1 protein. Enforced Cul1 expression or antisense neutralization of p27kip1 is capable of overcoming the slow-growth phenotype of c-Myc null primary mouse embryonic fibroblasts (MEFs). In reconstitution assays, the addition of in vitro translated Cul1 protein alone is able to restore p27kip1 ubiquitination and degradation in lysates derived from c-myc/MEFs or density-arrested human fibroblasts. These functional and biochemical data provide a direct link between c-Myc transcriptional regulation and ubiquitin-mediated proteolysis and together support the view that c-Myc promotes G1 exit in part via Cul1-dependent ubiquitination and degradation of the CDK inhibitor, p27kip1 (O'Hagan, 2000).

Cdc kinase subunit (Cks) proteins were originally identified through their ability to genetically suppress defective alleles of the cyclin-dependent kinases (CDKs) of both fission and budding yeast. Subsequent investigations demonstrate that these small (9-18 kDa) proteins are ubiquitous in eukaryotes and can directly bind to CDK/cyclin complexes, hence the designation Cks. Whereas lower eukaryotes express only one Cks protein, mammals, and possibly other vertebrates, express two orthologs, Cks1 and Cks2. Although the precise functions of Cks proteins have been elusive, they have been shown to be essential for viability in both fission and budding yeast. Furthermore, these proteins are sufficiently conserved at the structural and functional level that both human Cks orthologs can functionally substitute for Cks1 in yeast (Spruck, 2001 and references therein).

Most genetic and biochemical data point to a mitotic role for Cks proteins, although other cell cycle functions have been suggested. Loss of function of the Cks1 homolog of fission yeast (S. pombe) suc1, results in M phase arrest with condensed but unsegregated chromosomes, an extended spindle, and elevated levels of Cdc2/cyclin B kinase activity. Similarly, loss of Cks1 function in S. cerevisiae leads to a mitotic arrest with high Cdc28/cyclin B kinase activity. However, Cks1 has also been shown to have a G1 function in budding yeast. Studies using Xenopus egg extracts also suggest a mitotic role for the Cks homolog, Xe-p9. Immunodepletion of Xe-p9 from interphase egg extracts prevent entry into mitosis, whereas immunodepletion from mitotic extracts lead to M phase arrest with elevated levels of cyclin B and CDK1/cyclin B kinase activity. Taken together, these results suggest the Cks proteins may be required both for entry into and progression through mitosis (Spruck, 2001 and references therein).

The molecular function(s) performed by Cks proteins are less clear. Cks proteins can bind to MPF (CDK1/cyclin B), which controls entry and progression through mitosis. It has therefore been assumed that the ability of Cks proteins to bind to CDK1, the catalytic core of MPF, is the basis for Cks-dependent mitotic functions. It has been demonstrated that mitotic regulators Cdc25, Wee1, and Myt1 are regulated by MPF phosphorylation and that Cks binding to CDK1 can stimulate these phosphorylation events in vitro, consistent with the observed role of Cks proteins in mitotic entry (Spruck, 2001 and references therein).

Once cells have entered mitosis, progression depends on MPF-dependent activation of a protein ubiquitin ligase (E3) known as the cyclosome/APC. In particular, exit from mitosis requires APC-mediated ubiquitination of cyclin B and subsequent proteasomal degradation. It has been proposed that activation of the APC complex at the metaphase-anaphase transition requires phosphorylation of specific subunits by MPF. Consistent with a role in this regulation, Xe-p9 and Cks1 have been shown to stimulate in vitro CDK1/cyclin B-dependent phosphorylation of the APC subunit Cdc27 in Xenopus and S. cerevisiae, respectively. However, an additional role for Cks1 in cyclin B degradation has been suggested in S. cerevisiae. Both genetic and biochemical data indicate that Cks1 is required for efficient proteasomal targeting of ubiquitinated cyclin B (Spruck, 2001 and references therein).

The three-dimensional structure of human Cks1 bound to CDK2, determined by X-ray diffraction crystallography, has revealed that Cks proteins bind the C lobe of CDKs, distal from the active site. Specifically, Cks proteins interact solely with the 5 helix and L14 loop regions of CDKs. In addition, Cks protein binding causes little if any alteration of CDK structure. Based on these structural considerations, it is unlikely that Cks1 proteins directly affect catalysis by cognate CDK enzymes. However, the fact that Cks protein binding can stimulate phosphorylation of specific substrates suggests that Cks proteins may provide substrate-specific targeting or docking functions for CDK/cyclin complexes, particularly CDK1/cyclin B and its homologs in the context of mitotic entry and exit. To further characterize the functions of Cks proteins, mice nullizygous for the genes encoding each of the Cks orthologs, Cks1 and Cks2, were constructed (Spruck, 2001).

In this study, it has been shown that Cks1 directs the ubiquitin-mediated proteolysis of the CDK-bound substrate p27Kip1 by the protein ubiquitin ligase (E3) SCFSkp2. Cks1 associates with the F box protein Skp2 and is essential for recognition of the p27Kip1 substrate for ubiquitination in vivo and in vitro. Using purified recombinant proteins, p27Kip1 ubiquitination activity was reconstituted and this has been shown to be dependent on Cks1. CKS1-/- mice are abnormally small, and cells derived from them proliferate poorly, particularly under limiting mitogen conditions, possibly due to elevated levels of p27Kip1 (Spruck, 2001).

p27Kip1 accumulates in G0 and G1 and then achieves a lower steady-state level beginning at the G1/S phase transition. Presumably the lowering of p27Kip1 levels at this time in the cell cycle contributes to the cooperative activation of CDKs that is required for efficient initiation and maintenance of DNA replication. In part, the downregulation of p27Kip1 levels is achieved by an increase in ubiquitin-dependent proteolysis at the G1/S boundary. Previous studies have demonstrated that targeting phosphorylation of p27Kip1 on Thr-187 by CDK2/cyclin E, which is activated near the G1/S boundary, is one mechanism for coupling p27Kip1 degradation to cell cycle progression. Additionally, Skp2, the targeting F box protein for p27Kip1 ubiquitination, has been shown to accumulate specifically at the G1/ S transition, providing a second mechanism for coupling p27Kip1 turnover to the G1/S boundary. A third potential mechanism for cell cycle control of p27Kip1 turnover is revealed by these studies. Cks1 mRNA and protein accumulate at the G1/S boundary. Furthermore, the correlation of phenotype with CKS1 gene dosage suggests that Cks1 is rate limiting for function. Since Cks1 thus appears to be rate limiting for SCFSkp2 function, p27Kip1 turnover will be linked to accumulation of both Skp2 and Cks1. Why three distinct mechanisms establish the temporality of p27Kip1 degradation is not clear, although redundant regulation is often a hallmark of important cell cycle events (Spruck, 2001).

The cyclin-dependent kinase inhibitor p21WAF1/CIP1 is a key regulator of cell-cycle progression and its expression is tightly regulated at the level of transcription and by proteasome-dependent proteolysis. The turnover of p21WAF1/CIP1 by proteasomes does not always require the ubiquitylation of p21WAF1/CIP1 suggesting that there could be an alternative pathway into the proteasome. The C8 alpha-subunit of the 20S proteasome interacts with the C-terminus of p21WAF1/CIP1 and mediates the degradation of p21WAF1/CIP1. A small deletion in this region that disrupts binding to C8 increases the half-life of p21WAF1/CIP1 expressed in vivo. In contrast a deletion that increases the affinity between C8 and p21WAF1/CIP1 significantly reduces the stability of the latter. These data suggest that interaction with a 20S proteasome alpha-subunit is a critical determinant of p21WAF1/CIP1 turn-over and show how non-ubiquitylated molecules might bypass the 19S regulator of the proteasome and become targeted directly to the 20S core protease. Consistent with this, p21WAF1/CIP1 is degraded rapidly by purified 20S proteasomes in a manner that is dependent on the C8-interaction domain (Touitou, 2001).

The tumor suppressor protein, p53, plays a critical role in mediating cellular response to stress signals by regulating genes involved in cell cycle arrest and apoptosis. p53 is believed to be inactive for DNA binding unless its C terminus is modified or structurally altered. Unmodified p53 actively binds to two sites at -1.4 and -2.3 kb within the chromatin-assembled p21 promoter and requires the C terminus and the histone acetyltransferase, p300, for transcription. Acetylation of the C terminus by p300 is not necessary for binding or promoter activation. Instead, p300 acetylates p53-bound nucleosomes in the p21 promoter with spreading to the TATA box. Thus, p53 is an active DNA and chromatin binding protein that may selectively regulate its target genes by recruitment of specific cofactors to structurally distinct binding sites (Espinosa, 2001).

Surprisingly, p300 does not function by facilitating p53 binding to its DNA recognition sites within chromatin. Instead, p300 acts at a later step in the transcription process by acetylating nucleosomes within the proximal and distal p21 promoter when targeted by bound p53. This presumably renders the nucleosomes sufficiently fluid to allow interaction with other components of the transcription machinery. p300-mediated transcriptional activation has been described for other chromatin-assembled genes. These experiments demonstrate that a mechanism by which p300 can regulate the activity of natural promoters operates by acetylating chromatin over a long-range when recruited by a distal transcription factor. In the absence of p53, p300 cannot acetylate nucleosomes due to lack of template targeting, and the p21 promoter remains inactive. p53 proteins containing mutations in lysine residues acetylated by p300 are as active as wild-type p53 in regulating p21 transcription in vitro. This indicates that acetylation of p53 does not contribute to its transactivation potential, and that p300 does not mediate transcription by this mechanism in biochemical assays. This conclusion is in agreement with previous in vivo analyses in which p53 mutants lacking these lysine residues does not show a significant decrease in transcriptional activity. However, p53 acetylation may play a role in protein stabilization or subnuclear localization (Espinosa, 2001).

It is intriguing that p53 binds to the p21 promoter with higher affinity and with different kinetics when assembled into chromatin than it does to DNA. This is particularly interesting because this occurs in the absence of chromatin remodeling or modifying complexes and is not observed with other transcription factors that can also bind to nucleosomes. This could be explained if bending of the DNA, when wrapped around a nucleosome, generates a secondary structure that is more stable for p53 binding. Indeed, previous studies determined the importance of DNA bending for p53 high-affinity binding and predicted that some p53 binding sites would be exposed and accessible when incorporated into a nucleosome. Importantly, the structure recognized by p53 in p21 promoter DNA is preserved and improved or stabilized in chromatin. The physiological significance of the distinct kinetics of p53 occupancy observed on the p21 promoter as chromatin or DNA is unclear. The linear rate of association of p53 with chromatin may indicate that lower concentrations of p53 are required to fully occupy binding sites in vivo than the cooperative binding to DNA would indicate. This could be significant if the cell has to respond efficiently to activate p53-responsive pathways without waiting for a critical threshold of p53 concentration to be reached. It should be emphasized, however, that the nature of p53 binding to chromatin and the requirements for remodeling/modifying activities may vary with individual target promoters (Espinosa, 2001).

The role of Notch signaling in growth/differentiation control of mammalian epithelial cells is still poorly defined. Keratinocyte-specific deletion of the Notch1 gene results in marked epidermal hyperplasia and deregulated expression of multiple differentiation markers. In differentiating primary keratinocytes in vitro endogenous Notch1 is required for induction of p21WAF1/Cip1 expression, and activated Notch1 causes growth suppression by inducing p21WAF1/Cip1 expression. Activated Notch1 also induces expression of 'early' differentiation markers, while suppressing the late markers. Induction of p21WAF1/Cip1 expression and early differentiation markers occur through two different mechanisms. The RBP-Jkappa protein binds directly to the endogenous p21 promoter and p21 expression is induced specifically by activated Notch1 through RBP-Jkappa-dependent transcription. Expression of early differentiation markers is RBP-Jkappa-independent and can be induced by both activated Notch1 and Notch2, as well as the highly conserved ankyrin repeat domain of the Notch1 cytoplasmic region. Thus, Notch signaling triggers two distinct pathways leading to keratinocyte growth arrest and differentiation (Rangarajan, 2001).

The abundance of the cyclin-dependent kinase (CDK) inhibitor p57Kip2, an important regulator of cell cycle progression, is thought to be controlled by the ubiquitin-proteasome pathway. The Skp1/Cul1/F-box (SCF)-type E3 ubiquitin ligase complex SCFSkp2 has now been shown to be responsible for regulating the cellular level of p57Kip2 by targeting it for ubiquitylation and proteolysis. The elimination of p57Kip2 is impaired in Skp2-/- cells, resulting in abnormal accumulation of the protein. Coimmunoprecipitation analysis has also revealed that Skp2 interacts with p57Kip2 in vivo. Overexpression of WT Skp2 promotes degradation of p57Kip2, whereas expression of a dominant negative mutant of Skp2 prolongs the half-life of p57Kip2. Mutation of the threonine residue (Thr-310) of human p57Kip2, which is conserved between the COOH-terminal QT domains of p57Kip2 and p27Kip1, prevents the effect of Skp2 on the stability of p57Kip2, suggesting that phosphorylation at this site is required for SCFSkp2-mediated ubiquitylation. Finally, the purified recombinant SCFSkp2 complex mediates p57Kip2 ubiquitylation in vitro in a manner dependent on the presence of the cyclin E-CDK2 complex. These observations thus demonstrate that the SCFSkp2 complex plays an important role in cell-cycle progression by determining the abundance of p57Kip2 and that of the related CDK inhibitor p27Kip1 (Kimura, 2003).

Although Skp2 has been thought to mediate the degradation of p27 at the G1-S transition, Skp2−/− cells exhibit accumulation of p27 in S-G2 phase with overreplication. This study demonstrates that Skp2−/−p27−/− mice do not exhibit the overreplication phenotype, suggesting that p27 accumulation is required for its development. Hepatocytes of Skp2−/− mice enter the endoduplication cycle after mitogenic stimulation, whereas this phenotype is not apparent in Skp2−/−p27−/− mice. Cdc2-associated kinase activity is lower in Skp2−/− cells than in wild-type cells, and a reduction in Cdc2 activity is sufficient to induce overreplication. The lack of p27 degradation in G2 phase in Skp2−/− cells may thus result in suppression of Cdc2 activity and consequent inhibition of entry into M phase. These data suggest that p27 proteolysis is necessary for the activation of not only Cdk2 but also Cdc2, and that Skp2 contributes to regulation of G2-M progression by mediating the degradation of p27 (Nakayama, 2004).

The activity of the SCFskp2 E3 ligase is required for the proteolytic turnover of several proteins involved in cell cycle control and transcriptional regulation. Loss of skp2 in the mouse leads to a complex phenotype including changes in cell size and DNA content as well as severe proliferation defects. The loss of a single skp2 substrate, namely, the cyclin kinase inhibitor p27kip1, reverts the phenotype of skp2 knockout hepatocytes to normal. By comparing the kinetics of p27 turnover and cell cycle progression in skp2 knockout and p27T187A knock-in mice, a short period in G1 was defined in which p27 is able to block the cell cycle after the exit from quiescence. Loss of p27 turnover during this period prevents mitotic division and instead leads to compensatory cell growth, These observation makes p27 the essential target of skp2-dependent protein turnover (Kossatz, 2004).

Loss of the CDK inhibitor p27KIP1 is widely linked with poor prognosis in human cancer. In Wnt10b-expressing mammary tumors, levels of p27KIP1 were extremely low; conversely, Wnt10b-null mammary cells expressed high levels of this protein, suggesting Wnt-dependent regulation of p27KIP1. Interestingly it was found that Wnt-induced turnover of p27KIP1 was independent from classical SCFSKP2-mediated degradation in both mouse and human cells. Instead, turnover required Cullin 4A and Cullin 4B, components of an alternative E3 ubiquitin ligase induced in response to active Wnt signaling. This study found that CUL4A is a novel Wnt target gene in both mouse and human cells and that CUL4A physically interacts with p27KIP1 in Wnt-responding cells. It was further demonstrated that both Cul4A and Cul4B are required for Wnt-induced p27KIP1 degradation and S-phase progression. CUL4A and CUL4B are therefore components of a conserved Wnt-induced proteasome targeting (WIPT) complex that regulates p27KIP1 levels and cell cycle progression in mammalian cells (Miranda-Carboni, 2008).

The CRL4Cdt2 ubiquitin ligase targets the degradation of p21Cip1 to control replication licensing

The faithful replication of genomic DNA is crucial for maintaining genome stability. In eukaryotes, DNA rereplication is prevented by the temporal regulation of replication licensing. Replication-licensing factors are required to form prereplicative complexes during G1 phase, but are inactivated in S phase to prevent rereplication. A vertebrate CUL4 CRL ubiquitin ligase (CRL4) complex containing Cdt2 as the substrate recognition subunit promotes proper DNA replication, in part, by degrading the replication-licensing factor Cdt1 during S phase. This study shows that the C. elegans CRL4(Cdt2) complex has a conserved role in degrading Cdt1. Furthermore, CRL4(Cdt2) restrains replication licensing in both C. elegans and humans by targeting the degradation of the cyclin-dependent kinase (CDK) inhibitors CKI-1 and p21(Cip1), respectively. Human CRL4(Cdt2) targets the degradation of p21 in S phase, with the in vivo ubiquitylation of p21 by CRL4(Cdt2) dependent on p21 binding to PCNA. Inactivation of Cdt2 induces rereplication, which requires the presence of the CDK inhibitor p21. Strikingly, coinactivation of CRL4(Cdt2) and SCF(Skp2) (which redundantly targets p21 degradation) prevents the nuclear export of the replication-licensing factor Cdc6 during S phase, and the block on nuclear export is dependent on p21. This work defines the degradation of p21 as a critical aspect of replication licensing in human cells (Kim, 2008).

The CDK inhibitor p21 has a central role as an effector of cell cycle arrest when it is transactivated by the p53 tumor suppressor protein in response to DNA damage (Besson, 2008). While p21 is primarily considered an inhibitor of CDK/cyclin complexes, it also has CDK-independent functions involving the regulation of the cytoskeleton, transcription, and apoptosis (Besson, 2008). During the cell cycle, p21 levels are high in G1 phase, decrease significantly during S phase, and increase again during G2 phase. p21 has been shown to undergo proteasome-mediated degradation through the actions of a number of E3s: SCFSkp2, MDM2, MDMX, and APC/CCdc20. APC/CCdc20 targets the degradation of p21 during prometaphase (Amador, 2007). MDM2 and MDMX promote the proteasomal turnover of p21 predominantly in G1 and early S phases (Jin, 2008). SCFSkp2 has been shown to target p21 degradation in S-phase cells; however, the current study suggests that this degradation is not restricted to S phase. In contrast, CRL4Cdt2-mediated degradation of p21 occurs primarily in S phase (Kim, 2008).

This study shows that the in vivo ubiquitylation of p21 by CRL4Cdt2 is dependent on p21 binding to PCNA. The binding of Cdt1 to chromatin-associated PCNA has been proposed as a mechanism to ensure that Cdt1 degradation is S-phase-specific, and a similar mechanism may explain the S-phase specificity of the CRL4Cdt2-mediated p21 degradation. The significance of the PCNA-p21 interaction is underlined by the independent discovery that CRL4Cdt2 targets p21 degradation in response to DNA damage in a PCNA-dependent manner (Kim, 2008).

Table of contents

dacapo: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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