Cellular E2F Research Articles

Hsp90 chaperone facilitates E2F1/2-dependent gene transcription in human breast cancer cells

The 90 kDa heat shock protein, Hsp90, is involved in the conformational stabilization and functional maturation of diverse cancer-promoting proteins. To date, more than 300 Hsp90 clients have identified, suggesting that Hsp90 plays a central role in deciding cancer cell fate. In this study, we present the nuclear functions of Hsp90 in regulating the E2F-dependent gene transcription. We show that the conformation specific Hsp90 inhibitor, 17AAG decreases the total cellular E2F levels more selectively in cancer cells than transformed cells. With the help of coimmunoprecipitation experiments, we show that Hsp90 interacts with E2F1 and E2F2 in cancer cells, whereas in transformed cells, only E2F1 interacts with Hsp90. Retention of E2F2 in the nucleus of cancer cells upon MG132 combination with 17AAG has suggested that Hsp90 is required for E2F2 stability and function. The HDAC6 inhibitor tubacin treatment did not interfere with E2F1/2 stability and nuclear accumulation. However, the HDAC3 inhibitor, RGFP966 treatment, decreased nuclear E2F1/2 and its target gene expression. The nuclear accumulation of E2F1 and E2F2 upon cell cycle inhibition correlated with decreased acetylated Hsp90. We expose the nuclear functions of Hsp90 in facilitating the cell cycle progression through stabilizing E2F1/2.

Human herpesvirus 6 (HHV-6) alters E2F1/Rb pathways and utilizes the E2F1 transcription factor to express viral genes

E2F transcription factors play pivotal roles in controlling the expression of genes involved in cell-cycle progression. Different viruses affect E2F1/retinoblastoma (Rb) interactions by diverse mechanisms releasing E2F1 from its suppressor Rb, enabling viral replication. We show that in T cells infected with human herpesvirus 6A (HHV-6A), the E2F1 protein and its cofactor DP1 increased, whereas the Rb protein underwent massive degradation without hyperphosphorylation at three sites known to control E2F/Rb association. Although E2F1 and DP1 increased without Rb suppression, the E2F1 target genes-including cyclin A, cyclin E, and dihydrofolate reductase-were not up-regulated. To test whether the E2F1/DP1 complexes were used for viral transcription, we scanned the viral genome for genes containing the E2F binding site in their promoters. In the present work, we concentrated on the U27 and U79 genes known to act in viral DNA synthesis. We constructed amplicon-6 vectors containing a GFP reporter gene driven by WT viral promoter or by promoter mutated in the E2F binding site. We found that the expression of the fusion U27 promoter was dependent on the presence of the E2F binding site. Test of the WT U79 promoter yielded >10-fold higher expression of the GFP reporter gene than the mutant U79 promoter with abrogated E2F binding site. Moreover, by using siRNA to E2F1, we found that E2F1 was essential for the activity of the U79 promoter. These findings revealed a unique pathway in HHV-6 replication: The virus causes Rb degradation and uses the increased E2F1 and DP1 factors to transcribe viral genes.

TFDP3 inhibits E2F1-induced, p53-mediated apoptosis

By dimerizing with E2F proteins, TFDP has profound influence on cellular E2F activities. While TFDP1 and 2 enhance the DNA binding and the transcriptional activity of E2F, the newly identified member of the DP family, TFDP3 primarily functions as a negative regulator. To further characterize the inhibitory property of TFDP3, the present study specifically examined the modulatory role of TFDP3 on E2F1-induced cell death. HEK-293 cells underwent apoptosis following ectopic expression of E2F1. This effect was virtually abolished by co-transfection with TFDP3. In the meantime, the accumulation of p53 proteins and the increased expression of the pro-apoptotic molecules, including Bax, Puma, Noxa, and Bid were found to be suppressed. These data suggest a new mechanism for the regulation of E2F1-induced apoptosis and provide further evidence for the general involvement of TFDP3 in the regulation of E2F functions.

Inhibition of Cell Division by the Human Cytomegalovirus IE86 Protein: Role of the p53 Pathway or Cyclin-Dependent Kinase 1/Cyclin B1

The human cytomegalovirus (HCMV) IE86 protein induces the human fibroblast cell cycle from G(0)/G(1) to G(1)/S, where cell cycle progression stops. Cells with a wild-type, mutated, or null p53 or cells with null p21 protein were transduced with replication-deficient adenoviruses expressing HCMV IE86 protein or cellular p53 or p21. Even though S-phase genes were activated in a p53 wild-type cell, IE86 protein also induced phospho-Ser(15) p53 and p21 independent of p14ARF but dependent on ATM kinase. These cells did not enter the S phase. In human p53 mutant, p53 null, or p21 null cells, IE86 protein did not up-regulate p21, cellular DNA synthesis was not inhibited, but cell division was inhibited. Cells accumulated in the G(2)/M phase, and there was increased cyclin-dependent kinase 1/cyclin B1 activity. Although the HCMV IE86 protein increases cellular E2F activity, it also blocks cell division in both p53(+/+) and p53(-/-) cells.

Reconstitution of DNA Synthetic Capacity in Senescent Normal Human Fibroblasts by Expressing Cellular Factors E2F and Mdm2

Unraveling the mechanisms underlying cellular senescence will contribute to the understanding of processes involved in aging and cancer. We sought to determine whether expression of cellular factors in senescent WI-38 human fibroblasts was sufficient to induce nuclear DNA synthesis. Expression by recombinant adenovirus of E2F1, E2F2, E2F3, cyclin E/cdk2, and Mdm2 individually resulted in DNA synthesis in 10–30% of cells. However, combination of Mdm2 with E2F or cyclin E/cdk2 resulted in 50 to 75% of cells synthesizing DNA. DNA synthesis occurred approximately 30 h following infection. We conclude that expression of normal cellular factors is sufficient to induce DNA synthesis in senescent normal human fibroblasts.

They're Alive! Protein potion brings senescent cells back from the (un)dead

Like zombies, senescent cells join the ranks of the undead. They no longer divide, but they don't die either. Now it turns out that these cellular freaks possess the resources for resurrection to a more vigorous existence: The right brew of normal proteins can kick-start DNA replication in the stalled cells. Until now, scientists needed deviant proteins to shock senescent cells back to life: Cancer-causing molecules activate two cellular pathways that stimulate division. In the new work, Sarraj and colleagues mixed and matched benign proteins, looking for a combination that spurs senescent cells to start reproducing again. They tested Mdm2, which distracts the p53 protein from one of its usual jobs: inhibiting cell proliferation. Other candidates were three E2F proteins, which boost the production of molecules that encourage cells to multiply. Because senescent cells contain more p53 and less E2F than do normal cells, the researchers reasoned that providing additional Mdm2 or E2F might trigger cell duplication. To test this idea, they infected senescent human fibroblasts--cells that compose connective tissue--with various combinations of viruses, each of which is engineered to produce one of these proteins. By looking for incorporation of a tagged molecule into DNA, they were able to determine what fraction of cells copied their DNA--an indication that cell division had started up again. Serving up extra Mdm2 or E2F individually had a modest effect: 10% to 30% of the cells showed signs of replicative life. But Mdm2 plus any one of the three E2Fs jolted 50% to 70% of the cells back into action. Scientists have suspected that the right blend of cellular proteins could recharge senescent cells, but they hadn't shown that experimentally until now. The results provide evidence that supplying certain proteins lacking in senescent cells can indeed reverse the process. The study enhances our knowledge about possible links between senescence and cancer or aging. In animals, senescence might fight cancer by stunting the growth of damaged cells before they start dividing unchecked (see "Dangerous Liaisons" ). And accumulation of senescent cells might be to blame for some of the effects of aging (see "More Than a Sum of Our Cells" ). But no one's likely to start receiving antiaging E2F/Mdm2 injections any time soon: The treated cells might retain the harmful characteristics that sparked senescence in the first place and rejuvenating them might just produce more of a bad thing. But knowing how to bring senescent cells back to life will undoubtedly shed light on the spell that makes them zombies in the first place. --R. John Davenport; suggested by Chang-Su Lim S. Sarraj, R. Farb, R. Martell, Reconstitution of DNA synthetic capacity in senescent normal human fibroblasts by expressing cellular factors E2F and Mdm2. Exp. Cell Res. 270 , 268-276 (2001). [Abstract] [PDF]

G Protein-coupled Receptors Desensitize and Down-regulate Epidermal Growth Factor Receptors in Renal Mesangial Cells

Different types of plasma membrane receptors engage in various forms of cross-talk. We used cultures of rat renal mesangial cells to study the regulation of EGF receptors (EGFRs) by various endogenous G protein-coupled receptors (GPCRs). GPCRs (5-hydroxytryptamine(2A), lysophosphatidic acid, angiotensin AT(1), bradykinin B(2)) were shown to transactivate EGFRs through a protein kinase C-dependent pathway. This transactivation resulted in the initiation of multiple cellular signals (phosphorylation of the EGFRs and ERK and activation of cAMP-responsive element-binding protein (CREB), NF-kappaB, and E2F), as well as subsequent rapid down-regulation of cell-surface EGFRs and internalization and desensitization of the EGFRs without change in the total cellular complement of EGFRs. Internalization of the EGFRs and the down-regulation of cell-surface receptors in mesangial cells were blocked by pharmacological inhibitors of clathrin-mediated endocytosis and in HEK293 cells by transfection of cDNA constructs that encode dominant negative beta-arrestin-1 or dynamin. Whereas all of the effects of GPCRs on EGFRs were dependent to a great extent on protein kinase C, those initiated by EGF were not. These studies demonstrate that GPCRs can induce multiple signals through protein kinase C-dependent transactivation of EGFRs. Moreover, GPCRs induce profound desensitization of EGFRs by a process associated with the loss of cell-surface EGFRs through clathrin-mediated endocytosis.

Phosphorylation-dependent and -independent functions of p130 cooperate to evoke a sustained G1 block.

The retinoblastoma (pRb)-related p130 pocket protein is a regulator of cell growth and differentiation, and a candidate tumour suppressor. Both pRb and p130 operate through interactions with cellular proteins, including the E2F transcription factors. While such interactions are controlled by phosphorylation of multiple sites of pRb, regulation of p130 remains poorly understood. We now identify 22 in vivo phosphorylation sites of p130, targeted by diverse kinases, and present evidence for three cyclin-dependent kinase 4(6) [Cdk4(6)] specific phosphorylations, which appear critical for controlling the growth-restraining activity of p130. When expressed in U2OS cells, the phosphorylation-deficient mutant p130(Delta)(CDK4), in which the Cdk4 specific sites were mutated to alanine residues, imposed a more sustained G1 arrest than a constitutively active pRb(Delta)(CDK), known to repress all cellular E2F activity. Experiments using p130(Delta)(Cdk4) and another phosphorylation-deficient mutant, p130(PM19A), with 19 phosphorylation sites mutated, revealed that the p130-imposed G1 block reflects cooperative growth-suppressive effects of phosphorylation-regulated E2F binding and phosphorylation-independent sequestration of cyclin E(A)-Cdk2 through the N-terminal cyclin binding motif of p130.

Puralpha: a multifunctional single-stranded DNA- and RNA-binding protein.

Puralpha is a ubiquitous, sequence-specific DNA- and RNA-binding protein which is highly conserved in eukaryotic cells. Puralpha has been implicated in diverse cellular functions, including transcriptional activation and repression, translation and cell growth. Moreover, this protein has been shown to be involved in regulating several human viruses which replicate in the central nervous system (CNS), including human immunodeficiency virus type I (HIV-1) and JC virus (JCV). Puralpha exerts part of its activity by interacting with cellular proteins, including pRb, E2F, cyclin A, Sp1 and members of the Y-box family of proteins, including YB-1 and MSY1, as well as viral proteins such as polyomavirus large T-antigen and HIV-1 Tat. The ability of Puralpha to interact with its target DNA sequence and to associate with several viral and cellular proteins is modulated by RNA. Puralpha has also been shown to be involved in cell growth and proliferation. Its association with pRb, E2F and cyclin A coupled with its fluctuating levels throughout the cell cycle, position Puralpha as a crucial factor in the cell cycle. Moreover, microinjection studies demonstrate that Puralpha causes either a G(1) or G(2) arrest depending on the cell cycle time of injection. The gene encoding Puralpha has been localized to a human locus which is frequently deleted in myelogenous leukemias and other cancers and Puralpha gene deletions have been detected in many cases of lymphoid cancers. The following review details the structural characteristics of Puralpha, its family members and the involvement of this protein in regulating various cellular and viral genes, viral replication and cell growth.

Regulation of cyclin E gene expression by the human papillomavirus type 16 E7 oncoprotein.

In this study, we characterized the 5' regulatory region of the murine cyclin E gene and analysed activation of the gene by the E7 oncogene of human papillomavirus type 16 in transfection experiments. We found that the murine cyclin E promoter is composed of multiple regulatory elements, and we present evidence for at least two independent transcription units, designated P1 and P2. Overlapping binding sites for the cellular transcription factors Sp1 and E2F were identified in both promoters, and we found that E2F-mediated activation of transcription is inhibited by Sp1 in cotransfection experiments. The E2F/Sp1 binding sites contribute to transcriptional activation by E7, and the data suggest that the cyclin E gene is rendered E7-inducible through the combination of several cis-acting elements which display only weak intrinsic responsiveness to E7.

Differential Effects of Overexpression of PKCα and PKCδ/ϵ on Cellular E2F Activity in Late G1 Phase

Introduction of a reporter gene containing E2F binding sites linked to the luciferase gene permitted us to detect transient cellular E2F activity in late G1 phase rat 3Y1 fibroblasts. Overexpression of three major protein kinase C (PKC) isozymes expressed in 3Y1 cells caused differing effects on E2F activity depending on the isozymes overexpressed. Overexpression of PKCα inhibited E2F activity while the overexpression of PKCδ or PKCϵ enhanced it, suggesting that these PKC isozymes play different roles in the regulation of E2F activity. Consistent with previous findings that the activation of PKC by TPA in late G1 phase results in the inhibition of DNA synthesis (Huang, C., and Ives, H.E., 1987,Nature329,849–850), the addition of TPA in late G1 phase specifically inhibited E2F activity. Overexpression of PKC isozymes resulted in an enhancement of the TPA-induced inhibition of E2F in late G1 phase. This enhancement was observed for all three PKC isozymes examined, suggesting that these PKC isozymes all are potent mediators of the TPA-induced inhibition of E2F activity in late G1 phase.

Stimulation of Human Insulin Receptor Gene Expression by Retinoblastoma Gene Product

Multiple cis-acting elements have been defined to be important for the transcriptional regulation of the human insulin receptor (hIR) gene expression. We report here that one of these elements also mediated the stimulation of hIR promoter activity by the retinoblastoma gene product (Rb). The cis-element responsible for Rb stimulation was localized to the GA and GC boxes situated between -643 to -607 of the hIR gene. We have previously demonstrated that these GA and GC boxes bind Sp1 with high affinity and are responsible for E1a activation of hIR promoter activity. Mutation of these sequences completely abolished Rb-dependent enhancement of hIR promoter activity. In addition, we localized three regions in the N-terminal domain of Rb to be involved in stimulation of hIR promoter activity. Our results represent one of the first studies to demonstrate a functional importance assigned to the multiple phosphorylation sites in the N terminus of Rb. Finally, the mechanism by which Rb activates the hIR promoter are presented.

E2F1 overexpression in quiescent fibroblasts leads to induction of cellular DNA synthesis and apoptosis

Various experiments have demonstrated a role for the E2F transcription factor in the regulation of cell growth during the G0/G1/S phase transition. Indeed, overexpression of the E2F1 product, a component of the cellular E2F activity, induces DNA synthesis in quiescent fibroblasts. To provide an approach to a more detailed biochemical analysis of these events, we have made use of a recombinant adenovirus containing the E2F1 cDNA in order to efficiently express the E2F1 product in an entire population of cells. We demonstrate an induction of DNA synthesis when quiescent cells are infected with the E2F1 recombinant virus. However, we also find that the induction does not lead to a complete replication of the cellular genome, as revealed by flow cytometry. The incomplete nature of cellular DNA replication is due, at least in part, to the fact that E2F1 overexpression leads to massive cell death that is characteristic of apoptosis. This E2F1-mediated induction of apoptosis is largely dependent on endogenous wild-type p53 activity and can be subverted by introducing mutant forms of p53 into these cells or by overexpressing E2F1 in fibroblasts derived from p53-null mouse embryos. We conclude that E2F1 can induce events leading to S phase but that the process is not normal and appears to result from the activation of a cell death pathway.

An Inverted Repeat Motif Stabilizes Binding of E2F and Enhances Transcription of the Dihydrofolate Reductase Gene

An overlapping inverted repeat sequence that binds the eukaryotic transcription factor E2F is 100% conserved near the major transcription start sites in the promoters of three mammalian genes encoding dihydrofolate reductase, and is also found in the promoters of several other important cellular and viral genes. This element, 5'-TTTCGCGCCAAA-3', is comprised of two overlapping, oppositely oriented sites which match the consensus E2F site (5'-TTT(C/G)(C/G)CGC-3'). Recent work has shown that E2F binding activity is composed of at least six related cellular polypeptides which are capable of forming DNA-binding homo- and heterodimers. We have investigated the binding of cellular E2F activity and of homo- and heterodimers of cloned E2F proteins to the inverted repeat E2F element. We have demonstrated that mutations in this element that abolish its inverted repeat nature, while preserving a single consensus E2F site, significantly decrease the binding stability of all of the forms of E2F tested. The rate of association of E2F-1/DP-1 heterodimers with the inverted repeat wild type site was not significantly different from those with the two single site mutated probes. Furthermore, the mutations decrease in vitro transcription and transient reporter gene expression 2-5-fold, an effect equivalent to that of abolishing E2F binding altogether. These data suggest a functional role that may explain the conservation of inverted repeat E2F elements among the DHFR promoters and several other cellular and viral promoters.

Inhibition of apoptosis by the retinoblastoma gene product.

Tissue homeostasis and the prevention of neoplasia require regulatory co-ordination between cellular proliferation and apoptosis. Several cellular proteins, including c-myc and E2F, as well as viral proteins such as E1A, have dual functions as positive regulators of apoptosis and proliferation. The product of the retinoblastoma tumor suppressor gene, pRb, binds these proteins and is known to function in growth suppression. To examine whether pRb may function as a negative regulator of both proliferation and apoptosis, we analyzed apoptosis induced in transfected derivatives of the human osteosarcoma cell line SAOS-2. Ionizing radiation induced apoptosis in a time- and dose-dependent manner in SAOS-2 cells, which lack pRb expression. In both a transient and stable transfection assay, SAOS-2 derivatives expressing wild-type (wt) pRb exhibited increased viability and decreased apoptosis following treatment at a variety of radiation doses. Expression in SAOS-2 of a mutant pRb that fails to complex with several known binding partners of pRb, including E1A and E2F, did not protect SAOS-2 cells from apoptosis. Radiation exposure induced a G2 arrest in SAOS-2 and in derivatives expressing pRb. Inhibition of DNA synthesis and cell cycle progression by aphidicolin treatment failed to protect SAOS-2 cells or pRb-expressing isolates from undergoing apoptosis. Our data document a novel function for pRb in suppressing apoptosis and suggest that several proteins shown to induce apoptosis, including E1A, E2F and c-myc, may do so by interfering with the protective function of pRb.

The adenovirus E4-6/7 protein transactivates the E2 promoter by inducing dimerization of a heteromeric E2F complex.

Binding of the mammalian transcription factor E2F to the adenovirus E2a early promoter is modulated through interaction with the viral E4-6/7 protein. E4-6/7 induces the cooperative and stable binding of E2F in vitro to two correctly spaced and inverted E2F binding sites in the E2a promoter (E2F induction) by physical interaction in the protein-DNA complex. The E2a promoter is transactivated in vivo by the E4-6/7 product. The C-terminal 70 amino acids of E4-6/7 are necessary and sufficient for induction of E2F binding and for transactivation. To assess the mechanism(s) of E2a transactivation and the induction of cooperative E2F binding by the E4-6/7 protein, we have analyzed a series of point mutants in the functional C-terminal domain of E4-6/7. Two distinct segments of E4-6/7 are required for interaction with E2F. Additionally, and E4-6/7 mutant with a phenylalanine-to-proline substitution at amino acid 125 (F-125-P) efficiently interacts with E2F but does not induce E2F binding to the E2a promoter and is defective for transactivation. Induction of E2F stable complex formation at the E2a promoter by the F-125-P mutant protein is restored by divalent E4-6/7-specific monoclonal antibodies, but not a monovalent Fab fragment, or by appending a heterologous dimerization domain to the N terminus of the mutant protein. These and other data support the involvement of E4-6/7 dimerization in the induction of cooperative and stable E2F binding and transactivation of the E2a promoter. We present evidence that at least two cellular components are involved in E2F DNA binding activity and that both are required for E2F induction by the E4-6/7 product. The recently cloned E2F-related activities E2F-1 and DP-1 individually bind to an E2F binding site weakly, but when combined generate an activity that is indistinguishable from endogenous cellular E2F. Recombinant E2F-1, DP-1, and E4-6/7 are sufficient to form the induced E2F complex at the E2a promoter.

The Adenovirus E4-6/7 Protein Transactivates the E2 Promoter by Inducing Dimerization of a Heteromeric E2F Complex

Binding of the mammalian transcription factor E2F to the adenovirus E2a early promoter is modulated through interaction with the viral E4-6/7 protein. E4-6/7 induces the cooperative and stable binding of E2F in vitro to two correctly spaced and inverted E2F binding sites in the E2a promoter (E2F induction) by physical interaction in the protein-DNA complex. The E2a promoter is transactivated in vivo by the E4-6/7 product. The C-terminal 70 amino acids of E4-6/7 are necessary and sufficient for induction of E2F binding and for transactivation. To assess the mechanism(s) of E2a transactivation and the induction of cooperative E2F binding by the E4-6/7 protein, we have analyzed a series of point mutants in the functional C-terminal domain of E4-6/7. Two distinct segments of E4-6/7 are required for interaction with E2F. Additionally, and E4-6/7 mutant with a phenylalanine-to-proline substitution at amino acid 125 (F-125-P) efficiently interacts with E2F but does not induce E2F binding to the E2a promoter and is defective for transactivation. Induction of E2F stable complex formation at the E2a promoter by the F-125-P mutant protein is restored by divalent E4-6/7-specific monoclonal antibodies, but not a monovalent Fab fragment, or by appending a heterologous dimerization domain to the N terminus of the mutant protein. These and other data support the involvement of E4-6/7 dimerization in the induction of cooperative and stable E2F binding and transactivation of the E2a promoter. We present evidence that at least two cellular components are involved in E2F DNA binding activity and that both are required for E2F induction by the E4-6/7 product. The recently cloned E2F-related activities E2F-1 and DP-1 individually bind to an E2F binding site weakly, but when combined generate an activity that is indistinguishable from endogenous cellular E2F. Recombinant E2F-1, DP-1, and E4-6/7 are sufficient to form the induced E2F complex at the E2a promoter.

A Genetic Analysis of the E2F1 Gene Distinguishes Regulation by Rb, p107, and Adenovirus E4

The cellular transcription factor E2F appears to be a target for the regulatory action of the retinoblastoma tumor suppressor gene product. The recent isolation of the E2F1 cDNA clone, which encodes a polypeptide with properties characteristic of E2F, has now allowed a more detailed analysis of the regulation of E2F function by Rb as well as the Rb-related p107 protein and the adenovirus 19-kDa E4 gene product. Previous experiments have shown that each of these regulatory proteins can modulate the activity of cellular E2F. We find that each of these regulatory events can be mediated through the E2F1 product. Moreover, an examination of various E2F1 mutations reveals distinct specificities for these regulatory proteins. For instance, the ability of E4 to alter E2F1 function is dependent upon sequences within a putative leucine repeat of E2F1 as well as within the C-terminal acidic domain. In contrast, the leucine repeat element was not important for Rb- or p107-mediated inhibition of E2F1 activity. Although the C-terminal acidic domain of E2F1, previously shown to be important for Rb binding, appears to be a site for regulation of E2F1 by Rb and p107, point mutations within this region distinguish recognition by Rb and p107. These results underscore the complexity of E2F regulatory interactions and also demonstrate a qualitative distinction in the interactions of Rb and p107 with E2F1, perhaps reflective of functional differences.

A genetic analysis of the E2F1 gene distinguishes regulation by Rb, p107, and adenovirus E4.

The cellular transcription factor E2F appears to be a target for the regulatory action of the retinoblastoma tumor suppressor gene product. The recent isolation of the E2F1 cDNA clone, which encodes a polypeptide with properties characteristic of E2F, has now allowed a more detailed analysis of the regulation of E2F function by Rb as well as the Rb-related p107 protein and the adenovirus 19-kDa E4 gene product. Previous experiments have shown that each of these regulatory proteins can modulate the activity of cellular E2F. We find that each of these regulatory events can be mediated through the E2F1 product. Moreover, an examination of various E2F1 mutations reveals distinct specificities for these regulatory proteins. For instance, the ability of E4 to alter E2F1 function is dependent upon sequences within a putative leucine repeat of E2F1 as well as within the C-terminal acidic domain. In contrast, the leucine repeat element was not important for Rb- or p107-mediated inhibition of E2F1 activity. Although the C-terminal acidic domain of E2F1, previously shown to be important for Rb binding, appears to be a site for regulation of E2F1 by Rb and p107, point mutations within this region distinguish recognition by Rb and p107. These results underscore the complexity of E2F regulatory interactions and also demonstrate a qualitative distinction in the interactions of Rb and p107 with E2F1, perhaps reflective of functional differences.

Interaction of adenoviral proteins with pRB and p53.

The transforming gene products of the small DNA tumor viruses subvert host cell growth control mechanisms by binding to specific cell regulatory proteins. These include the retinoblastoma gene product (pRB) and p53. One indication of the pivotal roles played by these regulatory products is the observation that they are each targeted consistently by viruses of several groups, by adenoviruses, the human papillomaviruses, and the papovaviruses. In adenovirus, pRB and p53 are targeted by the E1A and E1B genes, respectively. The genetic probes made possible by manipulation of the virus genes in vitro have helped to illuminate the pathways in which pRB and p53 function. E1A studies have contributed to our current understanding that the retinoblastoma product is one of a family of related proteins, which with associated cyclins and kinases can modulate the activity of the cellular E2F transcription factor. E1B studies have helped explore models of p53 function, including the suggestion that p53, probably through aspects of its transcription regulating activity, can initiate a pathway in which programmed cell death can be invoked to stop unrestricted cell proliferation.