E2F


EVOLUTIONARY HOMOLOGS (part 3/3)

Target genes of E2F

The E2F transcription factor family is known to play a key role in the timely expression of genes required for cell cycle progression and proliferation, but only a few E2F target genes have been identified. The possibility that E2F regulators play a broader role was suggested by identifying additional genes bound by E2F in living human cells. A protocol was developed to identify genomic binding sites for DNA-binding factors in mammalian cells that combines immunoprecipitation of cross-linked protein-DNA complexes with DNA microarray analysis. Among ~1200 genes expressed during cell cycle entry, it was found that the promoters of 127 are bound by the E2F4 transcription factor in primary fibroblasts. A subset of these targets is also bound by E2F1. Most previously identified target genes known to have roles in DNA replication and cell cycle control and represented on the microarray were confirmed by this analysis. A remarkable cadre of genes was identifed with no previous connection to E2F regulation, including genes that encode components of the DNA damage checkpoint and repair pathways, as well as factors involved in chromatin assembly/condensation, chromosome segregation, and the mitotic spindle checkpoint. These data indicate that E2F directly links cell cycle progression with the coordinate regulation of genes essential for both the synthesis of DNA as well as its surveillance (Ren, 2002).

Eight genes were identified with a well-established role in cell cycle control. This cluster includes cyclin A, Cdc2, Cdc25A, CDK2, two members of the E2F family (E2F2 and E2F3), and two members of the RB family (RB and p107). Each of these genes had been regarded as E2F-responsive, and this work confirms that they are, indeed, direct, physiological targets of E2F (Ren, 2002).

A role for E2F in the activation of several DNA replication genes has been well established. Known E2F targets include genes encoding proteins involved in the initiation of replication (Orc1, Mcm proteins, Cdc6), nucleotide metabolism (thymidine kinase), and the enzymatic synthesis of DNA (DNA polymerase alpha). This study expands this list to include additional nucleotide synthesis and replication factor genes (Ren, 2002).

An emerging idea from recent studies suggests that E2F may be required to regulate cyclin gene expression beyond S phase, and expression-profiling has identified several potential downstream targets involved in mitosis. A significant number of genes that function in mitosis were identified. Among this list were genes known to play a role in cytokinesis (Plk, PRC1), chromosome condensation (SMC4, SMC2), and chromosome segregation (securin or PTTG1, CENP-E, HEC1). These experiments strongly suggest that E2F plays a direct role in regulating several genes involved in mitosis. It is interesting to note that several components of the mitotic spindle checkpoint were identified, including CENP-E, Bub3, and Mad2, proteins that interact with Bub1, which has been identified as an E2F target by Nevins and colleagues. Thus, this work establishes that E2F directly binds the promoters of genes linked to post-S-phase events and suggests that it plays an important role in their expression (Ren, 2002).

Interestingly, a large cluster of 12 genes required for DNA repair was identified. These genes are involved in the full spectrum of repair processes, including mismatch repair (MSH2, MLH1), base excision repair (UNG), nucleotide excision repair (RPA3), homologous recombination (RAD51 and RAD54), and nonhomologous end-joining (DNA-dependent protein kinase). These experiments may point to the conservation of a phenomenon first noted in Saccharomyces cerevisiae, namely, that genes involved in repair of DNA damage may be required during replication to ensure genomic integrity. Many of the genes isolated in this screen are members of multimeric complexes, and the data suggest that they may be coordinately regulated. For example, FEN1-PCNA, Rad51-Rad54, Msh2-Mlh1, and Bard1-Cstf1 interactions have been reported, and several pairs of interacting proteins were found among the other functional categories as well. Many of these genes appear to be bound by E2F4 in quiescent cells when each is inactive and by the E2F1 activator in G1/S phase cells when each is expressed. Furthermore, the analysis of wild-type and p107-/-; p130-/- mouse embryonic fibroblasts indicates that at least two of these genes (RAD54L and BARD1) are repressed in vivo by these pRB-related proteins during G0 and early G1. Consistent with these findings, murine Rad51 has been found to be induced severalfold in cells ectopically expressing E2F1 or E2F2. Therefore, the findings link the processes of DNA replication and repair in mammalian cells and suggest that their expression could be regulated through a common factor, E2F (Ren, 2002).

One of the most surprising findings was the identification of a cluster of genes involved in several different checkpoints. Two genes involved in the DNA damage checkpoint, p53 and Chk1, were identifed. p53 is induced in response to DNA damage and acts to enforce a cell cycle block in G1 phase. The identification of p53 as an E2F target was unanticipated, because the p53 promoter lacks a recognizable E2F consensus site. This finding may be explained by the indirect recruitment of E2F by additional promoter-bound factors. E2F has been shown previously to indirectly increase levels of p53 through activation of the p14ARF gene, a component of the p14ARF-Mdm2 stabilization pathway. These results suggest that E2F may also directly control p53 expression levels. This finding is also intriguing in light of previous reports implicating an essential role for both p53 and the pRB family in the G1 DNA damage arrest checkpoint. The mechanisms underlying the pRB requirement for this G1 block are not known, although a role for E2F-responsive genes has been postulated. A second checkpoint gene, Chk1, was also identified in the E2F location analysis. CHK1 is required for the G2 DNA damage (and perhaps an S phase) checkpoint. Interestingly, pRB is required for Chk1 down-regulation and resumption of G2 after DNA damage, suggesting that E2F could be involved in Chk1 gene expression (Ren, 2002).

Several components of the mitotic spindle checkpoint, including CENP-E, Bub3, Mad2, and the TTK kinase (a homolog of yeast Mps1p), were also identifed. These proteins have been shown to interact, again suggesting that this checkpoint may be controlled coordinately by E2F. Interestingly, two of these genes, MAD2L and TTK, as well as several mitotic genes (HEC, NEK2, and PTTG1), are significantly derepressed in quiescent and early G1 cells deficient for the E2F4-p107/p130 repressor (Ren, 2002).

B-myb (see Drosophila Myb oncogene-like) and cdc25C (see Drosophila String) exemplify different groups of genes whose transcription is consecutively up-regulated during the cell cycle. Both promoters are controlled by transcriptional repression via modules consisting of an E2F binding site (E2FBS) or the related CDE (cell cycle-dependent element) plus a contiguous CHR (cell cycle genes homology region) co-repressor element. The B-myb repressor module, which is derepressed early (mid G1), is preferentially recognized by E2F-DP complexes; a mutation that selectively abolishes E2F binding, impairs regulation. In contrast, the cdc25C repressor module, which is derepressed late (S/G2), interacts selectively with CDE-CHR binding factor-1 (CDF-1). E2F binding, but not CDF-1 binding, requires specific nucleotides flanking the E2FBS/CDE core, while CDF-1 binding, but not E2F binding, depends on specific nucleotides in the CHR. Swapping these nucleotides between the two promoters profoundly changes protein binding patterns and alters expression kinetics. Predominant CDF-1 binding leads to derepression in late S, predominant E2F binding results in up-regulation in late G1, while promoters binding both E2F and CDF-1 with high efficiency show intermediate kinetics. These results support a model where the differential binding of E2F and CDF-1 repressor complexes contributes to the timing of promoter activity during the cell cycle (Lucibello, 1997).

Many of the gene-encoding S-phase-acting proteins are E2F targets, including DNA polymerase alpha, thymidylate synthase, proliferating cell nuclear antigen and ribonucleotide reductase. In addition to the S-phase genes, several genes that play a role in cell cycle progression (such as cdc2, cyclin A and B-myb) (see Drosophila Myb oncogene-like) are also induced by E2F1 (a vertebrate homolog). Cyclin E is strongly induced by E2F1, thereby defining an autoregulatory circuit, since cyclinE-dependent kinase activity can stimulate E2F1 transcription, most likely through the phosphorylation and inactivation of Rb and other Rb family members (DeGregori, 1995).

Transient induction of the cyclin E gene in late G1 gates progression into S. This event is controlled via a cyclin E repressor module (CERM), a novel bipartite repressor element located near the cyclin E transcription start site. CERM consists of a variant E2F-binding site and a contiguous upstream AT-rich sequence that cooperate during G0/G1 to delay cyclin E expression until late G1. CERM binds the protein complex CERC, which disappears upon progression through G0-G1 and reappears upon entry into the following G1. CERC disappearance correlates kinetically with the liberation of the CERM module in vivo and cyclin E transcriptional induction. CERC contains E2F4/DP1 and a pocket protein, and sediments faster than classical E2F complexes in a glycerol gradient, suggesting the presence of additional components in a novel high molecular weight complex. Affinity purified CERC binds to CERM but not to canonical E2F sites, thus displaying behavior different from known E2F complexes. In cells nullizygous for members of the Rb family, CERC is still detectable and CERM-dependent repression is functional. Thus p130, p107 and pRb all function interchangeably in CERC. Notably, the CERC-CERM complex dissociates prematurely in pRb-/- cells in correspondence with the premature expression of cyclin E. Thus, a new regulatory module has been idenfied that controls repression of G1-specific genes in G0/G1 (Le Cam, 1999).

In addition to the E2F-dependent activation of a number of genes encoding DNA replication activities such as DNA Pol alpha, the majority of genes encoding initiation proteins, including Cdc6 and the Mcm proteins, are activated following the stimulation of cell growth and are regulated by E2F. The transcription of a subset of these genes, which includes Cdc6, cyclin E, and cdk2, is also regulated during the cell cycle. Whereas overall E2F DNA-binding activity accumulates during the initial G1 following a growth stimulus, only E2F3-binding activity reaccumulates at subsequent G1/S transitions, coincident with the expression of the cell-cycle-regulated subset of E2F-target genes. Immunodepletion of E2F3 activity inhibits the induction of S phase in proliferating cells. It is proposed that E2F3 activity plays an important role during the cell cycle of proliferating cells, controlling the expression of genes whose products are rate limiting for initiation of DNA replication, thereby imparting a more dramatic control of entry into S phase than would otherwise be achieved by post-transcriptional control alone (Leone, 1998).

Transcription of the human proto-oncogene MYC (see Drosophila Myc) is repressed in quiescent or non-dividing cells. Upon mitogenic stimulation expression of MYC is rapidly and transiently induced, maintained throughout G1, and declines to a basal level throughout further cell cycle transitions. Regulation of MYC promoter activity critically depends on the presence of a binding site for transcription factor E2F. Transcription from the MYC P2 promoter is induced efficiently by E2F-1, but repressed by RB. Furthermore, overexpression of cyclin A strongly activates the MYC promoter and this effect is further enhanced by coexpression of E2F-1 and cyclin A. Expression of G1-phase cyclin D1 leads to an E2F binding site-dependent trans-activation of the MYC promoter and this activation can be abrogated by overexpression of RB. The interaction of D-type G1 cyclins with RB resembles that of the adenovirus E1A protein with RB in that it can disrupt inhibitory E2F-RB complexes. These results support a model in which intervention of distinct cyclins and their respective associated kinases promotes transcriptional activation of MYC throughout the cell cycle either by conversion of E2F within multimeric complexes into an active transcription factor or by liberation of free functional E2F (Oswald, 1994).

Smad3 is a direct mediator of transcriptional activation by the TGFß receptor. Its target genes in epithelial cells include cyclin-dependent kinase inhibitors that generate a cytostatic reponse. This study defines how, in the same context, Smad3 can also mediate transcriptional repression of the growth-promoting gene c-myc. A complex containing Smad3, the transcription factors E2F4/5 and DP1, and the corepressor p107 preexists in the cytoplasm. In response to TGFbeta, this complex moves into the nucleus and associates with Smad4, recognizing a composite Smad-E2F site on c-myc for repression. Previously known as the ultimate recipients of cdk regulatory signals, E2F4/5 and p107 act here as transducers of TGFbeta receptor signals upstream of cdk. Smad proteins therefore mediate transcriptional activation or repression depending on their associated partners (Chen, 2002).

The v-abl oncogene of Abelson murine leukemia virus encodes a deregulated form of the cellular nonreceptor tyrosine kinase. v-Abl activates c-myc transcription, and c-Myc is an essential downstream component in the v-Abl transformation program. To explore the mechanism by which v-Abl activates c-myc transcription, a cotransfection assay was developed. Transactivation of a c-myc promoter by v-Abl requires the SH1 (tyrosine kinase) and SH2 domains of v-Abl; the C-terminal domains are not required for transactivation. The assay also identifies the E2F site in the c-myc promoter as a v-Abl-responsive element. In addition, multimerized E2F sites were shown to be sufficient to confer v-Abl-dependent activation on a minimal promoter. This is the first identification of a v-Abl response element for transcriptional activation. v-Abl tyrosine kinase-dependent changes in proteins binding the c-myc E2F site have also been demonstrated, including induction of a complex containing DP1, p107, cyclin A, and cdk2. Identification of v-Abl-dependent changes in E2F-binding proteins provides an important link between v-Abl, transcription, cell cycle regulation, and control of cellular growth (Wong, 1995).

In the human, accumulation of G1 cyclins is regulated by E2F1. E2F binding sites are found in both the cyclin E and cyclin D1 promoters: both promoters are activated by E2F gene products. In the case of cyclin E, the E2F sites contribute to cell cycle control. The endogenous cyclin E gene is activated following expression of the E2F1 product encoded by a recombinant adenovirus vector. These results suggest the involvement of E2F1 and cyclin E in a autoregulatory loop that governs the accumulation of critical activities affecting the progression of cells through G1 (Ohtani, 1995).

Orderly cell cycle progression is regulated by coordinated interactions among cyclin-dependent kinases (Cdks), their target "pocket proteins" (the retinoblastoma protein [pRB], p107, and p130), the pocket protein binding E2F-DP complexes, and the Cdk inhibitors. The cyclin D1 gene encodes a regulatory subunit of the Cdk holoenzymes, which phosphorylates the tumor suppressor pRB, leading to the release of free E2F-1. Overexpression of E2F-1 can induce apoptosis and may either promote or inhibit cellular proliferation, depending on the cell type. In these studies, overexpression of E2F-1 inhibits cyclin D1-dependent kinase activity, cyclin D1 protein levels, and promoter activity. The DNA binding domain, the pRB pocket binding region, and the amino-terminal Sp1 binding domain of E2F-1 are required for full repression of cyclin D1. Overexpression of pRB activates the cyclin D1 promoter, and a dominant interfering pRB mutant is defective in cyclin D1 promoter activation. Two regions of the cyclin D1 promoter are required for full E2F-1-dependent repression. The region proximal to the transcription initiation site at -127 binds Sp1, Sp3, and Sp4, and the distal region at -143 binds E2F-4-DP-1-p107. In contrast with E2F-1, E2F-4 induces cyclin D1 promoter activity. Differential regulation of the cyclin D1 promoter by E2F-1 and E2F-4 suggests that E2Fs may serve distinguishable functions during cell cycle progression. Inhibition of cyclin D1 abundance by E2F-1 may contribute to an autoregulatory feedback loop to reduce pRB phosphorylation and E2F-1 levels in the cell (Watanabe, 1998).

Expression of human Origin recognition complex 1 gene (HsOrc1) is regulated via the E2F transcription factor. HsOrc1 expression is low in quiescent cells, and it is then dramatically induced upon stimulation of cell growth. In contrast, expression of the HsOrc2 gene does not appear to be similarly regulated. The promoter that regulates HsOrc1 has been isolated and it confers cell growth-dependent expression. The cell growth control is largely the consequence of E2F-dependent negative transcription control in quiescent cells. Activation of HsOrc1 transcription following growth stimulation requires G1 cyclin-dependent kinase activity, and forced E2F1 expression can bypass this requirement. Expression of cyclin-dependent kinase inhibitor p21 inhibits G1 cyclin-dependent kinase activity and blocks S-phase entry. This p21-mediated inhibition of G1 cyclin-dependent kinase activity also eliminates the induction of HsOrc1 mRNA accumulation that is normally associated with the growth stimulation of quiescent cells. These results thus provide a direct link between the initiation of DNA replication and the cell growth regulatory pathway involving G1 cyclin-dependent kinases, and E2F (Ohtani, 1996).

E2F transcription activity is carried out by a family of heterodimers encoded by distinct genes. Through the overproduction of each of the five known E2F proteins in mammalian cells, it has been demonstrated that a large number of genes encoding proteins important for cell cycle regulation and DNA replication can be activated by the E2F proteins and that there are distinct specificities in the activation of these genes by individual E2F family members. Coexpression of each E2F protein with the DP1 heterodimeric partner does not significantly alter this specificity. E2F1 leads to the activation of four different p16INK4a-related transcripts, although not p16INK4a itself. Only E2F1 overexpression induces cells to undergo apoptosis, despite the fact that at least two other E2F family members, E2F2 and E2F3, are equally capable of inducing S phase. The ability of E2F1 to induce apoptosis appears to result from the specific induction of an apoptosis-promoting activity rather than the lack of induction of a survival activity, because co-expression of E2F2 and E2F3 does not rescue cells from E2F1-mediated apoptosis. It is concluded that E2F family members play distinct roles in cell cycle control and that E2F1 may function as a specific signal for the initiation of an apoptosis pathway that must normally be blocked for a productive proliferation event (DeGregori, 1997).

cdc25A is a tyrosine phosphatase that activates G1 cyclin-dependent kinases (Cdk's). In human keratinocytes, cdc25A expression is down-regulated after the initial drop in Cdk activity caused by cell exposure to the antimitogenic cytokine transforming growth factor beta (TGF-beta) or removal of serum factors. The TGF-beta-inhibitory-response element in the cdc25A promoter maps to an E2F site at nucleotides -62 to -55 from the transcription start site. This site is not required for basal transcription in keratinocytes. Evidence is provided that the cell cycle arrest program activated by TGF-beta in human keratinocytes includes the generation of E2F4-p130 complexes that in association with histone deacetylase HDAC1 inhibits the activity of the cdc25A promoter from this repressor E2F site. This mechanism is part of a program that places keratinocytes in the quiescent state following the initial drop in Cdk activity caused by cell exposure to TGF-beta (Iavarone, 1999).

Cell division is driven by cyclin-B-dependent kinase and anaphase-promoting complex (APC)-mediated proteolysis. Continuing transcription of E2F target genes beyond the G1/S transition is required for coordinating S-phase progression with cell division. Using an in vivo assay to measure protein stability in real time during the cell cycle, it has been shown that repression of E2F activity or inhibition of cyclin-A-dependent kinase in S phase triggers the destruction of cyclin B1 through the re-assembly of APC, the ubiquitin ligase that is essential for mitotic cyclin proteolysis, with its activatory subunit Cdh1. Phosphorylation-deficient mutant Cdh1 or immunodepletion of cyclin A results in assembly of active Cdh1-APC even in S-phase cells. These results implicate an E2F-dependent, cyclin A/Cdk2-mediated phosphorylation of Cdh1 in the timely accumulation of cyclin B1 and the coordination of cell-cycle progression during the G-1 phase post-restriction point period (Lukas, 1999).

E2F is a family of transcription factors that regulate both cellular proliferation and differentiation. To establish the role of E2F3 in vivo, an E2f3 mutant mouse strain was generated. E2F3-deficient mice arise at one-quarter of the expected frequency, demonstrating that E2F3 is important for normal development. To determine the molecular consequences of E2F3 deficiency, the properties of embryonic fibroblasts derived from E2f3 mutant mice were analyzed. Mutation of E2f3 dramatically impairs the mitogen-induced, transcriptional activation of numerous E2F-responsive genes. A number of genes, including B-myb, cyclin A, cdc2, cdc6, and DHFR, could be identified whose expression is dependent on the presence of E2F3 but not E2F1. A critical threshold level of one or more of the E2F3-regulated genes determines the timing of the G1/S transition, the rate of DNA synthesis, and thereby the rate of cellular proliferation. E2F3 is not required for cellular immortalization but is rate limiting for the proliferation of the resulting tumor cell lines. It is concluded that E2F3 is critical for the transcriptional activation of genes that control the rate of proliferation of both primary and tumor cells (Humbert, 2000a).

The retinoblastoma protein (pRB) and its two relatives, p107 and p130, regulate development and cell proliferation in part by inhibiting the activity of E2F-regulated promoters. High-density oligonucleotide arrays have been used to identify genes in which expression changes in response to activation of E2F1, E2F2, and E2F3. The E2Fs control the expression of several genes that are involved in cell proliferation and E2Fs regulate a number of genes involved in apoptosis, differentiation, and development. These results provide possible genetic explanations to the variety of phenotypes observed as a consequence of a deregulated pRB/E2F pathway (Muller, 2001).

The E2Fs regulate the expression of several proteins that are involved in early development, including homeobox proteins, transcription factors involved in cell fate decisions, a number of proteins that determine homeotic gene transcription, and signaling pathways such as the TGFbeta and Wnt pathways that are essential for early development. As an example of the relevance of these findings, it has been reported that position-effect variegation (PEV) in Drosophila depends on E2F activity. Loss of E2F activity enhances PEV, whereas overexpression of E2F activity suppresses PEV in Drosophila. These data suggested that the E2Fs themselves have an epigenetic effect by regulating chromatin structure or, more likely, that the E2Fs control PEV by regulating genes of the Polycomb group (PcG) family. In this screen, several PcG genes have been identified, like Enhancer of Zeste 2 (EZH2), Embryonic Ectoderm Development protein (EED) and Homolog of Polyhomeotic (EDR2/HPH2). The E2F-induced expression of these genes may provide an explanation for the role of E2F in the regulation of PEV and, more importantly, in development (Muller, 2001).

The pRB/E2F pathway is known to be central in the regulation of various types of cellular differentiation. For example, pRB is required for erythroid, neuronal, eye, muscle, and adipocyte differentiation. Both p107 and p130 are required for normal endochondrial bone development. In addition, E2F4 is known to contribute to hematopoetic lineage and to craniofacial development, whereas loss of E2f5 leads to overproduction of cerebrospinal fluid and to hydrocephalus. Although these phenotypes are complex, gene expression analysis may help to elucidate the role (or roles) played by the E2Fs in pRB/p107/p130 regulated differentiation. (1) A number of transcription factors were detected that are involved in cell fate decisions, such as Hairy/enhancer of split related (HEY1), Paired-like homeodomain (PTX1), ID4, MAF family members, and Sox9. These transcription factors have been associated with neurogenesis, morphogenesis, hindlimb, and craniofacial development (PTX1), block of differentiation of various tissues (ID4), the regulation of early differentiation (MAF family), and cartilage formation (Sox9). The expression of transcription factors that are involved in cell fate decisions is exquisitely controlled, and in several cases it has been described that the over- or mis-expression of such transcription factors leads to malformations and/or transformation. (2) E2F activation leads to a dramatic change in the expression of genes in the TGFbeta pathway. TGFbeta family members, which include TGFbetas, activins, and bone morphogenetic proteins (BMPs), are secreted molecules that regulate a plethora of cellular responses, such as proliferation, differentiation, migration, and apoptosis. E2F activation leads to strong suppression of Inhibin beta A and TGFbeta2, and to a strong induction of Follistatin expression. Inhibin beta A participates in the regulation of gametogenesis and craniofacial development, and mice that are homozygously null for Inhibin beta A die within 24 h of birth (Muller, 2001).

Interestingly, Inhibin beta A was also isolated as erythroid differentiation factor and the gene has been shown to be an important regulator of photoreceptor differentiation in the developing retina. Together with the fact that Follistatin is an inhibitor of Activin A (the homodimer of inhibin beta A that binds Activin receptors), these data suggest that the simultaneous induction of Follistatin and repression of inhibin beta A could be involved in specific differentiation defects observed in the eye and in the erythrocytes of the Rb- mice. For example, Rb- erythroid cells develop normally in high percentage Rb- chimeric mice, which suggests that the Rb wild-type cells rescued the Rb- cells, perhaps by secreting paracrine or endocrine erythroid differentiation factor (or factors) such as inhibin beta A or Follistatin (Muller, 2001).

There is a requirement for a normal pRB/E2F pathway in chondrocyte differentiation. Several genes have been identified that are involved in chondrocyte differentiation, such as the TGFbeta pathways genes, FGF receptor 2 and FGF receptor 3, Cartilage linking protein 1, and Connective Tissue Growth factor. Discrete changes in the expression of several of these genes as a result of deregulated E2F activity could lead to both the defects in chondrocyte differentiation observed in the p107/p130 double-knockout mice, and the craniofacial abnormalities observed in the p107/p130 and the E2f4 knockout mice. Moreover, it is striking that activating mutations of FGF receptor 3 causes achondroplasia (the most common genetic form of dwarfism in humans), considering the fact that p107/p130 knockout mice have short bones (Muller, 2001).

Eukaryotic DNA replication requires the previous formation of a prereplication complex containing the ATPase Cdc6 and the minichromosome maintenance (Mcm) complex. Although considerable insight has been gained from in vitro studies and yeast genetics, the functional analysis of replication proteins in intact mammalian cells has been lacking. Adenoviral vectors have been used to express normal and mutant forms of Cdc6 in quiescent mammalian cells to assess function. Cdc6 expression alone is sufficient to induce a stable association of endogenous Mcm proteins with chromatin in serum-deprived cells where cyclin-dependent kinase (cdk) activity is low. Moreover, endogenous Cdc6 is sufficient to load Mcm proteins onto chromatin in the absence of cdk activity in p21-arrested cells. Cdc6 synergizes with physiological levels of cyclin E/Cdk2 to induce semiconservative DNA replication in quiescent cells whereas cyclin A/Cdk2 is unable to collaborate with Cdc6. Cdc6 that cannot be phosphorylated by cdks is fully capable of inducing Mcm chromatin association and replication. Mutation of the Cdc6 ATP-binding site severely impairs the ability of Cdc6 to induce Mcm chromatin loading and reduces its ability to induce replication. Nevertheless, the ATPase domain of Cdc6 in the absence of the noncatalytic amino terminus is not sufficient for either Mcm chromatin loading or DNA replication, indicating a requirement for this domain of Cdc6 (Cook, 2002).

The best-characterized substrates of cyclin E/Cdk2 are the retinoblastoma family proteins, Rb, p130, and p107. Phosphorylation of Rb by cyclin D/Cdk4 and cyclin E/Cdk2 dissociates Rb from E2F and allows the induction of E2F target genes. The synergy between low-level cyclin E/Cdk2 expression and Cdc6 is only seen when cyclin E/Cdk2 activity is low enough to induce endogenous cdc6 expression minimally. Thus one function of cyclin E/Cdk2 in the induction of S phase is its well-documented role in transcriptional control of E2F target genes such as cdc6. The role of cyclin E/Cdk2 in Mcm chromatin loading is restricted to its function in E2F-dependent transcriptional control of the cdc6 gene because expression of Cdc6 in the absence of cdk activity (either by ectopic expression or by induction of the endogenous gene by E2F) bypasses the need for cdk activity in Mcm chromatin loading. Cyclin E/Cdk2 activity is not required for prereplication complex formation as long as Cdc6 is produced (Cook, 2002).

Clearly, cyclin E/Cdk2 plays additional roles in replication initiation downstream of Mcm chromatin loading because Cdc6-mediated Mcm chromatin loading is not sufficient for replication without cyclin E/Cdk2, and Cdk2 activity is still required for initiation in X. laevis extracts in which transcriptional control is not important. At least one of those functions is likely to be the loading of the Cdc45 protein onto the newly formed prereplication complex, although the precise mechanism of this aspect of cyclin E/Cdk2 function remains to be elucidated (Cook, 2002).

Cellular senescence is an extremely stable form of cell cycle arrest that limits the proliferation of damaged cells and may act as a natural barrier to cancer progression. A distinct heterochromatic structure is descibed that accumulates in senescent human fibroblasts, that is designated senescence-associated heterochromatic foci (SAHF). SAHF formation coincides with the recruitment of heterochromatin proteins and the retinoblastoma (Rb) tumor suppressor to E2F-responsive promoters and is associated with the stable repression of E2F target genes. Notably, both SAHF formation and the silencing of E2F target genes depend on the integrity of the Rb pathway and do not occur in reversibly arrested cells. These results provide a molecular explanation for the stability of the senescent state, as well as new insights into the action of Rb as a tumor suppressor (Narita, 2003).

SAHFs are observed in interphase nuclei and contain the heterochromatin-associated proteins H3 methylated on lysine 9 (K9M-H3) and HP1, exclude histones found in euchromatin (e.g., K9Ac-H3 and K4M-H3), and are not sites of active transcription. SAHFs are distinct from pericentric heterochromatin, and their appearance is accompanied by an increase in HP1 incorporation into senescent chromatin and an enhanced resistance of senescent DNA to nuclease digestion (Narita, 2003).

SAHF formation requires an intact Rb pathway, since expression of E1A, or inactivation of either p16INK4a or Rb, can prevent their appearance. During the initial phases of senescence, Rb might control the nucleation of heterochromatin at specific sites throughout the genome, which then spreads by the action of histone methyltransferases and recruitment of HP1 proteins. HP1 proteins have the capacity to dimerize and may interact to form higher order chromatin structures once a critical mass has been reached. A similar pattern of nucleation and spreading occurs during silencing of the mating type locus in S. pombe, position effect variegation in Drosophila, and X inactivation in mammalian cells, although HP1 proteins do not accumulate on the inactive X. Importantly, SAHF formation correlates precisely with cell cycle exit and the silencing of E2F target genes (Narita, 2003).

Much of what is known concerning the regulation of E2F activity comes from studies examining cell cycle transitions into and out of a quiescent state. These transitions are controlled in a reversible manner, in part, by the competing action of HATs and HDACs on the histones of E2F target promoters. This study compares the physical state and regulation of E2F target genes in quiescent and senescent cells. In both cell states, the amount of K9-aceylated histone H3 that associates with E2F target promoters declines, consistent with the downregulation of transcription that accompanies cell cycle exit. However, in senescent IMR90 cells, histone H3 acetylation is ultimately replaced by methylation at lysine 9, an apparently irreversible modification that prevents acetylation by HATs and is barely observed on E2F-responsive promoters in quiescent cells. Methylated lysine 9 forms a docking site for HP1 proteins and, accordingly, HP1gamma preferentially associates with E2F target promoters in senescent cells. These modifications are predicted to form a 'lock' on the transcription of E2F responsive promoters, making them less accessible to the transcription machinery. Accordingly, several E2F-responsive genes in senescent cells are stably repressed and insensitive to enforced E2F expression relative to quiescent cells. Although it remains to be determined whether every E2F target gene behaves as those studied here, their transition to a heterochromatin-like organization may contribute to the insensitivity of senescent cells to mitogenic signals and the apparent irreversibility of the senescence process (Narita, 2003).

EZH2, homolog of Drosophila E(z) is highly expressed in metastatic prostate cancer and in lymphomas. EZH2 is a component of the PRC2 histone methyltransferase complex, which also contains EED and SUZ12 and is required for the silencing of HOX gene expression during embryonic development. Both EZH2 and EED are essential for the proliferation of both transformed and non-transformed human cells. In addition, the pRB-E2F pathway tightly regulates their expression and, consistent with this, EZH2 is found to be highly expressed in a large set of human tumors. These results raise the question whether EZH2 is a marker of proliferation or if it is actually contributing to tumor formation. Significantly, it is proposed that EZH2 is a bona fide oncogene, since ectopic expression of EZH2 is found to be capable of providing a proliferative advantage to primary cells and, in addition, its gene locus is specifically amplified in several primary tumors (Bracken, 2003).

Geminin (see Drosophila Geminin) and Cdt1 (Drosophila homolog: Double parked) play an essential role in the initiation of DNA replication, by regulating the chromatin loading of the MCM complex. The transcription of human Geminin and Cdt1, as well as that of MCM7, is activated by transcription factors E2F1-4, but not by factors E2F5-7. Analysis of various Geminin and Cdt1 promoter constructs shows that an E2F-responsive sequence in the vicinity of the transcription initiation site is necessary for the transcriptional activation. The promoter activity for human Geminin was activated by the E7, but not E6, oncogene of human papillomavirus type 16. While E2F1-induced activation of human Cdt1 gene transcription was suppressed by pRb, but not by p107 or p130, its E2F4-induced activation was suppressed by pRb, p107, and p130. Furthermore, the promoter activities of human Geminin and Cdt1 were demonstrated to be growth-dependent. Taken together, the results demonstrate that Geminin and Cdt1 constitute targets for various members of the E2F family of transcription factors, and that expression of Geminin and Cdt1 is perhaps mediated by the activation of a pRb/E2F pathway (Yoshida, 2004).

E2F transcription factors play a critical role in the control of cell cycle progression, regulating the expression of genes involved in DNA replication, DNA repair, mitosis, and cell fate. This involves both positive-acting and negative-acting E2F proteins, the latter group including the E2F6 protein, which has been shown to function as an Rb-independent repressor of E2F-target gene transcription. In an effort to better delineate the context of E2F6 function, including the mechanisms of E2F6 functional specificity, chromatin immunoprecipitation assays were used to assess when and with what genes E2F6 associates during a cell cycle. E2F6 was found to associate specifically with the E2F target genes that are activated at G1/S; this interaction occurs during S phase of the cell cycle. In sharp contrast, E2F6 does not bind to E2F-regulated genes activated at G2/M. In the absence of E2F6, E2F4 can bind to the G1/S-regulated promoters and compensate for loss of E2F6 function. Indeed, inhibition of both E2F4 and E2F6 activity results in specific derepression of these genes during S phase. It is concluded that E2F6 functions as a repressor of E2F-dependent transcription during S phase and given the specificity for the G1/S-regulated genes, it is proposed that E2F6 functions to distinguish G1/S and G2/M transcription during the cell cycle (Giangrande, 2004).

E2F coregulates an essential HSF developmental program that is distinct from the heat-shock response

Heat-shock factor (HSF; see Drosophila Hsf) is the master transcriptional regulator of the heat-shock response (HSR) and is essential for stress resilience. HSF is also required for metazoan development; however, its function and regulation in this process are poorly understood. This study characterize the genomic distribution and transcriptional activity of Caenorhabditis elegans HSF-1 during larval development and showed that the developmental HSF-1 transcriptional program is distinct from the HSR. HSF-1 developmental activation requires binding of E2F/DP (see Drosophila E2f) to a GC-rich motif that facilitates HSF-1 binding to a heat-shock element (HSE) that is degenerate from the consensus HSE sequence and adjacent to the E2F-binding site at promoters. In contrast, induction of the HSR is independent of these promoter elements or E2F/DP and instead requires a distinct set of tandem canonical HSEs. Together, E2F and HSF-1 directly regulate a gene network, including a specific subset of chaperones, to promote protein biogenesis and anabolic metabolism, which are essential in development (Li , 2016).

Other E2F interactions

There are both physical and functional interactions among p53, E2F1 and DP1. As a consequence of the ability to physically associate, the expression of wild-type p53 can inhibit transcriptional activation by E2F, and the expression of both E2F1 and DP1 can also downregulate p53-dependent transcriptional activation. Transcriptional activation by p53 is know to be inhibited by the direct binding of mdm2, but either or both E2F1 and DP1 can inhibit p53 transcriptional activation independently of mdm2 (O'Conner, 1995).

Expression of the vertebrate cyclin A gene is characterized by repression of its promoter during the G1 phase of the cell cycle and its induction at S-phase entry. This regulation is mediated by the transcription factor E2F, which binds to a specific site in the cyclin A promoter. The E2F binding site differs from the prototype E2F site in nucleotide sequence and protein binding; it is bound by E2F complexes containing cyclin E and p107 but not pRB. Ectopic expression of cyclin D1 triggers premature activation of the cyclin A promoter by E2F, and this effect is blocked by the tumor suppressor protein p16INK4 (Schulze, 1995).

E2F-1 is phosphorylated more effectively by cyclin A-cdk2 than by cyclin E-cdk2. Phosphorylation of E2F-1 begins with the S phase. The binding of E2F-1 to the viral e2 promoter is reduced by the phosphorylation of E2F-1 by cyclin A-cdk2, suggesting that phosphorylation of E2F-1 may induce shut off of E2F-1 regulated gene expression at the transcriptional level (Kitagawa, 1995).

The c-Myc and E2F transcription factors are among the most potent regulators of cell cycle progression in higher eukaryotes. This report describes the isolation of a novel, highly conserved 434 kDa protein, designated TRRAP, which interacts specifically with the c-Myc N terminus and has homology to the ATM/PI3-kinase family. TRRAP also interacts specifically with the E2F-1 transactivation domain. Expression of transdominant mutants of the TRRAP protein or antisense RNA blocks c-Myc- and E1A-mediated oncogenic transformation. These data suggest that TRRAP is an essential cofactor for both the c-Myc and E1A/E2F oncogenic transcription factor pathways (McMahon, 1998).

During the G1 phase of the cell cycle, an E2F-RB complex represses transcription, via the recruitment of histone deacetylase activity. Phosphorylation of RB at the G1/S boundary generates a pool of 'free' E2F, which then stimulates transcription of S-phase genes. Given that E2F1 activity is stimulated by p300/CBP acetylase and repressed by an RB-associated deacetylase, would E2F1 be subject to modification by acetylation? The p300/CBP-associated factor P/CAF, and to a lesser extent p300/CBP itself, can acetylate E2F1 in vitro and intracellular E2F1 is acetylated. The acetylation sites lie adjacent to the E2F1 DNA-binding domain and involve lysine residues highly conserved in E2F1, 2 and 3. Acetylation by P/CAF has three functional consequences on E2F1 activity: increased DNA-binding ability, activation potential and protein half-life. These results suggest that acetylation stimulates the functions of the non-RB bound 'free' form of E2F1. Consistent with this, it has been found that the RB-associated histone deacetylase can deacetylate E2F1. These results identify acetylation as a novel regulatory modification that stimulates E2F1's activation functions (Martinez-Balbas, 2000).

The transcription factor E2F-1 interacts stably with cyclin A via a small domain near E2F-1's amino terminus and is negatively regulated by the cyclin A-dependent kinases. Thus, the activities of E2F, a family of transcription factors involved in cell proliferation, are regulated by at least two types of cell growth regulators: the retinoblastoma protein family and the cyclin-dependent kinase family. Using purified components in an in vitro system, it has been shown that the E2F-1-DP-1 heterodimer, the functionally active form of the E2F activity, is not a substrate for the active cyclin D-dependent kinases but is efficiently phosphorylated by the cyclin B-dependent kinases, which do not form stable complexes with the E2F-1-DP-1 heterodimer. Phosphorylation of the E2F-1-DP-1 heterodimer by cyclin B-dependent kinases, however, does not result in down-regulation of E2F's DNA-binding activity, as is readily seen after phosphorylation by cyclin A-dependent kinases, suggesting that phosphorylation per se is not sufficient to regulate E2F DNA-binding activity. Furthermore, heterodimers containing E2F-4, a family member lacking the cyclin A binding domain found in E2F-1, are not efficiently phosphorylated or functionally down-regulated by cyclin A-dependent kinases. However, addition of the E2F-1 cyclin A binding domain to E2F-4 confers cyclin A-dependent kinase-mediated down-regulation of the E2F-4-DP-1 heterodimer. Thus, both enzymatic phosphorylation and stable physical interaction are necessary for the specific regulation of E2F family members by cyclin-dependent kinases (Dynlacht, 1997).

The transcription factor E2F1 is believed to be involved in the regulated expression of the DNA replication genes. To gain insights into the transcriptional activation function of E2F1, a search was performed for proteins in HeLa nuclear extracts that bind to the activation domain of E2F1. DDB, a putative DNA repair protein, associates with the activation domain of E2F1. DDB was identified as a heterodimeric protein (48 and 127 kDa) that binds to UV-damaged DNA. The UV-damaged-DNA binding activity from HeLa nuclear extracts can associate with the activation domain of E2F1. The 48-kDa subunit of DDB, synthesized in vitro, binds to a fusion protein of E2F1, depending on the C-terminal activation domain. The interaction between DDB and E2F1 can also be detected by coimmunoprecipitation experiments. Immunoprecipitation of an epitope-tagged DDB from cell extracts results in the coprecipitation of E2F1. In a reciprocal experiment, immunoprecipitates of E2F1 were found to contain DDB. Fractionation of HeLa nuclear extracts also reveals a significant overlap in the elution profiles of E2F1 and DDB. For instance, DDB, which does not bind to the E2F sites, is enriched in the high-salt fractions containing E2F1 during chromatography through an E2F-specific DNA affinity column. There is also evidence for a functional interaction between DDB and E2F1 in living cells. For instance, expression of DDB specifically stimulates E2F1-activated transcription. The transcriptional activation function of a heterologous transcription factor containing the activation domain of E2F1 is stimulated by coexpression of DDB. Moreover, DDB expression can overcome the retinoblastoma protein (Rb)-mediated inhibition of E2F1-activated transcription. The results suggest that this damaged-DNA binding protein can function as a transcriptional partner of E2F1. It is speculated that in addition to its damaged-DNA binding function, DDB might serve as a negative regulator of E2F1-activated transcription, since damaged DNA will sequester DDB and make it unavailable for E2F1. The binding of DDB to damaged DNA might be involved in downregulating the replication genes during growth arrest induced by damaged DNA (Hayes, 1998).

Overexpression of c-Myc or E2F1 sensitizes host cells to various types of apoptosis. Overexpressed c-Myc or E2F1 induces accumulation of reactive oxygen species (ROS) and thereby enhances serum-deprived apoptosis in NIH3T3 and Saos-2. During serum deprivation, MnSOD mRNA is induced by NF-kappaB in mock-transfected NIH3T3, while this induction was inhibited in NIH3T3 overexpressing c-Myc or E2F1. In these clones, E2F1 inhibits NF-kappaB activity by binding to its subunit p65 in competition with a heterodimeric partner p50. In addition to overexpressed E2F1, endogenous E2F1 released from Rb is also found to inhibit NF-kappaB activity in a cell cycle-dependent manner by using E2F1+/+ and E2F1-/- murine embryonic fibroblasts. These results indicate that E2F1 promotes apoptosis by inhibiting NF-kappaB activity (Tanaka, 2002).

The rate of hepatocyte proliferation in livers from newborn C/EBPalpha knockout mice is increased. An examination of cell cycle-related proteins showed that the cyclin-dependent kinase (CDK) inhibitor p21 level is reduced in the knockout animals compared to that in wild-type littermates. Additional cell cycle-associated proteins are affected by C/EBPalpha. C/EBPalpha controls the composition of E2F complexes through interaction with the retinoblastoma (Rb)-like protein, p107, during prenatal liver development. S-phase-specific E2F complexes containing E2F, DP, cdk2, cyclin A, and p107 are observed in the developing liver. In wild-type animals these complexes disappear by day 18 of gestation and are no longer present in the newborn animals. In the C/EBPalpha mutant, the S-phase-specific complexes do not diminish and persist to birth. The elevation of levels of the S-phase-specific E2F-p107 complexes in C/EBPalpha knockout mice correlates with the increased expression of several E2F-dependent genes such as those that encode cyclin A, proliferating cell nuclear antigen, and p107. The C/EBPalpha-mediated regulation of E2F binding is specific, since the deletion of another C/EBP family member, C/EBPbeta, does not change the pattern of E2F binding during prenatal liver development. The addition of bacterially expressed, purified His-C/EBPalpha to the E2F binding reaction results in the disruption of E2F complexes containing p107 in nuclear extracts from C/EBPalpha knockout mouse livers. Ectopic expression of C/EBPalpha in cultured cells also leads to a reduction of E2F complexes containing Rb family proteins. Coimmunoprecipitation analyses reveal an interaction of C/EBPalpha with p107 but none with cdk2, E2F1, or cyclin A. A region of C/EBPalpha that has sequence similarity to E2F is sufficient for the disruption of the E2F-p107 complexes. Despite its role as a DNA binding protein, C/EBPalpha brings about a change in E2F complex composition through a protein-protein interaction. The disruption of E2F-p107 complexes correlates with C/EBPalpha-mediated growth arrest of hepatocytes in newborn animals (Timchenko, 1999).

To explore mechanisms for specificity of function within the family of E2F transcription factors, proteins that interact with individual E2F proteins have been identified. A two-hybrid screen identified RYBP (Ring1- and YY1-binding protein) as a protein that interacts specifically with the E2F2 and E2F3 family members, dependent on the marked box domain in these proteins. Previous work has demonstrated that the interaction of the adenovirus E4 ORF6/7 protein with E2F, mediated through the marked box domain, leads to a stimulation of E2F-dependent transcription. The Cdc6 promoter contains adjacent E2F- and YY1-binding sites, and both are required for promoter activity. In addition, YY1 and RYBP, in combination with either E2F2 or E2F3, can stimulate Cdc6 promoter activity synergistically, dependent on the marked box domain of E2F3. Using chromatin immunoprecipitation assays, it has been shown that both E2F2 and E2F3, as well as YY1 and RYBP, associate with the Cdc6 promoter at G1/S of the cell cycle. In contrast, no interaction of E2F1 with the Cdc6 promoter was detected. It is suggested that the ability of RYBP to mediate an interaction between E2F2 or E2F3 and YY1 is an important component of Cdc6 activation and provides a basis for specificity of E2F function (Schlisio, 2002).

Multicilin drives centriole biogenesis via E2f proteins

Multiciliate cells employ hundreds of motile cilia to produce fluid flow, which they nucleate and extend by first assembling hundreds of centrioles. In most cells, entry into the cell cycle allows centrioles to undergo a single round of duplication, but in differentiating multiciliate cells, massive centriole assembly occurs in G0 by a process initiated by a small coiled-coil protein, Multicilin. This study shows that Multicilin acts by forming a ternary complex with E2f4 or E2f5 and Dp1 that binds and activates most of the genes required for centriole biogenesis, while other cell cycle genes remain off. This complex also promotes the deuterosome pathway of centriole biogenesis by activating the expression of deup1 but not its paralog, cep63. Finally, this study shows that this complex is disabled by mutations in human Multicilin that cause a severe congenital mucociliary clearance disorder due to reduced generation of multiple cilia. By coopting the E2f regulation of cell cycle genes, Multicilin drives massive centriole assembly in epithelial progenitors in a manner required for multiciliate cell differentiation (Ma, 2014)

KDM4A coactivates E2F1 to regulate the PDK-dependent metabolic switch between mitochondrial oxidation and glycolysis

The histone lysine demethylase KDM4A/JMJD2A (see Drosophila KDM4A) has been implicated in prostate carcinogenesis through its role in transcriptional regulation. This study describes KDM4A as a E2F1 (see Drosophila E2F1) coactivator and demonstrate a functional role for the E2F1-KDM4A complex in the control of tumor metabolism. KDM4A associates with E2F1 on target gene promoters and enhances E2F1 chromatin binding and transcriptional activity, thereby modulating the transcriptional profile essential for cancer cell proliferation and survival. The pyruvate dehydrogenase kinases (PDKs; see Drosophila Pdk) PDK1 and PDK3 are direct targets of KDM4A and E2F1 and modulate the switch between glycolytic metabolism and mitochondrial oxidation. Downregulation of KDM4A leads to elevated activity of pyruvate dehydrogenase and mitochondrial oxidation, resulting in excessive accumulation of reactive oxygen species. The altered metabolic phenotypes can be partially rescued by ectopic expression of PDK1 and PDK3, indicating a KDM4A-dependent tumor metabolic regulation via PDK. These results suggest that KDM4A is a key regulator of tumor metabolism and a potential therapeutic target for prostate cancer (Wang, 2016).

E2F mutation

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 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, 1999).

Analysis of newborn mice deficient in E2F4, the major form of E2F in many cell types, reveals abnormalities in hematopoietic lineage development as well as defects in the development of the gut epithelium. Specifically, a deficiency of various mature hematopoietic cell types is observed together with an increased number of immature cells in several lineages. This is associated with an increased frequency of apoptotic cells. A substantial reduction is found in the thickness of the gut epithelium that normally gives rise to crypts as well as a reduction in the density of villi. These observations suggest a critical role for E2F4 activity in controlling the maturation of cells in a number of tissues (Rempeli, 2000).

The retinoblastoma protein (pRB) plays a key role in the control of normal development and proliferation through the regulation of the E2F transcription factors. A mutant mouse model was generated to assess the in vivo role of the predominant E2F family member, E2F4. Remarkably, loss of E2F4 has no detectable effect on either cell cycle arrest or proliferation. However, E2F4 is essential for normal development. E2f4-/- mice die of an increased susceptibility to opportunistic infections that appears to result from craniofacial defects. They also display a variety of erythroid abnormalities that arise from a cell autonomous defect in late stage maturation. This suggests that E2F4 makes a major contribution to the control of erythrocyte development by the pRB tumor suppressor (Humbert, 2000b).

E2F4 accounts for the majority of endogenous E2F DNA binding activity and is thought to play a key role in mediating the transcriptional repressive properties of pRB, p107, and p130. Consequently, comparison of the phenotypes of E2f4 and Rb, p107 and p130 mutant mice provides considerable insight into the biological roles of the E2F•pocket protein complexes. Mice deficient for p107 and p130 have a defect in long bone development that results from the failure of chondrocytes to arrest and differentiate at the correct developmental stage. In addition, p107-/-:p130-/- mutant MEFs enter S phase prematurely, and this correlates with the dramatic deregulation of many E2F-responsive genes. These data support the notion that p107•E2F and p130•E2F complexes play a key role in mediating the repression of E2F-responsive genes during G0/G1. However, loss of E2F-4, and therefore the vast majority of the p107•E2F and p130•E2F complexes, does not cause any defect in long bone formation or cell cycle regulation and it has little or no effect on the expression of E2F-responsive genes. Thus, loss of E2F4 is not equivalent to loss of p107 and p130. These findings support two alternative models of p107/p130 action. First, correct cell cycle regulation could be dependent upon the formation of transcriptionally repressive p107•E2F and p130•E2F complexes but E2F5 is sufficient to mediate this effect in the absence of E2F4. Alternatively, loss of p107 and p130 could lead to the deregulation of E2F-responsive genes and thereby S phase entry through the inappropriate release of 'free', transcriptionally active E2F4 and E2F5 complexes. This would be entirely analogous to the finding that ectopic S phase entry and aberrant apoptosis in Rb-deficient embryos or tumors is largely due to the inappropriate activation of E2F1, a pRB-specific E2F. However, it is inconsistent with the current belief that, at least in normal cells, E2F4 and E2F5 make little or no contribution to the activation of E2F-responsive genes (Humbert, 2000 and references therein).

In contrast to p107 and p130, significant similarities were found in the roles of E2F4 and pRB in vivo. pRb-deficient mice die at E13.5 from ineffective erythropoiesis. This phenotype is complex and results from defects in both RBCs and hepatocytes. However, there is evidence to support the presence of a cell-autonomous component to the erythroid defect. (1) Embryos with high Rb-/- ES cell contribution display abnormal nucleated erythrocytes peaking at E16.5 of development. (2) pRB-deficient erythroid progenitor cells can repopulate the bone marrow and periphery of wild-type adoptive transfer animals but there is a high frequency of nucleated erythrocytes, and these animals develop anemia within a year. E2F4 also plays a critical role in normal erythroid maturation in a cell intrinsic manner. Taken together, these data suggest that E2F4 makes a major contribution to the regulation of erythropoietic system development by the RB tumor suppressor (Humbert, 2000b and references therein).

Myc and E2f1 promote cell cycle progression, but overexpression of either can trigger p53-dependent apoptosis. Mice expressing an Eμ-Myc transgene in B lymphocytes develop lymphomas, the majority of which sustain mutations of either Arf (a tumor suppressor whose product inhibits Mdm2, thereby stabilizing p53) or p53. Eμ-Myc transgenic mice lacking one or both E2f1 alleles exhibit a slower onset of lymphoma development associated with increased expression of the cyclin-dependent kinase inhibitor p27Kip1 and a reduced S phase fraction in precancerous B cells. In contrast, Myc-induced apoptosis and the frequency of Arf and p53 mutations in lymphomas were unaffected by E2f1 loss. Therefore, Myc does not require E2f1 to induce Arf, p53, or apoptosis in B cells, but depends upon E2f1 to accelerate cell cycle progression and downregulate p27Kip1 (Baudino, 2003).

The inactivation of the retinoblastoma (Rb) tumor suppressor gene in mice results in ectopic proliferation, apoptosis, and impaired differentiation in extraembryonic, neural, and erythroid lineages, culminating in fetal death by embryonic day 15.5 (E15.5). The specific loss of Rb in trophoblast stem (TS) cells, but not in trophoblast derivatives, leads to an overexpansion of trophoblasts, a disruption of placental architecture, and fetal death by E15.5. Despite profound placental abnormalities, fetal tissues appeared remarkably normal, suggesting that the full manifestation of fetal phenotypes requires the loss of Rb in both extraembryonic and fetal tissues. Loss of Rb results in an increase of E2f3 expression, and the combined ablation of Rb and E2f3 significantly suppresses Rb mutant phenotypes. This rescue appears to be cell autonomous since the inactivation of Rb and E2f3 in TS cells restores placental development and extends the life of embryos to E17.5. Taken together, these results demonstrate that loss of Rb in TS cells is the defining event causing lethality of Rb–/– embryos and reveal the convergence of extraembryonic and fetal functions of Rb in neural and erythroid development. It is concluded that the Rb pathway plays a critical role in the maintenance of a mammalian stem cell population (Wenzel, 2007).

The E2F family is conserved from C. elegans to mammals, with some family members having transcription activation functions and others having repressor functions. Whereas C. elegans and Drosophila have a single E2F activator protein and repressor protein, mammals have at least three activator and five repressor proteins. Why such genetic complexity evolved in mammals is not known. To begin to evaluate this genetic complexity, the inactivation of the entire subset of activators, E2f1, E2f2, E2f3a and E2f3b, was targeted singly or in combination in mice. E2f3a is sufficient to support mouse embryonic and postnatal development. Remarkably, expression of E2f3b or E2f1 from the E2f3a locus (E2f3a3bki or E2f3a1ki, respectively) suppressed all the postnatal phenotypes associated with the inactivation of E2f3a. It is concluded that there is significant functional redundancy among activators and that the specific requirement for E2f3a during postnatal development is dictated by regulatory sequences governing its selective spatiotemporal expression and not by its intrinsic protein functions. These findings provide a molecular basis for the observed specificity among E2F activators during development (Tsai, 2008).

The E2f7 and E2f8 family members are thought to function as transcriptional repressors important for the control of cell proliferation. This study analyzed the consequences of inactivating E2f7 and E2f8 in mice, and showed that their individual loss had no significant effect on development. Their combined ablation, however, resulted in massive apoptosis and dilation of blood vessels, culminating in lethality by embryonic day E11.5. A deficiency in E2f7 and E2f8 led to an increase in E2f1 and p53, as well as in many stress-related genes. Homo- and heterodimers of E2F7 and E2F8 were found on target promoters, including E2f1. Importantly, loss of either E2f1 or p53 suppressed the massive apoptosis in double-mutant embryos. These results identify E2F7 and E2F8 as a unique repressive arm of the E2F transcriptional network that is critical for embryonic development and control of the E2F1-p53 apoptotic axis (Li, 2008).

The tumor suppressor PML (promyelocytic leukemia protein) regulates cellular senescence and terminal differentiation, two processes that implicate a permanent exit from the cell cycle. This study shows that the mechanism by which PML induces a permanent cell cycle exit and activates p53 and senescence involves a recruitment of E2F transcription factors bound to their promoters and the retinoblastoma (Rb) proteins to PML nuclear bodies enriched in heterochromatin proteins and protein phosphatase 1α. Blocking the functions of the Rb protein family or adding back E2Fs to PML-expressing cells can rescue their defects in E2F-dependent gene expression and cell proliferation, inhibiting the senescent phenotype. In benign prostatic hyperplasia, a neoplastic disease that displays features of senescence, PML was found to be up-regulated and forming nuclear bodies. In contrast, PML bodies were rarely visualized in prostate cancers. The newly defined PML/Rb/E2F pathway may help to distinguish benign tumors from cancers, and suggest E2F target genes as potential targets to induce senescence in human tumors (Vernier, 2011).

E2F and centrosome duplication

Centrosome duplication is a key requirement for bipolar spindle formation and correct segregation of chromosomes during cell division. In a manner highly reminiscent of DNA replication, the centrosome must be duplicated once, and only once, in each cell cycle. How centrosome duplication is regulated and coordinated with other cell-cycle functions remains poorly understood. A centrosome duplication assay has been established using mammalian somatic cells. Centrosome duplication is shown to require the activation of E2F transcription factors and Cdk2-Cyclin A activity (Meraldi, 1999).

The results clearly show that centrosome duplication in somatic mammalian cells requires the phosphorylation of Rb. This implies that both DNA replication and centrosome duplication are controlled through the same pathway. In normal proliferating cells, this would seem to provide an efficient means of ensuring the coordinate execution of these two key events. It also follows, however, that the loss of a functional Rb pathway, either through mutation or the action of viral oncogenes, might jeopardize the coordination between DNA replication and centrosome duplication and lead to genomic instability (Meraldi, 1999).

It is further shown that centrosome duplication requires the activity of E2F transcription factors. Rb has been reported to regulate the activities of several potential effector proteins, but overexpression of E2F is sufficient to induce centrosome duplication in cells expressing a non-phosphorylatable Rb mutant. This indicates that, among the Rb-binding proteins, E2F is the major downstream effector regulating centrosome duplication. Furthermore, the dependence of somatic cells on the transcriptional activity of E2F may explain why such cells need an intact nucleus and protein synthesis for centrosome duplication, whereas embryonic systems only require cytoplasmic components. In the case of DNA synthesis, E2F activity is required to synthesize several essential gene products, including key regulators of DNA replication such as Cdc6. By analogy, it seems plausible that E2F may activate the synthesis of gene products that are critical for centrosome duplication; these may include regulatory proteins as well as bona fide centrosomal components. In view of the results presented here, it is particularly intriguing that both the cyclin A and E genes are among the known target genes for E2F (Meraldi, 1999).

Centrosome duplication in somatic cells requires Cdk2 activity in addition to E2F. In contrast, no evidence could be obtained for involvement of Cdk1 or Cdk3, indicating that the Cdk2 requirement is specific. This conclusion is in excellent agreement with the results of two recent independent studies on centrosome duplication in cell-free systems based on Xenopus egg extracts. However, in Xenopus extracts cyclin E has been identified as the primary partner of Cdk2, whereas the current studies on somatic cells lead the authors to emphasize the role of cyclin A. This discrepancy cannot be explained but it is believed that it reflects a genuine difference in the regulation of centrosome duplication in the two systems. In fact, somatic cell cycles differ in several aspects from those of early embryonic cells. (1) They are characterized by the presence of prolonged G1 and G2 phases and they exhibit regulation at the transcriptional level, neither of which are seen in rapidly dividing embryonic cells. (2) The two systems differ markedly in the regulation of cyclins A and E; in somatic mammalian cells cyclin E levels peak sharply around G1/S, whereas they are almost constant during the cell cycles of early Xenopus embryos. Conversely, cyclin A is a prominent S-phase partner of Cdk2 in somatic mammalian cells, whereas in Xenopus embryos A-type cyclins do not associate with Cdk2 before the mid-blastula transition. In view of this, it is possible that cyclin E has a prominent role in centrosome duplication in early Xenopus embryos but that in somatic mammalian cells this function is performed primarily by cyclin A (Meraldi, 1999).

The experiments have revealed an unexpected but intriguing difference in the abilities of cyclins A and E to support Cdk2 activity with respect to either centrosome duplication or DNA replication. In particular, in cells in which E2F activation has been blocked by the expression of a non-phosphorylatable Rb mutant, only cyclin A is able to promote centrosome duplication; cyclin E is ineffective. Conversely, under very similar experimental conditions, cyclin E is more effective than cyclin A in inducing DNA replication. In somatic cells therefore, cyclin A may be the preferred partner of Cdk2 for centrosome duplication, whereas cyclin E may be primarily responsible for promoting the G1/S transition and initiating DNA replication. The temporal coincidence between centrosome duplication and cyclin A expression is consistent with such a model, but more work is required to substantiate or refute this hypothesis (Meraldi, 1999).

E2F and the Extracellular matrix

Fibronectin within the extracellular matrix plays a role in cell attachment, spreading, and shape, while it also affects aspects of cell proliferation. Transcription factors such as E2F1 are also known to regulate cell shape and cell proliferation. Yet, to date no linkage has been established between fibronectin expression and E2F1. Cells constitutively expressing a mutant E2F1 protein (E2F1d87) produce reduced amounts of fibronectin mRNA and protein. The altered expression of fibronectin seen in the E2F1d87 expressing cells is due, in part, to a reduction in transcription from the fibronectin promoter. Providing exogenous fibronectin (but not Type I collagen or laminin) as a substrate for cell adhesion is sufficient to revert the altered morphology and to reestablish actin-containing microfilaments that are lost in the mutant cell line. An additional characteristic of the cells expressing the mutant E2F1 is that they demonstrate slow growth and a doubling in S phase duration. While providing exogenous fibronectin as an adhesion substrate does not shorten the S phase duration in the mutant line, it does significantly shorten the S phase duration in the parental NIH3T3 cell line, implicating a role for the extracellular matrix in regulating S phase transit in normal cells (Jordan-Sciutto, 1997).

E2F and development

Using an expression cloning approach, a novel function for the transcription factor E2F has been unveiled. Xenopus E2F (xE2F) is required for patterning of the Xenopus embryonic axis. Overexpression of xE2F in embryos induces ectopic expression of ventral and posterior markers, including selected members of the Hox genes, and suppresses the development of dorsoanterior structures. Loss of xE2F function in embryos leads to the elimination of ventral and posterior structures. These observations suggest that xE2F acts as an important regulator of region-specific gene expression and in the formation of the embryonic axis. This study provides evidence for an additional embryonic function for E2F, independent of its well-documented role in cell cycle regulation, and suggests a novel mechanism of region-specific gene expression during vertebrate embryogenesis (Suzuki, 2000).

In contrast to the intensive scrutiny given to the role of E2Fs in cell cycle regulation, the function of the E2F family in early development is poorly understood. xE2F is able to induce the expression of ventral- and posterior-specific genes, including the Hox genes and the homeobox gene Xhox3; xE2F is essential for the formation of ventral and posterior tissues in the early Xenopus embryo. These findings suggest that the E2F family of transcription factors may play an important role in the regulation of cell differentiation along the D/V and A/P axes during early vertebrate development. Since the E2F family has been shown to act as a key regulator of cell cycle progression, one could argue that the effects of either overexpression or inactivation of xE2F on early development may be indirectly caused by deregulation of the cell cycle. However, several lines of evidence argue against this possibility. (1) The division rate of blastomeres injected with either xE2F or dnxE2F RNA appears normal; neither acceleration nor delay of the division is observed. In addition, inhibition of xE2F activity by dnxE2F in the ventral marginal zone causes the transformation of cell fate from ventral to dorsal, rather than leading to the arrest of cell division. Neither xE2F nor dnxE2F affects cell proliferation and cell death in embryos. (2) xE2F induces the expression of its target genes, such as Hox genes, without de novo protein synthesis, indicating that the gene expression induced by xE2F is a direct event that does not require changes in cell cycle. Third, neural development in Xenopus is largely independent of cell proliferation, as inhibition of cell division in gastrula embryos using DNA synthesis inhibitors does not interfere with neurulation as well as neuronal differentiation. Therefore, effects of xE2F or other factors on the cell cycle alone are not likely to result in a change of cell fate in the neural tissue. Thus, it is proposed that xE2F regulates the formation of the D/V and A/P axes independent of cell cycle regulation (Suzuki, 2000).

The in vivo function of xE2F is similar to that of BMP4, but not identical, based on the following observations. (1) Unlike xE2F, BMP4 is able to induce the expression of mesodermal genes such as Xwnt-8 and Xbra in addition to Xhox3 in ectodermal explants. (2) Ectoderm can be neuralized by the inhibition of BMP signaling, but not by antagonizing xE2F function. Therefore, xE2F may be involved only in a part of BMP signaling, acting downstream of, or parallel to, BMP signaling. It will be important to determine how xE2F regulates growth factor signaling and how it integrates these extracellular inputs into region-specific gene expression to determine cell fates along the A/P and D/V axes during embryogenesis (Suzuki, 2000).

The C/EBP transcription factor is required for differentiation of adipocytes and neutrophil granulocytes, and controls cellular proliferation in vivo. To address the molecular mechanisms of C/EBPalpha action, C/EBPalpha mutants defective in repression of E2F-dependent transcription have been identified; they are impaired in their ability to suppress cellular proliferation, and to induce adipocyte differentiation in vitro. Using targeted mutagenesis of the mouse germline, it has been shown that E2F repression-deficient C/EBPalpha alleles fail to support adipocyte and granulocyte differentiation in vivo. These results indicate that E2F repression by C/EBPalpha is critical for its ability to induce terminal differentiation, and thus provide genetic evidence that direct cell cycle control by a mammalian lineage-instructive transcription factor couples cellular growth arrest and differentiation (Porse, 2001).

When preadipocytes reenter the cell cycle, Peroxisome proliferator-activated receptor gamma (PPARgamma) expression is induced, coincident with an increase in DNA synthesis, suggesting the involvement of the E2F family of cell cycle regulators. E2F1 induces PPARgamma transcription during clonal expansion, whereas E2F4 represses PPARgamma expression during terminal adipocyte differentiation. Using a combination of in vivo experiments with knockout and chimeric animals and in vitro experiments, it has been demonstrated that the absence of E2F1 impairs, whereas depletion of E2F4 stimulates, adipogenesis. E2Fs hence represent the link between proliferative signaling pathways, triggering clonal expansion, and terminal adipocyte differentiation through regulation of PPARgamma expression. This underscores the complex role of the E2F protein family in the control of both cell proliferation and differentiation (Fajas, 2002).

The E2F and pRB family members appear to participate in the regulation of cell cycle events that are required for adipogenesis. In growth-arrested preadipocytes, E2F4 and E2F5 are complexed with p130, leading to repression of its target genes. Upon reentry into cell cycle of these growth-arrested preadipocytes, p130, as well as the other members of the retinoblastoma family, is phosphorylated by the cyclin/cdk holoenzymes, releasing the E2F complex, resulting in the activation of the E2F target genes. After several rounds of DNA synthesis, the cyclin-dependent kinase inhibitors, such as p21, p27, and p18, are induced, and they mediate cell cycle exit and maintain the irreversible growth arrest characteristic of terminal adipocyte differentiation. PPARgamma and C/EBPalpha have been shown to contribute to this permanent cell cycle exit by inhibiting the E2F DNA binding activity and upregulating the levels of p21, respectively. There is also evidence that pRB plays a positive role in adipocyte differentiation through association and activation of C/EBPalpha. In this study, it is shown that the E2F proteins play a direct role in the regulation of early adipocyte differentiation. E2F1 and 3 trigger the expression of PPARgamma during the early stages of adipogenesis, whereas E2F4 represses expression of PPARgamma at the terminal stage of adipocyte differentiation (Fajas, 2002).

Current models posit that E2F transcription factors can be divided into members that either activate or repress transcription, in part through collaboration with the retinoblastoma (pRb) tumor suppressor family. The E2f3 locus encodes E2f3a and E2f3b proteins, and available data suggest that they regulate cell cycle-dependent gene expression through opposing transcriptional activating and repressing activities in growing and quiescent cells, respectively. However, the role, if any, of E2F proteins, and in particular E2f3, in myogenic differentiation is not well understood. This study dissected the contributions of E2f3 isoforms and other activating and repressing E2Fs to cell cycle exit and differentiation by performing genome-wide identification of isoform-specific targets. E2f3a and E2f3b target genes are involved in cell growth, lipid metabolism, and differentiation in an isoform-specific manner. Remarkably, using gene silencing, it was shown that E2f3b, but not E2f3a or other E2F family members, is required for myogenic differentiation, and this requirement for E2f3b does not depend on pRb. These functional studies indicate that E2f3b specifically attenuates expression of genes required to promote differentiation. These data suggest how diverse E2F isoforms encoded by a single locus can play opposing roles in cell cycle exit and differentiation (Asp, 2009).

E2F and apoptosis

Strong stimulation of the T-cell receptor (TCR) on cycling peripheral T cells causes their apoptosis by a process called TCR-activation-induced cell death (TCR-AICD). TCR-AICD occurs from a late G1 phase cell-cycle check point, independent of the 'tumor suppressor' protein p53. Disruption of the gene for the E2F-1 transcription factor, an inducer of apoptosis, causes significant increases in T-cell number and splenomegaly. T cells undergoing TCR-AICD induce the p53-related gene p73, another mediator of apoptosis, which is hypermethylated in lymphomas. Introducing a dominant-negative E2F-1 protein or a dominant-negative p73 protein into T cells protects them from TCR-mediated apoptosis, whereas dominant-negative E2F-2, E2F-4 or p53 does not. Furthermore, E2F-1-null or p73-null primary T cells do not undergo TCR-mediated apoptosis either. It is concluded that TCR-AICD occurs from a late G1 cell-cycle checkpoint that is dependent on both E2F-1 and p73 activities. These observations indicate that, unlike p53, p73 serves to integrate receptor-mediated apoptotic stimuli (Lissy, 2000).

The transcription factor E2F-1 induces both cell-cycle progression and, in certain settings, apoptosis. E2F-1 uses both p53-dependent and p53-independent pathways to kill cells. The p53-dependent pathway involves the induction by E2F-1 of the human tumor-suppressor protein p14ARF, which neutralizes HDM2 (human homolog of MDM2) and thereby stabilizes the p53 protein. E2F-1 induces the transcription of the p53 homolog p73. Disruption of p73 function inhibits E2F-1-induced apoptosis in p53-defective tumor cells and in p53-/- mouse embryo fibroblasts. It is conclude that activation of p73 provides a means for E2F-1 to induce death in the absence of p53 (Irwin, 2000).

Neuronal death induced by a variety of means requires participation of the E2F family of transcription factors. E2F acts as a gene silencer in neurons and repression of E2F-responsive genes is required for neuronal survival. Moreover, neuronal death evoked by DNA damaging agents or trophic factor withdrawal is characterized by derepression of E2F-responsive genes. Such derepression, rather than direct E2F-promoted gene activation, is required for death. Among the genes that are derepressed in neurons subjected to DNA damage or trophic factor withdrawal are the transcription factors B- and C-myb. Overexpression of B- and C-myb is sufficient to evoke neuronal death. These findings support a model in which E2F-dependent gene repression and derepression play pivotal roles in neuronal survival and death, respectively (Liu, 2001).

Taken together, these findings suggest a pathway by which DNA damage or NGF withdrawal leads to E2F-dependent gene derepression and to neuronal death. The first well-defined step in this pathway is elevation of cyclin D-associated kinase activity. Past evidence indicates that such an increase is driven by a combination of events, including elevation of cyclin D-associated kinase activity as well as by translocation of cyclin D to the nucleus. The enhancement of cyclin D-associated kinase activity promotes hyperphosphorylation of pocket proteins bound to E2F: this compromises their capacity to mediate gene repression. The consequent deregulation of genes elevates cellular levels of proapoptotic proteins that contribute to neuronal death (Liu, 2001).

E2F and tumors

The p16(INK4a)-cyclin D-retinoblastoma tumor suppressor pathway is disrupted in most human cancers, and it has been suggested that the subsequent release of E2F transcription factors from inhibitory complexes may be a key event in tumor development. Transgenic mice were generated with E2F1 gene expression targeted to squamous epithelial tissues by a keratin 5 (K5) promoter. K5 E2F1 transgenic mice were crossed with p53 null mice to examine functional interactions between E2F1 and p53 in vivo. E2F1-induced apoptosis of epidermal keratinocytes is reduced in K5 E2F1 transgenic mice lacking p53, whereas E2F1-induced hyperproliferation is unaffected by p53 status. K5 E2F1 transgenic mice heterozygous or nullizygous for p53 develop spontaneous skin carcinomas, which normally are rare in p53-deficient mice. The timing of tumor development correlates with the level of E2F1 transgene expression and the status of p53. In primary transgenic keratinocytes, the major change in E2F1 DNA-binding activity is the generation of a complex also containing the retinoblastoma tumor suppressor protein. Nevertheless, the expression and associated kinase activity of cyclin E, a known target for E2F transcriptional activity, is elevated significantly in K5 E2F1 transgenic keratinocytes. These findings firmly establish that increased E2F1 expression can contribute to tumor development and suggest that p53 plays an important role in eliminating cells with deregulated E2F1 activity (Pierce, 1998).

Apoptosis induced by the p53 tumor suppressor can attenuate cancer growth in preclinical animal models. Inactivation of the pRb proteins in mouse brain epithelium by the T121 oncogene induces aberrant proliferation and p53-dependent apoptosis. p53 inactivation causes aggressive tumor growth due to an 85% reduction in apoptosis. E2F1 is shown to signal p53-dependent apoptosis since E2F1 deficiency causes an 80% apoptosis reduction. E2F1 acts upstream of p53 since transcriptional activation of p53 target genes is also impaired. Yet, E2F1 deficiency does not accelerate tumor growth. Unlike normal cells, tumor cell proliferation is impaired without E2F1, counterbalancing the effect of apoptosis reduction. These studies may explain the apparent paradox that E2F1 can act as both an oncogene and a tumor suppressor in experimental systems (Pan, 1998).

Return: see E2F: Evolutionary homologs part 1/3 | part 2/3


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

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