TRANSCRIPTION FACTOR E2F-4

Abstract: A novel transcription factor, belonging to the E2f gene family, is disclosed. This is called E2F-4. It interacts with DP-1 and canbe regulated by p107.

Description: TRANSCRIPTION FACTOR E2F- This invention relates to a novel transcription factor and to its production and uses. The molecular events that occur during the cell cycle need to be integrated with the transcription apparatus so that gene expression can be synchronised with cell cycle progression. Recently, a transcription factor called E2F (or DRTF1) has been identified and shown to bind to pRb, the protein product of the retinoblastoma susceptibility gene, an anti-oncogene or tumour suppressor gene (see for example Wagner and Green, Nature 3 2. 189-190, 1991). It is widely believed that the cellular transcription factor E2F functions as a key component in cell cycle control because it associates with important cell cycle regulating proteins, such as the retinoblastoma gene product (pRb), pl07, cyclins and cyclin-dependent kinases, and furtheπnore its transcriptional activity is modulated by certain viral oncoproteins, such as adenovirus Ela, SV40 large T antigen, and the human papilloma virus E7 protein. It is believed that the transcription factor E2F (or DRTF1) plays an important role in integrating cell cycle events with the transcription apparatus because, during cell cycle progression in mammalian cells, it undergoes a series of periodic interactions with molecules that are known to be important regulators of cellular proliferation. For example, the retinoblastoma tumour suppressor gene product (pRb), which negatively regulates progression from Gl into S phase, and is frequently modified in tumour cells, binds to E2F. Similarly, the pRb-related protein pl07 occurs predominantly in an S phase complex with E2F. Both pRb and pl07 repress the transcriptional activity of E2F, which is likely to be fundamentally important for regulating cellular proliferation because E2F binding sites (the E2F site) occur in the control regions of a variety of genes that are involved with proliferation, such as c-myc and p34 cdc2 . Furthermore, mutant Rb proteins, encoded by alleles isolated from tumour cells, fail to bind to E2F, and hence are unable to interfere with E2F site-dependent transcriptional activation. Another important feature of E2F is that certain viral oncoproteins, such as adenovirus Ela, SV40 large T antigen and human papilloma virus E7, modulate its activity by sequestering pRb and pl07 from the inactive transcription factor. This effect requires regions in these viral proteins that are necessary for transformation of tissue culture cells and hence to overcome growth control. Thus, the ability of these oncoproteins to regulate E may be the means by which they over-ride the normal mechanisms of cellular growth contr and, conversely, transcriptional repression by pRb may be the basis of pRb-mediated negati growth control. A potential mechanism for integrating the transcription-regulating properties of pRb and pl with other cell cycle events was suggested by the identification of cyclin A and the cdc related cyclin-dependent kinase pSS 0 "" 2 in the E2F complex. Cyclin A is necessary f progression through S phase, a function that could perhaps be mediated through its ability recruit the cyclin-dependent kinase p33 cd 2 to E2F. Taken together these data suggest th E2F is a transcription factor whose primary role may be to relay cell cycle events to t transcription apparatus via molecules such a pRb, pl07, cyclins and cdks, thus ensuring th gene expression is synchronised and integrated with cell cycle progression. More recently, a transcription factor with the properties of E2F has been cloned a sequenced (Helin g£ al, Cell 7Q (1992), 337-350 and Kaelin et al, Cell 70 (1992), 351-364 We have now surprisingly found a further new protein which appears to be a new memb of the E2F gene family, which we have called E2F-4. The cDNA sequence of E2F-4 presented in Figure 1 A, as is the amino acid sequence of this protein. This new protein referred to as E2F-4 and this nomenclature will be used in this specification. It has been found that E2F-4 is one of a family of related transcription factor component Members of this family are believed to interact with DP proteins to form a series transcription factors. DP proteins (or polypeptides) include DP-1, DP-2 and DP-3 althoug the first of these will usually be contemplated in preference to the other two. The invention in a first aspect provides a protein as shown in Figure 1A, homologues thereo and fragments of the sequence and its homologues, which is capable of functioning as mammalian transcription factor. In particular, the invention provides a polypeptid (preferably in substantially isolated form) comprising: (a) E2F-4; (b) the protein of Figure 1A; (c) a mutant, allelic variant or species homologue of (a) or (b); (d) a protein at least 70% homologous to (a) or (b); (e) a fragment of any one of (a) to (d) capable of forming a complex with a DP protein, pRb, pl07 and/or pl30; or (f) a fragment of any of (a) to (e) of at least 15 amino acids long. All polypeptides within this definition are referred to below as polypeptide(s) according to the invention. The proteins pRb, pl07, DP proteins and pl30 are referred to herein as complexing proteins or "complexors" as they may form a complex with the proteins of the invention. Under certain conditions E2F-4 may not bind, or may only bind weakly, to pRb. A polypeptide of the invention will be in substantially isolated form if it is in a form in which it is free of other polypeptides with which it may be associated in its natural environment (eg the body). It will be understood that the polypeptide may be mixed with carriers or diluents which will not interfere with the intended purpose of the polypeptide and yet still be regarded as substantially isolated. The polypeptide of the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 90% , eg. 95 % , 98% or 99% of the polypeptide in the preparation is a polypeptide of the invention. Mutant polypeptides will possess one or more mutations which are additions, deletions, or substitutions of amino acid residues. Preferably the mutations will not affect, or substantially affect, the structure and/or function and/or properties of the polypeptide. Thus, mutants will suitably possess the ability to be able to complex with DP proteins, pRb, pl07 and/or pl30. Mutants can either be naturally occurring (that is to say, purified or isolated from a natural source) or synthetic (for example, by performing site-directed mutagenesis on the encoding DNA). It will thus be apparent that polypeptides of the invention can be either naturally occurring or recombinant (that is to say prepared using genetic engineering techniques). An allelic variant will be a variant which will occur naturally in an, eg. murine, animal an which will function to regulate gene expression in a substantially similar manner to th protein in Figure 1A. Similarly, a species homologue of the protein will be the equivalent protein which occur naturally in another species, and which performs the equivalent function in that species to th protein of Figure 1A in another (eg. mammalian) animals. Within any one species, homologue may exist as several allelic variants, and these will all be considered homologues of the protein. Allelic variants and species homologues can be obtained by following the procedures described herein for the production of the protein of Figure 1A and performing such procedures on a suitable cell source, eg from a rodent, carrying an allelic variant o another species. Since the protein may be evolutionarily conserved it will also be possible to use a polynucleotide of the invention to probe libraries made from rodent or other cells in order to obtain clones encoding the allelic or species variants. The clones can be manipulated by conventional techniques to identify a polypeptide of the invention which can then be produced by recombinant or synthetic techniques known per se. Preferred species homologues include mammalian or amphibian species homologues. A protein at least 70% homologous to that in Figure 1A is included in the invention, as are proteins at least 80 or 90% and more preferably at least 95% homologous to the protein of Figure 1A. This will generally be over a region of at least 20, preferably at least 30, for instance at least 40, 60 or 100 or more contiguous amino acids. Methods of measuring protein homology are well known in the art and it will be understood by those of skill in the art that in the present context. Homology is usually calculated on the basis of amino acid identity (sometimes referred to as "hard homology"). Generally, fragments of Figure 1A or its allelic variants or species homologues thereof capable of forming a complex with the complexors will be at least 10, preferably at least 15, for example at least 20, 25, 30, 40, 50 or 60 amino acids in length. It will be possible to determine whether fragments form a complex with the complex of proteins by providing the complexor protein and the fragment under conditions in which they normally form a complex, such as a trans-activating transcription factor, and determining whether or not a complex has formed. The determination may be made by, for example, measuring the ability of the complex to bind an E2F binding site in vitro, or alternatively , detennining the molecular weight of the putative complex by methods such as SDS-PAGE. Preferred fragments include those which are capable of forming a trans-activation complex with DP-1 or other complexors. The examples herein describe a number of methods to analyse the function of the protein and these may be adapted to assess whether or not a polypeptide is capable of forming a trans-activation complex with the DP-1 protein. For example, the polypeptide can be added to the complexor in the presence of a reporter gene construct adapted to be activated by the DP-1/E2F-4 complex (for example, see Figure 10 of WO-A-94/10307 in the name of the Medical Research Council). Such an experiment can determine whether the polypeptide fragment has the necessary activity. A polypeptide of the invention may be labelled with a revealing or detectable label. The (revealing) label may be any suitable label which allows the polypeptide to be detected. Suitable labels include radioisotopes, e.g. 1M I, enzymes, antibodies and linkers such as biotin. Labelled polypeptides of the invention may be used in diagnostic procedures such as immunoassays in order to determine the amount of E2F-4 protein in a sample. A polypeptide or labelled polypeptide according to the invention may also be attached or fixed to a solid phase or support, for example the wall of an immunoassay dish. A second aspect of the invention relates to a polynucleotide which comprises : (a) a sequence of nucleotides shown in Figure 1A; (b) a sequence complementary to (a); (c) a sequence capable of selectively hybridising to a sequence in either (a) or (b); (d) a sequence encoding a polypeptide as defined in the first aspect; or (e) a fragment of any of the sequences in (a) to (d). The present invention thus provides a polynucleotide, suitably in substantially isolated or purified form, which comprises a contiguous sequence of nucleotides which is capable of selectively hybridizing to Figure 1A or to its complement. Polynucleotides of the invention include a DNA of Figure 1 A and fragments thereof capable of selectively hybridizing to sequence of Figure 1 A. A further embodiment of the invention provides a DNA coding the protein in Figure 1A or a fragment thereof. The polynucleotide may also comprise RNA. It may also be a polynucleotide which inclu within it synthetic or modified nucleotides. A number of different types of modification oligonucleotides are known in the art. These include methylphosphonate a phosphorothionate backbones, addition of acridine or polylysine chains at the 3' and/or ends of the molecule. For the purposes of the present invention, it is to be understood t the oligonucleotides described herein may be modified by any method available in the a Such modifications may be carried out in order to enhance the in vivo activity or lifespan oligonucleotides of the invention used in methods of therapy. A polynucleotide capable of selectively hybridizing to the DNA of Figure 1A will generally at least 70%, preferably at least 80 or 90% and optimally at least 95% homologo to the DNA of Figure 1 A over a region of at least 20, preferably at least 30, for instance least 40, 60 or 100 or more contiguous nucleotides. These polynucleotides are within t invention. A polynucleotide of the invention will be in substantially isolated form if it is in a form which it is free of other polynucleotides with which it may be associated in its natu environment (usually the body). It will be understood that the polynucleotide may be mix with carriers or diluents which will not interfere with the intended purpose of t polynucleotide and it may still be regarded as substantially isolated. A polynucleotide according to the invention may be used to produce a primer, e.g. a P primer, a probe e.g. labelled with a revealing or detectable label by conventional means usi radioactive or non-radioactive labels, or the polynucleotide may be cloned into a vect Such primers, probes and other fragments of the DNA of Figure 1A will be at least 1 preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are al encompassed within the invention. Polynucleotides, such as a DNA polynucleotides according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. It may be also cloned by reference to the techniques disclosed herein. The invention includes a double stranded polynucleotide comprising a polynucleotide according to the invention and its complement. A third aspect of the invention relates to an (eg. expression) vector suitable for the replication and expression of a polynucleotide, in particular a DNA or RNA polynucleotide, according to the invention. The vectors may be, for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the polynucleotide and optionally a regulator of the promoter. The vector may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector. The vector may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell. The vector may also be adapted to be used in vivo, for example in a method of gene therapy. Vectors of the third aspect are preferably recombinant replicable vectors. The vector may thus be used to replicate the DNA. Preferably, the DNA in the vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by a host cell. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences. Such vectors may be transformed or transfected into a suitable host cell to provide for expression of a polypeptide of the invention. A fourth aspect of the invention thus relates to host cells transformed or transfected with the vectors of the third aspect. This may be for the replication and expression of a polynucleotide according to the invention, including the sequence of Figure 1 A or the open reading frame thereof. The cells will be chosen to be compatible with the vector and may for example be bacterial, yeast, insect or mammalian. A polynucleotide according to the invention may also be inserted into the vectors describ above in an antisense orientation in order to provide for the production of antisense RN Antisense RNA or other antisense polynucleotides may also be produced by synthetic mea Such antisense polynucleotides may be used in a method of controlling the levels of the E2F protein in a cell. Such a method may include introducing into the cell the antisen polynucleotide in an amount effective to inhibit or reduce the level of translation of the E2F mRNA into protein. The cell may be a cell which is proliferating in an uncontrolled mann such as a tumour cell. Thus, in a fifth aspect the invention provides a process for preparing a polypeptide accordi to the invention which comprises cultivating a host cell transformed or transfected with (expression) vector of the third aspect under conditions providing for expression (by t vector) of a coding sequence encoding the polypeptide, and recovering the express polypeptide. The invention in a sixth aspect also provides (monoclonal or polyclonal) antibodies to polypeptide according to the invention. Antibodies of die invention include fragment thereof as well as mutants that retain the antibody's binding activity. The invention furth provides a process for the production of monoclonal or polyclonal antibodies to a polypepti of the invention. Monoclonal antibodies may be prepared by conventional hybrido technology using the proteins or peptide fragments thereof as an immunogen. Polyclon antibodies may also be prepared by conventional means which comprise inoculating a ho animal, for example a rat or a rabbit, with a polypeptide of the invention and recoveri immune serum. Fragments of monoclonal antibodies which can retain their antigen binding activity, such F F(ab') and F(al> 2 )' fragments are included in this aspect of the invention. In additio monoclonal antibodies according to the invention may be analyzed (eg. by DNA sequen analysis of the genes expressing such antibodies) and humanized antibody wi complementarity determining regions of an antibody according to the invention may be mad for example in accordance with the methods disclosed in EP-A-0239400 (Winter). The present invention further provides compositions comprising the antibody or fragment thereof of the invention together with a carrier or diluent. Such compositions include pharmaceutical compositions in which case the carrier or diluent will be pharmaceutically acceptable. Polypeptides of the invention can be present in compositions together with a carrier or diluent. These compositions include pharmaceutical compositions where the carrier or diluent will be pharmaceutically acceptable. Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. For example, formulations suitable for parenteral administration include aqueous and non- aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the polypeptide to blood components or one or more organs. Polypeptides according to the invention, antibodies or fragments thereof to polypeptides according to the invention and the above-mentioned compositions may be used for the treatment, regulation or diagnosis of conditions, including proliferative diseases, in a mammal including man. Such conditions include those associated with abnormal (eg at an unusually high or low level) and/or aberrant (eg due to a mutation in the gene sequence) expression of one or more transcription factors such as the DP or E2F proteins or related family members. The conditions also include those which are brought about by abnormal expression of a gene whose gene product is regulated by the protein of Figure 1A. Treatment or regulation o conditions with the above-mentioned peptides, antibodies, fragments thereof and composition etc. will usually involve administering to a recipient in need of such treatment an effectiv amount of a polypeptide, antibody, fragment thereof or composition, as appropriate. One group of preferred polypeptides according to the invention are those which are base upon the region of amino acids 382-413 of Figure 1A. This region of the protein is capabl of binding to pl07. The invention also provides antibodies, and fragments thereof, targeted to this region in orde to inhibit the activation of transcription factors via the disruption of the formation of th E2F-4-DP protein complex. The present invention further provides a method of performing an immunoassay for detectin the presence or absence of a polypeptide of the invention in a sample, the method comprising (a) providing an antibody according to the invention; (b) incubating the sample with the antibody under conditions that allow for th formation of an antibody-antigen complex; and (c) detecting, if present, the antibody-antigen complex. In another aspect, the invention provides a novel assay for identifying putativ chemotherapeutic agents for the treatment of proliferative or viral disease which comprise bringing into contact a DP protein or a derivative thereof, a polypeptide of the invention an a putative chemotherapeutic agent, and measuring the degree of inhibition of formation of th DP/E2F-4 protein complex caused by the agent. It may not be necessary to use complet DP-1 and/or E2F-4 protein in the assay, as long as sufficient of each protein is provided suc that under the conditions of the assay in the absence of agent, they form a heterodimer. The cloning and sequencing of DP-1 (and E2F 1,2 and 3) are known in the art and method for the recombinant expression and preparation of antibodies to DP-1 can be found in WO-A 94/10307. Thus, the invention provides a screening method for identifying putative chemotherapeutic agents for the treatment of proliferative disease which comprises: (A) bringing into contact: (i) a DP polypeptide; (ii) a polypeptide of the first aspect, and (iii) a putative chemotherapeutic agent; under conditions in which the components (i) and (ii) in the absence of (iii) form a complex; and (B) measuring the extent to which component (iii) is able to disrupt said complex. In the assay, any one or more of the three components may be labelled, eg with a radioactive or color-metric label, to allow measurement of the result of the assay. Putative chemotherapeutic agents include peptides of the invention. Variants, homologues and fragments of DP proteins are defined in a corresponding manner to the variants, homologues and fragments of the E2F-4 protein. The complex of (i) and (ii) may be measured, for example, by its ability to bind an E2F DNA binding site in vitro. Alternatively, the assay may be an in vivo assay in which the ability of the complex to activate a promoter comprising an E2F binding site linked to a reporter gene is measured. The in vivo assay may be performed for example by reference to the examples which show such an assay in yeast, insect, amphibian or mammalian cells. Candidate therapeutic agents which may be measured by the assay include fragments of 10 or more amino acids of (a) the protein of Figure 1A; (b) an allelic variant or species homologue thereof; or (c) a protein at least 70% homologous to (a). Vectors carrying a polynucleotide according to the invention or a nucleic acid encoding a polypeptide according to the invention may be used in a method of gene therapy. Such gene therapy may be used to treat uncontrolled proliferation of cells, for example a tumour cell. Methods of gene therapy include delivering to a cell in a patient in need of treatment an effective amount of a vector capable of expressing in the cell either an antisense polynucleotide of the invention in order to inhibit or reduce the translation of E2F-4 mR into E2F-4 protein or a polypeptide which interferes with the binding of E2F-4 to a protein or a related family member. The vector is suitably a viral vector. The viral vector may be any suitable vector availa in the art for targeting tumour cells. For example, Huber et al (Proc. Natl. Acac. Sci. U (1991) 8§, 8039) report the use of amphotrophic retroviruses for the transformation hepatoma, breast, colon or skin cells. Culver et al (Science (1992) 256; 1550-1552) al describe the use of retroviral vectors in virus-directed enzyme prodrug therapy, as do Ra et al (Cancer Research (1993) 52; 83-88). Englehardt et al (Nature Genetics (1993) 4; 27- describe die use of adenovirus based vectors in the delivery of the cystic fibro transmembrane conductance product (CFTR) into cells. The invention contemplates a number of assays. Broadly, these can be classified as follow 1. Conducting an assay to find an inhibitor of E2F-4 rra/ty-activation (that is to sa inhibition of activation of transcription). This inhibitor may therefore inhibit binding of E2 4 to DNA (usually at the E2F binding site). Potentially suitable inhibitors are proteins, a may have a similar or same effect as pl07. Thus suitable inhibitory molecules may compri fragments, mutants, allelic variants, or species homologues of pl07 in the same manner defined for proteins of the first aspect. 2. Assaying for inhibitors of (hetero)dimerisation. Such inhibitors may preve dimerisation of E2F-4 (or a polypeptide of the first aspect) with a complexor, for examp a DP protein, such as DP-1. Of course the inhibitor can be a fragment, mutant, allel variant or species homologue of a DP protein in a similar manner as defined for the protei of the first aspect. 3. A third category of assay is to find inhibitors of phosphorylation. It is thought th E2F-4 (and other proteins of the first aspect) might be activated by phosphorylatio Therefore, an inhibitor of phosphorylation is likely to inhibit E2F-4 frα/w-activation properti (and may therefore, ultimately have the same effect as the inhibitors found in either of t two previous assays). Phosphorylation is by cdk's and so an inhibitor of this phosphorylation is one that is contemplated by such assays. The invention contemplates a number of therapeutic uses. For example, gene therapy using a nucleic acid a sequence that is antisense to E2F-4. Molecules that can bind to a DP-1 protein and thereby form an inactive complex with the DP protein are additionally contemplated. Suitable molecules include those of the first aspect apart from E2F-4 itself. Such molecules may be mutants of E2F-4, and are often referred to as dominant negative molecules in the art. The invention contemplates the treatment or prophylaxis of diseases that are based on the uncontrolled proliferation of cells, or where uncontrolled proliferation is an important or essential pathological aspect of the disease. This includes cancer, viral disease, self proliferation itself as well as auto immune diseases such psoriasis. One may also wish to temporarily inhibit the growth of dividing cells, for example hematopoietic stem cells and/or bone marrow cells. In these aspects one is generally seeking to prevent, inhibit or interfere with the activity of E2F-4. In contrast some diseases and conditions can be treated by increasing E2F-4 expression, for example by promoting or inducing overexpression. This preferably results in apoptosis, sometimes known as programmed cell death. Overexpression of the E2F-4 protein can result in death of the cell, and therefore this aspect can also be used in the treatment of cancer. One aim is therefore to increase the activity of E2F-4. Similar uses are known for E2F-1 (Qin et al, PNAS USA 91 (in press)). It should be borne in mind that the E2F-4 gene might be mutated in tumour cells. In that event, the mutated gene could be used in diagnosis of a condition resulting from the mutation. It also lends itself to treatment via the mutated gene. The following examples describe the isolation and characterization of the novel protein and DNA of the invention from a human source. However, other e.g. mammalian sources are within the scope of the present invention and die other e.g. mammalian homologues of the protein may be isolated in an analogous manner. The invention will now be described, by way of example, with reference to the accompanyin Examples which should not be construed as being limiting. EXAMP g l The E2F gene family encodes a number of closely related transcription factors that control gene expression during the cell division cycle. E2F sites have been found in a number of genes whose expression is tightly regulated during the cell cycle (for review, see Nevins 5 1992; Farnham et al, 1993). Importantly, in a number of cases, it has been shown that mutation of the E2F-binding sites in the promoters of these genes leads to a loss of cell-cycle- regulated expression (Farnham et al, 1993; Lam and Watson 1993). Complexes have been found between E2F and the retinoblastoma protein (pRb), E2F and the pRb-related pl07, and E2F and a third member of the pRb gene family, pl30. These three complexes show a 0 different pattern of appearance during the cell cycle. Complexes between E2F and pRb are found mostly in the Gj phase of the cell cycle, but some E2F/pRb complexes persist during S phase. The complexes between E2F and pl07 show a more complex pattern of appearance during die cell cycle. In G„ DNA-binding complexes have been observed diat contain E2F, pl07, cyclin E, and cyclin-dependent kinases (cdk), cdk2. In S phase cyclin E is no longer 5 found in these complexes, but cyclin A is found associated with E2F, pl07 and cdk2 (Lees et al 1992; Shirodkar et al, 1992). The function of these cyclin/cdk complexes in the E2F/pl07 complex remains obscure as yet. In addition, complexes have been observed between E2F and pl30 primarily in quiescent cells (Cobrinik et al, 1993). E2F/pl30 complexes disappear quickly when cells emerge from quiescence and enter into the cell cycle. The activity of E2F is tightly regulated by association widi proteins of the pRb gene family. Two findings were critical for our understanding of die mechanism by which pRb regulates E2F activity. First, the demonstration diat pRb can inhibit -r/i s-activation by E2F showed d at proteins of the pRb gene family act as negative regulators of E2F activity (Chellappan et al, 1991; Hamel et al 1992; Hiebert et al, 1992; Weintraub et al; 1992, Helin 5 et al 1993). Second, die demonstration iat E2F binds preferentially to hypophosphorylated pRb suggested diat complex formation between pRb and E2F is regulated by phosphorylation of pRb (Buchkovich et al, 1989; Chen et al, 1989; DeCaprio et al, 1989). Since pRb can be phosphorylated by cyclin E and die D-type cyclins, when in complex with their respective cdk, diese cyclin/cdk complexes are thought to release E2F from pRb by phosphorylating 0 pRb. Recentiy, a second cyclin/cdk complex was shown to act as a negative regulator of E2F activity: Cyclin A can bind directly to E2F-1 and inhibit DNA binding by E2F-1 (Krek et al, 1994). As cyclins D and E are present primarily in late G, and cyclin A primarily in S phase, E2F-1 activity is likely to increase in late G, and decline in S phase when cyclin A is synthesized. It has been shown that pl07, like pRb, can inhibit E2F-dependent ge expression (Schwarz et al, 1993; Zamanian and La Thangue 1993; Zhu et al. 1993 However, very little is known about the regulation of the E2F activity that interacts wi pl07. E2F DNA-binding activity consist of heterodimeric complex of two molecules: E2F polypeptide and a dimerization partner named DP-1. cDNAs encoding E2F-1 and DP have been isolated by molecular cloning (Helin et al. 1992; Kaelin et al. 1992; Girling et a 1993; Huber et al. 1993). Recentiy the complexity of this intricate network of interacti proteins increased further when two homologues of E2F-1, named E2F-2 and E2F-3, we isolated (Ivey-Hoyle et al. 1993; Lees et al. 1993). E2F-1, E2F-2 and E2F-3 associate vivo only with pRb and not widi die related pl07. The presence of E2F activity associat witii pl07 (Cao et al. 1992; Lees et al. 1992, Schirodkar et al., 1992) would indicate th the family of E2F-like polypeptides is even larger and includes additional members that ha the ability to associate with pl07. Consistent with this hypothesis, it has been shown th pl07-associated E2F polypeptides are similar, but distinct from pRb-associated E2 polypeptides (Dyson et al. 1993). We report here the cloning of a novel member of the E2 gene family, E2F-4, that interacts with pl07 in vivo. E2F-4 can promote cell-cycl progression and can act as an oncogene when overexpressed in primary fibroblasts. Results Isolation of cDNA clones encoding E2F-4 To isolate cDNAs encoding proteins that interact with pl07, we screened a day-16 whol mouse embryo cDNA expression library with a 32 P-labelled pl07 protein probe. Thre independent phage were isolated whose encoded fusion protein bound to pi 07. Partial DN sequence analysis revealed diat all three were derived from the same gene. The shorte clone had a cDNA insert of 800 bp and contained an open reading frame of only 1 amin acids. These 31 amino acids showed significant homology (15 of 31 identities) to die pRb binding domain of E2F-1. The longer cDNA clones showed additional homology to die thre known E2Fs. We thus named me protein encoded by tiiis novel cDNA E2F-4. The parti mouse E2F-4 cDNA clones were then used to obtain longer mouse cDNAs and to isolate human E2F-4 cDNA. Despite extensive efforts, we were unable to obtain die extreme 5' en of die human E2F-4 cDNA. We did, however, obtain a full-length mouse E2F-4 cDNA. Comparison of the 5' ends of die mouse and human E2F-4 cDNAs indicated diat the human cDNA only lacked the first 16 codons of E2F-4. These 16 amino acids, however, are fully conserved between mouse and human E2F-4 (Ginsberg et al. , Genes & Development, 15 November 1994). Translation of the human E2F-4 cDNA (including die first 16 amino acids derived from mouse E2F-4) yields a 413 amino-acid protein with a predicted molecular mass of 44 kD. Figure 1A shows the E2F-4 cDNA sequence and die deduced amino acid sequence. E2F-4 and E2F-1 share three regions of homology (Fig. IB). The overall similarity between the two proteins is 63% (41 % identity). A striking difference between E2F-4 and E2F-1 is that E2F-1 has an additional 100 amino acids amino-terminal of the DNA-binding domain. This region of E2F-1 contains a cyclin A-binding site (amino acid residues 67-108; Krek et al.. 1994). The first region of homology is from amino acids 10-82 of E2F-4 and 120-190 of E2F-1 (similarity 71%), which coincides with die DNA binding region of E2F-1 (Helin et al. 1993b). E2F-1 can only bind DNA in a heterodimeric complex widi DP-1. The region required for DP-1 interaction is located downstream of die DNA-binding domain (amino acids 191 -284) . The similarity between E2F-4 and E2F- 1 in tiiis region is -64 % . E2F-4 and E2F- 1 diverge in the carboxyl terminus. Downstream of amino acid 194 of E2F-4 and 297 of E2F-1 there is a region with almost no sequence conservation. In this region, E2F-4 contains a remarkable stretch of 12 consecutive serines. Although E2F-1 does not contain this stretch of serines, it does contain a region that is serine-rich. The functional significance of mis repeat in E2F-4 is presently unknown. The last segment of homology is located at die extreme carboxyl terminus of me two proteins. This region of homology corresponds to die region of E2F-1 that is involved in binding pRb (Helin et al, 1992). Since die first E2F-4 cDNA that we isolated with the pl07 protein probe encoded only die last 31 amino acids of E2F-4, it is likely that this region, like its counterpart in E2F-1 diat is sufficient for pRb binding, is sufficient for binding to pl07. In this region, 15 of the 31 residues are identical between E2F-4 and E2F-1. To analyze the pattern of expression of E2F-4, a partial mouse E2F-4 cDNA was used to probe a Northern blot containing poly(A)' RNA from various tissues of die mouse. At high stringency, a single transcript of 2.1 kb was detected. The expression was high in kidney and tiiymus and relatively low in lung, brain, spleen, and testis (data not shown). E2F-4 requires DP-1 for DNA binding Efficient sequence-specific DNA binding by E2F-1 requires heterodimerization with a seco protein named DP-1 (Helin et al. 1993b; Krek et al., 1993). To investigate whether E2F can function like the otiier members of the E2F gene family, we investigated whetiier E2F was able to bind an E2F DNA consensus site, and whetiier DP-1 was required for D binding by E2F-4. We transfected U2-OS osteosarcoma cells with DP-1 and E2F expression vectors separately or together. After 2 days, gel shift extracts were made fro the transfected cells, and these extracts were incubated with a 32 P labelled oligonucleotide di specifies a consensus E2F site. DNA-protein complexes were then resolved on an acrylami gel and detected by autoradiography. As can be seen in Figure 2, transfection of U2-OS cells with DP-1 alone did not le to an increase in E2F DNA binding activity. Transfection of E2F-4 expression vector alon however, yielded a clearly detectable increase in E2F activity. The most striking increa was seen when E2F-4 and DP-1 expression vectors were introduced simultaneously in d osteosarcoma cells (Fig.2). These results show diat E2F-4 can bind to a consensus E2F si and d at binding is enhanced in d e presence of DP-1. E2F-4 and DP-1 are svnergistic in the activation of an E2F site reporter. Next we investigated whetiier E2F-4 has the ability to activate die transcription of a CA reporter gene diat was linked to a core promoter and four upstream E2F sites. cotransfected C33A cervical carcinoma cells with increasing amounts of E2F-4 (Fig. 3 A) DP-1 expression vectors (Fig.3B) and die CAT reporter plasmid. When transfected alon E2F-4 was unable to activate transcription of die CAT reporter. In contrast, expression DP-1 led to a clearly detectable increase in die activity of the reporter gene. When E2F and DP-1 were cotransfected, a strong increase in transcriptional activity was observed. T activation by E2F-4 and DP-1 togetiier was more than die sum of eitiier plasmid alon indicating that E2F-4 and DP-1 are cooperative in rra/w-activation. The cotransfection E2F-4 and DP-1 with a reporter plasmid containing a mutant E2F site did not result increased activation of die reporter (data not shown). Togetiier, these experimen demonstrate mat E2F-4 is able to activate transcription from a promoter containing E2 DNA-binding sites in the presence of DP-1. pl07 Inhibits E2F-4 7ra/ij-activation Transcriptional activation by E2F-1 can be suppressed by pRb, because the pRb-binding site of E2F-1 and die r w-activation domain overlap. Since E2F-4 was isolated wid pl07 protein probe, we studied die effect of pl07 on E2F-4 fra/tr-activation. C33A cervical carcinoma cells were cotransfected with E2F-4 and DP-1 expression vectors and die E2F-site containing CAT reporter plasmid in d e presence of increasing amount of pl07 expression vector. Figure 4 shows that ρl07 can efficiendy suppress E2F-4 fra/ir-activation. Maximal inhibition was already observed with 200ng of pl07 expression vector. As a control we used die pl07 mutant, pl07DE, that lacks most of the pocket region of pl07 (Zhu et al, 1993). Cotransfe ion of 1 μg of pCMVpl07DE did not significantly affect E2F-4 fra/w-activation (Fig 4). When pRb was expressed, a decrease in E2F-4-mediated fra/ir-activation was observed, although to a lesser extent than widi pl07. Inhibition of E2F-mediated trans- activation by transfection of either pi 07 or pRb has been observed previously (Zamaman and La Thangue 1993; Zhu et al 1993), indicating that both pocket proteins are able to associate with endogenous E2F's when transiently overexpressed. These experiments, however, show that -.rαΛs-activation by E2F-4 can be suppressed by overexpression of wild type pl07 or pRb and not by a mutant form of pl07 protein diat lacks growth-inhibitory activity (Zhu et al, 1993). E2F-4 specifically interacts with p!07 in vivo In nontransfected cells E2F-1 interacts only widi pRb and not widi pl07 (Dyson et al, 1993; Lees et al, 1993). However, when E2F-1 and pl07 are transiently transfected, an interaction can be observed (R.L.Beijersbergen and R. Bernards, unpubl.). This indicates diat overexpression of tiiese proteins may lead to interactions that are not observed under physiological conditions. E2F-4 was identified by virtue of its ability to bind pl07 in vitro. The data in Figure 4 suggest diat E2F-4 can interact with pl07 and pRb in transiently transfected cells, but they did not show d at these interactions also take place under physiological conditions. To address whetiier E2F-4 interacts with pi 07 and/or pRb in nontransfected cells, we generated E2F-4-specific polyclonal antiserum and used this in a sequential immunoprecipitation experiment. ML-1 leukemia cells were metabolically labeled widi [ 32 P]orthophosphate. Nonionic detergent lysates were prepared and subjected to immunoprecipitation widi eitiier pl07-specific monoclonal antibody or pRb-specific antibo Proteins that were coimmunoprecipitated with pRb and 107 were tiien released by heating SDS-containing buffer, diluted and reimmunoprecipitated with polyclonal E2F-4 antiseru E2F-1 -specific monoclonal antibody, or nonimmune control serum. Consistent with die d of others (Dyson et al, 1993; Lees et al, 1993) mAb E2F-1 recognized a single prot species that is present in the anti-pRb immunoprecipitate but not in the anti-pl immunoprecipitate (Fig. 5). Conversely, the E2F-4 antiserum clearly detected two clos migrating protein species that were present in the pl07 immunoprecipitate. A very fa signal was detected when die E2F-4 antiserum was used to reimmunoprecipitate protei associated widi pRb (Fig. 5, lane 4). This could indicate diat die E2F-4 antiserum show weak cross-reactivity with other E2Fs. Alternatively, some (<5%) E2F-4 may be bound pRb. We generated monoclonal antibodies diat specifically recognize E2F-4 and not E2 1, which recognize die same bands as detected by die polyclonal E2F-4 serum in ML-1 cel HeLa cells and BJAB cells (data not shown). Taken together, tiiese data strongly suggest d E2F-4 interacts preferentially with pl07, rather than widi pRb. E2F-4 is a phosphoprotein The E2F-4 monoclonal antibodies recognize a 32 P-labelled protein in a pl immunoprecipitate indicating that E2F-4 is phosphorylated in vivo (Fig.5). The finding di die E2F-4 antibodies recognized multiple closely migrating protein species (Fig.5) cou indicate diat different proteins are recognized by the same antibody or that E2F-4 protein subject to post-translational modifications d at result in altered mobility in an SDS gel. further investigate die nature of die different E2F-4 protein species that were present in pl immunoprecipitates, we labeled ML-1 cells widi [ 35 S]-methionine. mAb RK13 recogniz three protein species in a pi 07 immunoprecipitate (data not shown). The same pattern bands was also detected when U2-OS cells were transfected widi an E2F-4 expression vect (Fig.6), indicating diat die expression vector encodes the full-length E2F-4 protein. investigate whether tiiese protein species were all derived from die same protein differential phosphorylation, we performed die following experiment. U2-OS cells we transfected widi E2F-4, DP-1, and pi 07 expression vectors, labelled widi [ 35 S] methionin and immunoprecitated under low stringency conditions widi pl07 antibody. The pl immunoprecipitate was treated in the presence or absence of calf intestine phosphate, and pl07 associated proteins were reimmunoprecipitated widi mAb E2F-4. Figure 6 shows that following phosphatase treatment the three protein species that bind to pl07 and are recognized by mAb RK13 were reduced to mostly a single fast migrating protein species, although some slower migrating species remain. Taken together, our data suggest diat E2F-4 is a phosphoprotein and diat the different E2F-4 protein species arise by differential phosphorylation of E2F-4. Association of P107 widi E2F-4 requires dimerisation with DP-1. To investigate whether E2F-4 requires DP-1 for interaction with ρl07, we transfected U2-OS cells widi pi 07 and E2F-4 in the absence or presence of an hemagglutinin (HA) epitope- tagged DP-1 expression vector. Transfected cells were labelled widi f 3 P] orthophosphate and cell lysates were immunoprecipitated at low stringency wid pl07 antibody to allow coimmunoprecipitation of pl07-associated proteins. The E2F-4 and DP-1 proteins were equally expressed in all transfections (data not shown). The anti-pl07 immunoprecipitates were then boiled in 2% SDS and 15 mM DTT to release associated proteins and released proteins were reimmunoprecipitated widi die anti-E2F-4 mAb RK13 or with mAb 12CA5 directed against die HA-tagged DP-1. The transfection of pl07 with DP-1 did not result in complex formation between these two proteins (Fig.7, lane 3). The same result was obtained when pl07 was cotransfected widi E2F-4 (Fig.7, lane 5). In contrast, in cells transfected with all three expression vectors, pl07, E2F-4 and DP-1 both E2F-4 and DP-1 were found in complex widi pl07. Stimulation of Cell Proliferation bv E2F-4/DP-1 Dimer A number of cellular genes whose protein products are required for DNA synthesis contain E2F sites in tiieir promoters (Nevins 1992). Transcriptional activation of tiiese genes by E2F is believed to be an important and, in most cases, essential step in die controlled expression of tiiese genes during die cell cycle. It is expected, dierefore, that deregulated expression of active E2F would stimulate cell proliferation. Consistent with tiiis, it has been shown that E2F-1 can stimulate quiescent cells to initiate DNA syntiiesis (Johnson et al, 1993). In this study we investigated die growdi-promoting effects of E2F-4 and its dimerization partner DP-1 in cycling cells. Osteosarcoma cell line SaoS-2 was transiently transfected widi DP-1 or E2F-4 or cotransfected widi E2F-4 and DP-1 expression vectors. Three days later the cells were harvested and processed for flow cytometry analysis to determine die proliferation status of the population. Transfected cells were identified by d e cotransfected cell surface marker CD20 and analyzed in comparison to the control vector- transfected cells (Zhu et al 1993). Table 1 shows that overexpression of DP-1 stimulated cell-cycle progression of the transfected population, decreasing die G x population and increasing die S-phase population at die same time by almost 20% . Overexpression of E2F-4 alone also resulted in an increase of S-phase cells, although to a smaller extent. Overexpression of E2F-4 together widi DP-1 exhibited d e most dramatic effect by decreasing die G, population and increasing die G 2 +M-phase population as indicated in tiiree independent experiments. These results indicate eidier that overexpression of E2F-4 and DP- 1 promote entry into S phase or delay exit from later stages of the cell cycle. Since E2F-4 can act as a dominant oncogene in transformation of primary rodent fibroblasts we favour die explanation diat E2F-4 promotes cell-cycle progression rather tiian inhibiting exit from S phase and G 2 /M phase of the cell cycle. Table 1 : Overexpression of E2F-4/DP-1 Stimulates Cell Proliferation Phase Phase Phase Experiment G 0 G, S G2/M pCMV 1 51.1 31.7 17.2 2 44.6 33.3 22.1 3 55.3 23.9 20.7 pCMV-DP-1 1 33.4 48.5 18.1 pCMV-E2F-4 1 41.5 44.1 14.4 pCMV-E2F-4 + 1 28.5 43.1 28.4 pCMV-DP-1 2 25.4 40.6 34.0 3 30.3 40.6 29.1 SaoS-2 cells were transfected widi pCMV-CD20 CD 20 cell surface protein in combination with expression vectors pCMV, pCMV-DP-1, pCMV-E2F-4, or pCMV-E2F-4 together with pCMV-DP-1. Transfected cells were analyzed by FACS and cell cycle profiles of CD20- positive cells was determined. The percentage of cells in Go/G,,S, and G 2 /M phase are depicted. Overexpression of E2F-4 and DP-1 together with ras causes transformation The effect of E2F-4 on cell-cycle progression suggests a role for E2F-4 in normal cell-cycle control. If this is the case, overexpression of E2F-4 might result in deregulated growtii control. Proteins diat promote transition from Gi to S phase of the cell cycle may have immortalizing activity on primary cultures of fibroblasts. For example, both adenovirus El A and c-Myc promote S phase entry (Heikkila et al 1987; Moran and Madiews 1987; Eilers et al, 1991) and have immortalizing activity when introduced in primary rat embryo fibroblast (REF) cultures (Land et al, 1983; Ruley 1983). To study die-effect of E2F-4 on primary cultures of REFs, we transfected REFs with E2F-4, DP-1 expression vectors alone, or cotransfected with E2F-4 and DP-1. In all experiments a plasmid encoding a mutant Ha-ra? oncogene was cotransfected to obtain full oncogenic transformation (Land et al, 1983). Since both die E2F and DP-1 expression vectors contain a neomycin resistance gene, colonies of transformed cells could be selected in the presence of G418. After 4 weeks G418-resistant colonies were detected only in die E2F-4-, DP-1-, and /"αs-transfected cells and not in die E2F-4- plus ras- or DP-1- plus ras- transfected cells. Figure 8 shows the morphology of two colonies of REFs, E4R1 and E4R2, transfected with E2F-4, DP-1 and ras, in comparison to the primary REF culture. Botii transfectants have a transformed morphology. Consistent with their transformed morphology botii transfectants were able to form colonies in soft agar and gave rise to tumours with short latency periods when 5 x 10" cells were injected in nude mice (data not shown). The expression of E2F-4 and DP-1 in die transformed REF cell lines was measu in two independent assays. First, die REF cell lines were labelled widi [ 32 P]ortiιophospha and subjected to immunoprecipitation with mAb 12CA5 directed against the HA-tagged DP- Figure 9 shows diat both E2F-4- plus DP-1 -transfected REF cell lines, E4R1 and E4R2, b not control adenovirus 5-transformed REFs, expressed die 55-kD HA-tagged DP-1 prote (cf. lanes 1, 3 and 5). In die former two lines, mAb 12CA5 also immunoprecipitated a 6 kD protein diat could be reimmunoprecipitated widi mAb E2F-4 (Fig 9, lanes 7,9). The data indicate d at die immortalized REF cell lines express botii DP-1 and E2F-4. To further analyze the E2F DNA-binding activity in these REFs, we made nucle extracts of E4R2 and adenovirus 5-transformed REFs and used tiiese in a gel retardatio assay. The E2F-4 transfectant E4R2 showed a dramatic increase in total amount of E2 DNA-binding activity, which was completely supershifted by the addition of he E2F- antiserum (data not shown). Furthermore, most of die E2F activity in the E4R2 was fre E2F, whereas in tiie adenovirus-transfected line only slower migrating complexes we observed diat were not affected by die E2F-4 antiserum. We conclude diat overexpressio of E2F-4 and DP-1 widi an increased E2F-4 DNA-binding activity in REFs, in combinatio with an activated ras oncogene, results in the generation of transformed cell lines diat hav oncogenic activity. Discussion We have isolated a fourth member of the E2F gene family. E2F-4 differs from the thre known E2Fs in that E2F-4 associates in vivo preferentially with die pRb-related pi 07 an only weakly, if at all, with pRb itself. In contrast, E2F-1, E2F-2, and E2F-3 interact onl with pRb and not widi 107. In spite of this important difference, tiiere are a number o striking similarities between the pRb E2Fs and the pl07 E2F described here. First, there is significant structural homology between these two classes of proteins in the DNA-binding domain and die DP-1 dimerization domain. Consistent with this, we found d at E2F-4 can activate transcription of a promoter that harbours consensus E2F DNA-binding sites and diat E2F-4 requires DP-1 for efficient DNA binding. Transcriptional activation by E2F-4 can be repressed by overexpression of pl07. These results indicate diat die E2F-4/pl07 complex is transcriptionally inactive. Similarly, the pRb-associated E2Fs are also transcriptionally inactive when complexed by pRb. The finding that E2F-4 can bind a consensus E2F-binding site in cooperation with DP-1 does not rule out the possibility that in vivo E2F-4 will bind to a different subset of E2F site-containing promoters than the pRb/E2Fs. Regulation by the various pocket proteins may lead to activation of E2Fs at distinct points in the cell cycle and may therefore result in the sequential activation of promoters with a particular E2F site. The recent finding diat die thymidine kinase and b-myb promoters carry E2F sites that preferentially bind E2F/pl07 complexes supports die notion that pRb/E2Fs and pl07/E2Fs differ subtly in DNA-binding specificity (Lam and Watson 1993; Li et al, 1993a). Overexpression of pi 07 leads to a G, arrest in a number of cell types (Zhu et al, 1993). Since the pocket region of pl07 is required for growtii-suppressive activity, it is likely that pi 07 inhibits cell-cycle progression by binding to a number of cellular proteins that are involved in promoting progression from G, to S phase of d e cell cycle. We and others have recently shown that the c-Myc oncoprotein can form a specific complex with pi 07 in vivo (Beijersbergen et al, 1994; Gu et al, 1994). Importantly, binding of pl07 to d e c-Myc tra/w-activation domain resulted in a dramatic inhibition of c-Myc mww-activation. Since high level expression of c-Myc was able to override a pl07-induced cell-cycle block, it is likely diat pl07 inhibits cell-cycle progression, at least in part, by binding to and inactivating the c-Myc protein (Beijersbergen et al, 1994). Our present data add E2F-4 to the short list of pl07-interacting cellular proteins, and a striking similarity between c-Myc and E2 becomes apparent from the present study. First, both c-Myc and E2F-4 appear to hav frαrt- y -activation domain that can bind pl07, resulting in inhibition of -w-y-activatio Furthermore, our data indicate diat E2F-4, like c-Myc, may promote progression from G, S phase of the cell cycle (Table 1; Heikkila et al, 1987; Eilers et al, 1991). T interpretation is substantiated by the observation that E2F-4, like c-Myc, can cooperate w an activated ras oncogene in the transformation of REF cultures (Fig 8). Therefore, it w be worthwhile to study die possible involvement of E2F-4 in human cancer. Although pl07 can inhibit the activity of cell cycle regulatory proteins such as c-M and E2F-4, mutant forms of pi 07 do not appear to occur in human cancers. One possibili is that pl07 is functionally redundant. The pl07-related protein pl30, which was recent isolated by molecular cloning, is a candidate for a protein having pl07-like activity. pl is structurally more related to pl07 than to pRb (Hannon et al, 1993; Li et al, 1993b). pl also has been observed in complexes widi E2F DNA-binding activity and higher ord complexes between pl30 and cyclin E/cdk2 and cyclin A/cdk2 have also been observ (Cobrinik et al, 1993). However, pl30/E2F complexes occur in different stages of the c cycle as compared widi d e pl07/E2F complexes. An important question that remains to be resolved concerns the regulation of E2F activity in tiie cell cycle. From the study of die E2F-l-pRb interaction it is clear that n only phosphorylation of pRb but probably also of E2F-1 and DP-1 by cdks is important controlling activity (Krek et al, 1994). In contrast, very little is known about the regulati of pl07 by phosphorylation. The cdks play a key role in regulating pl07/E2F compl formation is, however, quite likely. Lees et al, (1992) have detected cell-cycle-regulated complex formation among E2 pl07, and cyclin E/cdk2 in the Gj phase of the cell cycle. In S phase, a complex of E2 pl07, and cyclin A cdk2 was detected. The appearance of the different cyclin/cdk complexes in die E2F/pl07 complex follows the kinetics of the synthesis of these cyclins. The role of these two cyclin/cdk complexes in controlling the E2F/pl07 interaction is presently not known. It is possible that these kinases phosphorylate pl07 and/or E2F-4. Our finding that E2F-4 is a phosphoprotein is consistent with a role for cyclin/cdk complexes in controlling E2F-4 activity. Whatever the targets of these cyclin/cdk complexes may be, their presence does not appear to lead to die dissociation of die pl07/E2F complex. Since complexes between E2F and pi 07 are found tiiroughout the cell cycle, it is not obvious when pi 07- interacting E2Fs are free to activate transcription of their target genes. We are currently studying die effect of the various cyclin/cdk complexes on E2F-4/pl07 complex formation and die effect of these kinases on E2F-4 activity. The availability of the first pi 07 specific E2F should be a helpful tool in understanding die regulation of pi 07 by cdks and die differences between regulation of pRb and pl07 by tiiese kinases. MATERIAL AND METHODS FOR EXAMPLE 1 Screening of cDNA libraries with pi 07 The pi 07 protein probe used in die screening of cDNA expression libraries was GST-2TK- pl07, containing the pocket region of human pl07. The GST-2TK vector contains a consensus phosphorylation site for protein kinase A (Blanar and Rutter 1992). GST-pl07 fusion protein was made in Escherichia coli, purified and labelled in vitro with [γ- 32 P]ATP as described to a sp. act. of > 1 x 10 8 cpm/μg of protein. A mouse embryo cDNA library in λEX/lox (obtained from Novagen, Madison, WI) was screened widi die pi 07 protein probe as described (Ayer et al, 1993). In a screening of 5 x 10 s phages, 3 positive phages were identified. The partial mouse E2F-4 cDNA was used to screen additional mouse and human cDNA libraries. The human E2F-4 cDNA described here was isolated from die T84 colon carcinoma library (obtained form Stratagene). Plasmids pGST-E2F-4 was constructed by cloning a fragment of the human E2F-4 cDNA encoding amino acids 108-300 in pGEX3X. For transfection experiments the following plasmids were used: pCMV E2F-4 was constructed by linking die 5' end of mouse E2F-4 (encoding amino acids 1-16) to a fragment of die human E2F-4 cDNA encoding amino acids 17-413 at die conserved Nael site in pRc/CMV (Invitrogen). pCMV-llA-DP-1 has been described (Helin etal, 1993b). pCMV-E2F-l was generated by inserting the coding sequence of pSP72 RBAP- 1 (Kaelin et al, 1992) in pRc/CMV. The plasmids pCMV-pRb, pCMV-pl07, pCMV- pl07DE were described previously (Zhu et al. 1993). Cell lines Human C33A, ML-1, and U2-OS were cultured in Dulbecco's modified Eagle medium (DMEM) with 10% fetal calf serum (FCS). REFs were isolated from 13 day-old rat embryos and cultured in DMEM in die presence of 10% FCS. Transfections were performed using die calcium phosphate precipitation technique (Van de Eb and Graham 1980). Stable transfectants were obtained after selection widi 0.5 mg/ml of G418 for 4 weeks. Immunological reagents To generate antibodies against E2F-4, a bacterial expression plasmid containing glutathione S-transferase (GST)-E2F-4 (amino acids 108-300) was generated. GST-E2F-4 protein was made in E. coli, purified over glutathione-Sepharose™ beads, and used to immunise mice. After three rounds of immunization with GST-E2F-4, polyclonal serum was obtained. Monoclonal antibodies were prepared by fusion of splenocytes of immunized BALB/c mice to Sp 2 O myeloma cells four days after the final boost. Positive tissue culture supematants were identified in an ELISA. Supematants of 19 different ELISA positive hybridoma cell lines were tested in immunoprecipitation. For the experiments described we used die polyclonal serum pell and die mAb RK13. Antibodies against pl07 (SD2, SD4, SD9, SD6, SD15), pRb (XZ77 and C36) and against the HA epitope (12CA5) have been described previously (Hu et al. 1991; Zhu et al, 1993). Metabolic labelling Cells were incubated for 1 hr in phosphate free DMEM or methionine-free DMEM in die presence of 10% FCS, followed by an incubation in medium containing 5 mCi [ 32 P]oιthophosρhate or 250 Ci Trans [ 35 S]label for 4 hr. Immunoprecipitations Cells were lysed in ELB + buffer[containing 5 mM EDTA, ImM DTT, 10 mM NaF, 10 mM sodium orthovanadate, 0.2 mM sodium pyrophosphate, 1 μg/ml chymostatin and aprotinin, and 1 mM phenylmethylsulphonyl fluoride (PMSF) and immunoprecipitated as described previously (Beijersbergen et al, 1994). Phgspha^e Treatment Transientiy transfected U2-OS cells were labelled with [ 35 S]methionine and precipitated wi mAb 12CA5 as described. After die first immunoprecipitation, the precipitated proteins we washed tiiree times in ELB + buffer followed by a wash widi 1 ml of phosphatase buffer [ mM Tris-HCI (pH 9.0), 5mM MgCl 2 containing 1 mM PMSF, and lμg/ml of bo chymostatin and aprotininj. The immunoprecipitated proteins were split into two a resuspended in 50 μl of phosphatase buffer and incubated for 30 min at 30°C in the absen or presence of 5 units of calf intestine alkaline phosphatase (Boehringer). Reactions we terminated by addition of 50 μl of 2 x releasing buffer (ELB + containing 4% SDS and 30 m DTT) and boiled for 10 min. After this, protein A-Sepharose™ beads were removed centrifugation and d e supernatant diluted to 1 ml in ELB buffer and precleared with prote A-Sepharose™ beads. The released proteins were then immunoprecipitated with mAb RK1 The immunoprecipitated proteins were collected by binding to protein A-Sepharose™ bead boiled in SDS-containing buffer, separated on a 7.5% SDS-polyacrylamide gel, dried, a subjected to fluorography. CAT Assays C33A cells were transiently transfected widi die expression vectors as indicated togetiier wi 2 μg of E2F 4 CAT (Helin et al, 1993b) 0.2 μg of RSV luciferase, and pRc/CMV to a tot of 20 μg/lOcm composition plate. Cells were harvested 40 hr after transfection, and d transfected cells were resuspended in lOOμl of 0.1 M Tris-HCI (pH 8.0). The cells we freeze-thawed three times and centrifuged at 15,000g, at 4°C for 10 min. Supematants we assayed for luciferase activity (Promega, Luciferase system) and CAT activity using the phase extraction assay (Seed and Sheen 1988). Gel Retardation Assays Transfected U2-OS cells were washed three times with PBS and the cells were collected in PBS. After centrifugation at lOOOg, the cells were resuspended in 100 μl of 5 x binding buffer [100 mM HEPES (pH 7.4), 0.5 M KCl, 5 mM MgCl 2 , 0.5 mM EDTA, 35% glycerol, 5mM NaF]. After one freeze-thaw step, the cells were kept on ice for 30 min. The extracts were centrifuged at 100,000g for 30 min at 4°C. The supernatant was used in gel retardation assays. Gel retardation assays for transientiy transfected U2-OS cells were performed as described previously- (Helin et al, 1993] with minor modifications. Ten micrograms of cell extract was used in 20 μl reactions containing 1 x binding buffer and 1 μg of sonicated salmon sperm DNA. Reactions were incubated for 10 min at room temperature, after which 0.5 mg of 32 P-labelled oligonucleotide containing die consensus E2F DNA-binding site (Santacruz) was added and the reaction was incubated for an additional 20 min at room temperature. The reaction products were separated on a 3.5% polyacrylamide gel in 0.25 x TBE at 90V for 2.5 hr. The gel was then dried, and reaction products were visualized by autoradiography. EXAMPLE 2 The orderly progression tiirough die cell cycle is mediated by die sequential activation of several cyclin/cyclin-dependent kinase (cdk) complexes. These kinases phosphorylate a number of cellular substrates, amongst which is the product of die retinoblastoma gene, pRb. Phosphorylation of pRb in late Gl results in the release of the transcription factor E2F from pRb, resulting in die transcriptional activation of E2F responsive genes. This Example shows that phosphorylation of the pRb-related pl07 is also cell cycle regulated. pl07 is first phosphorylated at 8 hours following serum stimulation of quiescent fibroblasts, which coincides widi an increase in cyclin DI protein levels. Consistent widi this, we show that a cyclin Dl/cdk4 complex, but not a cyclin E/cdk2 complex, can phosphorylate pl07 in vivo. Furthermore, phosphorylation of pl07 can be abolished by die overexpression of a dominant negative form of cdk4. Phosphorylation of pi 07 results in the loss of the ability to associate with E2F-4, a transcription factor with growth promoting and oncogenic activity. A pl07- induced cell cycle block can be released by cyclin Dl/cdk4, but not by cyclin E/cdk2. These data indicate diat die activity of pl07 is regulated by phosphorylation through D- type cyclins. The cell division cycle in mammalian cells is regulated by die cyclical activation of a number of kinases whose activity depends on their association with a cyclin subunit (Sherr, 1993). In the Gl phase of cell cycle, cyclins D and E are expressed. D type cyclins are encoded by a family of three closely related genes, cyclin DI, D2 and D3, whereas only a single gene for E type cyclin has been isolated (Matsushime et al., 1991; Koff et al., 1991). Several lines of evidence indicate diat D and E type cyclins are rate-limiting for passage through Gl. Antibodies to cyclin DI block S phase entry in several cell types (Baldin et al., 1993; Lukas et al., 1994) and over-expression of botii cyclin DI and E shortens Gl (Ohtsubo and Roberts, 1993; Quelle et al. , 1993; Resnitzky et al., 1994; Wimmel et al., 1994). Since induction of cyclin DI or E expression in serum-starved cells does not result in S phase entry, it appears that the expression of these cyclins is necessary but not sufficient to progress through Gl into S phase (Resnitzky et al., 1994). An important difference between cyclins D and E is diat D type cyclins are implicated as a causal agent in cancer: cyclin DI gene amplification has been found in breast cancer, esophageal carcinoma and squamous cell carcinoma (Lammie et al., 1991; Jiang et al., 1992; Schuuring et al., 1992a, b). Furthermore, cyclin DI is translocated in parathyroid adenomas and in centrocytic lymphomas (Withers et al., 1991; Rosenberg et al., 1991; Motokura et al., 1991; Seto et al., 1992). More recently, die product of die mts-l tumor suppressor gene was identified as pl6, a strong inhibitor of cyclin Dl-associated kinase activity (Serrano et al., 1993; Kamb et al., 1994). Finally, in vitro, cyclin DI can cooperate with other oncogenes to transform fibroblasts (Hinds et al., 1994; Lovec et al., 1994) and in transgenic mice over-expression of cyclin DI in breast epithelium results in breast cancer (Wang et al., 1994). Because of the involvement of D type cyclins, and not of E type cyclins in die genesis of several types of human cancer, important functional differences are likely to exist between tiiese two Gl cyclins. One such difference may be die kinetics of induction of D versus E type cyclins. Cyclin E is a "generic" cyclin in that its expression is induced in a cyclical fashion in die cell cycle, reaching maximal levels towards the end of Gl and cyclin E-associated kinase activity has been shown to peak at die Gl/S transition (Koff et al., 1992; Dulic et al., 1992). In contrast, cyclin DI appears to be induced most strongly following mitogen stimulation of quiescent cells (Matsushime et al., 1991, 1994; Won et al., 1992; Ajchenbaum et al., 1993; Sewing et al., 1993). Whether DI cyclin is also expressed in a cyclical fashion in exponentially growing cells is controversial. Some have shown invariant expression of cyclin DI during die cell cycle, whereas others have seen cell cycle-dependent variation in cyclin DI levels (Matsushime et al., 1991; Baldin et al., 1993; Sewing et al., 1993; Lukas et al., 1994). Since several strong inhibitors of cyclin Dl-associated kinase activity exist, an important question diat has so far not been addressed is whetiier the cyclin Dl- associated kinase activity varies during the cell cycle. The product of die retinoblastoma gene, pRb, is a substrate for Gl cyclin/cdk complexes. Extracts from insect cells infected with recombinant baculoviruses that express either cyclin E and cdk2 or D type cyclins and cdk4 efficiently phosphorylate pRb in vitro (Ewen et al., 1993; Kato et al., 1993). In human osteosarcoma cells, expression of cyclin E, D2 and D3, but not of DI, resulted in hypeφhosphorylation of pRb (Hinds et al., 1992; Ewen et al., 1993). Significantly, a pRb-induced cell cycle block could be released by ectopic expression of cyclin E, D2 and D3, but not effectively by cyclin DI expression (Hinds et al., 1992; Ewen et al., 1993). The retinoblastoma protein and the related pl07 and pl30 interact with several cellular polypeptides including E2F, a transcription factor diat controls gene expression during the cell cycle (Cao et al., 1992; Cobrinik et al., 1993). E2F DNA binding activity consists of a heterodimeric complex containing an E2F component complexed to a DP component (Bandara et al., 1993; Helin et al., 1993; Krek et al., 1993). The E2F component of the heterodimer is encoded by at least five closely related polypeptides. E2F-1, 2 and 3 associate in vivo only with pRb and not widi die related ρl07 (Lees et al., 1993). E2F-4 in contrast seems to interact with pl07 and pl30, but not widi pRb (Beijersbergen et al., 1994b; Ginsberg et al., 1994, Vairo et al, 1995, R.L.B. and R.B., unpublished data), whereas E2F-5 interacts only with pl30 (M. Hijmans, et al, 1995). E2F binds preferentially to hypophosphorylated pRb suggesting that complex formation between pRb and E2F is regulated by phosphorylation of pRb by cyclin/cdk complexes (Buchkovich et al. , 1989; DeCaprio et al. , 1989; Chen et al. , 1989; Chellappan et al., 1991). Hypophosphorylated pRb is found mostiy in the Gl phase of cell cycle, whereas the hypeφhosphorylated form of pRb is first observed at die Gl- to S transition (Buchkovich et al., 1989; Chen et al., 1989; DeCaprio et al., 1989). The complexes between E2F and pi 07 show a more complex pattern of appearance during d e cell cycle. In late Gl, DNA-binding complexes have been observed diat contain E2F, pl07, cyclin E and cdk2. In S phase cyclin E is no longer found in tiiese complexes, instead E2F is found associated widi pl07, cyclin A and cdk2 (Lees et al., 1992; Shirodkar et al., 1992). In spite of the presence of cyclin/cdk complexes in association with pi 07, very little is known about the regulation of pi 07 by phosphorylation. In contrast to pRb, cell cycle regulated phosphorylation of pi 07 has not been observed. We show here diat die growtii inhibitory activity of pl07 is subject to regulation by phosphorylation by cyclin/cdk complexes. Results. Effect of cyclin E on pl07. In late Gl phase of the cell cycle, pl07 is found in a multi protein complex tiiat contains E2F DNA binding activity, cyclin E and cdk2 (Lees et al., 1992). To study die effect of cyclin E and its associated kinase cdk2 on the E2F/pl07 complex, we used a transient transfection assay with E2F-4. We have shown recently diat E2F-4 associates in vivo specifically witii ρl07 and not with pRb. As a control, we studied the effect of cyclin E/cdk2 on the pRb/E2F-l complex. We co-transfected a CAT reporter gene harboring upstream E2F sites together widi either E2F-4 and DP-1 expression vectors or with E2F-1 and DP-1 expression vectors. Fig. 10A and B show that both die E2F-4/DP-1 and the E2F-1/DP-1 heterodimers efficientiy activated the CAT reporter gene (tracks 3). As expected, co-transfection of pl07 inhibited E2F-4 transactivation and pRb expression suppressed E2F-1 transactivation (tracks 5). When cyclin E/cdk2 and pRb expression vectors were co-transfected, a release of E2F-1 inhibition by pRb was observed, presumably as a result of phosphorylation of pRb by cyclin E/cdk2 (Fig. 10A, track 6) (Hinds et al., 1992; Ewen et al., 1993). Suφrisingly, co-transfection of cyclin E/cdk2 with pl07 failed to relieve ρl07 inhibition of E2F-4 (Fig. 10B. track 6). We conclude that cyclin E cdk2 acts differently on pRb and pl07 in diat cyclin E/cdk2 can release pRb, but not pl07 inhibition of E2F. Effect of D type cyclins on pl07 Next, we studied die effect of the cyclin DI on pl07. Fig. 11 shows diat, in contrast to cyclin E/cdk2, cyclin DI, togetiier widi its major catalytic partner cdk4, can release pl07 inhibition of E2F-4 and pRb-inhibition of E2F-1 (Fig. 11 A, B, compare tracks 4 and 7). The inactivation of pl07 as an inhibitor of E2F-4 activity could be caused by several mechanisms. First, cyclin Dl/cdk4 could phosphorylate pl07, thereby releasing active E2F-4. An alternative mechanism of cyclin DI action could be de direct binding of cyclin DI to die pocket region of pi 07, since cyclin DI shares a motif (LXCXE) with a number of viral transforming proteins that bind avidly to pl07 and pRb through this motif (Dowdy et al., 1993). However, neitiier cyclin DI alone nor cdk4 alone were able to release pl07 and pRb inhibition of E2F activity (Fig. 11A, B). This suggests that a cyclin D/cdk complex is required to release pi 07 inhibition of E2F- 4. To study die kinase requirement of D type cyclins to inactivate pl07, we co- transfected E2F-4 and pl07 widi otiier combinations of D type cyclins and cdks. To address die specificity of the kinase, we expressed cyclin D3, instead of cyclin DI, in combination with either cdk2 or cdk4. Both cdk2 and cdk4, when bound to cyclin D3, have significant kinase activity towards pRb, whereas cyclin DI only forms an active complex widi cdk4 (Ewen et al., 1993) . Fig. 12 shows that alώough cyclin Dl/cdk4 and cyclin D3/cdk4 effectively rescued E2F-4 inhibition by pl07, no release of inhibition was observed widi cyclin D3/cdk2 (Fig. 12, compare tracks 6, 8, and 9). These data indicate diat not only die cyclin, but also die associated kinase contributes to die activity of the cyclin/cdk complex towards pl07. The data shown above are consistent with a model in which some, but not all, cyclin D/cdk complexes can phosphorylate and functionally inactivate pl07. To further study die molecular mechanism by which cyclin DI acts on pl07, we used two mutants of cyclin DI . The first mutant, cyclin Dl-GH carries a mutation in the LXCXE motif that is required for high affinity binding of cyclin DI to pocket proteins. As a result, the GH mutant of cyclin DI is defective for binding to pocket proteins (Dowdy et al., 1993; Hinds et al., 1994). The second mutant, cyclin Dl-KE carries a mutation in tiie cyclin box, a domain diat mediates association widi cdks. The KE mutant is therefore unable to form a stable complex with cdk4 (Hinds et al., 1994). Fig. 13 shows diat die cyclin DI GH mutant retained die ability to release E2F-4 from pi 07 inhibition, whereas the KE mutant was inactive in this assay. These data are consistent widi d e notion that cyclin Dl/cdk4 phosphorylates pl07 and do not support the hypothesis that cyclin DI competes with E2F-4 for binding to die pl07 pocket. To further substantiate that phosphorylation is essential for the inactivation of pl07, we used a dominant negative mutant of cdk4 (Van den Heuvel and Harlow, 1993). Fig. 14A shows that this kinase inactive cdk4, in combination widi cyclin DI, was not able to release die pl07 inhibition of E2F-4, indicating diat kinase activity of the cyclin D/cdk4 complex is essential for the release of the pi 07 mediated inhibition. Recently a strong inhibitor of cyclin Dl/cdk4 kinase activity, p27 k ' pl , was isolated (Polyak et al., 1994; Toyoshima and Hunter, 1994). We also studied d e effect of p27 ipl expression on cyclin Dl/cdk4 in the assay described above. Fig. 14B shows diat whereas cyclin Dl/cdk4 effectively released pl07 inhibition of E2F-4, co-transfection of a p27 kipl expression vector completely blocked die release by cyclin Dl/cdk4. Thus, inhibition of kinase activity of cyclin Dl/cdk4 by either mutation of cdk4 or co- expression of p27 kipl prevented die release of pi 07 inhibition. These data lend further RECTIFIED SHEET (RULE 91) support to the notion that cyclin Dl/cdk4 kinase activity is required to release pl07 inhibition of E2F-4. Cyclin Dl/cdk4 complexes phosphorylate pl07. The effect of cyclin Dl/cdk4 could be die result of phosphorylation of the E2F- 4 protein, the pl07 protein, or both. Although we have recently shown that E2F-4 is a phosphoprotein, we did not see any alterations in E2F-4 phosphorylation upon expression of cyclin Dl/cdk4 neither in the absence nor presence of pl07 (Beijersbergen et al., 1994b and data not shown). We therefore focused on the effect of cyclin Dl/cdk4 on pl07. The cell cycle-regulated phosphorylation of pRb can be readily detected because of die increased apparent molecular weight of hypeφhosphorylated pRb in SDS-acrylamide gels (Buchkovich et al., 1989; Ludlow et al., 1989). To study die effect of cyclin Dl/cdk4 on pl07 phosphorylation, we transfected die osteosarcoma cell line U2-OS with eitiier an epitope-tagged pl07 expression vector alone, or cotransfected widi die dominant negative form of cdk4, cyclin Dl/cdk4. Two days after transfection cells were lysed and whole cell extracts were subjected to 7.5% polyacrylamide SDS gel electrophoresis and die pl07 protein was detected by Western blot analysis with 12CA5 antibody directed against HA- tagged pl07. Fig. 15 shows diat pl07 migrates as a doublet when expressed in U2-OS cells (lane 2). This doublet is reduced to a single band diat migrates with the lower apparent molecular weight species when the dominant-negative form of cdk4 is co- expressed (lane 3). This result suggests that the pl07 protein is phosphorylated upon expression in U2-OS cells and diat this phosphorylation can be abolished by die co- expression of a dominant-negative form of cdk4. Cotransfection of cyclin Dl/cdk4 resulted in an increase of pi 07 species with higher apparent molecular weight (lane 4), most likely as a result of phosphorylation of pi 07 by cyclin D/cdk4. U2-OS cells express multiple pi 07 species (Fig. 15, lane 2) probably as a result of endogenous kinase activity present in these cells. We searched for additional cell lines that lack significant pl07 kinase activity. Figure 15, lane 6, shows that C33A cervical carcinoma cells only express a single species of pi 07, which co-migrates with the fastest migrating species of pl07 in U2-OS cells (compare lanes 2 and 6). As expected, co-transfection of cyclin Dl/cdk4 resulted in a slower migrating species of pi 07 (lane 7). In contrast, cotransfection of pi 07 witii cyclin E/cdk2, or cyclin A/cdk2 did not result in a significant increase in pl07 mobility (compare Fig. 15, lanes 6, 8 and 9). This lack of phosphorylation of pl07 by cyclin E/cdk2 in C33A cells correlates with die absence of the release of the pl07 mediated inhibition of E2F-4 transactivation in the same cells (Fig. 10A). U2-OS cells that were labeled widi [ 32 P]-ortiιophosρhate after transfection with HA tagged pl07 alone or togetiier widi the different cyclin/cdk combinations, showed an increase in die amount of [ 3 P]-labeled pl07 only in the presence of cyclin Dl/cdk4 and not cyclin E/cdk2 or cyclin A/cdk2 (data not shown). To show that the increase in apparent molecular weight of pi 07 is the result of phosphorylation, we treated pl07 immunoprecipitates with lambda phoshatase. pl07, immunoprecipitated from U2-OS cells transfected widi HA tagged pl07, displays different pl07 molecular weight species (Fig. 16, lane 1). Lambda phosphatase treatment of pl07 immunoprecipitates resulted in die appearance of a single band diat co-migrates with the faster migrating pl07 species (Fig. 16 lane 3). A pl07 immunoprecipitate treated widi lambda phosphatase in the presence of phosphatase inhibitors contained botii die slower and faster migrating species of pl07 protein (Fig. 16 lane 2). We conclude diat pl07 is phosphorylated in vivo, most likely due to cyclin D-associated kinase activity and diat expression of cyclin Dl/cdk4 leads to hypeφhosphorylation of pi 07. Phosphorylation of p!07 prevents interaction with E2F-4 To address whetiier pl07 hypeφhosphorylation results in a loss in association with the E2F-4/DP-1 heterodimeric complex, we performed die following experiment. An epitope tagged DP-1 expression vector was co-transfected widi E2F-4 and pl07 expression vectors in die presence or absence of cyclin/cdk expression vectors. Two days after transfection, cells were metabolically labeled widi [ 35 S]-methionine and lysed in non ionic detergent. One half of each lysate was immunoprecipitated wid pi 07 antibodies, the other half with the 12CA5 antibody directed against HA tagged DP-1. Immunoprecipitated proteins were resolved on a low percentage acrylamide SDS gel. Fig. 17, lane 4 shows diat in the absence of co-transfected cyclin/cdks, die DP-1/E2F-4 heterodimer is bound to pi 07. Expression of cyclin Dl/cdk4, completely abolished this interaction, whereas expression of cyclin E/cdk2 did not cause a release of pl07 from E2F-4. (Fig. 17, lanes 5 and 6). Thus, cyclin Dl/cdk4-mediated phosphorylation of SUBSTITUTE SHEET (RULE 26. pl07 prevents an interaction between pl07 and the E2F-4/DP-1 heterodimer, whereas the expression of cyclin E/cdk2 does not affect the complex. U2-OS cells contain both hypo-and hypeφhosphorylated species of ρl07 protein. The experiments described above, indicate that phosphorylation of pl07 prevents association with E2F-4, therefore pl07 species d at are associated widi E2F-4 should be in the hypophosphorylated state. To address die phosphorylation status of pl07 protein that is bound to E2F-4, we transfected U2-OS cells with HA-tagged E2F-4 alone or together with DP-1. Cells were lysed and E2F-4 protein was immunoprecipitated with 12CA5 antibody directed against HA-E2F-4. As a control for the ρl07 species present in cells transfected widi both HAE2F-4 and DP-1, pl07 protein was immunoprecipitated widi pl07 antibody SD4. The immunoprecipitated proteins were separated on a low percentage polyacrylamide SDS gel and pl07 protein was detected by Western blot with a pl07 antibody. As can be seen in Figure 18, lane 1, both species of pl07 were again present in these cells. In contrast, pl07 that was co-immunoprecipiated widi HAE2F-4 only consisted of die faster migrating species (Fig.18, lanes 3 and 4). These results indicate diat E2F-4 preferentially associates with the hypophoshorylated pl07 species. Together we can conclude diat pl07 is phosphorylated, most likely by cyclin D/cdk4 kinase complexes, and diat phosphorylation of pl07 prevents the association between E2F-4 and pl07. pl07 phosphorylation is cell cycle-regulated. To study whether pl07 is phosphorylated in a cell cycle-dependent fashion, we studied the phosphorylation status of pl07 in untransfected NIH 3T3 fibroblasts. NIH 3T3 cells were made quiescent by culturing for 40 hours in low serum (0.2%). After this period die cells were re-stimulated widi 10% serum and serum-stimulated cells were harvested at different time points. Cell extracts were subjected to low percentage polyacrylamide SDS-gel electrophoresis and pl07 protein was detected by Western blot analysis. As shown in Figure 19, quiescent cells express only the fastest- migrating species of pl07. After 8 hours of serum stimulation, part of the pl07 protein has shifted to a higher apparent molecular weight. After 10 hours, most pl07 protein is of the slower-migrating species (Fig. 19). In addition, we observed an increase in die amount of pl07 after 10 hrs of serum stimulation. At this stage, we do not know whether this is the result of increased protein synthesis or protein stabilization as a result of phosphorylation. To examine the role of the D-type cyclins in die phosphorylation of pl07, we analyzed the same extracts for cyclin DI expression. As can be seen in Figure 19, middle panel, cyclin DI expression is induced at 6-8 hours after serum stimulation and remains constant at later time points. As a control we also analyzed die same extracts for the expression of cyclin E. Cyclin E is not induced until 12 to 14 hours after serum stimulation (Fig. 19, lower panel). These results indicate diat phosphorylation of pl07 is regulated in a cell cycle-dependent manner and that this phosphorylation takes place in the cell cycle shortly after cyclin DI induction and well before cyclin E induction, suggesting diat cyclin D is d e most likely cyclin candidate to control pl07 phosphorylation. To investigate how these difference in pl07 phosphorylation affect E2F-pl07 complex formation during die cell cycle, we analyzed E2F DNA binding complexes in the same 3T3 cells after serum stimulation. The most predominant complex in GO cells is E2F in complex with pl30 (Cobrinik et al, 1993). In our experiments, the complexes did not change until 8 hours after serum stimulation (Fig. 20A, lanes 1-4) and contain predominantly ρl30 as confirmed by pl30 antibody dependent supershifts (data not shown, Cobrinik et al, 1993). A more slowly migrating complex was first detected at 10 to 12 hours after serum stimulation (lanes 5-7). Only after time points of more than 18 to 24 hours (lane 8), as cells begin to pass from Gl into S-phase of the cell cycle (as determined by flow cytometry, data not shown) the slowest migrating complex became prominent. To determine whetiier pi 07 was present in this slowly migrating complex, the same extracts were analyzed in die presence of a pi 07 antibody. Between 10 and 16 hours after serum stimulation, very small amounts of the supershifted complexes were detectable and remained constant (Fig. 20B. lanes 1-4). At 24 hours after serum stimulation, d e majority of the higher order E2F complexes were supershifted by pl07 antibody. When die presence of E2F complexes containing pi 07 is compared to die presence of pi 07 protein in these cells (compare Fig. 19 and Fig. 20), one can see that although there is a strong increase in the expression of phosphorylated pl07 protein at time points 10-14 hours after stimulation this does not result in the increase in E2F/pl07 complexes. The increase in E2F complexes containing pl07 at later timepoints could indicate that at these timepoints, newly synthesized pi 07 protein is no longer phosphorylated and can therefore associate with E2F and form the E2F/pl07 complex that is detected in S-phase cells. RECTIFIED SHEET (RULE 91) Although pi 07 is phosphorylated when cells progress from Gl into S-phase of the cell cycle, E2F activity can be found associated with pl07 in S-phase cells and asynchronously growing cells. We therefore asked whether hypophosphorylated pl07, capable of forming a complex with E2F, is present in asynchronously growing cells. Both quiescent- and asynchronously growing 3T3 cells were analyzed for the presence of the different pl07 species. As can be seen in Figure 21, die level of pl07 is much lower in GO cells and only consists of the hypophosphorylated form. In asynchronously growing cells, much higher levels of pl07 were detected and these cells contain both hypo-and hypeφhosphorylated forms of pl07. These results indicate diat although pl07 is phosphorylated in die Gl phase of the cell cycle after serum stimulation, a detectable amount of pl07 remains undeφhosphorylated in die later stages of the cell cycle. This hypophosphorylated pl07 is able to associate widi E2F's as is shown in Figure 18, and is likely responsible for the pl07-associated E2F activity detected in S-phase and in asynchronous cells. Cyclin Dl/cdk4 can rescue a pl07-induced cell cycle block. E2F transcription factors play an important role in the regulation of the Gl- to S-phase transition. When transiently transfected, botii pRb and pl07 have the ability to arrest SAOS-2 cells in the Gl-phase of die cell cycle (Zhu et al., 1993). The pRb- induced cell cycle block can be rescued by over-expression of cyclin A, cyclin E, cyclin D2 or D3, resulting in hypeφhosphorylation of pRb (Hinds et al., 1992; Ewen et al., 1993). In contrast, a ρl07-induced cell cycle block cannot be rescued effectively by either cyclin A or cyclin E expression (Zhu et al., 1993). The data described above are in agreement with this, in that they show diat pl07 cannot be inactivated by ectopic expression of cyclin E. Since pl07 inhibition of E2F-4 can be released by cyclin Dl/cdk4, we asked whetiier expression of cyclin Dl/cdk4 was able to release SAOS-2 cells from a pl07-induced cell cycle block. SAOS-2 cells were transfected with the cell surface marker CD20 alone, with CD20 and pl07 or with CD20, pl07 and cyclin/cdk expression vectors. Three days after transfection, cells were selected for CD20 expression by FACS analysis and die cell-cycle distribution of transfected (CD20 + ) cells was determined by propidium iodide staining. Table 2 shows that expression of pi 07 in SAOS-2 cells resulted in a significant accumulation of CD20-positive cells in Gl. Expression of cyclin Dl/cdk4 completely released the pl07-induced cell cycle block, decreasing die Gl population and increasing both S and G2/M phase cells. Consistent with the results of Zhu et al (1993), we observed only a partial release of the pl07 cell cycle block by cyclin E/cdk2 (Table 2). Additional controls with cyclin D3 alone or in combination with cdk2 or cdk4 showed diat, as expected, only the cyclin D3/cdk4 complex was able to release the pl07 cell cycle block. Taken togetiier, our data indicate that pl07 growth inhibitory activity in the cell cycle is regulated by D-type cyclins in association with cdk4, but not effectively by cyclin E and its associated kinase. TIFIED SHEET (RULE 91) TABLE 2 Rescue of pl07 induced cell cycle arrest in SA-OS2 cells by Gl cyclins. vector pl07 Rescue Gl G2/M Gl G2 M 47 12 41 73 6 21 42 15 43 67 9 24 44 14 42 61 10 29 Cyclins +pCMVcylinDl/cdk4 35 22 42 38 24 38 37 16 23 • pCMVcyclin E/cdk2 39 22 39 58 19 23 54 15 31 +pCMVcyclinD3 54 14 31 +pC-MVcyclinD3/cdk4 34 18 48 +pCMVcyclinD3/cdk2 56 13 31 Table 2 Rescue pl07 induced cell cycle arrest in SAOS-2 cells by Gl cyclins. SAOS-2 cells were transfected widi pCMV CD20 (2 μg) expression vector in combination witii expression vectors pCMV or pCMVpl07 (5 mg) together widi different cyclins and cdks (10 μg each) as indicated. Transfected cells were analyzed by FACS and die cell cycle profile of CD20 positive cells was determined. The percentage of cells in G0/G1- phase, S-phase and G2/M-phase of die cell cycle are depicted. DISCUSSION. In this paper we present several lines of evidence to indicate diat pl07 is negatively regulated as a result of phosphorylation. The pl07 protein is found to be differentially phosphorylated and tiiis phosphorylation dramatically changes during die Gl to S-phase of the cell cycle in 3T3 fibroblasts (Fig.20). pl07 is a "pocket" protein that can form a complex with the celluar transcription factor E2F-4 in vivo. E2F-4 plays a role in the regulation of cell cycle progression (Beijersbergen et al, 1994, Ginsberg et al, 1994). The activity of the transcription factor E2F-4 can be suppresssed by die expression of pl07. Overexpression of pl07 results in a Gl cell cycle arrest in certain cell types (Zhu et al, 1993). We show here that botii die ability of pl07 to interact widi E2F-4 and die ability to induce a Gl cell cycle arrest are both abolished upon phosphorylation of pl07. Furthermore, our experiments indicate diat pl07 is a target for cyclin D/cdk4 complexes. In a first set of experiments, we studied die effects of cyclin/cdk complexes on the interaction between pl07 and its E2F partner, E2F-4 (Beijersbergen et al., 1994b, Ginsberg et al., 1994). Transactivation by E2F-4 can be suppressed by expression of pl07. Importantly, pl07 suppression of E2F-4 could be released by co-expression of both cyclin DI and cdk4, but not by either protein alone. This activity of cyclin Dl/cdk4 was completely abolished when a dominant negative form of cdk4 was used or by co-expression of p27 kipl , a strong inhibitor of the cyclin Dl/cdk4 kinase complex. Both these results indicate that cyclin Dl-associated kinase activity is required to release pi 07 inhibition of E2F-4. Expression of cyclin Dl/cdk4 caused hypeφhosphorylation of pl07 and resulted in a loss of association with E2F-4. In agreement with a role for cyclin DI in phosphorylating pl07 is the finding diat pl07 can be phosphorylated in vitro by cyclin Dl/cdk4. Li et al, (1993) showed diat pi 07 is found in complex witii D type cyclins in vivo and in vitro kinase reactions with tiiese complexes show that pi 07 can be phosphorylated by D type cyclin associated kinase activity. The binding of pl07 and cyclin D, in analogy to cyclin D and pRb, suggest an alternative mechanism of D type cyclin action in that D type cyclins might interfere with both pl07 and pRb function through direct binding to die pocket (Dowdy et al., 1993, Ewen et al., 1993, Hinds et al., 1993), thereby functionally inactivating pl07 or pRb in the SUBSTITUTE SHEET (RULE 26. absence of phosphorylation. As discussed above, our data support a model in which pl07 is inactivated dirough phosphorylation by the cyclin D/cdk4 complex and do not support the hypothesis diat cyclin DI acts by competing with E2F for pl07 pocket binding. Further evidence diat phosphorylation of pl07 is mediated by cyclin D complexes was obtained when we serum-stimulated quiescent fibroblasts: Phosphorylation of pl07 occurs shortly after cyclin DI is induced but well before cyclin E induction (Fig. 20). Also, over-expression of a dominant negative form of cdk4 resulted in the disappearance of the hypeφhosphorylated forms of pl07 in U2-OS cells, strongly suggesting that D type cyclin associated kinase activity is responsible for this modification. Togetiier, these data provide strong evidence diat pl07 is an in vivo substrate for cyclin D/cdk4. In agreement with the hypothesis diat pl07 is a target for cyclin D associated kinase activity is the finding that ρl07 phosphorylation as observed in U2-OS cells is absent in both SAOS-2 and C33A cells (Fig. 15, data not shown). The absence of pl07 phosphorylation in C33A and SAOS-2 cells (which have only very little cyclin D-associated kinase activity), showing a correlation between the presence of cyclin D-associated kinase activity and die phosphorylation status of pl07 (RLB and RB, unpublished data). Recent data show diat cyclin DI can cooperate with a mutant form of adenovirus EIA and an activated rag oncogene in the transformation of primary fibroblasts. Importantly, the GH mutant of cyclin DI that had retained die ability to transform in this assay, was also able to release pi 07 inhibition of E2F-4 (Fig.5, Hinds et al., 1994). Conversely, the KE mutant of cyclin DI, which is unable to form a stable complex with cdk4, did not transform and did not release pl07 inhibition of E2F- 4. Thus, the ability of cyclin DI mutants to transform segregates with the ability to inactivate pl07. This suggests that cyclin DI derives its oncogenic activity, at least in part, by releasing pl07-associated proteins from their inactive, pl07-bound state. We and others have recently shown that pl07 interacts with at least two cellular proteins, E2F-4 and c-MYC, both of which are endowed widi oncogenic activity (Beijersbergen et al., 1994a,b; Ginsberg et al., 1994; Gu et al., 1994). To our suφrise, the cyclin E/cdk2 complex was unable to affect the complex between E2F-4 and pi 07. This result was unexpected because pi 07 forms a stable complex with cyclin E in vivo. In contrast to pRb, pl07 contains within its pocket region a spacer that allows the formation of higher order complexes widi cyclins and cdk's. These complexes contain apart from pl07 and E2F (that interacts with the pocket region) cyclin A or cyclin E, together with their associated kinase cdk2. In spite of this, cyclin E cdk2 mediated phosphorylation of pl07 does not appear to occur in the E2F containing complex in vivo, at least not with die result that pl07 is released from E2F. The role of the cyclin E/cdk2 complex in the pl07/E2F complex therefore remains to be elucidated. It is a possibility that cyclin E/cdk2 is present in die pl07/E2F complex to phosphorylate the E2F and or die DP-1 component. Alternatively, cyclin E could be bound and inactivated by pl07. In this scenario, pl07 would act as a "molecular sink" to bind and inactivate cyclin E. Active cyclin E/cdk2 could then only occur above a certain threshold level of cyclin E. A similar threshold-setting role has recently been proposed for cdk inhibitory molecules (Peters, 1994) and we have recently obtained evidence to support a role for pl07 in down-modulating cyclin A and E activity (Zhu et al 1995). In S phase, pl07 is found in complex with E2F and cyclin A (Lees et al., 1992). Cyclin A can also interact directly widi E2F-1 resulting in the phosphorylation of at least one member of the E2F/DP complex (Krek et al., 1994, Dynlacht et al., 1994). This phosphorylation results in die down modulation of E2F-1 activity presumably through loss of DNA binding of die E2F-1/DP-1 complex. We did not find a release of pi 07 inhibition by cyclin A/cdk2 overexpression (data not shown). This indicates that like cyclin E/cdk2, cyclin A/cdk2 is also unable to inactivate pl07 by phosphorylation. Although hypophosphorylated pl07 is present in GO, E2F complexes containing pl07 are not readily detected. Instead E2F is found in complex with pl30. Only after disappearance of the E2F-pl30 complex (Fig. 20, lane 5), E2F/pl07 complexes can be detected albeit at very low amounts. This could indicate that pi 30 can effectively compete with hypophosphorylated pl07, which is present at this stage of the cell cycle, for binding to E2F-4. That pi 30 can bind E2F-4 was recendy shown by us and others (RLB and RB, submitted for publication, Vairo et al, in press). The observation tiiat pl07 is phosphorylated at 8 hours after serum stimulation would suggest that at this moment newly syntiiesized E2F-4 would not be inactivated through the association witii pl07. At the same moment E2F/pl30 complexes disappear. This would suggest a scenario in which E2F's that can be inactivated by pl30 and ρl07 would now be free to activate transcription. On the other hand an increase in pi 07 containing E2F complexes is observed at the Gl/S transition and tiiroughout S-phase of the cell cycle, in our experiments at 18 hours post serum stimulation (Fig.20). The result of this regulation is that the pl30 and pl07 associated E2Fs are only free to activate transcription during a very small time frame from 8 to 18 hours following serum stimulation, before and after which the higher order complexes are present. The formation of these higher order complexes may play an important role in the active repression of genes when cells progress to the later stages of die cell cycle. A possible explanation for the re-occurrence of E2F/pl07 complexes at S-phase, is diat there is a strong increase in pi 07 protein expression. The majority of this newly synthesized protein is phosphorylated, but as a result of a decrease in cyclin D-associated kinase activity, a significant amount of pl07 remains hypophosphorylated in S-phase. This newly synthesized hypophosphorylated pl07 can form a complex with E2F-4. Overexpression of pl07 in SAOS-2 cells results in a growth arrest in the Gl phase of the cell cycle (Zhu et al., 1993). This pl07-induced growth arrest could be rescued by cotransfection of cyclin Dl/cdk4 or cyclin D3/cdk4 (Table 2). Thus, phosphorylation of pl07 by cyclin Dl/cdk4 does not only lead to die release of E2F-4, but inactivates virtually completely the growtii suppressive activity of pl07. In contrast, cyclin E/cdk2 only resulted in a partial release of die pl07 cell cycle block. Thus, the different ability of cyclins D and E to release E2F-4 from pl07 is correlated widi their ability to inactivate the growth inhibitory activity of pl07. Cellular targets of pl07 may therefore play an important role in the progression of Gl to S phase. Consistent widi this, both E2F-4 and c-MYC can stimulate S phase entry (Eilers et al., 1991; Beijersbergen et al., 1994b). Our data reveal significant differences in substrate specificity between die two most prominent Gl cyclin/cdk complexes: pl07 can be inactivated by D type cyclins and not by cyclin E, whereas pRb that can be inactivated by cyclin D2, D3 and E. Since cyclin DI is induced prior to cyclin E in d e cell cycle, our data also suggests that the pl07-associated E2Fs are activated at an early point in Gl, when cyclin D- associated kinase activity is induced. That pl07 E2Fs can function early in Gl is also supported by the recent findings diat, in contrast to E2F-1, E2F-4 mRNA is already present in quiescent cells (Ginsberg et al., 1994; Johnson et al., 1994). Furthermore, E2F-4 is transcriptionally induced prior to E2F-1 when cells progress tiirough Gl into S (RLB and RB, unpublished data). Even tiiough cyclin E/cdk2 and cyclin Dl/cdk4 complexes share many functional properties (Resnitzky et al., 1994; Ewen et al., 1993), a striking difference is diat cyclin DI is frequently involved in the genesis of human cancer, whereas little evidence for deregulated cyclin E expression in cancer exists to date. Our present data show that cyclin DI, when in complex with cdk4, can functionally inactivate pl07, whereas cyclin E/cdk2 was inactive towards pl07. It is possible that the higher oncogenic activity of cyclin DI stems from its ability to inactivate the growth inhibitory activity of both pRb and pl07, whereas over-expression of cyclin E would leave die pl07-mediated growth inhibitory circuit intact. Materials and Methods for Example 2 Cell lines Human C33A, U2-OS and SAOS-2 were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum. NIH 3T3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) widi 10% newborn calf serum. Transfections were performed using die calcium phosphate precipitation technique (Graham and Van der Eb, 1973). Synchronization of cells NIH 3T3 cells were allowed to grow 24 hrs, and were then incubated widi DMEM containing 0.2% serum for 40 hours. Re-stimulation of quiescent cells was performed by incubation widi DMEM containing 10% serum for the indicated times. Plasmids pCMVp27 was generated by die polymerase chain reaction using p27-specific primers on DNA isolated from a BALB/c brain cDNA library . DNA Sequencing showed that the amplified p27 cDNA was identical to die published sequence (Toyoshima and Hunter, 1994). PCMVHA E2F-4 was generated by cloning die haemagglutinin epitope tag at the NH 2 -terminus of E2F-4 in pCMV. The plasmids pCMVpRb, pCMVρl07, pCMVpl07DE, pCMVCD20, pCMVE2F-4, pCMV-E2F-l , pCMV-DP-1 , pCMVcyclin E, cyclin A, cyclin DI, cyclin D2, cdk2, cdk4 , cdk4 dominant negative, cyclin Dl-GH and cyclin Dl-KE have all been described elsewhere (Hinds et al., 1992, 1994; Zhu et al., 1993; Van den Heuvel and Harlow, 1993; Beijersbergen et al., 1994b). Immunological reagents For the experiments described here we used die monoclonal antibody RK13 directed against E2F-4. Antibodies against pl07 (SD2, SD4, SD9, SD6 and SD15) and against the HA epitope (12CA5) have been described previously (Field et al., 1988; Zhu et al., 1993). The antibody used for the detection of endogenous ρl07 in western, C-18, and cyclin E, C-19, were obtained from Santa Cruz. Cyclin DI was detected by DSC-6 antibody from Sanbio. CAT assays C33A cells were transiently transfected with die expression vectors as indicated togetiier with 2 μg E2F 4 CAT (Helin et al., 1993), 0.2 μg RSVluciferase. pRc/CMV was added to a total of 20 μg DNA per 10-cm plate. Cells were harvested forty hours after transfection. Cells were collected and resuspended in 100 μl 0.1 M Tris-HCI, pH 8.0. Cells were freeze/diawed three times and centrifuged at 15,000g at 4°C for 10 min. Supematants were assayed for luciferase activity (Promega, Luciferase system) and CAT activity using the phase extraction assay (Seed and Sheen, 1988). In all experiments, CAT activity was normalized to luciferase activity. Phosphatase treatment Immunoprecipitates were washed twice in phosphatase buffer (50mM Tris-HCI, pH 7.8 and 5mM DTT) and resuspended in 60μl phosphatase buffer. Lambda phosphatase (NEB,400 units) were added, where indicated, and incubated for 60 min at 30°C. Phosphatase inhibitors NaF (5mM) and Na 3 VO 4 (5mM) were added where indicated. Metabolic labeling and immunoprecipitations Cells were incubated for 1 hour in methionine-free DMEM in the presence of 10% fetal calf serum, followed by an incubation in methionine-free DMEM supplemented widi 250 μCi Trans [ 35 S]-label for 4 hours. After labeling, cells were lysed in ELB + buffer (ELB buffer containing 5 mM EDTA, 1 mM DTT, 10 mM NaF, 10 mM sodium orthovanadate, 0.2 mM sodium pyrophosphate, 1 μg/ml chymostatin and aprotinin and 1 mM phenylmethylsulfonylfluoride) and immunoprecipitated as described previously (Beijersbergen et l., 1994a). Western blot analysis Cells were lysed in SDS-containing sample buffer and sonicated for 10 seconds. The cell extracts were then separated on SDS polyacrylamide gels and transferred to nitrocellulose. The membrane was blocked in TBST (15 mM NaCl, 10 mM Tris-HCI pH 8.0 and 0.05% Tween-20) containing 5% dried milk for 1 hour at RT and C18 antibody diluted 1:2000 in TBST containing 1 % dried milk for 16 hours at 4'C. The membrane was then washed and die antibody was detected using horseradish peroxidase-linked goat anti rabbit IgG and enhanced chemiluminescence (Amersham). HA tagged pl07 was detected widi die 12CA5 monoclonal antibody at 1:1000 dilution, cyclin DI witii DSC-6 in 1:250 dilution and cyclin E widi C- 19 in 1:500 dilution in TBST containing 1 % dried milk. Mobility shift assays For die preparation of whole cell extracts of quiescent or serum stimulated 3T3 cells, 10cm tissue culture dishes were washed with cold PBS, cells were collected and resuspended in lOOμl of lysis buffer (20 mM Hepes, pH7.9, 0.4 MNaCl, ImM EDTA, lOmM DTT, ImM phenyhnetiiylsulfonyl fluoride (PMSF) and 25 % glycerol. The cells were kept on ice for 20 min and lysed by two freeze thaw cycles (-80°C and 0°C). After centrifugation for 10 min at 15.000 φm at 4°C, the supernatant was stored at - 80°C. Gel shifts were performed using lOμg of cells extract in a 20μl reaction volume containing lOmM Hepes, pH7.9, lOOmM KCl, ImM EDTA, ImM DTT, 4% Ficoll and lμg sonicated and boiled salmon sperm DNA. Where indicated SD9 antibody (Santa cruz) was added to die reaction mixture. Reactions were incubated for 10 min at room temperature (RT) after which 0.5ng of 32 P-labeled oligonucleotide containing the consensus E2F DNA binding site (Santa cruz) was added and die reaction was incubated for an additional 20 min at RT. The reaction products were separated on a 3.5 % polyacrylamide gel in 0.25x TBE at 100V for 3hrs at room temperamre. The gel was then dried, and subjected to autoradiography. SAOS-2 cell cycle block rescue SAOS-2 cells were transfected using calcium phosphate precipitates containing 30 μg of plasmid DNA for each 15 cm dish. After 16 hours the cells were washed twice with DMEM/10% FCS and incubated witii fresh medium at 37 * C. Forty-eight hours later the cells were collected in PBS containing 3 mM EDTA, spun down and incubated widi 20 μl of FITC conjugated anti CD20 monoclonal antibody (Becton-Dickinson) for 20 min. on ice. The cells were washed and fixed in 80% ethanol at 4 * C. Before FACS analysis the cells were washed widi PBS supplemented witii 0.1 % serum, and incubated with 20 μg/ml propidium iodide and 200 μg/ml RNAse A. The FACS analysis was performed on a Becton-Dickinson FACScan apparatus. A gate was set to select CD20- positive cells with a fluorescence intensity of more than 20-fold the control population. The propidium iodide signal was used to determine die cell cycle distribution of the selected cells. 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Ruley, W.E. 1983 Adenovirus EIA enables viral and cellular transforming genes to transform primary cells in culture Nature. 304: 602-606. Schuuring, E., Verhoeven, E., Mooi, W. J. and Michalides, R. J. (1992a). Identification and cloning of two overexpressed genes, U21B31/PRAD1 and EMS1, within the amplified chromosome llql3 region in human carcinomas. Oncogene 7, 355-361. Schuuring, E., Verhoeven, E., van, T. H., Peterse, J. L., Nunnink, B., Thunnissen, F. B., Devilee, P., Comelisse, C. J., van de Vijver, M.J, Mooi, W. J. et. al. (1992b). Amplification of genes within the chromosome 1 lql3 region is indicative of poor prognosis in patients with operable breast cancer. Cancer Res. 52, 5229-5234. Schwarz, J.K., S.H. Devoto, E.J. Smith, S.P.Chellappan. L. Jakoi and J.R. Nevins. 1993. Interactions of thep 107 and Rb proteins wid E2F during die cell proliferation response. EMBO J. 12: 1013-1020. Seed, B. and Sheen, J.Y. (1988). A simple phase-extaction assay for chloramphenicol acetyltransferase activity. Gene 67, 271-277. Serrano, M., Hannon, G. and Beach, D. (1993). A new regulatory motif in cell cycle control causing specific inhibition of cyclin D/cdk4. Nature 366, 704-707. Seto, M., Yamamoto, K., Iida, S., Akao, Y., Utsumi, K. R., Kubonishi, I., Miyoshi, I., Ohtsuki, T., Yawata, Y., Namba, M. et. al. (1992). Gene rearrangement and overexpression of PRAD1 in lymphoid malignancy with t(l 1 ; 14)(ql3;q32) translocation. Oncogene 7, 1401-1406. Sewing, A., Burger, C, Brusselbach, S., Schalk, C, Lucibello, F. C. and Muller, R. (1993). Human cyclin DI encodes a labile nuclear protein whose synthesis is directly induced by growth factors and suppressed by cyclic AMP. J Cell Sci. 104, 545-554. Sherr, C. J. (1993). Mammalian Gl cyclins. Cell. 73, 1059-1065. Shirodkar, S., M. Ewen, J.A. DeCaprio. J. Morgan, D.M. Livingstone and T. Chittenden. 1992. the transcription factor E2F interacts with the retinoblastma product and a pl07 cyclin A complex in a cell cycle regulated manner. Cell 68: 157-166. Tam, S.W., Theodoras, A.M., Shay, J.W., Draetta, G.F. and Pagano, M. (1994). Differential expression and regulation of cyclin DI protein in normal and tumor human cells: association with cdk4 is required for cyclin DI function in Gl progression. Oncogene 9; 2663-2674. Toyoshima, H. and Hunter, T. (1994). p27, a novel inhibitor of Gl cyclin-Cdk protein kinase activity, is related to p21. Cell. 78, 67-74. Vairo, G., Livingston, D.M. and Ginsberg D. (1995). Functional interaction between E2F-4 and pl30: evidence for distinct mechanismns underlying growth suppression by different retinoblastoma protein family members. Genes & Dev. In press. van den Heuvel, S. , and Harlow, E. (1993). Distinct roles for cyclin dependent kinases in cell cycle control. Science 262, 2050-2054. Van der Eb A.J. and F. L. Graham. 1980. Assay of transforming activity of tumor virus DNA. Methods Enzymol. 65: 826-839. Wang, T. C, Cardiff, R. D., Zukerberg, L., Lees, E., Arnold, A. and Schmidt, E. V. (1994). Mammary hypeφlasia and carcinoma in MMTV-cyclin DI transgenic mice. Nature. 369, 669-671. Weintraub, S.J., CA. Prater, and D.C Dean 1992. Retinoblastoma protein switches the E2F site from positive to negative element. Nature. 358: 259-261. Wimmel, A., Lucibello, F. C, Sewing, A., Adolph, S. and Muller, R. (1994). Inducible acceleration of Gl progression tiirough tetracycline-regulated expression of human cyclin E. Oncogene 9, 995-997. Withers, D. A., Harvey, R. C, Faust, J. B., Melnyk, O., Carey, K. and Meeker, T. C (1991). Characterization of a candidate bcl-1 gene. Mol Cell Biol. 11, 4846- 4853. Won, K. A., Xiong, Y., Beach, D. and Gilman, M. Z. (1992). Growth-regulated expression of D-type cyclin genes in human diploid fibroblasts. Proc Nad Acad Sci U S A. 89, 9910-9914. Zamanian, M. and N. B. La Thangue, 1993. Transcriptional repression by the Rb related protein pl07. Mol. Biol. Cell 4: 389-396. Zhu, L., S.van den Heuvel, K. Helin, A Fattaey, M. Ewen, D. Livingston, N. Dyson and E. Harlow. 1993. Inhibition of cell proliferation by pl07, a relative of the retinoblastoma protein. Genes & Dev. 7: 1111-1125. Zhu, L., Enders, G., Lees, J.A., Beijersbergen, R.L., Bernards, R. and Harlow, E. (1995). The pRb related protein pl07 contains two growth suppression domains: Indepedent interactions with E2F and cyclin/cdk complexes. EMBOJ. In press. DESCRIPTION OF FIGURES FIGURE 1 (A) Nucleotide sequence of the E2F-4 cDNA. The first 74 bp was derived from mouse E2 4, and die remainder from human E2F-4. (B) Schematic representation of E2F-4 comparison with E2F-1. Black and shaded boxes are regions of high homology; hatch regions are specific for E2F-4 or E2F-1. FIGURE 2 E2F-4 requires DP-1 for DNA binding. U2-OS cells were transfected with expressi plasmids as indicated above each lane, and cell extracts were prepared for gel retardati assays as described earlier. Cell extracts were tested for binding to a 32 P-labeI oligonucleotide that specifies a consensus E2F DNA-binding site. FIGURE 3 E2F-4 and DP-1 cooperate in tr-i/--. -activation. (A) C33A cells were transfected wi increasing amounts of pCMV-E2F-4 (lower curve) or with increasing amounts pCMV-E2F- together with 100 ng of pCMVDP-1 (upper curve) together widi 2 μg of reporter constru (E2F 4 CAT) and 0.2μg of RSV luciferase as an internal control. CAT activity was normalise to the luciferase activity for each sample (corrected counts). CAT activities are the avera of duplicate samples. (B) C33A cells were transfected as described in (A), except that pCM DP-1 was transfected in increasing amounts (lower curve) or increasing amounts of pCM DP-1 in combination witii lOOng of pCMV-E2F-4 (upper curve). CAT activity was calculated as in A. FIGURE 4 pl07 inhibits E2F-4-mediated fra/tr-activation. C33 Λ cells were transfected witii 100 ng of pCMV-E2F-4 and 100 ng of pCMV-DP-1. In combination with increasing amounts of pCMV-pl07, 100 ng of pRb, or 1000 ng of pCMV-pl07-DE. Together with the expression plasmids, the cells were transfected witii 2μg of E2F 4 CAT and 0.2 μg of pRSV luciferase. CAT activity was normalized to die luciferase internal control. Fold activation was calculated relative to the basal level of E2F 4 CAT, which was set to unity (1.0). Data was representative of at least three independent experiments performed in duplicate. FIGURE S E2F-4 specifically interacts with pl07 in vivo. ML-1 cells were labeled widi [ 32 P]orthophosphate and subjected to sequential immunoprecipitation. In the first immunoprecipitation, lysates were incubated widi pl07 antibodies or pRb antibodies as indicated, pi 07- and pRb-associated proteins were released as described in Materials and methods and reimmunoprecipitated witii E2F-4 polyclonal serum (lanes 1 ,4) E2F-1 mAb KH20, or normal mouse serum as a control. Immune complexes were separated on 7.5% SDS-polyacrylamide gels, die gels were dried, and protein were detected by autoradiography. FIGURE 6 E2F-4 is differentially phosphorylated. U2-OS cells were transfected widi 10μg pCMV-E2 4 and 10 μg of pCMV-HA-DP-1. Cells were labeled with [ 35 S]methionine, and cell lysate were subjected to sequential immunoprecipitation. First, lysates were immunoprecipated wit mAb 12CA5 directed against the HA-tagged DP-1. The immunoprecipitated proteins we treated in die absence (-) or presence (* ) of calf intestine alkaline phosphatase. The protein were then released as described in Material and methods and subjected t reimmunoprecitation with anti-E2F-4mAb RK13. Immunocomplexes were separated on 7.5 SDS-polyacrylamide gels and subjected to fluorography. FIGURE 7 E2F-4/DP-1 dimerization is required for pl07 association. The osteosarcoma cell line U2-O was transfected widi die indicated expression plasmids. Cells were labeled wid [ 32 P]orthophosphate 40 hr after transfection. The lysates were then subjected to sequentia immunoprecipitation. First, the lysates were immunoprecipitated with anti-pl07 antibodies. pl07 associated proteins were then released as described in Material and methods an reimmunoprecipitated witii control antibody (lanes 1,4,7). E2F-4 mAb RK13 (lanes 2,5,8), or mAb 12CA5 against the HA-tagged DP-1 (lanes 3,6,9). Equal amounts of 32 P-labeled cel lysates were used for each immunoprecipitation. FIGURE 8 E2F-4/DP-1 overexpression causes transformation. Overexpression of E2F-4 in combinatio with DP-1 and mutant ras results in tiie outgrowth of G418-resistant colonies having transformed moφhology. (A) Primary REFs; (B) REFs transformed by E2F-4, DP-1, and ras (E4R1); (C) REFs transformed by E2F-4, DP-1, and ras (E4R2). Magnification, 52x. FIGURE 9 E2F-4 expression in transformed REFs. Adenovirus-transformed primary fibroblasts (Ad5) and E2F-4/DP-1 transformed primary fibroblasts (E4R1 and E4R2) were labelled witii [ 32 P] orthophosphate and subjected to sequential immunoprecipitation. First, the lysates were immunoprecipitated with mAb 12CA5 against HA-tagged DP-1 (lanes 1,3,5) or control antibody (lanes 2,4,6). The associated proteins were released as described in Materials and methods and subjected to reimmunoprecipitation with E2F-4 mAb RK13 (lanes 7,9,11) or control antibody (lanes 8,10,12). Immune complexes were separated on 7.5% SDS- polyacrylamide gels and detected by autoradiography. Equal amounts of 32 P-labelled cell lysates were used for immunoprecipitation in this experiment. FIGURE 10 Effect of Cyclin E/cdk2 on ρl07-E2F and pRb-E2F interactions. (A) C33A cells were transfected with 100 ng pCMVE2F-4 and 100 ng pCMVDP-1 in combination with 200 ng pCMVpl07 and widi 2.0 μg pCMVcyclin E and 2.0 μg pCMVcdk2 as indicated. Togetiier with the expression plasmids, die cells were transfected witii 2 μg E2F -CAT and 0.2 μg pRSV Luciferase. CAT activity was normalized to die luciferase internal control. Fold activation was calculated relative to the basal level of E2F 4 -CAT which was set to unity (1.0). The experiments were performed in duplicate and activity was also assayed in duplicate. Data are representative for at least three independent experiments. (B) C33A cells were transfected with 100 ng pCMVE2F-l and 100 ng pCMVDP-1 in combination with 200 ng pCMVpRB and witii 2.0 μg pCMVcyclin E and 2.0 μg pCMVcdk2 as indicated. Fold activation was determined as described in A. FIGURE 11 Effect of cyclin DI and cdk4 on pl07 and pRb. (A) C33A cells were transfected with 100 ng pCMVE2F-4 and 100 ng pCMVDP-1 in combination with 200 ng pCMVpl07 and widi 2.0 μg pCMVcyclin DI and 2.0 μg pCMVcdk4 as indicated. Togetiier with the expression plasmids, the cells were transfected with 2 μg E2F 4 -CAT and 0.2 μg pRSV Luciferase. (B) C33A cells were transfected with 100 ng pCMVE2F-l and 100 ng pCMVDP-1 in combination with 200 ng pCMVpRB and with 2.0 μg pCMVcyclin DI and 2.0 μg pCMVcdk4 as indicated. Experiments were performed as described in Figure 1A. Fold activation was calculated as described in Figure 1A. FIGURE 12 Cyclin DI and D3 in combination with cdk4 can release pl07 inhibition. Cells were transfected as described in Figure 3. 2.0 μg cyclin DI, cyclin D3, cdk2 or cdk4 were co-transfected as indicated. FIGURE 1? Effect of Cyclin DI mutants on pl07/E2F-4. C33A cells were transfected as described in Figure 3. 2.0 μg pCMVcyclin DI KH, mutant cyclin DI that carries a mutation in the LXCXE domain, or 2.0 μg pCMVcyclin DI KE, mutant cyclin DI diat lacks the cdk4 interaction site, were transfected as indicated. FIGURE 14 (A) Cyclin DI in combination with a dominant negative cdk4 cannot release pl07 inhibition. C33A cells were transfected as described in Figure 2, 2 μg cdk4 dominant negative (cdk4DN) was transfected as indicated. (B) Release of pl07 inhibition by cyclin Dl/cdk4 is blocked by p27 kipl . C33A cells were transfected as described in figure 2. 2.0 μg p27 kipl was transfected as indicated. FIGURE 15 Effect of cdk4 dominant negative and cyclins on the phosphorylation of pl07. U2-OS cells (lanes 1-4) were transfected witii HA-tagged pl07 alone (lane 2) or in combination with dominant negative cdk4 (lane 3), or cyclin Dl/cdk4 (lane 4). C33A cells (lanes 5-9) were transfected witii HA-tagged pl07 alone (lane 6) or in combination with cyclin Dl/cdk4 (lane 7), cyclin E/cdk2 (lane 8) or cyclin A/cdk2 (lane 9). 40 hrs after transfection, the cells were lysed and cell extracts were subjected to low percentage polyacrylamide SDS gel electrophoresis. The separated proteins were transferred to nitrocellulose and HA tagged pi 07 protein was detected by western widi 12CA5 monoclonal antibody. FIGURE 16 Electrophoretic heterogeneity of pi 07 is due to differential phosphorylation. U2-OS cells were transfected widi HA tagged pl07. 40 hrs after transfection the cells were lysed and die lysate was split in tiiree. HA tagged pl07 protein was immunoprecipitated and resuspended in SDS containing sample buffer (lane 1) or treated widi λ-phosphatase in the presence (lane 2) or absence of phosphatase inhibitors (lane 3) as described in material and mediods. The immunoprecipiates were subjected to low percentage polyacrylamide SDS gel electrophoresis and transferred to nitrocellulose. HA tagged pl07 protein was detected by western using 12CA5 monoclonal antibody. FIGURE 17 Cyclin Dl/cdk4 phosphorylates pl07 and causes dissociation of E2F-4. U2-OS cells were transfected widi pCMVE2F-4 and pCMVDP-1 together with pCMVpl07 in combination with control vector pCMV (lanes 1 and 4) or pCMVcyclin DI and pCMV cdk4 (lanes 2 and 5) or pCMV cyclin E and pCMVcdk2 (lanes 3 and 6). The cells were labeled witii [ 35 S] methionine 40 hrs after transfection. Cell lysates were split and one half was immunoprecipiated with pl07 antibodies (lanes 1-3), die otiier half with 12CA5 directed against HA tagged DP-1 (lanes4-6). Immunecomplexes were separated on a 7.5% SDS polyacrylamide gel, dried and subjected to fluorography. DP-1 was added in these transfections because the association between E2F-4 and pi 07 depends on die presence of DP- 1 (Beijersbergen et al., 1994b). FIGV S 18 E2F-4 associates preferentially with the undeφhosphorylated species of pl07. U2-OS cells transfected with pCMV HAE2F-4 alone or together with pCMVDP-1. 40 hrs after transfection the cells were lysed and 90% of the lysate was immunoprecipiated widi 12CA5 antibody directed against HAE2F-4. As a control 10% of the lysate from cells transfected with both HAE2F-4 and DP-1 was immunoprecipitated with pl07 antibody SD-4. Immunoprecipitated proteins were separated by electrophoresis in 7.5% polyacrylamide SDS gels and pl07 species were detected by western blotting with pl07 antibody C18. FIGURE 19 pl07 is phosphorylated in a cell cycle dependent manner. Extracts of equal numbers of NIH3T3 cells were prepared after serum starvation (lane 1) or after serum-stimulation at different time points as indicated (lanes 2-10) and subjected to gel electrophoresis followed by Western blot detection of pi 07 (upper panel), cyclin DI (middle panel) or cyclin E (lower panel). F1GURE 20 Analysis of E2F-4 DNA binding activity in G 0 and serum stimulated 3T3 fibroblasts. (A) Extracts were prepared from NIH 3T3 fibroblasts after serum starvation (lane 1) or at different time points after serum stimulation (lanes 2-8) and subjected to mobility shift analysis as described in Materials and Methods. The position of free E2F and die different E2F-complexes, E2F-pl30, E2F-P10-7 and E2F-pRb are indicated. (B) The same extracts that were used in panel A for the time points, 8, 10, 12, 16 and 24 hours after serum stimulation (lanes 1-5) were subjected to mobility shift analysis in the presence of pl07 antibody SD9. The position of free E2F and die pl07 antibody-dependent supershift complexes are indicated. * indicates a non-specific background band. FIGURE 21 Hypo-and hypeφhosphorylated pl07 species are present in asynchronous 3T3 cells. 80 μg of total protein from quiescent or asynchronously growing NIH3T3 cells was subjected to electrophoresis through a 7.5% SDS polyacrylamide gel. pl07 protein was detected by Western analysis.

Claims: CI^MS 1. A polypeptide comprising: (a) E2F-4; (b) die protein of Figure 1A; (c) a mutant, allelic variant or species homologue of (a) or (b); (d) a protein at least 70% homologous to (a) or (b); (e) a fragment of any one of (a) to (d) capable of forming a complex with a B protein, pRb, pl07 and or pl30; (f) a fragment of any of (a) to (e) of at least 15 amino acids long. 2. A polypeptide according to claim 1 carrying a revealing or detectable label. 3. A polypeptide according to claim 1 or 2 fixed to a solid phase. 4. A composition comprising a polypeptide according to claim 1 or 2 together with a carrier or a diluent. 5. A polynucleotide which comprises: (a) a sequence of nucleotides shown in Figure 1 A; (b) a sequence complementary to (a); (c) a sequence capable of selectively hybridising to a sequence in either (a) or (b); (d) a sequence encoding a polypeptide as defined in claim 1; or (e) a fragment of any of the sequences in (a) to (d). 6. A polynucleotide according to claim 1 which is a DNA polynucleotide. 7. A polynucleotide according to claim 5 or 6 wherein the sequence (or fragment) comprises at least 20 nucleotides. 8. A polynucleotide according to any of claims 5 to 7 which comprises the cDNA sequence shown in Figure 1A. 9. A double stranded polynucleotide comprising a polynucleotide according to any claims 5 to 9 and its complementary sequence. 10. A polynucleotide according to any of claims 5 to 9 carrying a revealing or detectab 5 label. 11. A vector comprising a polynucleotide according to any of claims 5 to 10. 12. A vector according to claim 11 which is a recombinant replicable vector comprisi 10 a coding sequence which encodes a polypeptide as defined in claim 1. 13. A host cell comprising a vector according to claim 11. 14. A host cell according to claim 13 transformed by, or transfected with, a recombina 15 vector according to claim 12. 15. A host cell transformed by a recombinant vector according to claim 11 wherein t coding sequence is operably linked to a control sequence capable of providing for t expression of the coding sequence by the host cell. 20 16. A process for preparing a polypeptide as defined in claim 1, the process comprisi cultivating a host cell according to claim 13 or 14 under conditions providing for expressio of the recombinant vector of the coding sequence, and recovering the expressed polypeptid 25 17. An antibody (which includes a fragment or mutant thereof), capable of binding to protein as defined in claim 1. 18. An antibody or fragment thereof according to claim 17 which is, or is part of, monoclonal antibody. 30 19. An antibody or fragment or mutant thereof according to claim 17 or 18 carrying revealing or detectable label. 20. An antibody or fragment or mutant thereof according to any of claims 17 to 19 bound or fixed to a solid phase. 21. A hybridoma cell line which produces a monoclonal antibody according to claim 18. 22. A method of performing an immunoassay for detecting the presence or absence of a polypeptide as defined in claim 1 in a sample, the method comprising: (a) providing an antibody according to any of claims 17 to 20; (b) incubating the sample with the antibody under conditions that allow for the formation of an antibody-antigen complex; and (c) detecting, if present, the antibody-antigen complex. 23. A test kit suitable for performing an immunoassay as defined in claim 22 comprising a carrier having at least one well containing an antibody according to claim 17. 24. A screening method for identifying putative chemotherapeutic agents for the treatment of proliferative or viral disease which comprises (A) bringing into contact: (i) a DP polypeptide; (ii) a polypeptide as defined in claim 1; and (iii) a putative chemotherapeutic agent; under conditions in which the components (i) and (ii) in the absence of (iii) form a complex, and (B) measuring the extent to which component (iii) is able to disrupt, interfere with or inhibit the activity of the complex. 25. An assay according to claim 24 wherein the complex of (i) and (ii) is measured by its ability to bind an E2F DNA binding site in vitro. 26. An assay according to claim 24 wherein die complex of (i) and (ii) is measured by its ability to activate in vivo a promoter comprising an E2F binding site linked to a reporter gene. 27. An assay according to claim 26 wherein the assay is performed in a yeast cell, insect cell or a mammalian cell. 28. An assay according to any of claims 23 to 27 wherein the putative chemotherapeutic agent is a fragment of 10 or more amino acids of a polypeptide as defined in parts (a) to (e) of claim 1. 29. A polypeptide as defined in claim 1, a vector as defined in any of claims 10 to 12, a host cell as defined in claim 14 or 15, or an antibody as defined in any of claims 17 to 19 for use in a method of treatment of the human or animal body. 30. A pharmaceutical composition comprising a polypeptide as defined in claim 1, a vector as defined in any of claims 10 to 12, a host cell as defined in claim 14 or 15, or an antibody as defined in any of claims 17 to 19 and a pharmaceutically acceptable carrier or excipient.

Inventors: BEIJERSBERGEN RODERICK LEONARD, BERNARDS RENE, BEIJERSBERGEN, RODERICK, LEONARDUS, BERNARDS, RENE
Applicants: BEIJERSBERGEN RODERICK LEONARD, BERNARDS RENE, PROLIFIX LTD, VER NL KANKER INST, BEIJERSBERGEN, RODERICK, LEONARDUS, BERNARDS, RENE, PROLIFIX LIMITED, VERENIGING HET NEDERLANDS KANKER INSTITUUT
Application year: 1995
Main classification: C12N15
IPC codes: A61K38, C07K14, C12N15