Journal of Cell Science, Supplement 19, 91-94 (1995)Printed in Great Britain © The Company of Biologists Limited 1995

91

Regulation of transcription by E2F1/DP1

Klaus Martin, Didier Trouche, Christian Hagemeier and Tony KouzaridesWellcome/CRC Institute, and Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK

SUMMARY

The E2F1 transcription factor, in co-operation with DPI, controls the expression of several S-phase specific genes. This activity is most likely responsible for the oncogenic and S-phase inducing properties of E2F1, suggesting that this transcription factor plays a key role in regulating the cell cycle. The transcriptional activation functions of E2F1 are resident in a small C-terminal domain which can inde­

pendently activate transcription. Here we review the protein-protein interactions which impinge upon and regulate this activation domain and put forward some models on their mechanism of action.

Key words: transcription, E2F, D PI, retinoblastoma

E2F TRANSCRIPTIONAL ACTIVITY AND THE CELL CYCLE

The E2F binding site is found in a num ber of S-phase specific genes, suggesting that the transcription factors which occupy it m ay regulate S-phase entry (Nevins, 1992). Intense activity over the last few years has revealed that this site is occupied by a heterodim er of E2F1 and D PI which represents the pro­totypes of distinct families o f transcription factors (Girling et al., 1993; Helin et al., 1992; Kaelin et al., 1992; Shan et al.,1992). These two proteins bind to the E2F site co-operatively and also stim ulate transcription in a co-operative fashion (Bandara, 1993; Krek, 1993). However, the transcriptional activation functions o f the E2F1/DP1 heterodim er seem to reside prim arily in the E2F1 protein, in the sense that E2F1 contains a C-term inal domain that will activate transcription independently when targeted to the prom oter (Flemington, 1993; Hagem eier et al., 1993a; Helin, 1993). Such experiments have not been successful in identifying an independent activa­tion dom ain in D PI.

A central role for E2F1 in cell cycle progression has com e from experim ents which show that E2F1 can, when overex­pressed, induce quiescent cells to go into S-phase (Johnson et al., 1993). These experiments support the notion that E2F1- induced transcriptional activation co-ordinates the induction of genes required for S-phase. This activity may also be the expla­nation for the oncogenic properties of E2F1 (Johnson et al., 1994; M elillo et al., 1994; Singh, 1994). However, what is less clear is how the other five E2F family m em bers fit into this process (Beijersbergen et al., 1994; Ginsberg et al., 1994; Hijm ans et al., 1995; Ivey-Hoyle et al., 1993; Lees et al., 1993). The target prom oters regulated by these family members (if different from E2F1/DP1 targets) are not yet known. However, there is some evidence that different E2F family members are regulated by distinct members o f the Rb superfamily of tum our suppressor proteins (Beijersbergen et al., 1994; Ginsberg et al., 1994). Since these tum our suppressors may be functional at

distinct (but overlapping) windows o f tim e during Gi and early S-phase (reviewed by M uller, 1994), the prediction would be that different E2F/DP family m em bers may be functional at distinct points in the G i-phase.

The transcriptional activation capacity of the E2F1/DP1 com plex is under tight control. A num ber of influences, both positive and negative, im pinge on the activity of the C-term inal activation domain in E2F1. In the next few sections we review the mechanism s by which this activation dom ain functions and how this activity may be regulated.

REPRESSION OF E2F1/DP1 BY THE RETINOBLASTOMA PROTEIN

The Retinoblastom a (Rb) tum our suppressor protein can be found in protein com plexes bound to E2F sites. Rb can directly contact both E2F1 and D PI and its binding results in the silencing o f E2F1/DP1 activity. Since the binding site for Rb in E2F1 maps to the activation dom ain at the C-term inus of the protein, Rb is thought to silence E2F1/DP1 activity by masking activation functions of this domain. The binding site for Rb in the E2F activation domain has been narrowed down to 18 residues (409-426) which can independently bind Rb (Helin et al., 1992). M utation o f a tyrosine residue w ithin this sequence elim inates both Rb binding and Rb-induced repression o f E2F (Hagem eier et al., 1993b; Helin et al., 1993).

The domain o f Rb required to contact the E2F1/DP1 com plex maps to the C terminus o f the Rb protein and overlaps the Rb ‘packet’ domain (Kaelin et al., 1991). D isruption o f Rb pocket sequences, by deletion or point mutation, appears to be a com monly used mechanism for the elim ination o f Rb function in the cell. Such m utations are com monly found in tum ours suggesting that rem oval o f Rb function is a step towards loss o f growth control (reviewed by W einberg, 1991).

The Rb packet contains two subdomains, A and B, separated by a ‘spacer’ sequence. These tw o subdom ains are frequently

92 K. Martin and others

mutated in tum ours and their sequence is highly conserved in the Rb-related proteins p 107 and p 130 (Hannon et al., 1993; Li et al., 1993). In addition, each of these two subdomains has a lower level o f sequence similarity to a general transcription factor: domain A is hom ologous to the C-term inal half o f the TATA-box binding protein TBP and domain B is homologous to the C-term inal half of TFIIB (Hagem eier et al., 1993b).

The significance o f the TBP/TFIIB similarity in the Rb packet domain is the following. The TBP and TFIIB proteins are part o f the basal transcriptional machinery. The TBP protein (com plexed with TBP associated factors) along with TFIIB forms the pre-initiation com plex, which binds to the TATA box region and recruits the rest o f the transcriptional machinery. The major contributors to the pre-initiation com plex, TBP and TFIIB, can contact the E2F activation domains (Hagem eier et al., 1993a, and our unpublished results). The domain o f TBP and TFIIB required for the binding to E2F is precisely the domain found to be hom olo­gous in the packet domain o f Rb. This therefore leads to the suggestion that the Rb packet domain may be a structural mim ic o f the TBP and TFIIB proteins enabling Rb to com pete for E2F binding. Consistent with this model the Rb and TBP proteins recognise sim ilar residues within the E2F activation domain. Deletion and point m utational analysis of E2F cannot discrim inate the binding site for Rb from the binding for TBP (Hagem eier et al., 1993a). These results suggest a model for Rb-induced repression of E2F activity, whereby Rb binds to E2F and masks its TBP and TFIIB binding potential. Since residues o f E2F involved in TBP binding are also required for transcriptional activation in vivo, binding of Rb to these residues would result in the silencing o f this activity. However, it is likely that the binding of Rb may not merely prevent activity of this E2F domain. There is evidence suggesting that Rb can positively repress activity below basal level when bound to E2F (W eintraub et al., 1992).

359 407 426 437E2F A.D. I I 615

Module 1 Module 2 Module 3

i------------- ... i 1

2I I 8

I 1 ^ — — 70

J 120

Fig. 1. Activation modules present within the E2F activation domain (AD). The relative activity capacity o f E2F sequences linked to the GAL4 DNA binding domain are shown (Hagemeier et al., 1993). This analysis defines 3 co-operative activation modules. Proteins interacting with modules 1 and 2 are shown on top.

between E2F and TBP may be necessary but not sufficient for activation. Recently a protein has been identified whose binding site resides entirely within module 1. The identity o f this protein becam e obvious following the observation that E2F has an activation domain with sequence sim ilarity to the activation domain of p53 (M artin et al., 1995). The sequence conservation is extensive (26% identity over 57 residues) and overlaps a region o f p53 required to bind the M DM 2 onco­protein. Consistent with this sequence similarity, mutation of E2F residues within module 1 which show homology to p53, abolishes the interaction with MDM2. In addition to binding E2F, M DM 2 can contact the heterodim eric partner o f E2F, the DPI protein.

STIMULATION OF E2F1/DP1 ACTIVITY BY MDM2

E2F HAS A MODULAR ACTIVATION DOMAIN WITH SIMILARITY TO p53

A domain of E2F1, containing only C-term inal residues, can activate transcription very efficiently when directed to the prom oter via the GAL4 DNA binding domain (Flemington et al., 1993; Hagem eier et al., 1993a; Helin et al., 1993). The fact that Rb can silence the activation functions o f this domain suggests that all the determinants for Rb-induced repression lie within this 57 residue domain.

Dissection o f the E2F activation domain indicates that it has a m odular structure. The domain can be divided into three sectors or m odules (Fig. 1), each o f which cannot activate tran­scription independently as a GAL4 fusion (Hagem eier et al.,1993). However, when modules 1 and 2 or 2 and 3 are fused, synergistic transcriptional activation is observed. Co-operative activation m odules have been observed in other transcription factors. A lthough the mechanism of such co-operativity has not been established for any protein, one speculation is that co­operation reflects the ability of different modules to influence different steps in transcription, for exam ple by contacting distinct proteins.

The binding site for the TBP protein coincides with module 2 of the E2F activation domain. This suggests that contact

The M DM 2 gene is found amplified in a third o f all sarcomas (Oliner et al., 1992). The product of the M DM 2 gene has oncogenic capacity as m easured by its ability to transform prim ary cells in co-operation with activated Ras (Finlay, 1993). The oncogenic activity of M DM 2 is attributed, at least partly, to its ability to bind the activation domain o f p53 and silence its activation potential (M omand et al., 1992).

In contrast to its repressive effect on p53, M DM 2 stimulates the activation capacity of the E2F1/DP1 com plex (M artin et al., 1995). This stimulation is mediated via the E2F activation domain and requires the M DM 2 binding residues in module 1.

How the M DM 2 protein can repress p53 yet stimulate E2F is not yet understood. These opposing effects may be due to the ability o f M DM 2 to bind p53 and E2F under distinct con­form ational states. M DM 2 contains an activation domain w hich is only evident when this domain is taken out o f the context of the full length MDM2 protein (Oliner et al., 1993). This result points to the possibility that M DM 2 has a cryptic activation domain which is only used following contact with E2F. This would fit in with a ‘co-activator’ model for M DM 2- m ediated stimulation of E2F activity. In this model, M DM 2 would bind module 1 sequences and mediate transcriptional activation functions using its own transcriptional activation domain. In this way, M DM 2 may act as a bridge between E2F1/DP1 and the basal transcriptional machinery. It is less

Transcription by E2F1/DP1 93

Repression of activation functions

Relief o f S-phase block

Promotion of S-phase

Fig. 2. Possible role of MDM2 on the cell cycle taking into account its opposing effect on p53 and E2F transcriptional activation capacity.

V ----

Stimulation of activation functions

likely that M DM 2 functions by displacing the Rb repressor protein from the E2F activation domain since the MDM 2- mediated stimulation of E2F activity can be seen in Rb- negative SAOS2 cells. Furtherm ore, the binding sites for M DM 2 and Rb on E2F are distinct (Fig. 1) and M DM 2 over­expression does not release Rb-induced repression of E2F (unpublished observations). The ability o f M DM 2 to bind Rb (Xiao et al., 1995) may be an event which leads to Rb m odu­lating M DM 2 rather than the reverse. Rb is an abundant m ulti­functional protein which may bind and sequester M DM 2 in a non-productive com plex away from the promoter. In this way excess Rb could be free to bind and repress E2F in the absence of the stimulating activity o f M DM 2.

W hichever the mechanism by which M DM 2 stimulates E2F1/DP1 activity, the net result should be stimulation of S- phase entry. The same is true for the repressive effect of M DM 2 on the p53 transcription factor - the net result is relief o f p53-m ediated growth arrest, leading to stimulation of S- phase entry (Fig. 2). A role for M DM 2 in cell cycle control fits well with its oncogenic capacity. These results suggest that M DM 2 may be an oncogene because it negatively regulates a tum our suppressor, p53, and also positively regulates an oncogene, E2F. If M DM 2 does play a central role in the reg­ulation o f cell cycle events it would be interesting to establish w hether overexpression o f M DM 2 is sufficient to induce quiescent cells to enter S-phase.

REGULATION OF E2F1/DP1 ACTIVITY BY PHOSPHORYLATION

The activity of the E2F1/DP1 com plex is also controlled by events which do not directly im pinge on the E2F activation domain (Fig. 3). These are mainly regulatory phosphorylations which indirectly affect proteins w hich bind the activation domain.

These regulatory phosphorylations are m ediated by cell cycle regulated kinases (CDKs). The most studied of these events involves the phosphorylation o f Rb (reviewed by Sherr,1994). The net effect o f such phosphorylation is the inability o f Rb to bind E2F leading to a relief o f Rb induced repression. Presumably this event (which occurs at the G |/S boundary) allows co-activator proteins (such as M DM 2?) access to the E2F activation domain.

A different set o f phosphorylation events which also results in preventing Rb from binding E2F, involves E2F residues 332 and 337. Phosphorylation at these sites, which fall outside the E2F activation domain, prevents binding o f Rb but facilitates binding of the adenovirus E4 protein (Fagan et al., 1994). It is tem pting to speculate that such phosphorylation transm its a confirmational charge which then regulates protein interactions at the activation domain. W hether this phosphorylation then facilitates the binding of cellular co-activators such as M DM 2 (by analogy to E4) is still to be determined.

The timing of the phosphorylation events discussed here is within the G i-phase and the cyclin/CDK com plexes involved are predom inant during this phase. This is com patible with the notion that the primary role o f these phosphorylation events is to mediate the removal of the Rb repressor protein from E2F. This cell cycle regulated process appears to be circumvented by viral transforming products. Proteins such as the adenovirus E l A have evolved to target the binding and rem oval o f Rb from E2F thus activating S-phase entry (Nevins, 1992; Zam anian and La Thangue, 1992). This emphasises the central role played by Rb in keeping E2F1/DP1 activity under control and highlights the need to understand more about the precise mechanism of this silencing event.

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Fig. 3. Regulation of the E2F activation domain. Events which impinge on the activity of the E2F activation domain are shown. Arrows do not indicate a direction but merely a consequence of the events.

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