ELIZABETH MORAN GENE EXPRESSION

Cycles within cycles The discovery that the product of the retinoblastoma gene is a

component of E2F transcription complexes is a major breakthrough in understanding the function of this ‘tumor suppressor’ gene product.

The successful cloning of the retinoblastoma gene prod- uct (RR) barely hve years ago (reviewed in. [l I) raised hopes that the mechanism by which this negative growth regulator restricts excessive cellproliferation would soon be understood. The intense concentration of work in- spired by the identification of the gene has led to an im- portant milestone: a recent series of experiments demon- strating that the RR gene product is a component of cellular EZF transcription factor complexes.

The clues that first suggested a link between RB and E2F came from studies using the adenovirus transform- ing gene product, Elk The ElA proteins contain three independent active sites able to intervene in host cell functions. The N-terminus and region-2 - the two ac- tive sites sufficient for mitogenic activity - are able, at least indirectly, to activate expression of cell-cycle- speciIic products, suggesting&at these sites help to actl- vate tmnscrlption factors specllically involved in cell cycle activation. The E2F transcription, factor was first identihed as a DNA-binding activity ln HeIa cells, where it seems to exist In a monomeric form, but an important advance was made when Nevins and co-workers [2] showed that E2F in most cells is found in protein complexes that can be dissociated by ElA in a manner dependent on region-2. This observation established a link between ElA-medlated cell cycle activation and a specific transcription factor.

The link between E2F and region-2 of ElA suggested a link between E2F and RB, because the N-termI- nus and region-2 of ElA are also the binding sites for a variety of cellular products. The identification of RB as one of the proteins associated with region-2 [3,4], had already made ElA an important tool in the ar- senal of reagents bell developed to probe RB function. The realization that ElA atfects E2F specifically, by disso- ciating E2F complexes to release free E2F, suggested that ElA-associated proteins might include products that had originally been part of the E2F complexes. Thus arose the impetus to search for RR in these complexes.

When oligonucleotides encoding the DNA sequence to which the E2F transcription factor binds are Incubated with whole cell extracts, two distinct protein complexes form around the DNA fragments, retarding their progress through a polyacrylamide gel. To learn whether the RR product is a component of either of these complexes, Chellappan et al [5] added RR-speciIic antibodies to the cell extracts before incubation with the DNA frag- ment. Addition of the antibodies specilically eliminated formation of the faster-migrating complex, indicating the

Volume 1 Number 5 1991

presence there of RR protein. E2F DNA-binding activity could also be specifically immunoprecipitated using RR- specific antibodies. The opposite approach would be to ask whether RR can be detected in immunoprecipitated E2F complexes, but E2Fqecific antibodies are not yet available. However, Chellappan et al went on to show that the RB protein copuriiies with E2F DNA-binding activity isolated on a DNA affinity column. This type of experiment not only confirms the presence of RB protein ln the E2F com- plexes, it has the added advantage of separating the E2F- bound RI3 from the total cell RR population, and that re- vealed something of great interest. Asynchronously grow- ing cells, such as those used to prepare the extracts for the E2F DNA al&&y columns, contain several forms of RB protein in various stages of phosphorylation that can be resolved in polyacrylamide gels, When the RB that coputies with E2F was visualized using western blots, it was apparent that only the least phosphorylated form of RB detectably copurifies with the E2F DNA-binding ac- tivity. The extent of RB phosphorylation is tightly con- trolled during the cell cycle. RR is largely unphosphoty- lated throughout the Gl phase; it becomes highly phos- phorylated shortly before S phase, and is dephospho- tylated some time during mitosis (Fig. 1) (reviewed in [ 11). It is therefore likely that E2F complexes containing RB protein are most prevalent during Gl, and that RR association with E2F occurs at specific times during the cell cycle. This observation will probably be a key point in understanding the exact mechanism by which RI3 modulates E2F activity. In differentiating cells, a DNA-binding activity that recog- nizes the same DNA sequence as E2F has been termed DRTFI. It is thought to be closely related to E2F. Anti- bodies speci6c for RR can alter the mobiity of one of the complexes observed in DRTFI mobility shift experl- ments [ 61, corroborating the evidence suggesting that RR is a component of E2F complexes. Additional evidence that RB associates with E2F comes from an entirely differ- ent approach. Chlttenden et al, [7] immobilized a pep- tide representing the portion of RB that bmds to region-2 of the ElA protein (and related DNA tumor virus trans- forming gene products), and tested whether it could se- lect specific DNA fragments with a preferred sequence out of a random pool. They found that the RB peptide by itself has DNA-binding activity, but little ability to rec- ognize specilic DNA sequences unless it is lirst Incubated with a whole cell extract. The preincubation apparently allows the RR peptide to associate with additional cellular

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proteins to form complexes which then have sequence- specific DNA-binding activity. Cloning and sequencing of the selected DNA fragments revealed that thq tiere highly homologous to one another, and closely related to the consensus site to which E2F binds. These re- sults strongly support the evidence demonstrating a di- rect physical association between RB and E2F complexes.

In each of the studies demonstrating the association of RB with E2F, RB protein is detected as part of an E2F DNA-binding complex. Thus, although RB is expected to inhibit the transcriptional activity of the complex, it does not eliminate the DNA-binding ability of E2F. However, additional evidence indicates that RB is a component of a specifk E2F inhibitory complex. To study the forma- tion of E2F complexes, the Nevins group [8] searched for cellular factors that can influence the activity of free E2F. They partklly purified an activity that associates with free E2F to form a larger complex, and termed it E2F-BF (Fig. 1). In addition, they found an activity that inhibits the DNA-binding activity of free E2F. The inhibitory ac- tivity was termed E2F-I, and it has been further purified in an independent study 191, E2F-I purifies as a group of four major protein species, including one the size of RB that is directly recognized by RB-specilic antibodies. Purilied ElA can speciftcally remove RB from the E2F-I complex, and nullity E2F-I inhibitory activity.

These results suggest that RB can interact with E2F in more than one way. Rl3-E2F complexes that retain DNA- binding activity are readily demonstrable, but RB as a component of E2F-I can also eliminate E2F DNA-biding activity. E2F DNA-binding activity is very low in growth- arrested (GO) cells versus cycling cells [lo], so it is pos- sible that an E2F-tE2F complex is characteristic of GO, whereas a merent kind of RB-E2F complex is prevalent in Gl (Fig. 1).

The proteins associated with ElA region-2 include cy- clin A (for review, see [ 1 l] >, an intimate participant in cell-cycle control mechanisms, and a subunit of the cell cycle regulating kinase, p34uic2. A discovery of compa- rable significance to the identification of RB in the E2F complexes, is the demonstration from both the Nevins and the La Thangue groups [ 10,121 that cyclin A is also a component of E2F and DRTFI complexes. Never before have such close connections been realized between the cell mechanisms that control proliferation and those that control gene expression. Cyclin A and RB are detected to- gether in DRTFI complexes, but the Nevins group iinds cyclin A in a different E2F complex than that which con- tains RB. Cyclin A is present in the complex that forms when E2F-BF is incubated with free E2F [8] (Fig. 1). Per- haps the most fascinating aspect of the E2F complexes is that they seem to alternate during the cell cycle: the cyclin A-E2F complexes appear only during S phase, whereas the RB-E2F complex is inferred from the RB phosphorylation state to appear only during Gl [lo].

The list of potential components of E2F complexes is surprisingly long, but certainly intriguing. At least seven uncharacterized proteins have been visualized in mate- rial specifically bound by immobilized RB [13,14]. These

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could include one or more of the proteins that co purify with RB in the E2F-I complex; some species are of similar sizes. A physical association between the c- myc protein and RB has recently been demonstrated in vitro [ 151, and may be biologically signiEcant. DeFeo-Jones et ul. [16], have isolated partial cDNA clones for at least two RB-binding proteins, each encoding an amino acid region homologous to ElA region-2. It is not yet known whether these products are involved in E2F function, but they are novel proteins, and it is an exciting possibility that ad- ditional members of the RB-E2F control complex may already have been cloned.

Fig. 1. Schematic representation of postulated E2F complex for- mation during the cell cycle. RtLE2F and cyclin A-EZF complexes seem to alternate during the cell cycle, roughly as RB phosphory- lation/dephosphorylation and cyclin A synthesis/degradation are regulated. Each complex may have additional components but these are not yet identified. In synchronized cells, the free form of E2F can be observed at phase transitions; however, the cellu- lar signals for E2F dissociations and the exact time in the cycle at which they occur are not yet defined.

Cyclin A does not seem to bind to ElA directly, but rather through an intermediary protein, ~107 (B Faha and E Harlow, M Ewan and DM Livingston, unpublished data), which has recently been cloned and found to be closely related to RB in the ElA-binding domain [ 171. By infer- ence, ~107 and RB are related proteins that may interact with E2F at alternating times during the cell cycle. The

0 1991 Current Biology

best known of the cellular proteins with which cyclin A associates is the p34d kinase. There is no evidence to indicate that p34d is a member of E2F complexes, but it is conceivable that it participates in their regulation: several lines of evidence indicate that p34&, or a re- lated kinase that also associates with cyclin A, is the RI3 kinase [x3].

In a typical adenovirus infection, ElA binds RB and cy- clin 4 dissociating both types of complexes and releas- ing free E2F from each. The free E2F is commandeered by the viral E4 product, and enjoined to form a very sta- ble, transcriptional& active comple% on the virus E2 pro- moter. However, one point that is still not clear, despite so much new information, is the speciIic identify of the cellular targets of E2F-mediated transactivation. Cellular promoters with sites of demonstrated functional signif- icance to which E2F bids, include those of immediate early genes such as c-myc, and those of late Gl prod- ucts such as dihydrofolate reductase, but it is not clear at what point(s) in the normal cell cycle E2F becomes active in transcription. Disruption of either the S phase (cyclin A> or Gl (RR) E2F complex seems to increase E2F transcription activity [lo], implying that both com- plexes are inhibitory, and the functional form of E2F is the free form that may occur at phase transitions. How- ever, it is also possible that both complexes are active in transcription, but that the complex components con- fer some limiting characteristic such as promoter speci- ficity. Formation of the complexes does not seem to be essential for cell cycle progression, because cells trans- formed by DNA tumor virus transforming genes such as ElA show no evidence of containing these complexes.

Another basic question not yet answered is whether the participation of RB in transcription complexes is limited to E2F, or whether RI3 perhaps acts as a more general in- hibitor. Both the Nevins and La Thangue groups demon- strated a certain degree of specificity by showing that RB was not detected in complexes formed by reaction with ATF [6] or octamer [5] DNA motifs. The DNA-binding site selection technique [7] should, in principle, detect any DNA-binding complexes formed around the RB pep- tide. Nevertheless, in this experiment 44 out of 46 se- lected DNA fragments contained a sequence very closely related to the consensus sites to which E2F binds. The re- maining two fragments showed similarities to each other but not to the E2F site. They may represent very prelimi- nary evidence of another DNA-binding activity that can as- sociate with RB. It is also important to remember that, al- though some portions of RB-E2F complexes retain DNA- binding activity, RB is also a component of an activity, E2F-I, that eliminates DNA binding by E2F. Therefore, by extrapolation, RB might function as an inhibitor of some complexes in which it would not be seen if detection depended on a DNA-binding activity.

This remarkable series of papers lays a new foundation for a wonderfully detailed analysis of the control network governing gene expression and cellular proliferation. Un- ravelling the implications of these studies should keep researchers busy for some time to come.

Volume 1 Number 5 1991

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WEINBERG RA: The retinoblastoma gene and cell growth con- trol. Trend Bkxbem Sci 1990, 15:199-202. BAGCHI S, RAYCHAUDHUR~ P, NEVINS JR: Adenovirus ElA pro- teins can dissociate heteromeric complexes involving the E2F transcription factor: a novel mechanism for Ela tins- activation. Cell 1990, 62:659-669. WHITE P, WKLLUiso N NM, HARIDW E: CeRuk targets for transformation by the adenovirus ElA proteins. G?ii 1989, 56~67-75. WHYTE P, BUCHKOVICH KJ, Ho~ownz JM, FRIEND SH, R.~YBUCR M, WEINBERG R& HARUJW E: Association between an oncogene and an antioncogene: the adenovirus ElA proteins bind to the retinobkwoma gene product Nature 1988, 334124-129. ~HELLAPPAN sp, HJE8ERT S, MuDRyl M, HOROWK? JR, NE% m The E2F transcription factor is a cellular target for the RB protein. CeU 1991, 65:105flO61. BANDARA LR, IA THANGUE NB: Menovirus ElA prevents the retinoblastoma gene product fkom complex@ with a ceRu- kir transcription Victor. Nafure 1991, 351:4*97. CHITIENDEN T, INNGSTON DM, KKSLIN WG JR: The T/ElA-bind- lng domain of the retinoblastoma product can interact se- lectively with a sequence-specific DNA-binding protein. Cell 1991, 65:107>1082. RAYCHAUDHURI P, BAGCHI S, DEV~TO SH, KRAUSS VB, MORAN E, NEVINS JR: Domains of the adenovirus ElA protein re- quired for oncogenic activity are also required for dissocia- tion of E2F @nscription factor complexes. Genes Da, 1991, 5:1200-1211. BAGCHI S, WEIN~~ANN R, RAYCH~DHLJRI P: The retinobkstoma protein copurifies with EZF-I, an ElA-regulated inhibitor of the transcription factor EZF. Cell 1991, 65:1063-1072. MUDRIJ M, DEV~TO SH, HIEBERT SW, HUNTER T, PINES J, Nm JR: CeU cycle regulation of the E2F transcription factor in- volves an interaction with cyclin A. CeZI 1991, 65:1243-1253. NORBURY C, NURSE P: Cyclins and cell cycle control. Cwr Bid 1991, 1:2%24.

BANDARA LR, ADAM(X?++XI JP, HUNT T, LA THANGUE NB: Cy- clin A and the retinoblastoma gene product complex with a common transcription factor. Nature 1991, 352:24+251. HUANG S, LEE W-H, LEE J?YP: A cellular protein that competes with SV40 T antigen for binding to the retinoblastoma gene product. Nature 1991, 350:160-162. KAEW WG JR, PALLU DC, DECAPRIO JA, KAYE FJ, IMNGSON DM: Identitication of cellular proteins that can interakt specifically witb the T/ElA-binding region of the retinoblastoma gene product. Celi 1991, 64521-532. RUSTGI AK, DYSON N, BERNARDS R: Amino-terminal domains of c-myc and N-myc proteins mediate binding to the retinoblas- toma gene product. Nahrre 1991, 352541-544. DEFEO-JONES D, HUANG PS, JONES RE, HASKEJL KM, Vuocom GA, HANOBK MG, HLIBER HE, OLIFF A: cloning of cDNAs for cellular proteins that bid to the retinoblastoma gene prod- uct. Nature 1991, 352:251-254. EWAN M, XING W, BENTLEY LAWRENCE J, IIVINGST~N DM: Molec- ular cloning, chromosomal mapping, and expression of the cDNA for ~107, a retinoblastoma gene product related pro- tein CeN 1991, in press.

LIN BT-Y, GRUENWAUI S, MORJA AO, mu W-H, WANG m: Retinoblastoma cancer suppressor gene product is a sub- strate of the cell cycle regulator cdc2 ldnase. EMBO] 1991, 10:857+X%.

Elizabeth Moran, Cold Spring Harbor Iaboratory, Cold Spring Harbor, New York 11724, USA.

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