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American Society of Plant Biologists
Ins and Outs of E2Fsneckardt{at}aspb.org Precise control of the mitotic cell cycle is a critical feature of eukaryotic life. In humans, mutations in cell cycle regulatory genes lead to the uncontrolled cell proliferation and tumor formation associated with the most dreaded of all diseases, cancer. E2F transcription factors are key components of an elaborate mechanism of cell cycle control in higher eukaryotes. For example, the promoter regions of numerous cell cycle genes that are active during S-phase (S-phase regulatory genes and genes associated with DNA replication and cell proliferation) contain multiple E2F binding sites that are involved in the repression or activation of transcription at different stages of development and in different organs or tissues.
E2F regulation involves the interaction of E2F proteins with another key cell cycle regulator, the Retinoblastoma (Rb) protein (reviewed by Harbour and Dean, 2000
The activities of cyclin-dependent kinases (CDKs) and CDK inhibitors exert major control over transitions from one cell cycle phase to the next in all eukaryotes. In higher eukaryotes that contain an Rb–E2F pathway, CDK-dependent phosphorylation of Rb in mid to late G1 disrupts the binding of Rb to E2F, thereby relieving the repression of E2F-regulated genes that are required for S-phase. However, not all E2Fs are created equal, and this simple view may require revision as we discover more about the expression patterns and functions of the various E2F proteins. For example, of the six human E2Fs, E2F-1, E2F-2, and E2F-3 are involved in cell cycle progression to S-phase (Lavia and Jansen-Dürr, 1999
There is growing evidence that Rb-related (RBR) and E2F proteins are involved in cell cycle regulation in both plants and animals. RBR genes have been identified in several plant species and in Chlamydomonas (Gutierrez et al., 2002  ). The Arabidopsis genome contains a single RBR gene (Kong et al., 2000; Vandepoele et al., 2002), whereas maize has at least two RBR genes (Ach et al., 1997). Although little is know about plant RBR function, plant RBRs contain marked domain conservation with their animal counterparts and some similarity in binding characteristics, and it is likely that RBR–E2F interactions play an important role in the regulation of the plant cell cycle (reviewed by Gutierrez, 1998; Gutierrez et al., 2002). Two articles in this issue of The Plant Cell, by del Pozo et al. (pages 3057–3071) and Egelkrout et al. (pages 3225–3236), shed additional light on E2F trans-acting factors and cis-acting elements in plants.
Animal E2F proteins heterodimerize with members of another family of proteins called dimerization partners (DPs), and dimerization is necessary for efficient DNA binding. Arabidopsis contains six E2F genes (Kosugi and Osahi, 2002a , 2002b; Mariconti et al., 2002) and two DP genes (Magyar et al., 2000). Three of the E2F genes, AtE2Fa, AtE2Fb, and AtE2Fc (also known as AtE2F3, AtE2F1, and AtE2F2, respectively), share conserved domains with animal E2Fs, including a DNA binding domain, a Leu zipper dimerization domain, a "marked box," and an Rb binding domain. The proteins encoded by these three genes also contain putative transactivation domains at the C terminus that show low homology with the human E2F1 transactivation domain (de Jager et al., 2001), and each has shown (to varying degrees) transactivation potential when cotransfected into plant cells with a reporter gene containing E2F binding sites in a minimal promoter (Kosugi and Osahi, 2002a; Mariconti et al., 2002). It also has been shown that the interaction of these three E2Fs with one of the two DP proteins (AtDPa or AtDPb) is required for DNA binding (Kosugi and Osahi, 2002a). The remaining three E2F-like proteins, AtE2Fd, AtE2Fe, and AtE2Ff, contain a duplicated DNA binding domain and none of the other conserved E2F domains; these proteins appear to function as repressors of E2F-regulated genes independently of RBR and DP proteins (Kosugi and Osahi, 2002b; Mariconti et al., 2002).
Despite some evidence for the transactivation potential of AtE2Fc (Kosugi and Osahi, 2002a ; Mariconti et al., 2002), the weight of evidence suggests that AtE2Fc functions principally as a repressor. In yeast one-hybrid assays, AtE2Fa and AtE2Fb have exhibited strong transactivation potential, whereas AtE2Fc showed no activity (de Jager et al., 2001; Kosugi and Osahi, 2002a). Kosugi and Osahi (2002a) further showed that in vitro–translated AtE2Fc exhibited only minor transactivation capability in transfected plant cells, likely because of a weak or nonfunctional transactivation domain, in contrast to AtE2Fa and AtE2Fb, which demonstrated potent transactivation potential. These authors hypothesized that the E2Fc-DPb complex functions as a negative regula-tor of E2F-regulated genes (Kosugi and Osahi, 2002a).
The results presented by del Pozo et al. (2002)
In localization studies, del Pozo et al. (2002)
In animal cells, the abundance of E2F1 is regulated by targeted degradation via the ubiquitin–proteasome pathway (Marti et al., 1999 ). del Pozo et al. (2002) found that AtE2Fc increased in abundance significantly in cultured cells after the addition of the proteasome inhibitor MG132, suggesting that AtE2Fc stability also is regulated by proteasome-mediated degradation. In addition, an E2Fc-GUS fusion protein expressed from the AtE2Fc promoter showed high GUS expression in dark-grown seedlings that decreased dramatically in a proteasome-dependent manner after the seedlings were transferred to light. These results suggest that the abundance of AtE2Fc is regulated, in part, by ubiquitin–proteasome degradation in response to changes in light.
Targeted protein degradation via the ubiquitin–proteasome pathway involves the action of an F-box protein that forms part of the SCF (Skp1-Cdc53/Cullin-F-box protein) ubiquitin ligase complex and that binds and "recruits" phosphorylated protein targets for proteasome-mediated degradation (reviewed by Pickart, 2001
A number of plant cell cycle genes contain E2F binding sites in their promoter regions, including rice and tobacco PROLIFERATING CELL NUCLEAR ANTIGEN (PCNA), the tobacco ribonucleotide reductase genes RNR2 and RNR1b, and the Arabidopsis CDC6 and telomerase genes (Gutierrez et al., 2002 ). Egelkrout et al. (2002) examined the activity of two E2F cis elements (named E2F1 and E2F2) in the promoter of a Nicotiana benthamiana PCNA gene, which encodes a DNA polymerase  processivity factor required for DNA replication. Many E2F-regulated genes contain at least two E2F elements in their promoters. Egelkrout et al. (2002) provide evidence that these sites may act either synergistically or antagonistically to regulate transcription at different stages of development. In binding assays using N. benth-amiana nuclear extracts and recombinant Arabidopsis E2F proteins, they show that the E2F1 and E2F2 elements exhibit different binding specificities. Next, they conducted a series of experiments in which N. benthamiana plants were transformed to express the luciferase reporter gene under the control of the N. benthamiana PCNA promoter containing various mutations in the E2F elements. Reporter activity was examined for constructs with mutations in E2F1, E2F2, or both elements in young versus mature leaf tissue. The results showed a significant increase in reporter activity for all combinations of mutations relative to the wild type in mature leaves, suggesting that both elements function as repressors in mature leaf tissue. By contrast, in young leaves, the activities of promoters with mutations in the E2F2 element alone or in both the E2F1 and E2F2 elements were similar to those in the wild type, and the activity of the promoter with mutations only in the E2F1 element was decreased relative to the wild type. Overall, these results suggested that both E2F elements, and principally E2F2, contribute to the repression of PCNA expression in mature leaves, whereas the E2F1 element plays a role in promoting PCNA expression in young leaves. These findings also are consistent with the results of previous experiments that showed high PCNA expression in young N. benthamiana leaves and low expression in mature leaves (Egelkrout et al., 2001).
Geminiviruses are small DNA viruses that rely on plant DNA replication machinery to amplify their genomes. In recent years, it has become clear that geminiviruses and mammalian DNA tumor viruses use similar mechanisms to alter host transcriptional controls to induce the synthesis of DNA replication enzymes in quiescent cells. A previous report demonstrated that the geminivirus Tomato golden mosaic virus (TGMV) activates the transcription of the PCNA promoter in mature N. benthamiana leaves (Egelkrout et al., 2001 ). In this issue, Egelkrout et al. (2002) show that the E2F binding sites in the N. benthamiana PCNA promoter are essential for TGMV-mediated activation. The TGMV replication protein AL1 (or Rep) is sufficient to induce PCNA expression in differentiated plant cells (Nagar et al., 1995). AL1 binds to the RBR protein (Ach et al., 1997), and this interaction is required for PCNA induction in mesophyll and epidermal cells of mature leaves (Kong et al., 2000). These observations are consistent with a model in which TGMV relieves the repression of the PCNA promoter by interacting with RBR and disrupting the E2F-RBR complexes bound to the E2F elements in the promoter. The ability of TGMV to induce chromosomal DNA replication in mature plant cells, as described in this issue by Nagar et al. (pages 2995–3007), may reflect perturbation of the E2F-RBR regulatory network leading to altered host transcriptional and cell cycle controls.
Together, the studies of Egelkrout et al. (2002)
Ach, R.A., Durfee, T., Miller, A.B., Taranto, P., Hanley-Bowdoin, L., Zambriski, P.C., and Gruissem, W. (1997). RRB1 and RRB2 encode maize retinoblastoma-related proteins that interact with a plant D-type cyclin and geminivirus replication protein. Mol. Cell. Biol. 17, 5077–5086.[Abstract] Cartwright, P., Muller, H., Wagener, C., Holm, K., and Helin, K. (1998). E2F-6: A novel member of the E2F family is an inhibitor of E2F-dependent transcription. Oncogene 17, 611–623.[CrossRef][ISI][Medline]
Castellano, M.M., del Pozo, J.C., Ramirez-Parra, E., Brown, S., and Gutierrez, C. (2001). Expression and stability of Arabidopsis CDC6 are associated with endoreduplication. Plant Cell 13, 2671–2686. de Jager, S.M., Menges, M., Bauer, U.-M., and Murray, J.A.H. (2001). Arabidopsis E2F1 binds a sequence present in the promoter of S-phase-regulated gene AtCDC6 and is a member of a multigene family with differential activities. Plant Mol. Biol. 47, 555–568.[CrossRef][ISI][Medline]
del Pozo, J.C., Timpte, C., Tan, S., Callis, J., and Estelle, M. (1998). The ubiquitin-related protein RUB1 and auxin response in Arabidopsis. Science 280, 1760–1763.
del Pozo, J.C., Boniotti, M.B., and Gutierrez, C. (2002). Arabidopsis E2F2 functions in cell division and is degraded by the ubiquitin-SCFAtSKP2 pathway in response to light. Plant Cell 14, 3057–3071. De Veylder, L., Beeckman, T., Beemster, G.T.S., Engler, J.D., Ormenese, S., Maes, S., Naudts, M., Van Der Schueren, E., Jacqmard, A., Engler, G., and Inzé, D. (2002). Control of proliferation, endoreduplication and differentiation by the Arabidopsis E2Fa-DPa transcription factor. EMBO J. 21, 1360–1368.[CrossRef][ISI][Medline]
Egelkrout, E.M., Mariconti, L., Settlage, S.B., Cella, R., Robertson, D., and Hanley-Bowdoin, L. (2002). Two E2F elements regulate the proliferating cell nuclear antigen promoter differently during leaf development. Plant Cell 14, 3225–3236.
Egelkrout, E.M., Robertson, D., and Hanley-Bowdoin, L. (2001). Proliferating cell nuclear antigen transcription is repressed through an E2F consensus element and activated by geminivirus infection in mature leaves. Plant Cell 13, 1437–1452. Gaubatz, S., Lindeman, G.J., Ishida, S., Jakoi, L., Nevins, J.R., Livingston, D.M., and Rampel, R.E. (2000). E2F4 and E2F5 play an essential role in pocket protein-mediated G1 control. Mol. Cell 6, 729–735.[CrossRef][ISI][Medline] Gutierrez, C. (1998). The retinoblastoma pathway in plant cell cycle and development. Curr. Opin. Plant Biol. 1, 492–497.[CrossRef][ISI][Medline] Gutierrez, C., Ramirez-Parra, E., Castellano, M.M., and del Pozo, J.C. (2002). G1 to S transition: More than a cell cycle engine switch. Curr. Opin. Plant Biol. 5, 480–486.[CrossRef][ISI][Medline]
Harbour, J.W., and Dean, D.C. (2000). The Rb/E2F pathway: Expanding roles and emerging paradigms. Genes Dev. 14, 2393–2409.
Kepinsky, S., and Leyser, O. (2002). Ubiquitination and auxin signaling: A degrading story. Plant Cell 14 (suppl.), S81–S95. Kong, L.J., Orozco, B.M., Roe, J.L., Nagar, S., Ou, S., Feiler, H.S., Durfee, T., Miller, A.B., Gruissem, W., Robertson, D., and Hanley-Bowdoin, L. (2000). A geminivirus replication protein interacts with retinoblastoma through a novel domain to determine symptoms and tissue-specificity of infection in plants. EMBO J. 19, 3485–3495.[CrossRef][ISI][Medline]
Kosugi, S., and Osahi, Y. (2002a). Interaction of the Arabidopsis E2F and DP proteins confers their concomitant nuclear translocation and transactivation. Plant Physiol. 128, 833–843.
Kosugi, S., and Osahi, Y. (2002b). E2Ls, E2F-like repressors of Arabidopsis that bind to E2F sites in a monomeric form. J. Biol. Chem. 277, 16553–16558. Lavia, P., and Jansen-Dürr, P. (1999). E2F target genes and cell cycle checkpoint control. Bioessays 21, 221–230.[CrossRef][ISI][Medline] Magyar, Z., Atanassova, A., De Veylder, L., Rombauts, S., and Inzé, D. (2000). Characterization of two distinct DP-related genes from Arabidopsis thaliana. FEBS Lett. 486, 79–87.[CrossRef][ISI][Medline]
Mariconti, L., Pellegrini, B., Cantoni, R., Stevens, R., Bergounioux, C., Cella, R., and Albani, D. (2002). The E2F family of transcription factors from Arabidopsis thaliana. J. Biol. Chem. 277, 9911–9919. Marti, A., Wirbelauer, W., Scheffner, M., and Krek, W. (1999). Interaction between ubiquitin-protein ligase SCFSKP2 and E2F-1 underlies the regulation of E2F-1 degradation. Nat. Cell Biol. 1, 14–19.[CrossRef][ISI][Medline]
Nagar, S., Hanley-Bowdoin, L., and Robertson, D. (2002). Host DNA replication is induced by geminivirus infection of differentiated plant cells. Plant Cell 14, 2995–3007. Nagar, S., Pedersen, T.J., Carrick, K., Hanley-Bowdoin, L., and Robertson, D. (1995). A geminivirus induces expression of a host DNA synthesis protein in terminally differentiated plant cells. Plant Cell 7, 705–719.[Abstract] Pickart, C.M. (2001). Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 70, 503–533.[CrossRef][ISI][Medline]
Vandepoele, K., Raes, J., De Veylder, L., Rouzé, P., Rombauts, S., and Inzé, D. (2002). Genome-wide analysis of core cell cycle genes in Arabidopsis. Plant Cell 14, 903–916. Related articles in Plant Cell:
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RBR/E2F PATHWAY IN PLANTS
ACTIVATION AND REPRESSION AMONG...
-glucuronidase (GUS) gene under the control of the E2Fc promoter, GUS expression was high in cotyledons and shoot and root meristems in young seedlings and was restricted to meristematic regions, vascular tissues, and specialized cells (such as in trichomes and flowers) in older seedlings and mature plants (