A second Drosophila E2F

Mammalian E2F transcription factors comprise a family of proteins encoded by distinct genes that function in the form of heterodimers with DP proteins. In Drosophila melanogaster, only a single E2F-related transcription factor, dE2F, has been reported. A cDNA encoding another E2F family member in Drosophila, termed dE2F2, has been identified and characterized. The predicted amino acid sequence shares 38.8% identity with dE2F, including the QKRRIYDITNVLEGI motif, which is highly conserved in mammalian E2F family members and dE2F. The 18 amino acids, located in the carboxy-terminal region of the mammalian E2F family, sufficient for binding to pRb, are also conserved in dE2F2. Band mobility shift analyses with glutathione S-transferase fusion proteins reveals that dE2F2 binding to E2F-recognition sites is dependent on the presence of dDP protein, in apparent contrast to dE2F. Furthermore, cotransfection experiments in Kc cells demonstrate dE2F2 repression of the PCNA gene promoter activity, while dE2F causes activation, the target site for the repression being identical to the dE2F-recognition site (Sawado, 1998).

Invertebrate E2Fs

Early C. elegans embryos exhibit protein asymmetries that allow rapid diversification of cells. Establishing these asymmetries requires the novel protein MEX-5. Mutations in the efl-1 and dpl-1 genes cause defects in protein localization resembling defects caused by mutations in mex-5. efl-1 and dpl-1 encode homologs of vertebrate E2F and DP proteins that regulate transcription as a heterodimer. efl-1 and dpl-1 mutants have elevated levels of activated Map kinase in oocytes. Their mutant phenotype and that of mex-5 mutants can be suppressed by reducing Ras/Map kinase signaling. It is proposed that this signaling pathway has a role in embryonic asymmetry and that EFL-1/DPL-1 control the level of Map kinase activation. The biochemical function of MEX-5 has not been established; however, PIE-1 localization is controlled by protein degradation. MEX-5 could have an important role in this process because MEX-5 expression is reciprocal to PIE-1 (a protein involved in embryonic polarity) in wild-type embryos, and ectopic expression of MEX-5 is sufficient to repress the expression of proteins similar to PIE-1. If MEX-5 controls PIE-1 degradation, this activity must be stimulated by events that occur after fertilization; oocytes show high, uniform levels of both PIE-1 and MEX-5 prior to fertilization (Page, 2001).

MAPK activation in C. elegans oocytes appears to depend on the presence of sperm or seminal fluid. Since MAPK becomes activated in oocytes that do not physically contact the spermatheca, where sperm are stored, an activating signal may either diffuse from the spermatheca or be relayed from the oocytes or somatic cells that contact the spermatheca. Once MAPK is activated in oocytes, it is proposed that the level of activation is determined by one or more transcriptional targets of EFL-1 and DPL-1. MAPK appears to be activated to an abnormally high level in efl-1 and dpl-1 mutants, and the phenotypic defects of efl-1 and dpl-1 mutants can be suppressed by reduction-of-function mutations in the Ras/MAPK pathway. Analysis of MAPK activation in oocytes from dpl-1;fem-1 mutants suggests two general models: dpl-1 oocytes either lack negative regulators of MAPK or contain excessive amounts of positive regulators. Wild-type oocytes that are near the spermatheca appear to be transcriptionally quiescent, suggesting that the level of MAPK activation is determined by gene products that are transcribed in another region of the syncytial gonad. EFL-1 and DPL-1 are both expressed in a band of syncytial germ nuclei before the transition zone where nuclei first cellularize and become oocytes, consistent with the view that EFL-1 and DPL-1 function in the transcription of gene products important for oogenesis (Page, 2001).

The region where EFL-1 is expressed in adult gonads corresponds closely to the region of MAPK activation in pachytene-stage nuclei. This localization pattern, combined with the analysis of MAPK in oocytes, suggests the possibility that MAPK activation might stimulate EFL-1 expression; transcriptional targets of EFL-1 might then, in turn, repress MAPK activation. Gonads in old fem-1 adults eventually lose all detectable activated MAPK but continue to express EFL-1. These results suggest that MAPK activation is not required to maintain EFL-1 expression but do not address the possibility that MAPK activation is involved in initiating EFL-1 expression (Page, 2001).

The tumor suppressor Rb and the NuRD (nucleosome remodeling and histone deacetylation) complex have been implicated in transcriptional repression during cell cycle progression and cell fate specification. The Rb/E2F complex physically interacts with and thus recruits the NuRD complex to actively repress transcription. C. elegans counterparts of Rb, E2F/DP, and some NuRD complex components appear to function in a common class B synthetic Multivulva (synMuv) pathway to antagonize RTK/Ras signaling during vulval fate specification. Therefore, it has been suggested that they function together in a single complex to repress vulva-specific gene transcription. However, little is known about the in vivo interactions between these class B synMuv genes and their relationships with other pathways in specific cellular processes during vulval development. It has been shown that C. elegans Rb/E2F and NuRD complexes antagonize Ras activity by controlling a lin-39 Hox-mediated cell fusion event that regulates the competence of vulval cells. Interestingly, Rb/E2F and NuRD complexes exhibit very different genetic properties. While the NuRD complex negatively regulates lin-39 Hox activity, likely by downregulating its expression, RB/E2F appears to play dual roles in regulating lin-39: a negative role in controlling its activity and a previously uncharacterized positive role in regulating its expression (Chen, 2001).

The synthetic multivulva (synMuv) genes define two functionally redundant pathways that antagonize RTK/Ras signaling during C. elegans vulval induction. The synMuv gene lin-35 encodes a protein similar to the mammalian tumor suppressor pRB and has been proposed to act as a transcriptional repressor. Studies using mammalian cells have shown that pRB can prevent cell cycle progression by inhibiting DP/E2F-mediated transcriptional activation. C. elegans genes that encode proteins similar to DP or E2F have been identified. Loss-of-function mutations in two of these genes, dpl-1 DP and efl-1 E2F, cause the same vulval abnormalities as do lin-35 Rb loss-of-function mutations. It is proposed that rather than being inhibited by lin-35 Rb, dpl-1 DP and efl-1 E2F act with lin-35 Rb in transcriptional repression to antagonize RTK/Ras signaling during vulval development (Ceol, 2001).

Considerable evidence indicates that mammalian DP and E2F proteins can promote the entry of cells into S phase, thereby stimulating cell cycle progression. Observations of dpl-1(n3316 RNAi) animals, which are presumably deficient in both the zygotic and maternal contribution of DPL-1, suggest that DPL-1 activity is not essential in every cell for cell cycle progression. Since dpl-1 is the only predicted C. elegans DP family member, and since DP protein function is thought to be necessary for DP/E2F heterodimer function, DP/E2F activity in C. elegans may not be generally required for cell cycle progression. However, dpl-1 and efl-1 may be required to promote S phase entry in some cell types. Specifically, the Pn.a neuroblasts of dpl-1(n3316 RNAi) animals do not complete their divisions and sometimes generate large polyploid descendants. It is concluded that dpl-1 and possibly efl-1 may act in but are not essential for the cell cycle in C. elegans. It is possibile, however, that undetectable levels of dpl-1 activity and DPL-1 protein may be present in dpl-1(n3316 RNAi) animals and may fulfill a broader requirement for dpl-1 in promoting cell cycle progression. Loss-of-function mutations in dpl-1 and efl-1, like loss-of-function mutations in lin-35 Rb, cause cell-fate transformations that result in supernumerary cell divisions in the P(3,4,8).p lineages. The extra cell divisions in these mutants are consistent with the possibility that a DPL-1/EFL-1/LIN-35 protein complex normally inhibits cell cycle progression in P(3,4,8).p. Thus, while dpl-1 and efl-1 promote cell division in some cell types, e.g., in Pn.a descendants, dpl-1 and efl-1 may prevent cell division in other cell types, e.g., in P(3,4,8).p descendants. While it is tempting to speculate that dpl-1 and efl-1 are cell cycle regulators in C. elegans, it is not yet known if the effects of these genes on cell division are caused by the direct regulation of the cell cycle machinery or are caused by the regulation of cell fate-determining factors that subsequently impinge on the cell cycle machinery (Ceol, 2001).

Retinoblastoma (Rb)/E2F complexes repress expression of many genes important for G1-to-S transition, but also appear to regulate gene expression at other stages of the cell cycle. In C. elegans, lin-35/Rb and other synthetic Multivulva (SynMuv) group B genes function redundantly with other sets of genes to regulate G1/S progression, vulval and pharyngeal differentiation, and other unknown processes required for viabilty. lin-35/Rb, efl-1/E2F, and other SynMuv B genes negatively regulate a component of the anaphase-promoting complex/cyclosome (APC). The APC/C is a multisubunit complex that promotes metaphase-to-anaphase progression and G1 arrest by targeting different substrates for ubiquitination and proteasome-mediated destruction. The C. elegans APC/C gene mat-3/APC8 has been defined by temperature-sensitive embryonic lethal alleles that strongly affect germline meiosis and mitosis but only weakly affect somatic development. Severe nonconditional mat-3 alleles and a hypomorphic viable allele (ku233) are described, all of which affect postembryonic cell divisions including those of the vulval lineage. The ku233 lesion is located outside of the mat-3 coding region and reduces mat-3 mRNA expression. Loss-of-function alleles of lin-35/Rb and other SynMuv B genes suppress mat-3(ku233) defects by restoring mat-3 mRNA to wild-type levels. Therefore, Rb/E2F complexes appear to repress mat-3 expression (Garbe, 2004).

The genes egl-1, ced-9, ced-4, and ced-3 play major roles in programmed cell death in Caenorhabditis elegans. To identify genes that have more subtle activities, mutations were sought that confer strong cell-death defects in a genetically sensitized mutant background. Specifically, a screen was performed for mutations that enhance the cell-death defects caused by a partial loss-of-function allele of the ced-3 caspase gene. Mutations were identified in two genes not previously known to affect cell death, dpl-1 and mcd-1 (modifier of cell death). dpl-1 encodes the C. elegans homolog of DP, the human E2F-heterodimerization partner. By testing genes known to interact with dpl-1, roles were identified in cell death for four additional genes: efl-1 E2F, lin-35 Rb, lin-37 Mip40, and lin-52 dLin52. mcd-1 encodes a novel protein that contains one zinc finger and that is synthetically required with lin-35 Rb for animal viability. dpl-1 and mcd-1 act with efl-1 E2F and lin-35 Rb to promote programmed cell death and do so by regulating the killing process rather than by affecting the decision between survival and death. It is proposed that the DPL-1 DP, MCD-1 zinc finger, EFL-1 E2F, LIN-35 Rb, LIN-37 Mip40, and LIN-52 dLin52 proteins act together in transcriptional regulation to promote programmed cell death (Reddien, 2007).

Mammalian E2Fs

Members of the E2F transcription factor family (E2F-1-E2F-5) are believed to be critical positive regulators of cell cycle progression in eukaryotes although the in vivo functions of the individual E2Fs have not been elucidated. Mice were generated that lack E2F-1 and, surprisingly, these mice develop and reproduce normally. However, E2F-1-/- mice exhibit a defect in T lymphocyte development leading to an excess of mature T cells due to a maturation stage-specific defect in thymocyte apoptosis. As E2F-1-/- mice age they exhibit a second phenotype marked by aberrant cell proliferation. These findings suggest that while certain members of the E2F family may positively regulate cell cycle progression, E2F-1 functions to regulate apoptosis and to suppress cell proliferation (Field, 1996).

E2F transcription factors are essential for regulating the correct timing of activation for several genes whose products are implicated in cell proliferation and DNA replication. The E2Fs are targets for negative regulation by the retinoblastoma protein family, which includes pRB, p107, and p130, and they are in a pathway that is frequently found to be altered in human cancers. There are five members of the E2F family, and they can be divided into two functional subgroups: overexpression of E2F-1, -2, or -3 induces S phase in quiescent fibroblasts and overrides G1 arrests mediated by the p16INK4A tumor suppressor protein or neutralizing antibodies to cyclin D1; overexpression of E2F-4 or -5 does not. By constructing a set of chimeric proteins using E2F-1 and E2F-4 as representatives of the two subgroups, it has been shown that the amino terminus of E2F-1 is sufficient to confer S-phase-inducing potential as well as the ability to efficiently transactivate an E2F-responsive promoter to E2F-4. The E2F-1 amino terminus directs chimeric proteins to the nucleus. Surprisingly, a short nuclear localization signal derived from simian virus 40 large T antigen can perfectly substitute for the presence of the E2F-1 amino terminus in these assays. Thus, nuclearly localized E2F-4, when overexpressed, displays biological activities similar to those of E2F-1. Nuclear localization of endogenous E2F-4 is cell cycle regulated, with E2F-4 being nuclear in the G0 and early G1 phases and mainly cytoplasmic after the pRB family members have become phosphorylated. A novel mechanism is proposed for the regulation of E2F-dependent transcription in which E2F-4 regulates transcription only from G0 until mid- to late G1 phase, whereas E2F-1 is active in late G1 and S phases, until it is inactivated by cyclin A-dependent kinase in late S phase (Muller, 1997).

The E2F family of transcription factors consists of two subgroups termed E2F and DP. E2F is required for cell proliferation, and is necessary for fruit fly development. E2F activity is a target for regulation by the retinoblastoma gene family, which includes pRB, p107 and p130. Mutant RB-/-, RB-/-:p107-/- and p107-/-:p130-/- mice develop abnormally, probably as a result of dysregulation in the activity of E2F, indicating the importance of E2F in mammalian development. To investigate the role of E2F in murine development, the patterns of expression of E2F-1 through E2F-5, and DP-1 were examined in the developing nervous system by in situ hybridization. E2F-1, E2F-2 and E2F-5 are first detected in the 9.5 days post-coitus (dpc) forebrain. Expression of these E2F forms extends caudally thereafter and includes the developing brain and the upper half of the 10.5 dpc spinal cord. By 11.5 dpc, these E2F factors are expressed throughout the central nervous system. In 12.5 dpc embryos, E2F-1, E2F-2 and E2F-5 are highly expressed in proliferating, undifferentiated neuronal precursors. As neurons differentiate and migrate to the outer marginal zones in the nervous system, expression of these E2F members is extinguished. In the developing retina, another neuronal tissue, E2F-1 expression is also confined to the proliferating, undifferentiated retinoblastic layer. In contrast, E2F-3 expression is up-regulated as retinoblasts differentiate into the ganglion cell layer. In non-neuronal tissues, high E2F-4 transcript levels are present in regions corresponding to proliferative chondrocytes, whereas E2F-2 and E2F-4 transcripts are very abundant in the thymic cortex, which contains immature thymocytes. It is concluded that individual E2F forms are differentially regulated during the development of distinct tissues, and especially during neuronal development (Dagnino, 1997).

The E2F family of proteins is required to establish the correct cell-cycle-dependent transcription of genes that direct the process of cell division. All previously identified E2F proteins can act in a similar manner; depending on whether or not they are associated with the cell cycle inhibitors the retinoblastoma protein (pRB), p107, or p130, they can either repress or activate the transcription of E2F-responsive genes. The structure of another E2F family member, E2F-6, is reminiscent of the dominant inhibitors of other transcription factor families. The dimerization and DNA binding properties of E2F-6 are similar to those of the other E2F family members. However, E2F-6 is not regulated by pRB, p107, or p130, and it is unable to activate transcription. Instead, it can act to repress the transcription of E2F responsive genes by countering the activity of the other E2F complexes via a pRB-, p107-, or p130-independent mechanism (Trimarchi, 1998).

Mice mutant for the Rb tumor suppressor gene die in mid-gestation with defects in erythropoiesis, cell cycle control, and apoptosis. Embryos mutant for both Rb and its downstream target E2f-1 demonstrate significant suppression of apoptosis and S phase entry in certain tissues as compared to Rb mutants, implicating E2f-1 as a critical mediator of these effects. Up-regulation of the p53 pathway, required for cell death in these cells in Rb mutants, is also suppressed in the Rb/E2f-1 double mutants. However, double mutants have defects in cell cycle regulation and apoptosis in some tissues and die at approximately E17.0 with anemia and defective skeletal muscle and lung development, demonstrating that E2F-1 regulation is not the sole function of pRB in development (Tsai, 1998).

E2F transcription factors play an important role in the regulation of cell cycle progression. This paper reports the cloning and characterization of an additional member of this family, E2F-6. E2F-6 lacks pocket protein binding and transactivation domains, and it is a potent transcriptional repressor that contains a modular repression domain at its carboxyl terminus. Overproduction of E2F-6 had no specific effect on cell cycle progression in asynchronously growing Saos2 and NIH 3T3 cells, but it inhibited entry into S phase of NIH 3T3 cells stimulated to exit G0. Taken together, these data suggest that E2F-6 can regulate a subset of E2F-dependent genes whose products are required for entry into the cell cycle but not for normal cell cycle progression (Gaubatz, 1998).

Much evidence strongly suggests a positive role for one or more E2F species in the control of exit from G0/G1. Results described here provide direct evidence that endogenous E2F-1, as predicted, contributes to progression from G0 to S. By contrast, cycling cells lacking an intact E2F-1 gene demonstrate normal cell cycle distribution. Therefore, E2F-1 exerts a unique function leading to timely G0 exit of resting cultured primary cells, while at the same time being unnecessary for normal G1 to S phase progression of cycling cells (Wang, 1998).

Maintenance of cells in a quiescent state after terminal differentiation occurs through a number of mechanisms that regulate the activity of the E2F family of transcription factors. Changes in the subcellular compartmentalization of the E2F family proteins are required to prevent nuclei in terminally differentiated skeletal muscle from reentering S phase. In terminally differentiated L6 myotubes, E2F-1, E2F-3, and E2F-5 are primarily cytoplasmic, E2F-2 is nuclear, whereas E2F-4 is partitioned between both compartments. In these same cells, pRB family members, pRB, p107, and p130 are also nuclear. This compartmentalization of the E2F-1 and E2F-4 in differentiated muscle cells grown in vitro reflects their observed subcellular location in situ. Exogenous E2F-1 or E2F-4 expressed in myotubes at levels fourfold greater than endogenous proteins compartmentalize identically to their endogenous counterparts. Only when overexpressed at higher levels is inappropriate subcellular location for these proteins observed. At these levels, induction of the E2F-regulated genes, cyclins A and E, and suppression of factors associated with myogenesis, myogenin, and p21 Cip1 are observed. Only at these levels of E2F expression do nuclei in these terminally differentiated cells enter S phase. These data demonstrate that regulation of the subcellular compartmentalization of E2F-family members is required to maintain nuclei in a quiescent state in terminally differentiated cells (Gill, 2000).

DP, E2F's dimerization partner

The E2F and DP protein families form heterodimeric transcription factors that play a central role in the expression of cell cycle-regulated genes. The crystal structure of an E2F4-DP2-DNA complex shows that the DNA-binding domains of the E2F and DP proteins both have a fold related to the winged-helix DNA-binding motif. This motif consists of three alpha helices and a beta sheet, each contributing to a compact hydrophobic core. DP2 has the same overall structure as E2F4, except that the beta 2 and beta 3 helices of DP2 are longer by about two turns each, and E2F4 has an amino-terminal helical extension (beta N) that is not present in DP2. The remaining regions of E2F4 and DP2 superimpose quite well, with a 1.4-Å root mean square deviation for 59 residues. The structure-based alignment of E2F4 and DP2 shows that the regions corresponding to the beta 1 and beta 2 helices have only 8% sequence identity, and this, coupled with the greater length of the beta 2 and beta 3 helices of DP2, presumably complicated previous efforts to align the two families. The 30-residue region of clear homology between the E2F and DP families, termed the DEF box, coincides with the beta 3 helix and the beta2 and beta3 strands. The structure shows that the alpha3 helices of E2F4 and DP2 bind in the major groove of the DNA and make critical contacts to the edges of the bases. In both cases, the amino-termini of the alpha1 helices and portions of the beta sheets contact the phosphodiester backbone of the DNA. This overall DNA-binding arrangement for each protein is analogous to the other winged-helix proteins. However, winged-helix proteins typically bind DNA as monomers, whereas E2F4 and DP2 form an extensive hetero-oligomerization interface and present a continuous protein surface to the DNA. Recognition of the central c/gGCGCg/c sequence of the consensus DNA-binding site is symmetric, and amino acids that contact these bases are conserved among all known E2F and DP proteins. The asymmetry in the extended binding site TTTc/gGCGCc/g is associated with an amino-terminal extension of E2F4, in which an arginine binds in the minor groove near the TTT stretch. This arginine is invariant among E2Fs but not present in DPs. E2F4 and DP2 interact through an extensive protein-protein interface, and structural features of this interface suggest it contributes to the preference for heterodimers over homodimers in DNA binding (Zheng, 1999).

The product of the retinoblastoma (Rb) susceptibility gene, Rb-1, regulates the activity of a wide variety of transcription factors, such as E2F, in a cell cycle-dependent fashion. E2F is a heterodimeric transcription factor composed of two subunits, each encoded by one of two related gene families, denoted E2F and DP. Five E2F genes (E2F-1 through E2F-5) and two DP genes (DP-1 and DP-2) have been isolated from mammals, and heterodimeric complexes of these proteins are expressed in most, if not all, vertebrate cells. It is not yet clear whether E2F/DP complexes regulate overlapping and/or specific cellular genes. Little is known about whether Rb regulates all or a subset of E2F-dependent genes. Using recombinant E2F, DP, and Rb proteins, prepared in baculovirus-infected cells and a repetitive immunoprecipitation-PCR procedure (CASTing), consensus DNA-binding sites have been identified for E2F-1/DP-1, E2F-1/DP-2, E2F-4/DP-1, and E2F-4/DP-2 complexes as well as an Rb/E2F-1/DP-1 trimeric complex. The data indicate that (1) E2F, DP, and Rb proteins each influence the selection of E2F-binding sites; (2) E2F sites differ with respect to their intrinsic DNA-bending properties; (3) E2F/DP complexes induce distinct degrees of DNA bending, and (4) complex-specific E2F sites selected in vitro function distinctly as regulators of cell cycle-dependent transcription in vivo. These data indicate that the specific sequence of an E2F site may determine its role in transcriptional regulation and suggest that Rb/E2F complexes may regulate subsets of E2F-dependent cellular genes (Tao, 1997).

Release of E2F1/DP1 heterodimers from repression mediated by the retinoblastoma tumor suppressor (pRB) triggers cell cycle entry into S phase, suggesting that E2F1 and DP1 proteins must act in unison, either to facilitate or to suppress cell-cycle progression. In stark contrast to the milder phenotypes that result from inactivation of E2Fs, loss of Dp1 leads to death in utero because of the failure of extra-embryonic development. Loss of Dp1 compromises the trophectoderm-derived tissues -- specifically, the expansion of the ectoplacental cone and chorion, and endoreduplication in trophoblast giant cells. Inactivation of p53 is unable to rescue the Dp1-deficient embryonic lethality. Thus, DP1 is absolutely required for extra-embryonic development and consequently embryonic survival, consistent with E2F/DP1 normally acting to promote growth in vivo (Kohn, 2003).

Transcription factor E2F plays an important role in orchestrating early cell cycle progression through its ability to co-ordinate and integrate the cell cycle with the transcription apparatus. Physiologically active E2F arises when E2F and DP interact as E2F-DP heterodimers, in which the E2F component mediates transcriptional activation and the physical interaction with pocket proteins, such as the tumor suppressor protein pRb. In contrast, a discrete role for the DP subunit has not been defined. DIP is a novel mammalian protein that can interact with the DP component of E2F. DIP contains a BTB/POZ domain and shows significant identity with the Drosophila melanogaster germ cell-less gene product. The product of the germ cell-less gene, Gcl, which is required for proper germ cell fate specification during embryogenesis, has 36% identity and 56% similarity with mDIP. The similarity is distributed throughout both proteins, suggesting that mDIP is a close mammalian homolog of Drosophila Gcl. Furthermore, searching the available database of expressed sequence tags has identified several human sequences with >90% homology to mDIP at the amino acid level, providing further evidence that DIP is highly conserved within mammals. In mammalian cells, DIP is distributed in a speckled pattern at the nuclear envelope region, and can direct certain DP subunits and the associated heterodimeric E2F partner into a similar pattern. DIP-dependent growth arrest is modulated by the expression of DP proteins, and mutant derivatives of DIP that are compromised in cell cycle arrest exhibit reduced binding to the DP subunit. This study defines a new pathway of growth control that is integrated with the E2F pathway through the DP subunit of the heterodimer. The mechanism of action of Drosophila Gcl is not clear, although it is known to be necessary for germ cells to complete their differentiation program. However, two interesting and relevant properties of Drosophila Gcl have been described previously: (1) it is known that Gcl is a nuclear protein localized in the proximity of nuclear pores of the germ cell precursors, and (2) the overexpression of Gcl in embryos causes a delay in mitosis during the pole bud nuclear divisions at the syncytial blastoderm stage, suggesting a role for Gcl in regulating cell cycle control. It is not known whether the conservation between GCL and DIP reflects a common function, although the high level of DIP RNA during murine spermatogenesis is consistent with a role for Dip in germ cell specification. However, other properties of DIP, such as the low but widespread expression of DIP mRNA in tumour cell lines, together with its effects on cell proliferation imply perhaps a more widespread role for DIP than previously assigned to Gcl (de la Luna, 1999).

LAP2beta is an integral membrane protein of the nuclear envelope involved in chromatin and nuclear architecture. Using the yeast two-hybrid system, a novel LAP2beta-binding protein, mGCL, has been cloned that contains a BTB/POZ domain and is the mouse homolog of the Drosophila Germ-cell-less (Gcl) protein. In Drosophila embryos, Gcl is essential for germ cell formation and is localized to the nuclear envelope. In mammalian cells, Gcl co-localizes with LAP2beta to the nuclear envelope. Nuclear fractionation studies reveal that mGCL acts as a nuclear matrix component and not as an integral protein of the nuclear envelope. mGCL has been found to interact with the DP3alpha component of the E2F transcription factor. This interaction reduces the transcriptional activity of the E2F-DP heterodimer, probably by anchoring the complex to the nuclear envelope. LAP2beta is also capable of reducing the transcriptional activity of the E2F-DP complex and it is more potent than mGCL in doing so. Co-expression of both LAP2beta and mGCL with the E2F-DP complex results in a reduced transcriptional activity equal to that exerted by the pRb protein (Nili, 2001).

Interaction of E2F with cyclins

Human cyclin A1, a newly discovered cyclin, is expressed in testis and is thought to function in the meiotic cell cycle. The expression of human cyclin A1 and cyclin A1-associated kinase activities is regulated during the mitotic cell cycle. In the osteosarcoma cell line MG63, cyclin A1 mRNA and protein are present at very low levels in cells at the G0 phase. They increase during the progression of the cell cycle and reach the highest levels in the S and G2/M phases. Furthermore, the cyclin A1-associated histone H1 kinase activity peaks at the G2/M phase. Cyclin A1 can bind to important cell cycle regulators: the Rb family of proteins, the transcription factor E2F-1, and the p21 family of proteins. The in vitro interaction of cyclin A1 with E2F-1 is greatly enhanced when cyclin A1 is complexed with CDK2. Associations of cyclin A1 with Rb and E2F-1 are observed in vivo in several cell lines. When cyclin A1 is coexpressed with CDK2 in sf9 insect cells, the CDK2-cyclin A1 complex has kinase activities for histone H1, E2F-1, and the Rb family of proteins. These results suggest that the Rb family of proteins and E2F-1 may be important targets for phosphorylation by the cyclin A1-associated kinase. Cyclin A1 may function in the mitotic cell cycle in certain cells (Yang, 1999).

The E2F promoter and the regulation of E2F transcription

The E2F1 gene is subject to autoregulatory control during progression from G0 to S phase, primarily reflecting a negative control in G0 and early G1, when the majority of E2F exists as a complex with Rb family members. Deregulation of G1 cyclins in quiescent cells stimulates the E2F1 promoter and this is augmented by coexpression of cyclin-dependent kinases in an E2F-dependent manner. E2F1 gene is a downstream target for G1 cyclin-dependent kinase activity, most likely as a consequence of phosphorylation of Rb family members (Johnson, 1994). The presence of an E2F DNA-binding complex containing the Rb-related p130 protein (Rb2) correlates with E2F-1 gene expression, and overexpression of p130 inhibits transcription from the E2F-1 promoter. D-type cyclin-dependent kinase activity specifically activates the E2F-1 promoter by relieving E2F-mediated promoter (Johnson, 1995).

Previous studies have indicated that the presence of an E2F site is not sufficient for G1/S phase transcriptional regulation. For example, the E2F sites in the E2F1 promoter are necessary, but not sufficient, to mediate differential promoter activity in G0 and S phase. The E2F1 minimal promoter was used to test several hypotheses that could account for these observations. To test the hypothesis that G1/S phase regulation is achieved via E2F-mediated repression of a strong promoter, a variety of transactivation domains were brought to the E2F1 minimal promoter. Although many of these factors cause increased promoter activity, growth regulation is not observed, suggesting that a general repression model is incorrect. However, constructs having CCAAT or YY1 sites or certain GC boxes cloned upstream of the E2F1 minimal promoter display E2F site-dependent regulation. Further analysis of the promoter activity suggests that E2F requires cooperation with another factor to activate transcription in S phase. However, it was found that the requirement for E2F to cooperate with additional factors to achieve growth regulation could be relieved by bringing the E2F1 activation domain to the promoter via a Gal4 DNA binding domain. These results suggest a model that explains why some, but not all, promoters that contain E2F sites display growth regulation (van Ginkel, 1997).

Genomic sequences were isolated flanking the 5' region of the E2F2 coding sequence. Various assays demonstrate promoter activity in this sequence that reproduces the normal control of E2F2 expression during a growth stimulation. Sequence comparison reveals the presence of a variety of known transcription factor binding sites, including E-box elements (that are consensus Myc (Drosophila homolog: Myc) binding sites), as well as E2F binding sites. The E-box elements, which can function as Myc-responsive sites, contribute in a positive fashion to promoter function. E2F-dependent negative regulation in quiescent cells plays a significant role in the cell growth-dependent control of the promoter, similar to the regulation of the E2F1 gene promoter (Sears, 1997).

Myc transcription factor induces transcription of the E2F1, E2F2, and E2F3 genes. Using primary mouse embryo fibroblasts deleted for individual E2F genes, it has now been shown that Myc-induced S phase and apoptosis requires distinct E2F activities. The ability of Myc to induce S phase is impaired in the absence of either E2F2 or E2F3 but not E2F1 or E2F4. In contrast, the ability of Myc to induce apoptosis is markedly reduced in cells deleted for E2F1 but not E2F2 or E2F3. From this data, it is proposed that the induction of specific E2F activities is an essential component in the Myc pathways that control cell proliferation and cell fate decisions (Leone, 2001).

Deregulated Myc expression results in both the induction of S phase and the induction of apoptosis if survival factors are limiting. How these processes are linked is not well understood. A variety of possible Myc target genes have been identified, including the recent description of Id2. Like E2F2 and E2F3, Id2 appears to be essential for Myc-induced cell proliferation but not for Myc-induced apoptosis. In addition, these studies have also identified a role for Id2 in the control of Rb function, since the loss of Id2 function can partially suppress the phenotype resulting from loss of Rb. As such, it has been proposed that one role for Myc in the stimulation of cell growth is the induction of Id2, leading to inactivation of Rb. Nevertheless, since the loss of Id2 does not fully suppress an Rb null phenotype (mice die at birth), and previous work has shown a suppression of Rb phenotype as a result of loss of either E2F1, E2F2, or E2F3 function, it seems likely that the control of E2F proteins and interactions with Id2 are both important for Rb function. Moreover, given the ability of Myc to induce both Id2 and E2Fs, it is concluded that the induction of both groups of activities is likely to be an important function of Myc (Leone, 2001).

In addition to results showing the requirement for distinct E2Fs to mediate Myc-induced S phase versus apoptosis, other work has also suggested that distinct downstream events mediate these two functions of Myc. In particular, cdk activation has been shown to be necessary for the Myc-mediated induction of proliferation but not apoptosis. Although the lack of a cdk requirement for Myc-induced apoptosis might suggest an E2F-independent event, since E2F accumulation is normally regulated by Rb through cdk-mediated phosphorylation of Rb, previous work has demonstrated an ability of Myc to induce E2F1 accumulation in the absence of cdk activity, presumably by bypassing the normal Rb control. Thus, Myc-induced apoptosis could bypass the need for the cell cycle machinery by directly activating E2F1 (Leone, 2001).

E2F transcription factors play a major role in controlling mammalian cell cycle progression. A potential tumor suppressor, prohibitin, interacts with retinoblastoma protein (Rb), regulates E2F function and this activity correlates with its growth-suppressive activity. Prohibitin recruits Brg-1/Brm to E2F-responsive promoters, and this recruitment is required for the repression of E2F-mediated transcription by prohibitin. Expression of a dominant-negative Brg-1 or Brm releases prohibitin-mediated repression of E2F and relieves prohibitin-mediated growth suppression. Although prohibitin associates with, and recruits, Brg-1 and Brm independently of Rb, prohibitin/Brg-1/Brm-mediated transcriptional repression requires Rb. A viral oncoprotein, SV40 large T antigen, can reverse prohibitin-mediated suppression of E2F-mediated gene transcription, and targets prohibitin through interruption of the association between prohibitin and Brg-1/Brm without affecting the prohibitin-E2F interaction (Wang, 2002).

E2F1 corepresser recruits a co-repressor complex

TopBP1 (DNA topoisomerase IIbeta binding protein I) contains multiple BRCT domains and is involved in replication and the DNA damage checkpoint. Through its BRCT domain, TopBP1 interacts with and represses exclusively E2F1 but not other E2F factors. This regulation of E2F1 transcriptional activity is mediated by a pRb-independent, but Brg1/Brm-dependent mechanism. TopBP1 recruits Brg1/Brm, a central component of the SWI/SNF chromatin-remodeling complex, to E2F1-responsive promoters and represses the activities of E2F1, but not E2F2 or E2F3. This regulation is crucial in the control of E2F1-dependent apoptosis during normal cell growth and DNA damage. Interestingly, TopBP1 is induced by E2F and interacts with E2F1 during G1/S transition. Thus, TopBP1 functions as a critical modulator and serves as a negative feedback regulator of E2F1 by inhibiting E2F1-dependent apoptosis during G1/S transition as well as DNA damage to promote cell survival (Liu, 2004).

To identify E2F1-specific regulators, an E2F1-specific fragment (N terminus) was used as a bait and TopBP1 was isolated in a yeast two-hybrid screen. TopBP1 contains eight BRCA1 C-terminal (BRCT) motifs and interacts with several other proteins, including human papilloma virus type 16 (HPV16) transcription/replication factor E2, DNA polymerase epsilon, checkpoint protein hRad9, and Miz-1. It appears to be involved in DNA replication because incubation of an antibody against the sixth BRCT motif of TopBP1 inhibits DNA replication in an in vitro HeLa nuclei replication assay. TopBP1 is induced during DNA damage and is also involved in DNA damage checkpoint. Upon gamma-irradiation, TopBP1 colocalizes with Nbs1, BRCA1, and 53BP1 in the ionizing radiation-induced foci representing stalled replication forks. In addition to the control of DNA replication, TopBP1 is also required for cell survival. Inhibition of TopBP1 expression by antisense Morpholino oligomers induces apoptosis. Thus, TopBP1 is involved in several important aspects of growth control. So far, the detailed mechanism by which TopBP1 regulates these signaling events remains poorly understood (Liu, 2004 and references therein).

TopBP1 interacts with E2F1 through the sixth BRCT motif of TopBP1 and the N terminus of E2F1. This interaction is induced by ATM-mediated phosphorylation of E2F1 at Ser 31 during DNA damage. The interaction between BRCT domains and phosphopeptides is a general phenomenon. Through this interaction, the transcriptional and apoptotic activities of E2F1 are repressed, and E2F1 is recruited to DNA damage-induced foci. Moreover, the interaction between TopBP1 and E2F as well as the repression of E2F activity are specific to E2F1, but not seen in E2F2, E2F3, and E2F4, suggesting that TopBP1 is an E2F1-specific regulator (Liu, 2004).

E2F1 is shown to be regulated by a novel Retinoblastoma protein (pRb)-independent mechanism, in which TopBP1 recruits Brg1/Brm, a central subunit of the SWI/SNF chromatin-remodeling complex, to inhibit E2F1 transcriptional activity. This regulation is specific for E2F1 and is critical for the control of E2F1-dependent apoptosis during S phase and DNA damage. TopBP1 is induced by E2F and interacts with E2F1 during G1/S transition. Thus, E2F1 and TopBP1 form a feedback regulation to prevent apoptosis during DNA replication (Liu, 2004).

The mechanism by which TopBP1 represses E2F1 is through recruiting Brg1/Brm. Evidence to support this assertion: (1) TopBP1-mediated repression of E2F1 is defective in a Brg1/Brm-deficient cell line, C33A. The repression is restored by reconstitution with Brg1/Brm. (2) Dominant-negative mutants of Brg1 or Brm inhibit TopBP1 to repress E2F1. (3) TopBP1 interacts with Brg1/Brm and facilitates the interaction between E2F1 and Brg1/Brm. (4) TopBP1 recruits Brg1/Brm to E2F1-responsive promoters. (5) Dominant-negative mutants of Brg1/Brm derepress E2F1 activity during DNA damage. (6) Whereas TopBP1 siRNA induces E2F1-dependent apoptosis in HEK293 cells and wild-type MEFs, it fails to induce apoptosis in Brg1/Brm-deficient C33A cells (Liu, 2004).

Regulation of E2F dependent transcription - The role of Retinoblastoma family proteins, cyclins and cdk inhibitors

Continued: see E2F: Evolutionary homologs part 2/3 | part 3/3

E2F: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.