Regulation Of E2F1 Research Articles

Glycogen synthase kinase-3β binds to E2F1 and regulates its transcriptional activity

GSK3β and E2F1 play an important role in the control of proliferation and apoptosis. Previous work has demonstrated that GSK3β indirectly regulates E2F activity through modulation of cyclin D1 levels. In this work we show that GSK3β phosphorylates human E2F1 in vitro at serine 403 and threonine 433, both residues localized at its transactivation domain. This phosphorylation was not detected in vivo. However, co-immunoprecipitation experiments do reveal in vivo binding of these proteins. Moreover, uninhibitable and catalitycally inactive GSK3β forms inhibit the transcriptional activity of a fusion protein containing E2F1 transactivation domain. Both forms of GSK3β inhibit E2F1 with similar efficiency. Interestingly the effect was independent of the mutation of serine 403 and threonine 433 to alanine. This suggests that this transcriptional modulation is independent of GSK3β kinase activity and phosphorylation state of serine 403 and threonine 433. The re-targeting of these GSK3β forms to the nucleus results in a higher capacity to regulate E2F1 transcriptional activity. Depletion of the levels of GSK3β protein using siRNA activates E2F1 transcriptional activity. The data presented in this study offer a new mechanism of regulation of E2F1 by direct binding of GSK3β to its transactivation domain.

Nucleophosmin/B23 regulates transcriptional activation of E2F1 via modulating the promoter binding of NF-κB, E2F1 and pRB

Expression of nucleophosmin/B23 and E2F1 and E2F1-dependent transcription increased in U1 bladder cancer cells upon serum stimulation from quiescence. Nucleophosmin/B23-siRNA treatment abrogated such increase of E2F1-dependent transcriptional activity. In identifying physiologically important factors that may occupy E2F1 promoter and regulate its activity in vivo, we found that the pattern of NF-κB, E2F1 and pRB recruitment to E2F1 promoter changed in a strikingly dynamic fashion as cells progressed from quiescence into serum-stimulated growth. E2F1 promoter activity in quiescent cells was associated with recruitment of NF-κB. NF-κB was replaced largely by E2F1 in concert with gene activation during the early stage (12 h) of serum stimulation. At late stage (24 h) of serum stimulation, pRB was then recruited to the E2F1-promoter complex to counterbalance its activity. Upon siRNA-mediated reduction of intracellular nucleophosmin/B23, E2F1 and pRB were recruited to the promoter with the dissociation of NF-κB concomitant with gene inactivation. Based on immunoprecipitation experiments, nucleophosmin/B23 was found to be associated with NF-κB in cells grown in serum-supplemented but not in serum-deprived medium. Furthermore, nucleophosmin/B23 could also be co-immunoprecipitated with ppRB at the early stage (12 h) but not at the late stage (24 h) of serum stimulation. The results demonstrate a novel mechanism for transcriptional regulation of E2F1 and identify the functional role of nucleophosmin/B23 in modulating the binding of NF-κB, E2F1 and pRB to activate E2F1 promoter.

IKKα Regulates Estrogen-induced Cell Cycle Progression by Modulating E2F1 Expression

The IkappaB kinase (IKK) complex consists of the catalytic subunits IKKalpha and IKKbeta and a regulatory subunit, IKKgamma/NEMO. Even though IKKalpha and IKKbeta share significant sequence similarity, they have distinct biological roles. It has been demonstrated that IKKs are involved in regulating the proliferation of both normal and tumor cells, although the mechanisms by which they function in this process remain to be better defined. In this study, we demonstrate that IKKalpha, but not IKKbeta, is important for estrogen-induced cell cycle progression by regulating the transcription of the E2F1 gene as well as other E2F1-responsive genes, including thymidine kinase 1, proliferating cell nuclear antigen, cyclin E, and cdc25A. The role of IKKalpha in regulating E2F1 was not the result of reduced levels of cyclin D1, as overexpression of this gene could not overcome the effects of IKKalpha knock-down. Furthermore, estrogen treatment increased the association of endogenous IKKalpha and E2F1, and this interaction occurred on promoters bound by E2F1. IKKalpha also potentiated the ability of p300/CBP-associated factor to acetylate E2F1. Taken together, these data suggest a novel mechanism by which IKKalpha can influence estrogen-mediated cell cycle progression through its regulation of E2F1.

MicroRNAs Add to Cancer Complexity

MicroRNAs are small (20 to 25 nucleotides) RNA molecules that inhibit gene expression by targeting the messenger RNAs and either inhibiting translation or promoting degradation of the mRNA (see Meltzer). Not only are these small but numerous microRNAs involved in normal gene regulation during cell proliferation and differentiation, they are also implicated in cancer. Three groups provide new insight into the roles of microRNAs in this complex disease. Lu et al. systematically analyzed the abundance and identity of microRNAs in hundreds of human tissue and cancer samples using a new technique based on complementary oligonucleotides coupled to beads carrying fluorescent dyes. They found that the microRNA profiles were effective in classifying tumors based on their developmental history. For example, epithelial samples of the gastrointestinal tract all showed a similar profile, which may reflect their common origin from cells of the embryonic endoderm. In addition, cancer cells had lower abundance of microRNAs when compared with normal cells, which may provide an effective test for classifying samples as normal or tumorigenic diagnostically. He et al. analyzed an amplified region of chromosome 13 that is associated with some types of B cell lymphoma. The amplified region includes the expressed region c13orf25 , which encodes the microRNA cluster mir-17-92 . Increased expression of the transcripts from the mir-17-02 cluster was observed in B cell lymphoma samples compared with that in normal samples or samples from tumors that did not have the amplified region (colorectal carcinomas). To directly test whether overexpression of the microRNAs in the mir-17-92 cluster contributed to cancer progression, the authors introduced hematopoetic stem cells engineered to overexpress the oncogene myc , or myc plus a portion of the mir-17-92 cluster, into recipient mice. Latency to lymphoma development was substantially decreased, and penetrance was 100% for lymphoma development in the mice receiving the cells with the microRNA cluster. O'Donnell et al. revealed an explicit connection between c-Myc and cancer. Using cells in which c-myc could be inducibly expressed, the authors showed that six microRNAs were up-regulated when c-Myc was increased. The six microRNAs were encoded by the mir-17 cluster (which was also the subject of the He et al. report), the mir-106a cluster, and the mir-106b cluster. Cells deficient for Myc showed decreased abundance of the mir-17 microRNAs, and wild-type levels were restored by reconstitution of c-Myc. Chromatin immunoprecipitation analysis indicated that c-Myc bound directly to the mir-17 locus. The transcription factor E2F1 is a target of two of the microRNAs encoded by mir-17 , and the gene encoding E2F1 is also a target of c-Myc. The regulation of E2F1 by the microRNAs was confirmed using antisense oligonucleotides to the microRNAs, which increased the abundance of E2F1, and by overexpression of the mir-17 cluster, which decreased E2F1 abundance. In cells induced to overexpress c-Myc, which also induced the microRNAs, E2F1 mRNA was markedly increased (~7 times that in low c-Myc cells); unexpectedly, the abundance of the E2F1 protein showed a smaller increase (1.5 times that in low c-Myc cells). One possible explanation is that the coordinated induction by c-Myc of E2F1 (a positive regulator of the cell cycle) and the microRNAs that inhibit E2F1 translation allows fine-tuning of the system. This may prevent the formation of a runaway positive feedback loop whereby c-Myc increases E2F1 abundance, which in turn increases c-Myc abundance, leading to uncontrollable cell proliferation. P. S. Meltzer, Cancer genomics: Small RNAs with big impacts. Nature 435 , 745-746 (2005). [PubMed] J. Lu, G. Getz, E. A. Miska, E. Alvarez-Saavedra, J. Lamb, D. Peck, A. Sweet-Cordero, B. L. Ebert, R. H. Mak, A. A. Ferrando, J. R. Downing, T. Jacks, H. R. Horvitz, T. R. Golub, MicroRNA expression profiles classify human cancers. Nature 435 , 834-838 (2005). [PubMed] L. He, J. M. Thomson, M. T. Hemann, E. Hernando-Monge, D. Mu, S. Goodson, S. Powers, C. Cordon-Cardo, S. W. Lowe, G. J. Hannon, S. M. Hammond, A microRNA polycistron as a potential human oncogene. Nature 435 , 828-833 (2005). [PubMed] K. A. O'Donnell, E. A. Wentzel, K. I. Zeller, C. V. Dang, J. T. Mendell, c-Myc-regulated microRNAs modulate E2F1 expression. Nature 435 , 839-843 (2005). [PubMed]

Regulation of E2F-1 after DNA Damage by p300-Mediated Acetylation and Ubiquitination

Here we report a novel, non-competitive mechanism that links acetylation and ubiquitination, in which the association of transcription factor E2F-1 with the cellular co-activator and acetyltransferase p300 determines its acetylation and subsequent ubiquitination. By using an antibody specifically recognizing the acetylated form of E2F-1 (AcE2F-1), we found that, after DNA damage, AcE2F-1 accumulates in the cells in a time-dependent manner, and that acetylation is increased by the expression of p300. Remarkably, the same DNA damaging conditions also induce the accumulation of ubiquitinated E2F-1, an event that is again markedly stimulated by p300 overexpression. The effects of p300 on E2F-1 ubiquitination require the integrity of the HAT domain of p300 and of the three acetylated lysines in E2F-1. Of note, p300-induced E2F-1 ubiquitination does not depend on the p45Skp2 E3 ligase, since it does not extend to other p45Skp2 targets and also occurs with an E2F-1 mutant devoid of the p45Skp2-binding domain but still retaining the acetylated region. Finally, p300-induced E2F-1 ubiquitination is not influenced by RB.

E2F1 regulation of the human myo-inositol 1-phosphate synthase ( ISYNA1) gene promoter

Human myo-inositol 1-phosphate synthase (IP synthase; E.C. 5.5.1.4), encoded by ISYNA1, catalyzes the de novo synthesis of inositol 1-phosphate from glucose 6-phosphate. It is a potential target for mood-stabilizing drugs such as lithium and valproate. But, very little is known about the regulation of human IP synthase. Here, we have characterized the minimal promoter of ISYNA1 and show that it is upregulated by E2F1. Upregulation occurs in a dose-dependent fashion and can be suppressed by ectopic expression of Rb. EMSA and antibody supershift analysis identified a functional E2F binding motif at −117. Complex formation at this site was competed by an excess of unlabeled Sp1 oligo consistent with the −117 E2F site overlapping an Sp1 motif. Because the −117 E2F motif is not a high-affinity binding site, we propose that the upregulation of ISYNA1 occurs through the cooperative interaction of several low-affinity E2F binding motifs present in the minimal promoter.

TopBP1 recruits Brg1/Brm to repress E2F1-induced apoptosis, a novel pRb-independent and E2F1-specific control for cell survival.

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.

Tumor necrosis factor-mediated E2F1 suppression in endothelial cells: differential requirement of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase signal transduction pathways.

After balloon angioplasty, locally expressed tumor necrosis factor (TNF)-alpha disrupts endothelial cell (EC) proliferation and reendothelialization of the injured vessel. We have previously reported that TNF inhibits the EC cycle and downregulates the transcription factor E2F1. Ectopic expression of E2F1 at the site of injury improves reendothelialization of the injured vessel. In this study, we report that c-Jun N-terminal kinase (JNK) 1 and p38 mitogen-activated protein kinases (MAPKs) are differentially required for E2F1 expression and activity in ECs. Overexpression of constitutively active JNK1 mimicked TNF-mediated inhibitory events, whereas dominant-negative JNK1 prevented these effects. E2F cis elements in the promoter of E2F1 gene mediate suppressive actions of TNF, because removal of these sites rendered E2F1 promoter activity insensitive to TNF. JNK1 physically interacted with E2F1 and inactivated it via direct phosphorylation. Additionally, TNF inhibited Rb phosphorylation and dissociation from E2F1. Overexpression of constitutively active p38 MAPK facilitated Rb-E2F1 dissociation, whereas that of dominant-negative p38 MAPK did not. Taken together, these data suggest a differential requirement of JNK1 and p38 MAPK in TNF regulation of E2F1. Targeted inactivation of JNK1 at arterial injury sites may represent a potential therapeutic intervention for ameliorating TNF-mediated EC dysfunction.

Regulation of E2F1 by BRCT domain-containing protein TopBP1.

The E2F transcription factor integrates cellular signals and coordinates cell cycle progression. Our prior studies demonstrated selective induction and stabilization of E2F1 through ATM-dependent phosphorylation in response to DNA damage. Here we report that DNA topoisomerase IIbeta binding protein 1 (TopBP1) regulates E2F1 during DNA damage. TopBP1 contains eight BRCT (BRCA1 carboxyl-terminal) motifs and upon DNA damage is recruited to stalled replication forks, where it participates in a DNA damage checkpoint. Here we demonstrated an interaction between TopBP1 and E2F1. The interaction depended on the amino terminus of E2F1 and the sixth BRCT domain of TopBP1. It was specific to E2F1 and was not observed in E2F2, E2F3, or E2F4. This interaction was induced by DNA damage and phosphorylation of E2F1 by ATM. Through this interaction, TopBP1 repressed multiple activities of E2F1, including transcriptional activity, induction of S-phase entry, and apoptosis. Furthermore, TopBP1 relocalized E2F1 from diffuse nuclear distribution to discrete punctate nuclear foci, where E2F1 colocalized with TopBP1 and BRCA1. Thus, the specific interaction between TopBP1 and E2F1 during DNA damage inhibits the known E2F1 activities but recruits E2F1 to a BRCA1-containing repair complex, suggesting a direct role of E2F1 in DNA damage checkpoint/repair at stalled replication forks.

Differential Regulation of E2F1, DP1, and the E2F1/DP1 Complex by ARF

Differential Regulation of E2F1, DP1, and the E2F1/DP1 Complex by ARF

Evolving intricacies and implications of E2F1 regulation.

E2F transcription factors may play a pivotal role in the transcriptional regulation of several cellular processes far beyond the originally described cell cycle and proliferation. Among the six E2F family members, only E2F1 is noted for its role in apoptosis. The pocket protein family members Rb, p107, and p130 act as the main regulators of E2F activity. Nonetheless, in recent years other protein-protein interactions have been described for E2Fs. The post-translational modifications resulting from such protein interactions may have significant implications in the stability, half-life, and functional activity of E2Fs. In human diseases the significance of E2Fs is still under appreciated and is primarily recognized only as a consequence of the impairment in retinoblastoma gene product (Rb). However, with increasing knowledge of other protein interactions, the derailment of E2F activity could be anticipated to stem from an abnormality of any node in the complex network governing their availability and activity. The present review is intended to provide a perspective on the diversity of biochemical mechanisms underlying abnormal E2F expression and activity, understanding of which may have significant clinical implications.

Differential regulation of E2F1, DP1, and the E2F1/DP1 complex by ARF.

The tumor suppressor protein ARF inhibits MDM2 to activate and stabilize p53. Recent studies provided evidence for p53-independent tumor suppression functions of ARF. For example, it has been shown that ARF induces proteolysis of certain E2F species, including E2F1. In addition, ARF relocalizes E2F1 from the nucleoplasm to nucleolus and inhibits E2F1-activated transcription. Because DP1 is a functional partner of the E2F family of factors, we investigated whether DP1 is also regulated by ARF. Here we show that DP1 associates with ARF. Coexpression of ARF relocalizes DP1 from the cytoplasm to the nucleolus, suggesting that DP1 is also a target of the ARF regulatory pathways. Surprisingly, however, the E2F1/DP1 complex is refractory to ARF regulation. Coexpression of E2F1 and DP1 blocks ARF-induced relocalization of either subunit to the nucleolus. The E2F1/DP1 complex localizes in the nucleoplasm, whereas ARF is detected in the nucleolus, suggesting that ARF does not interact with the E2F1/DP1 complex. Moreover, we show that E2F1 is more stable in the presence of ARF when coexpressed with DP1. These results suggest that ARF differentially regulates the free and heterodimeric forms of E2F1 and DP1. DP1 is a constitutively expressed protein, whereas E2F1 is mainly expressed at the G(1)/S boundary of the cell cycle. Therefore, the E2F1/DP1 complex is abundant only between late G(1) and early S phase. Our results on the differential regulation E2F1, DP1, and the E2F1/DP1 complex suggest the possibility that ARF regulates the function of these cell cycle factors by altering the dynamics of their heterodimerization during progression from G(1) to S phase.

Apaf-1 Is a Mediator of E2F-1-induced Apoptosis

E2F-1 is capable of promoting both cell cycle progression and apoptosis. The latter is important for suppressing untoward expansion of proliferating cells. In this study, we investigated its underlying mechanisms. E2F-1-induced apoptosis was accompanied by caspase-9 activation and inhibited by a specific inhibitor of caspase-9 in K562 sublines overexpressing E2F-1. E2F-1 enhanced the expression of Apaf-1 without the cytosolic accumulation of cytochrome c. Apaf-1-deficient melanoma cell lines were resistant to E2F-1, indicating that Apaf-1 is an essential element of E2F-1-mediated apoptosis. Finally, we isolated the promoter region of the Apaf-1 gene and found a putative binding site for E2F. A chromatin immunoprecipitation assay revealed that E2F-1 bound to Apaf-1 promoter upon E2F-1 overexpression, suggesting that Apaf-1 is under transcriptional regulation of E2F-1. These data demonstrate a novel mechanism of apoptosis in which an increase in Apaf-1 levels results in direct activation of caspase-9 without mitochondrial damage, leading to the initiation of a caspase cascade.

Lovastatin-induced E2F-1 modulation and its effect on prostate cancer cell death.

Lovastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, induces growth arrest in a variety of cancer cell lines. Its mechanism of action, however, has not been completely elucidated. E2F-1 is thought to act as an oncogene and a tumour suppressor, with its action probably dependent upon the cellular context. We have shown in this study that transcriptional regulation and proteasomal degradation of E2F-1 are critical regulatory events in lovastatin-induced cell death. Accompanying this is a reduction in the E2F-1-regulated expression of cell cycle genes such as c-myc, cyclin D1, cyclin A and cyclin B1. Cell cycle analysis demonstrated that the accumulation of apoptotic cells was preceded by a progressive decrease in the S-phase cell population in response to lovastatin. Although expression of E2F-1 was reduced in three prostate cancer cell lines-PC-3, LNCaP and DU-145-the p21 and p27 protein levels were not increased in all the cell lines treated, suggesting that increase in p21 and p27 protein expression per se is not responsible for lovastatin-mediated down-regulation of E2F-1. The subsequent apoptotic death of these cells in the presence of lovastatin can be prevented by forced ectopic expression of E2F-1. Taken together, these facts imply that E2F-1 is the target of an HMG-CoA inhibitor and critical cell death mediator in prostate cancer cells.

Adhesion-regulated G1 cell cycle arrest in epithelial cells requires the downregulation of c-Myc.

Adhesion to the extracellular matrix is required for the expression and activation of the cyclin-cyclin-dependent kinase (CDK) complexes, and for G1 phase progression of non-transformed cells. However, in non-adherent cells no molecular mechanism has yet been proposed for the cell adhesion-dependent up-regulation of the p27 cyclin-dependent kinase inhibitor (CKI), and the associated inhibition of cyclin E-CDK2. We now show that in epithelial cells the expression of c-Myc is tightly regulated by cell-substrate adhesion. When deprived of adhesion, two independently derived mammary epithelial cell lines, 184A1N4 and MCF-10A, rapidly decrease their level of c-Myc mRNA and protein. This decrease in levels of c-Myc correlates with G1 phase arrest, as indicated by hypophosphorylation of pRb and inhibition of the activity of the cyclin E-CDK2 complex. In 184A1N4 cells, cell-substrate adhesion is required for the suppression of p27, and induction of cyclin E, E2F-1, but not cyclins D1 and D3. Enforced expression of c-Myc in non-adherent 184A1N4 and MCF-10A cells reverses the adhesion-dependent inhibition of cell cycle progression. Restoration of c-Myc in non-adherent cells induces the expression of E2F-1, and hyperphosphorylation of pRb in response to EGF treatment. In addition, expression of c-Myc results in the anchorage-independent activation of the CDK2 complex, the associated upregulation of cyclin E, and the destabilization and degradation of p27 by the ubiquitin-proteasome pathway. Our study thus suggests that c-Myc is the link between cell adhesion and the regulation of p27 and cyclin E-CDK2. Furthermore, we describe a role for c-Myc in adhesion-mediated regulation of E2F-1.

Transcriptional regulation of E2F-1 and eIF-2 genes by α-Pal: a potential mechanism for coordinated regulation of protein synthesis, growth, and the cell cycle

α-Pal regulates the basal transcription of the α and β subunits of eukaryotic initiation factor two (eIF-2), a rate-limiting enzyme for the initiation of protein biosynthesis. We recently showed that its global function may be to modulate the expression of key metabolic genes in response to cellular proliferation. In this paper, we examined a potential molecular mechanism by which α-Pal may achieve this function. When overexpressed, α-Pal upregulated protein synthesis and growth, but downregulated the cell cycle. The mechanism for the increased protein synthesis and growth appeared to be a transcriptional upregulation of the eIF-2α and eIF-2β genes. The mechanism for the cell cycle downregulation appeared to be a transcriptional downregulation of E2F-1, a transcription factor that regulates genes required for cell cycle progression beyond the G1/S interphase. Specifically, an apparently modified species of α-Pal bound to the eIF-2 promoters and induced transcriptional upregulation, whereas, an apparently unmodified species of the α-Pal bound to the E2F-1 promoter and induced transcriptional downregulation. By this mechanism, α-Pal may participate in coordinating the regulation of global protein synthesis, growth and the cell cycle; a regulation that is essential to cellular differentiation.

Regulation and Deregulation of E2F1 in Postmitotic Neurons Differentiated from Embryonal Carcinoma P19 Cells

Neurons withdraw from the cell cycle immediately after differentiation from their proliferative precursors. E2F1, a principal transcription factor that promotes cell cycle progression, must be silenced in neurons. We investigated the E2F1 system in postmitotic neurons derived from murine embryonal carcinoma P19 cells. P19 cells highly expressed the E2F1 gene during neural differentiation, and enriched neurons contained a high abundance of E2F1 mRNA. In contrast, postmitotic neurons possessed extremely low levels of E2F1 protein as assessed by the electrophoretic mobility shift assay and Western blotting. A recombinant E2F1 fusion protein was ubiquitinated in vitro when incubated with neuronal lysates. In addition, treatment with the proteasome inhibitor MG132 increased the endogenous level of E2F1 protein in neurons. These results suggest that the ubiquitin–proteasome pathway contributes, at least in part, to the downregulation of E2F1 protein in postmitotic neurons. Adenovirus-mediated transfer of E2F1 cDNA into postmitotic neurons induced both bromodeoxyuridine incorporation and chromatin condensation, suggesting that deregulated E2F1 expression causes both aberrant S-phase entry and apoptosis of postmitotic neurons. Thus, downregulation of endogenous E2F1 protein in postmitotic neurons may be indispensable for the prevention of their reentry into the cell cycle.

Phenotypic differentiation without permanent cell-cycle arrest by skeletal myocytes with deregulated E2F-1.

Skeletal muscle terminal differentiation includes expression of muscle cell-specific proteins and concomitant cell-cycle arrest. These two processes require functional retinoblastoma protein (RB). E2F-1 is an RB-associated transcriptional factor and an effector of RB in the regulation of G1 to S-phase transition. Here, we show that proper regulation of E2F-1 is crucial for differentiation-coupled cell-cycle arrest by skeletal myocytes. On induction to differentiate, C2 myoblasts constitutively expressing E2F-1 synthesized muscle cell-specific proteins, fused into myotubes, and upregulated the cdk inhibitor p21. However, unlike control cells, differentiated myocytes expressing exogenous E2F-1 incorporated bromodeoxyuridine into nuclei, indicating S-phase entry. This S-phase entry was accompanied by expression of cyclin A. Our results support the view that RB regulates cell-cycle arrest and muscle cell differentiation through separable mechanisms.

Transcriptional repression of the E2F-1 gene by interferon-alpha is mediated through induction of E2F-4/pRB and E2F-4/p130 complexes.

E2F is a heterodimeric transcription factor composed of one of five E2F subunits (E2F-1 to E2F-5) and a DP subunit. E2F regulates the expression of several growth-promoting genes, and thus, can be a target of antiproliferative action of interferons (IFNs). In this study, we investigated the mechanisms whereby IFN-alpha suppresses transcription of the E2F-1 gene. Transfection studies revealed that E2F-1 promoter was functionally divided into two parts: upstream activation sequences (UAS) and a downstream negative-regulatory element (E2F-binding sites). When cells were proliferating, transcription of the E2F-1 gene was primarily driven by the UAS, while E2F sites were not involved in activation. IFN-alpha markedly reduced E2F-1 promoter activity, but introduction of non-binding mutation at the E2F sites completely abrogated the inhibition. Free E2F4 was found to be the predominant species bound to the E2F sites in proliferating cells. IFN-alpha induced upregulation of E2F-4 along with dephosphorylation of pRB and p130, which resulted in the formation of E2F-4/pRB and E2F-4/p130 complexes on the E2F-1 promoter. These complexes function as transcriptional repressors to inhibit E2F-1 mRNA expression. Our findings indicate that E2F-4 is a critical regulator of E2F-1, which offer an excellent paradigm for understanding functional diversity within the E2F family.

Mutation of E2f-1 Suppresses Apoptosis and Inappropriate S Phase Entry and Extends Survival of Rb-Deficient Mouse Embryos

Mice mutant for the Rb tumor suppressor gene die in mid-gestation with defects in erythropoiesis, cell cycle control, and apoptosis. We show here that 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.