The E2F Family of Transcription Factors from Arabidopsis thaliana
2002; Elsevier BV; Volume: 277; Issue: 12 Linguagem: Inglês
10.1074/jbc.m110616200
ISSN1083-351X
AutoresLuisa Mariconti, Barbara Pellegrini, Rita Cantoni, Rebecca Stevens, Catherine Bergounioux, Rino Cella, Diego Albani,
Tópico(s)Cancer Research and Treatments
ResumoThe E2F transcription factors are key components of the cyclin D/retinoblastoma/E2F pathway. Here we demonstrate thatArabidopsis thaliana contains six functional AtE2F genes that are all expressed in cell suspension culture but show different patterns of expression during cell cycle progression. According to their structural and functional features, the six AtE2Fs can be divided into two distinct groups; although the three members of the first group, AtE2Fa, AtE2Fb and AtE2Fc, possess all the conserved domains found in other plant and animal E2Fs, the remaining AtE2Fs are novel proteins, which reveal a duplication of the DNA binding domain but lack any other conserved region. Furthermore, the AtE2Fs of the first group are functional transcription factors that in association with AtDP proteins can recognize specifically an E2F cis-element and can transactivate an E2F-responsive reporter gene in plant cells. In contrast, the AtE2Fs of the second group can bind specifically the E2F site without interacting with DP partners but cannot activate gene expression and, instead, are able to inhibit E2F-dependent activation of gene expression in Arabidopsis cells. These findings suggest distinctive roles for the plant E2F proteins and point to a complex concerted regulation of E2F-dependent gene expression in plant cells. The E2F transcription factors are key components of the cyclin D/retinoblastoma/E2F pathway. Here we demonstrate thatArabidopsis thaliana contains six functional AtE2F genes that are all expressed in cell suspension culture but show different patterns of expression during cell cycle progression. According to their structural and functional features, the six AtE2Fs can be divided into two distinct groups; although the three members of the first group, AtE2Fa, AtE2Fb and AtE2Fc, possess all the conserved domains found in other plant and animal E2Fs, the remaining AtE2Fs are novel proteins, which reveal a duplication of the DNA binding domain but lack any other conserved region. Furthermore, the AtE2Fs of the first group are functional transcription factors that in association with AtDP proteins can recognize specifically an E2F cis-element and can transactivate an E2F-responsive reporter gene in plant cells. In contrast, the AtE2Fs of the second group can bind specifically the E2F site without interacting with DP partners but cannot activate gene expression and, instead, are able to inhibit E2F-dependent activation of gene expression in Arabidopsis cells. These findings suggest distinctive roles for the plant E2F proteins and point to a complex concerted regulation of E2F-dependent gene expression in plant cells. retinoblastoma protein glutathione S-transferase electrophoretic mobility shift assay α-naphthaleneacetic acid 4-morpholineethanesulfonic acid β-glucuronidase cauliflower mosaic virus nuclear localization signal reverse transcription Recent studies have shown that the basic regulatory circuits governing cell cycle progression in animal cells are remarkably conserved in higher plants. In particular, plant cells possess all the key components of the cyclin D/retinoblastoma/E2F pathway, which in animal cells is a major regulator of cell proliferation and is part of a critical checkpoint controlling the progression from G1to S phase of the cell cycle (1Stals H. Inzé D. Trends Plant Science. 2001; 6: 359-364Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The cyclin D-cdk2 or cyclin D-cdk4 complexes regulate the activity of the pRB1 tumor suppressor protein and the related p107/p130 pocket proteins, which in their hypophosphorylated state bind to the E2F family of transcription factors and block their transactivating potential (2Black A.R. Azizkhan-Clifford J. Gene (Amst.). 1999; 237: 281-302Crossref PubMed Scopus (105) Google Scholar, 3Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (955) Google Scholar, 4Muller H. Helin K. Biochim. Biophys. Acta. 2000; 1470: M1-M12PubMed Google Scholar). Furthermore, the pocket proteins have been shown to recruit to the E2Fs chromatin remodeling activities, such as histone deacetylases, histone methyltransferase, or SWI·SNF complexes, which once tethered to the DNA can actively repress the transcription of E2F-regulated genes by blocking the access of other transcription factors in the proximity of the E2F binding sites (5Luo R.X. Postigo A.A. Dean D.C. Cell. 1998; 92: 463-473Abstract Full Text Full Text PDF PubMed Scopus (836) Google Scholar, 6Ferreira R. Magnaghi-Jaulin L. Robin P. Harel-Bellan A. Trouche D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10493-10498Crossref PubMed Scopus (224) Google Scholar, 7Vandel L. Nicolas E. Vaute O. Ferreira R. Ait-Si-Ali S. Trouche D. Mol. Cell. Biol. 2001; 21: 6484-6494Crossref PubMed Scopus (179) Google Scholar, 8Trouche D. Le Chalony C. Muchardt C. Yaniv M. Kouzarides T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11268-11273Crossref PubMed Scopus (260) Google Scholar, 9Zhang H.S. Gavin M. Dahiya A. Postigo A.A. Ma D. Luo R.X. Harbour J.W. Dean D.C. Cell. 2000; 101: 79-89Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar). It is now widely believed that in quiescent cell or during the early G1 phase of the cell cycle the E2Fs are mainly involved in the repression of several cell cycle-regulated promoters, whereas during the transition from G1 to S phase the release of transcriptionally active E2Fs, resulting from the phosphorylation of the pocket proteins, leads to an E2F-dependent activation of several genes coding for regulatory proteins and for enzymes involved in nucleotide and DNA synthesis. This dual function of the E2F complexes clearly explains why these transcription factors, depending on the cellular and developmental context, can be either positive or negative regulators of cell proliferation and can act as both oncogenes and tumor suppressors (2Black A.R. Azizkhan-Clifford J. Gene (Amst.). 1999; 237: 281-302Crossref PubMed Scopus (105) Google Scholar, 4Muller H. Helin K. Biochim. Biophys. Acta. 2000; 1470: M1-M12PubMed Google Scholar). Moreover, highlighting the remarkable functional complexity of animal E2Fs, in Drosophila the DmE2F1 protein has been shown to affect directly the localization and the DNA-replicating activity of the origin replication complex (10Bosco G. Du W. Orr-Weaver T.L. Nat. Cell Biol. 2001; 3: 289-295Crossref PubMed Scopus (202) Google Scholar) and in animal cells this family of proteins is believed to participate in the regulation of several cellular processes (3Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (955) Google Scholar). Indeed, according to the broad range of mammalian genes, which change their expression in response to the activation of some of the E2Fs, these transcription factors have been shown to be involved in the control of differentiation, development, proliferation, and apoptosis (11Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Crossref PubMed Scopus (628) Google Scholar). In mammalian cells some E2Fs have been actually shown to induce apoptosis through the regulation of p53 activity as well as in a p53-independent pathway (3Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (955) Google Scholar, 4Muller H. Helin K. Biochim. Biophys. Acta. 2000; 1470: M1-M12PubMed Google Scholar).The various E2F proteins can recognize specific DNAcis-elements forming heterodimers with partially related proteins called DP. So far, six E2Fs and two DPs have been found in human cells, and, according to a comparative analysis of the genome ofArabidopsis thaliana, at least six putative E2F genes and two DP genes appear to be present in Arabidopsis cells as well. Plant E2F genes have been described in carrot, tobacco, and wheat (12Albani D. Mariconti L. Ricagno S. Pitto L. Moroni C. Helin K. Cella R. J. Biol. Chem. 2000; 275: 19258-19267Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 13Ramirez-Parra E. Xie Q. Boniotti M.B. Gutierrez C. Nucleic Acids Res. 1999; 27: 3527-3533Crossref PubMed Scopus (99) Google Scholar, 14Sekine M. Ito M. Uemukai K. Maeda Y. Nakagami H. Shinmyo A. FEBS Lett. 1999; 460: 117-122Crossref PubMed Scopus (81) Google Scholar), and three of the Arabidopsis E2Fs have been recently described (15Magyar Z. De Atanassova A. Veylder L. Rombauts S. Inzé D. FEBS Lett. 2000; 486: 79-87Crossref PubMed Scopus (69) Google Scholar, 16de Jager S.M. Menges M. Bauer U.M. Murray J.A. Plant Mol. Biol. 2001; 47: 555-568Crossref PubMed Scopus (86) Google Scholar), whereas DP homologues have been reported in wheat and Arabidopsis (15Magyar Z. De Atanassova A. Veylder L. Rombauts S. Inzé D. FEBS Lett. 2000; 486: 79-87Crossref PubMed Scopus (69) Google Scholar, 17Ramirez-Parra E. Gutierrez C. FEBS Lett. 2000; 486: 73-78Crossref PubMed Scopus (49) Google Scholar). All the E2F proteins described so far possess a highly conserved DNA-binding domain, forming a winged helix motif, which is flanked toward the C-terminal side by a DP dimerization domain containing a leucine heptad repeat. Next to the dimerization domain, all the E2Fs possess another conserved region called marked box, which in human cells is recognized by the adenovirus E4 protein and may be involved in heterodimerization and DNA bending (18Vidal M. Braun P. Chen E. Boeke J.D. Harlow E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10321-10326Crossref PubMed Scopus (147) Google Scholar, 19Cress W.D. Nevins J.R. Mol. Cell. Biol. 1996; 16: 2119-2127Crossref PubMed Scopus (59) Google Scholar). Based on their primary structures and on their functional features, the six mammalian E2Fs can be divided into three groups. E2F1, E2F2, and E2F3, which belong to the first group, possess a conserved cyclin A-binding domain in their N-terminal region and a transactivating domain, overlapping a conserved pRB-binding region, at their C-terminal end. These three E2Fs are believed to be direct activators of S phase genes; accordingly, their expression is not detectable in quiescent cells but it is strongly up-regulated during G1/S transition. In contrast, E2F4 and E2F5, the members of the second group, are expressed in quiescent cells as well as throughout the cell cycle and lack the cyclin-A-binding domains but possess pRB-binding and transactivating C-terminal regions. E2F6, the last known member of the mammalian E2F family, lacks the N-terminal cyclin-A binding domain as well as the transactivating C-terminal region and it has been shown to bind directly polycomb group proteins and to act as a repressor of E2F-dependent transcriptional activation (20Trimarchi J.M. Fairchild B. Wen J. Lees J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1519-1524Crossref PubMed Scopus (214) Google Scholar). The plant E2Fs described so far, do not possess evident cyclin A-binding domains in the N-terminal region but show a conserved pRB-binding domain in their C-terminal region. In this respect, wheat, tobacco, and Arabidopsis E2Fs have been shown to interact with plant pRBR proteins (13Ramirez-Parra E. Xie Q. Boniotti M.B. Gutierrez C. Nucleic Acids Res. 1999; 27: 3527-3533Crossref PubMed Scopus (99) Google Scholar, 14Sekine M. Ito M. Uemukai K. Maeda Y. Nakagami H. Shinmyo A. FEBS Lett. 1999; 460: 117-122Crossref PubMed Scopus (81) Google Scholar, 16de Jager S.M. Menges M. Bauer U.M. Murray J.A. Plant Mol. Biol. 2001; 47: 555-568Crossref PubMed Scopus (86) Google Scholar) and the carrot E2F factor has been shown to be a transcriptional activator that, heterodimerizing with a DP partner, can bind a consensus E2Fcis-element and can transactivate a synthetic E2F-responsive promoter in both plant and animal cells (12Albani D. Mariconti L. Ricagno S. Pitto L. Moroni C. Helin K. Cella R. J. Biol. Chem. 2000; 275: 19258-19267Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In this work we report on the isolation and functional characterization of the cDNA clones of all six E2Fs of A. thaliana. Although the previously described AtE2F sequences show the typical features of other plant and animal E2F proteins, the three remaining clones reveal unique structural features and possess duplicated conserved DNA-binding domains but lack any of the additional regions which are conserved in other plant and animal E2Fs. These Arabidopsis E2F genes are all expressed in cell suspension culture but show different patterns of expression during cell cycle progression. Furthermore, DNA binding assays and transactivation experiments suggest distinctive roles for these plant E2F proteins pointing to a complex concerted regulation of E2F-dependent gene expression in plant cells.DISCUSSIONThe discovery of at least six E2F genes in A. thaliana, all of which are differentially expressed during cell cycle progression, suggests that, in plant cells, like in mammalian cells, the activity of the pRB/E2F pathway relies on the concerted regulation of several E2F members. Furthermore, according to their structural and functional characteristics, these six AtE2Fs can be divided into two distinctive groups. The AtE2Fa, AtE2Fb, and AtE2Fc proteins possess all the typical regions that are conserved in other plant E2Fs and are able to bind efficiently to a E2F site forming heterodimers with AtDP proteins. Additionally, as previously described for carrot DcE2F (12Albani D. Mariconti L. Ricagno S. Pitto L. Moroni C. Helin K. Cella R. J. Biol. Chem. 2000; 275: 19258-19267Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), these three AtE2Fs can transactivate a synthetic E2F-responsive promoter in plant cells, confirming their ability to act as positive regulators of gene expression. However, these studies have shown that the three members of the first group possess different transactivating capabilities. Even though AtE2Fa is able to bind canonical DNA target sites interacting in vitro with either AtDPa or AtDPb, it can transactivate slightly the E2F-responsive reporter gene only when it is coexpressed with AtDPa. In this respect, it is known that in human cells the activity of E2F4 and E2F5, which lack NLS sequences, is regulated at the level of subcellular localization. Coexpression of E2F4 together with human DP2, which possess a functional NLS, but not with DP1, has been shown to enable its nuclear translocation (27Magae J. Wu C.L. Illenye S. Harlow E. Heintz N.H. J. Cell Sci. 1996; 109: 1717-1726Crossref PubMed Google Scholar, 28de la Luna S. Burden M.J. Lee C.W. La Thangue N.B. J. Cell Sci. 1996; 109: 2443-2452Crossref PubMed Google Scholar) and its transactivation of a E2F-responsive reporter construct (29Lindeman G.J. Gaubatz S. Livingston D.M. Ginsberg D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5095-5100Crossref PubMed Scopus (164) Google Scholar,30Verona R. Moberg K. Estes S. Starz M. Vernon J.P. Lees J.A. Mol. Cell. Biol. 1997; 17: 7268-7282Crossref PubMed Scopus (178) Google Scholar). Concerning the Arabidopsis E2F·DP complexes, it is not known whether the activity of the various AtE2F and AtDP proteins is regulated at the level of their subcellular localization but it is interesting to note that whereas AtE2Fa possesses a putative nuclear localization signal in its N terminus, this sequence is only partially conserved in the AtE2Fb and AtE2Fc proteins. It remains to be confirmed whether this region is actually involved in the nuclear targeting of these proteins.In this study we have also demonstrated that Arabidopsiscells possess three novel and remarkable E2F members, called AtE2Fd, AtE2Fe, and AtE2Ff, that contain duplicated DNA-binding domains. This feature enables these proteins to recognize a consensus E2F site independently of a DP partner, and their DNA binding specificity proves that these factors are actual components of the ArabidopsispRB/E2F pathway. However, although they are likely to be targeted to the nucleus, these AtE2Fs cannot activate gene expression; instead, they appear to be able to interfere with the activity of the other AtE2F members, possibly by competing for the same DNA target sites. This effect is particularly clear with the AtE2Fd effector and is less pronounced with AtE2Ff. The latter factor, however, lacks part of the second DNA-binding domain, and EMSA analyses indicated that it binds less efficiently to the canonical E2F site. Furthermore, AtE2Ff lacks also the putative NLS sequence, which is located near the C terminus of both AtE2Fd and AtE2Fe, and its translocation to the nucleus could occur differently.In mammalian cells, depending on their interaction with pocket proteins, the various E2Fs can behave as activators or as repressors of gene transcription. The remarkably different pattern of expression of the various AtE2F genes during the passage from the G1 to the G2 phase of cell cycle indicates that also inArabidopsis cells at least some of the E2F activities are likely to be involved in both activation or repression of S phase-specific gene expression. This possibility has been already suggested for the carrot DcE2F protein, which is expressed ubiquitously in all plant tissues (12Albani D. Mariconti L. Ricagno S. Pitto L. Moroni C. Helin K. Cella R. J. Biol. Chem. 2000; 275: 19258-19267Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). A dual function of the plant E2F activities has also been suggested by the recent studies of two plant E2F-regulated promoters. In both the tobacco RNR2 promoter and the Nicotiana benthamiana PCNApromoter, two distinct E2F binding sites have been identified, and in both promoters the functional disruption of the downstream site has been shown to increase the transcriptional activity in either cycling cells or in mature leaves, respectively (31Chaboute M.E. Clement B. Sekine M. Philipps G. Chaubet-Gigot N. Plant Cell. 2000; 12: 1987-2000Crossref PubMed Scopus (85) Google Scholar, 32Egelkrout E.M. Robertson D. Hanley-Bowdoin L. Plant Cell. 2001; 13: 1437-1452Crossref PubMed Scopus (98) Google Scholar).The three AtE2Fs of the first group are good candidates for pocket protein-mediated control of E2F activity because they possess a conserved pRBR-binding region in their C terminus. In this context, AtE2Fa and AtE2Fb have been recently shown to interact with pRBR proteins in a yeast two-hybrid system (16de Jager S.M. Menges M. Bauer U.M. Murray J.A. Plant Mol. Biol. 2001; 47: 555-568Crossref PubMed Scopus (86) Google Scholar). Additionally, wheat TmE2F and tobacco NtE2F have been shown to bind plant pocket proteins and, in the case of the wheat factor, this interaction is strictly dependent on the presence of the conserved C-terminal region (13Ramirez-Parra E. Xie Q. Boniotti M.B. Gutierrez C. Nucleic Acids Res. 1999; 27: 3527-3533Crossref PubMed Scopus (99) Google Scholar, 14Sekine M. Ito M. Uemukai K. Maeda Y. Nakagami H. Shinmyo A. FEBS Lett. 1999; 460: 117-122Crossref PubMed Scopus (81) Google Scholar). These findings contribute to link the activity of the plant E2Fs to the mode of action of plant DNA viruses, which encode proteins that interact with pRBR proteins (33Gutierrez C. Plant Mol. Biol. 2000; 43: 763-772Crossref PubMed Google Scholar). On the other hand, the AtE2Fd, AtE2Fe, and AtE2Ff proteins lack the pRBR-binding domain and therefore are unlikely to be subjected to a control by pocket proteins. A similar situation is seen with the mammalian E2F6 protein, which is also not regulated by pocket proteins. However, unlike the three AtE2Fs of the second group, E2F6 binds a DP partner and requires this interaction to recognize efficiently E2F consensus sites. Moreover, E2F6 has been shown to actively repress gene expression by interacting directly with polycomb group proteins, which recognize specifically its marked box region (20Trimarchi J.M. Fairchild B. Wen J. Lees J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1519-1524Crossref PubMed Scopus (214) Google Scholar). The AtE2Fd, AtE2Fe, and AtE2Ff proteins do not contain a marked box region, and, although the first two members of this group possess discrete C-terminal regions that could potentially harbor unknown functional domains, the AtE2Ff protein is truncated in the second DNA-binding domain and does not appear to possess other discrete regions outside those potentially involved in DNA recognition. Hence, the AtE2Ff protein and possibly the other AtE2Fs of the second group might not be able to recruit repressing activities to the E2F-regulated promoters, as in the case of human E2F6, but could act as dominant inhibitors of E2F-dependent gene activation by simply blocking the access of other AtE2Fs to shared DNA target sites.Although the distribution of the various AtE2F proteins inArabidopsis cells remains to be verified, considering their functional features and in view of the expression pattern of their transcripts, we can envision a cascade of events that could contribute to the regulation of the pRB/E2F pathway during the cell cycle in plants. More specifically, the peak of accumulation of the AtE2Fb transcripts at the G1/S transition and their constitutive distribution during cell cycle progression suggest that this factor, similarly to the mammalian E2F4 and E2F5 proteins, could be a key target of the pRBR proteins in early G1. Its eventual release, following the phosphorylation of the pocket proteins, could lead to the subsequent activation of the AtE2Fa gene, which is maximally expressed in early S phase. On the other hand, we speculate that AtE2Fa, like the mammalian E2F1, E2F2, and E2F3 factors, could be a direct and specific activator of S phase genes, whereas AtE2Fc could have important regulatory functions during both the S phase and the following G2 phase. Finally, the AtE2F members of the second group are clearly adding a new level of complexity to the control of E2F-dependent gene expression by possibly inhibiting, in different phases of the plant cell cycle, the DNA binding capacity of the first group of AtE2Fs. In this respect, it is worth noting that in mammalian cells overexpression of a partial E2F protein containing only the DNA-binding domain or introduction of peptides that antagonize E2F DNA binding have been shown to trigger apoptosis in a p53-independent manner (34Hsieh J.K. Fredersdorf S. Kouzarides T. Martin K. Lu X. Genes Dev. 1997; 11: 1840-1852Crossref PubMed Scopus (263) Google Scholar, 35Phillips A.C. Bates S. Ryan K.M. Helin K. Vousden K.H. Genes Dev. 1997; 11: 1853-1863Crossref PubMed Scopus (242) Google Scholar, 36Bandara L.R. Girling R. La Thangue N.B. Nat. Biotechnol. 1997; 15: 896-901Crossref PubMed Scopus (34) Google Scholar). Whether or not any of the AtE2F members is involved in the control of apoptosis in plants, it is evident, from the data obtained so far, that they are likely to be complex regulators of the plant cell cycle. Future investigations will help to verify the proposed model of AtE2F activity and will reveal whether in plants, like in animals, these transcription factors are involved in the control of other cellular processes and/or can regulate unique plant developmental programs. Recent studies have shown that the basic regulatory circuits governing cell cycle progression in animal cells are remarkably conserved in higher plants. In particular, plant cells possess all the key components of the cyclin D/retinoblastoma/E2F pathway, which in animal cells is a major regulator of cell proliferation and is part of a critical checkpoint controlling the progression from G1to S phase of the cell cycle (1Stals H. Inzé D. Trends Plant Science. 2001; 6: 359-364Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The cyclin D-cdk2 or cyclin D-cdk4 complexes regulate the activity of the pRB1 tumor suppressor protein and the related p107/p130 pocket proteins, which in their hypophosphorylated state bind to the E2F family of transcription factors and block their transactivating potential (2Black A.R. Azizkhan-Clifford J. Gene (Amst.). 1999; 237: 281-302Crossref PubMed Scopus (105) Google Scholar, 3Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (955) Google Scholar, 4Muller H. Helin K. Biochim. Biophys. Acta. 2000; 1470: M1-M12PubMed Google Scholar). Furthermore, the pocket proteins have been shown to recruit to the E2Fs chromatin remodeling activities, such as histone deacetylases, histone methyltransferase, or SWI·SNF complexes, which once tethered to the DNA can actively repress the transcription of E2F-regulated genes by blocking the access of other transcription factors in the proximity of the E2F binding sites (5Luo R.X. Postigo A.A. Dean D.C. Cell. 1998; 92: 463-473Abstract Full Text Full Text PDF PubMed Scopus (836) Google Scholar, 6Ferreira R. Magnaghi-Jaulin L. Robin P. Harel-Bellan A. Trouche D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10493-10498Crossref PubMed Scopus (224) Google Scholar, 7Vandel L. Nicolas E. Vaute O. Ferreira R. Ait-Si-Ali S. Trouche D. Mol. Cell. Biol. 2001; 21: 6484-6494Crossref PubMed Scopus (179) Google Scholar, 8Trouche D. Le Chalony C. Muchardt C. Yaniv M. Kouzarides T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11268-11273Crossref PubMed Scopus (260) Google Scholar, 9Zhang H.S. Gavin M. Dahiya A. Postigo A.A. Ma D. Luo R.X. Harbour J.W. Dean D.C. Cell. 2000; 101: 79-89Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar). It is now widely believed that in quiescent cell or during the early G1 phase of the cell cycle the E2Fs are mainly involved in the repression of several cell cycle-regulated promoters, whereas during the transition from G1 to S phase the release of transcriptionally active E2Fs, resulting from the phosphorylation of the pocket proteins, leads to an E2F-dependent activation of several genes coding for regulatory proteins and for enzymes involved in nucleotide and DNA synthesis. This dual function of the E2F complexes clearly explains why these transcription factors, depending on the cellular and developmental context, can be either positive or negative regulators of cell proliferation and can act as both oncogenes and tumor suppressors (2Black A.R. Azizkhan-Clifford J. Gene (Amst.). 1999; 237: 281-302Crossref PubMed Scopus (105) Google Scholar, 4Muller H. Helin K. Biochim. Biophys. Acta. 2000; 1470: M1-M12PubMed Google Scholar). Moreover, highlighting the remarkable functional complexity of animal E2Fs, in Drosophila the DmE2F1 protein has been shown to affect directly the localization and the DNA-replicating activity of the origin replication complex (10Bosco G. Du W. Orr-Weaver T.L. Nat. Cell Biol. 2001; 3: 289-295Crossref PubMed Scopus (202) Google Scholar) and in animal cells this family of proteins is believed to participate in the regulation of several cellular processes (3Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (955) Google Scholar). Indeed, according to the broad range of mammalian genes, which change their expression in response to the activation of some of the E2Fs, these transcription factors have been shown to be involved in the control of differentiation, development, proliferation, and apoptosis (11Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Crossref PubMed Scopus (628) Google Scholar). In mammalian cells some E2Fs have been actually shown to induce apoptosis through the regulation of p53 activity as well as in a p53-independent pathway (3Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (955) Google Scholar, 4Muller H. Helin K. Biochim. Biophys. Acta. 2000; 1470: M1-M12PubMed Google Scholar). The various E2F proteins can recognize specific DNAcis-elements forming heterodimers with partially related proteins called DP. So far, six E2Fs and two DPs have been found in human cells, and, according to a comparative analysis of the genome ofArabidopsis thaliana, at least six putative E2F genes and two DP genes appear to be present in Arabidopsis cells as well. Plant E2F genes have been described in carrot, tobacco, and wheat (12Albani D. Mariconti L. Ricagno S. Pitto L. Moroni C. Helin K. Cella R. J. Biol. Chem. 2000; 275: 19258-19267Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 13Ramirez-Parra E. Xie Q. Boniotti M.B. Gutierrez C. Nucleic Acids Res. 1999; 27: 3527-3533Crossref PubMed Scopus (99) Google Scholar, 14Sekine M. Ito M. Uemukai K. Maeda Y. Nakagami H. Shinmyo A. FEBS Lett. 1999; 460: 117-122Crossref PubMed Scopus (81) Google Scholar), and three of the Arabidopsis E2Fs have been recently described (15Magyar Z. De Atanassova A. Veylder L. Rombauts S. Inzé D. FEBS Lett. 2000; 486: 79-87Crossref PubMed Scopus (69) Google Scholar, 16de Jager S.M. Menges M. Bauer U.M. Murray J.A. Plant Mol. Biol. 2001; 47: 555-568Crossref PubMed Scopus (86) Google Scholar), whereas DP homologues have been reported in wheat and Arabidopsis (15Magyar Z. De Atanassova A. Veylder L. Rombauts S. Inzé D. FEBS Lett. 2000; 486: 79-87Crossref PubMed Scopus (69) Google Scholar, 17Ramirez-Parra E. Gutierrez C. FEBS Lett. 2000; 486: 73-78Crossref PubMed Scopus (49) Google Scholar). All the E2F proteins described so far possess a highly conserved DNA-binding domain, forming a winged helix motif, which is flanked toward the C-terminal side by a DP dimerization domain containing a leucine heptad repeat. Next to the dimerization domain, all the E2Fs possess another conserved region called marked box, which in human cells is recognized by the adenovirus E4 protein and may be involved in heterodimerization and DNA bending (18Vidal M. Braun P. Chen E. Boeke J.D. Harlow E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10321-10326Crossref PubMed Scopus (147) Google Scholar, 19Cress W.D. Nevins J.R. Mol. Cell. Biol. 1996; 16: 2119-2127Crossref PubMed Scopus (59) Google Scholar
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