Timing of cyclin E gene expression depends on the regulated association of a bipartite repressor element with a novel E2F complex.
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Transient induction of the cyclin E gene in late G1 gates progression into S. We show that this event is controlled via a cyclin E repressor module (CERM), a novel bipartite repressor element located near the cyclin E transcription start site. CERM consists of a variant E2F-binding site and a contiguous upstream AT-rich sequence which cooperate during G0/G1 to delay cyclin E expression until late G1. CERM binds the protein complex CERC, which disappears upon progression through G0-G1 and reappears upon entry into the following G1. CERC disappearance correlates kinetically with the liberation of the CERM module in vivo and cyclin E transcriptional induction. CERC contains E2F4/DP1 and a pocket protein, and sediments faster than classical E2F complexes in a glycerol gradient, suggesting the presence of additional components in a novel high molecular weight complex. Affinity purified CERC binds to CERM but not to canonical E2F sites, thus displaying behavior different from known E2F complexes. In cells nullizygous for members of the Rb family, CERC is still detectable and CERM-dependent repression is functional. Thus p130, p107 and pRb function interchangeably in CERC. Notably, the CERC-CERM complex dissociates prematurely in pRb-/- cells in correspondence with the premature expression of cyclin E. Thus, we identify a new regulatory module that controls repression of G1-specific genes in G0/G1. (+info)
cdc25A is necessary but not sufficient for optimal c-myc-induced apoptosis and cell proliferation of vascular smooth muscle cells.
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Increasing evidence indicates that the control of cell proliferation and apoptosis are linked. The c-myc proto-oncogene is induced early after cell-cycle entry in vascular smooth muscle cells (VSMCs) in vitro and after arterial injury and regulates both cell proliferation and apoptosis. Although both proliferation and apoptosis are likely to be mediated via transcriptional activation of target genes, few c-myc targets have been identified. Therefore, the recent identification that cdc25A, a cell-cycle phosphatase involved in G1 progression, is transcriptionally activated by c-myc and regulates c-myc-induced apoptosis has suggested that cdc25A may be the principal mediator of c-myc in VSMCs. We examined cdc25A regulation of c-myc-induced proliferation and apoptosis by expressing cdc25A or antisense cdc25A in primary rat VSMCs or in VSMCs expressing deregulated c-myc or adenovirus E1A. Ectopic c-myc increased cdc25A expression, but cdc25A was still responsive to serum components, which indicated that c-myc alone is not the main determinant of cdc25A expression. Antisense cdc25A inhibited c-myc-induced proliferation and apoptosis; however, drug and metabolic blocks indicated that this effect was limited to G1. Ectopic cdc25A augmented the proproliferative and proapoptotic action of c-myc but did not increase cell proliferation or apoptosis in the absence of ectopic c-myc. In contrast, E1A/E2F-induced apoptosis was independent of cdc25A. We conclude that cdc25A expression modulates the ability of c-myc to induce apoptosis in G1. However, cdc25A alone does not induce apoptosis and cannot substitute for c-myc in VSMCs. Additional targets of c-myc are therefore involved in apoptosis of both G1 and post-G1 VSMCs. (+info)
Modulation of E2F complexes during G0 to S phase transition in human primary B-lymphocytes.
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The pocket protein-E2F complexes are convergence points for cell cycle signaling. In the present report, we identified and monitored the pocket protein-E2F complexes in human primary B-lymphocytes after activation by phorbol 12-myristate 13-acetate. Consistent with previous data from human and mouse fibroblasts and T-lymphocytes, E2F4 and DP1 form the predominant E2F heterodimers both in G0 and G1 phases of the human B-lymphocyte cell cycle, whereas E2F1 and -3 are first detected in late G1, and their expression levels increase towards S phase. Intriguingly, the major E2F complex that we detected in quiescent human B-lymphocytes is consisted of pRB, E2F4, and DP1. Though the levels of DP1 and -2 increase when cells progress from G0 to S, the proportion of DP1 to DP2 remains relatively constant during the cell cycle. We also observed an increase in electrophoretic mobility of the predominant E2F components, DP1 and E2F4, as B-lymphocytes progressed from G0 into early G1. This increase in mobility was attributable to dephosphorylation, as lambda phosphatase treatment could convert the slower migrating forms into the corresponding faster mobility forms. We further demonstrated that this change in phosphorylation status correlates with a decrease in DNA binding activity. This modulation of DNA binding activity mediated through the dephosphorylation of DP1 and E2F4 could help to explain the lack of in vivo DNA footprinting in late G1 and S phases of gene promoters negatively regulated through E2F sites and suggests a novel mechanism for controlling E2F transcriptional activity during the transition from quiescence to proliferation. (+info)
Cumulative effect of phosphorylation of pRB on regulation of E2F activity.
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The product of the retinoblastoma susceptibility gene, pRB, is a nuclear phosphoprotein that controls cell growth by binding to and suppressing the activities of transcription factors such as the E2F family. Transactivation activity is inhibited when E2F is bound to hypophosphorylated pRB and released when pRB is phosphorylated by cyclin-dependent kinases (CDKs). To determine which of 16 potential CDK phosphorylation sites regulated the pRB-E2F interaction, mutant pRB proteins produced by site-directed mutagenesis were tested for the ability to suppress E2F-mediated transcription in a reporter chloramphenicol acetyltransferase assay. Surprisingly, no one CDK site regulated the interaction of pRB with E2F when E2F was bound to DNA. Instead, disruption of transcriptional repression resulted from accumulation of phosphate groups on the RB molecule. (+info)
Transcription factor E2F-1 is upregulated in response to DNA damage in a manner analogous to that of p53.
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The transcription factor E2F-1 directs the expression of genes that induce or regulate cell division, and a role for E2F-1 in driving cells into apoptosis is the subject of intense discussion. Recently it has been shown that E2F-1 binds and coprecipitates with the mouse double-minute chromosome 2 protein (Mdm2). A domain of E2F-1 (amino acids 390 to 406) shows striking similarity to the Mdm2 binding domain of the tumor suppressor protein p53. It is known that interaction of Mdm2 with p53 through this domain is required for Mdm2-dependent degradation of p53. We show here that E2F-1 protein is upregulated in response to DNA damage. The kinetics of induction are dependent upon the source of DNA damage, i.e., fast and transient after irradiation with X rays and delayed and stable after irradiation with UVC, and thus match the kinetics of p53 induction in response to DNA damage. We show further that E2F-1 is also upregulated by treatment with the transcription inhibitor actinomycin D and with the kinase inhibitor DRB, as well as by high concentrations of the kinase inhibitor H7, all conditions which also upregulate p53. In our experiments we were not able to see an increase in E2F-1 RNA production but did find an increase in protein stability in UVC-irradiated cells. Upregulation of E2F-1 in response to DNA damage seems to require the presence of wild-type p53, since we did not observe an increase in the level of E2F-1 protein in several cell lines which possess mutated p53. Previous experiments showed that p53 is upregulated after microinjection of an antibody which binds to a domain of Mdm2 that is required for the interaction of Mdm2 with p53. Microinjection of the same antibody also increases the expression of E2F-1 protein, while microinjection of a control antibody does not. Furthermore, microinjection of Mdm2 antisense oligonucleotides upregulates E2F-1 protein, while microinjection of an unrelated oligonucleotide does not. These data suggest that E2F-1 is upregulated in a similar way to p53 in response to DNA damage and that Mdm2 appears to play a major role in this pathway. (+info)
Transcriptional repression of the E2F-1 gene by interferon-alpha is mediated through induction of E2F-4/pRB and E2F-4/p130 complexes.
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E2F is a heterodimeric transcription factor composed of one of five E2F subunits (E2F-1 to E2F-5) and a DP subunit. E2F regulates the expression of several growth-promoting genes, and thus, can be a target of antiproliferative action of interferons (IFNs). In this study, we investigated the mechanisms whereby IFN-alpha suppresses transcription of the E2F-1 gene. Transfection studies revealed that E2F-1 promoter was functionally divided into two parts: upstream activation sequences (UAS) and a downstream negative-regulatory element (E2F-binding sites). When cells were proliferating, transcription of the E2F-1 gene was primarily driven by the UAS, while E2F sites were not involved in activation. IFN-alpha markedly reduced E2F-1 promoter activity, but introduction of non-binding mutation at the E2F sites completely abrogated the inhibition. Free E2F4 was found to be the predominant species bound to the E2F sites in proliferating cells. IFN-alpha induced upregulation of E2F-4 along with dephosphorylation of pRB and p130, which resulted in the formation of E2F-4/pRB and E2F-4/p130 complexes on the E2F-1 promoter. These complexes function as transcriptional repressors to inhibit E2F-1 mRNA expression. Our findings indicate that E2F-4 is a critical regulator of E2F-1, which offer an excellent paradigm for understanding functional diversity within the E2F family. (+info)
Balancing proliferation and apoptosis in vivo: the Goldilocks theory of E2F/DP action.
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Stimulation of both proliferation and apoptosis by E2F-1 provides a mechanistic basis for how E2F-1 functions as an oncogene and a tumor suppressor in vivo. In each normal tissue, a precise balance of proliferation versus apoptosis must be maintained, and in many tissues this appears to be controlled by E2F-1 levels. Presumably, variable expression of all E2F family members in each tissue dictates a tissue-specific sensitivity to loss or overexpression of any one family member. At sites where E2F-1 contributes mainly to proliferation and p53 levels remain low, loss of E2F-1 expression may lead to tissue atrophy and overexpression may lead to hyperplasia or tumors. Hence, E2F-1 would act as an oncogene. At other sites where E2F-1 levels induce p19, which stabilizes p53 leading to apoptosis, E2F-1 overexpression may lead to tissue atrophy and loss of expression may lead to hyperplasia or tumors. And thus, E2F-1 would act as a tumor suppressor. Perhaps it is the unique property of E2F-1 within the E2F family to stimulate both proliferation and apoptosis which makes it a bimodal switch that pRB must control so carefully. It is a delicate equilibrium that must be maintained throughout embryonic development and adult life. However, it is easy to envision that mutations which deregulate other E2F family members and which ultimately lead to changes in E2F-1 levels could lead to similar growth aberrations. In summary, although pRB interacts with numerous transcription factors, pRB minimally must restrain the E2F/DP transcription factor family to prevent the cell cycle from whirling onwards out of control. (+info)
Cell-autonomous and non-autonomous growth-defective mutants of Drosophila melanogaster.
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During animal development, growth of the various tissues and organs that make up the body must be coordinated. Despite recent progress in understanding growth control within the cell unit, the mechanisms that coordinate growth at the organismal level are still poorly understood. To study this problem, we performed a genetic screen for larval growth-defective mutants in Drosophila melanogaster. Characterization of these mutants revealed distinct types of larval growth defects. An allelic series for the translation initiation factor, Eif4A, showed different growth rates and suggests that Eif4A could be used as a dose-dependent growth regulator. Two mutants that fail to exit cellular quiescence at larval hatching (milou and eif4(1006)) have a DNA replication block that can be bypassed by overexpression of the E2F transcription factor. A mutation (bonsai) in a homolog of the prokaryotic ribosomal protein, RPS15, causes a growth defect that is non-cell-autonomous. Our results emphasize the importance of translational regulation for the exit from quiescence. They suggest that the level of protein synthesis required for cell cycle progression varies according to tissue type. The isolation of non-cell-autonomous larval growth-defective mutants suggests that specialized organs coordinate growth throughout the animal and provides new tools for studies of organismal growth regulation. (+info)