Deregulated E2F transcriptional activity in autonomously growing melanoma cells.
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Inactivation of the retinoblastoma tumor suppressor protein (pRb) has been implicated in melanoma cells, but the molecular basis for this phenotype has not yet been elucidated, and the status of additional family members (p107 and p130, together termed pocket proteins) or the consequences on downstream targets such as E2F transcription factors are not known. Because cell cycle progression is dependent on the transcriptional activity of E2F family members (E2F1-E2F6), most of them regulated by suppressive association with pocket proteins, we characterized E2F-pocket protein DNA binding activity in normal versus malignant human melanocytes. By gel shift analysis, we show that in mitogen-dependent normal melanocytes, external growth factors tightly controlled the levels of growth-promoting free E2F DNA binding activity, composed largely of E2F2 and E2F4, and the growth-suppressive E2F4-p130 complexes. In contrast, in melanoma cells, free E2F DNA binding activity (E2F2 and E2F4, to a lesser extent E2F1, E2F3, and occasionally E2F5), was constitutively maintained at high levels independently of external melanocyte mitogens. E2F1 was the only family member more abundant in the melanoma cells compared with normal melanocytes, and the approximately fivefold increase in DNA binding activity could be accounted for mostly by a similar increase in the levels of the dimerization partner DP1. The continuous high expression of cyclin D1, A2, and E, the persistent cyclin-dependent kinase 4 (CDK4) and CDK2 activities, and the presence of hyperphosphorylated forms of pRb, p107, and p130, suggest that melanoma cells acquired the capacity for autonomous growth through inactivation of all three pocket proteins and release of E2F activity, otherwise tightly regulated in normal melanocytes by external growth factors. (+info)
Expression of the E2F family of transcription factors during murine development.
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The E2F family of transcription factors plays a crucial role in the control of cell cycle progression and regulation of cellular proliferation, both processes fundamental to mammalian development. In the present study, we have examined the levels of expression of the six currently identified E2F proteins in murine embryos/fetuses as a function of gestational age, compared the expression of these six proteins in selected developing and adult tissues, and examined E2F expression in the embryonic murine palate, a tissue in which perturbation of proliferation is associated with induction of cleft palate. Our results indicate that: 1) multiple forms of individual E2F family members are present in embryonic, fetal and adult cells/tissues; 2) each of the six E2Fs is expressed in a tissue specific manner in both adult and embryonic/fetal organs; 3) certain forms of individual E2F family members are preferentially detected in adult tissues, whereas others are preferentially expressed in embryonic/fetal tissues; 4) expression of the various E2Fs and their isoforms follows distinct temporal patterns during murine gestation; and 5) individual E2F family members also exhibit differential patterns of temporal expression during murine palatogenesis. (+info)
Activity of the human cytochrome c1 promoter is modulated by E2F.
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The human cytochrome c(1) promoter is strongly activated in transfected Drosophila SL2 cells expressing exogenous human E2F1. Transfection-deletion experiments, DNase I protection by E2F1 and gel mobility-shift experiments locate E2F1 activation sites to two regions on either side of the transcription start site. Deletion of either region prevents E2F1 activation in transfected SL2 cells, suggesting a co-operative interaction between them. E2F6, a member of the E2F family that lacks transactivation domains but contains specific suppressor domains, inhibits cytochrome c(1) promoter activity when co-transfected into HeLa cells, indicating that the E2F proteins modulate the cytochrome c(1) promoter in mammalian cells. However, E2F is not a general regulator of oxidative phosphorylation genes since three additional nuclear-encoded mitochondrial genes were unaffected by E2F1 or E2F6. (+info)
E2F transcription factors are differentially expressed in murine gametes and early embryos.
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We have examined the murine genes encoding transcription factors E2F1, -3, -5 and -6 in gametes and early embryos. All genes are expressed as maternal transcripts and all are efficiently transcribed after the blastocyst stage. Between those two stages, each E2F mRNA is transcribed with a distinctive and unique pattern. E2F proteins are also differentially expressed and compartmentalized in pre-implantation embryos. (+info)
Caspase-dependent apoptosis by ectopic expression of E2F-4.
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E2F is a family of transcription factors which regulates cell cycle and apoptosis of mammalian cells. E2F-1-3 localize in the nucleus, and preferentially bind pRb, while E2F-4 and 5 have no nuclear localization signal and preferentially bind p107/p130. E2F-6 suppresses the transcriptional activity of other E2F proteins. DP-1 and 2 are heterodimeric partners of each E2F protein. Using tetracycline-responsive promoters, here we compared the effects of ectopic expression of E2F-1, DP-1 and E2F-4 on cell cycle progression and apoptosis in Chinese hamster cell lines. We found that E2F-4, as well as DP-1 and E2F-1, induced growth arrest and caspase-dependent apoptosis. E2F-4 did not have a marked effect on cell cycle progression, while E2F-1 induced DNA synthesis of resting cells and DP-1 arrested cells in G1. Ectopic expression of E2F-4 did not activate E2F-dependent transcription. Our results suggest that expression of E2F-4 at elevated levels induces growth arrest and apoptosis of mammalian cells through a mechanism distinct from E2F-1 and DP-1. (+info)
The E2F6 transcription factor is a component of the mammalian Bmi1-containing polycomb complex.
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The E2F transcription factors play a key role in the regulation of cellular proliferation and terminal differentiation. E2F6 is the most recently identified and the least well understood member of the E2F family. It is only distantly related to the other E2Fs and lacks the sequences responsible for both transactivation and binding to the retinoblastoma protein. Consistent with this finding, E2F6 can behave as a dominant negative inhibitor of the other E2F family members. In this study, we continue to investigate the possible role(s) of E2F6 in vivo. We report the isolation of RYBP, a recently identified member of the mammalian polycomb complex, as an E2F6-interacting protein. Mapping studies indicate that RYBP binds within the known "repression domain" of E2F6. Moreover, we demonstrate that endogenous E2F6 and polycomb group proteins, including RYBP, Ring1, MEL-18, mph1, and the oncoprotein Bmi1, associate with one another. These findings suggest that the biological properties of E2F6 are mediated through its ability to recruit the polycomb transcriptional repressor complex. (+info)
p19ARF targets certain E2F species for degradation.
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p19ARF suppresses the growth of cells lacking p53 through an unknown mechanism. p19ARF was found to complex with transcription factors E2F1, -2, and -3. Levels of endogenous or ectopically expressed E2F1, -2, and -3, but not E2F6, were reduced after synthesis of p19ARF, through a mechanism involving increased turnover. p19ARF-induced degradation of E2F1 depended on a functional proteasome, and E2F1 was relocalized to nucleoli when coexpressed with p19ARF. Consistent with reduced levels of E2F1 and E2F3, the proliferation of cells defective for p53 function was suppressed by p19ARF, and the effect was partially reversed by ectopic overexpression of E2F1. These results suggest a broader role for p19ARF as a tumor suppressor, in which targeting of certain E2F species may cooperate with stimulation of the p53 pathway to counteract oncogenic growth signals. (+info)
A complex with chromatin modifiers that occupies E2F- and Myc-responsive genes in G0 cells.
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E2F-6 contributes to gene silencing in a manner independent of retinoblastoma protein family members. To better elucidate the molecular mechanism of repression by E2F-6, we have purified the factor from cultured cells. E2F-6 is found in a multimeric protein complex that contains Mga and Max, and thus the complex can bind not only to the E2F-binding site but also to Myc- and Brachyury-binding sites. Moreover, the complex contains chromatin modifiers such as a novel histone methyltransferase that modifies lysine 9 of histone H3, HP1gamma, and Polycomb group (PcG) proteins. The E2F-6 complex preferentially occupies target promoters in G0 cells rather than in G1 cells. These data suggest that these chromatin modifiers contribute to silencing of E2F- and Myc-responsive genes in quiescent cells. (+info)