Cell, Vol. 62, 659-669,

August

24, 1990, Copyright

0 1990 by Cell Press

Acienovirus EIA Proteins Can Dissociate Heteromeric Complexes Involving the E2F Transcription Factor: A Novel Mechanism for EIA Trans-Activation Srilata Bagchi, and Joseph R. Howard Hughes and Department Duke University Durham, North

Pradip Raychaudhuri, Nevins Medical Institute of Microbiology and Immunology Medical Center Carolina 27710

Summary Adenovirus infection activates the E2F transcription factor, in part through the formation of a heteromeric protein complex involving a 19 kd E4 gene product that then allows cooperative and stable promoter binding. We now find that cellular factors are complexed to E2F in extracts of several uninfected cell lines. ElA proteins can dissociate these complexes, releasing free E2F. This activity of ElA is independent of conserved domain 3 but is dependent on conserved domain 2 sequence. The ElAmediated dissociation of the complexes allows the E4 protein to interact with E2F, generating a stable DNA-protein complex with the E2 promoter and a stimulation of transcription. These experiments demonstrate a function for ElA in mediating a dissociation of transcription factor complexes, allowing new interactions to form and thus changing the transcriptional specificity. Introduction The control of transcription initiation is the critical first event in the regulation of gene expression. A variety of studies have now shown that alterations in factors that interact with specific DNA elements upstream of the transcription initiation site can control the rate of transcription initiation (Maniatis et al., 1987; Mitchell and Tjian, 1989; Johnson and McKnight, 1989). Thus, an understanding of a gene regulatory pathway must focus on the mechanisms controlling the activity of these specific DNA binding proteins. In this regard, adenovirus ElA-dependent Pans-activation of transcription has provided a system to explore mechanisms of transcription factor regulation (Berk, 1986; Nevins, 1989). The ElA protein does not act directly to stimulate transcription since it is not a DNA binding protein (Ferguson et al., 1985). Therefore, ElA must exert its action indirectly, presumably by altering the activity of cellular transcription factors. Indeed, it is now clear that transcription of the various viral and cellular genes that are stimulated by ElA action depends on the interaction of a variety of cellular transcription factors with upstream promoter elements (Nevins, 1989). Attempts to define regulatory sites by promoter mutational analysis have been complicated by the fact that sequences important for regulation are also important for basal transcription of the gene. Nevertheless, through a combination of promoter assays as well as DNA binding studies, there are clear indications of target sequences

and factors that are required for ElA-dependent transactivation. A TATAA-specific factor appears to allow stimulation of the ElB (Wu and Berk, 1988), hsp70 (Simon et al., 1988), and c-fos genes (Simon et al., 1990). The TFlllC transcription factor appears to be responsible for the ElAdependent stimulation of polymerase Ill transcription (Berger and Folk, 1985; Hoeffler and Roeder, 1985; Gaynor et al., 1985; Hoeffler et al., 1988; Yoshinaga et al., 1986). The E4F transcription factor is induced by adenovirus infection, dependent on ElA function, and is a good candidate for activation of E4 transcription (Raychaudhuri et al., 1987; Rooney et al., submitted), although other experiments have suggested a role for an ATF transcription factor in the regulation of E4 transcription (Lee and Green, 1987; Lillie and Green, 1989). The Apl recognition site in the E3 promoter can confer ElA inducibility weeks and Jones, 1983), and recent studies demonstrate an induction of Apl activity by adenovirus infection (Muller et al., 1989; Buckbinder et al., 1989). Finally, there is strong evidence linking the E2 promoter-specific factor E2F to the process of ElA-dependent Vans-activation (Kovesdi et al., 1986; Yee et al., 1989). Recent studies of the activation of E2F reveal a multistep process involving an increase in the DNA binding activity of the factor as well as an alteration that allows the factor to bind cooperatively to the adjacent E2 promoter sites (Hardy et al., 1989; Raychaudhuri et al., 1990). This stable binding of E2F requires the action of a 19 kd product of the E4 gene (Huang and Hearing, 1989; Neil1 et al., 1990). Indeed, it appears that the E4 19 kd protein interacts with E2F and can be found in the stable DNA-protein complex (Huang and Hearing, 1989; Neil1 et al., 1990; Raychaudhuri et al., 1990). This suggests the possibility that other proteins might interact with E2F in the uninfected cell. Such associations do indeed exist. Of most interest was the finding that the ElA proteins possess the ability to dissociate these complexes, which then allows the interaction of the viral E4 protein and the formation of a functional transcription complex. Results Cell Type Variation in E2F Gel Mobility A variety of previous experiments have demonstrated that adenovirus infection of HeLa cells results in the stimulation of the DNA binding activity of the E2F transcription factor (Kovesdi et al., 1986; Reichel et al., 1988). E2F can be detected in extracts of uninfected HeLa cells, but the level of binding activity is reduced. Furthermore, the E2F activity in uninfected cells is distinctly different from that found in virus-infected cells in that it only forms an unstable complex on the E2 promoter, occupying only a single site at low concentrations (Hardy et al., 1989; Raychaudhuri et al., 1990). In contrast, the E2F from infected cells rapidly occupies both recognition sites and forms a very stable complex (Hardy et al., 1989; Raychaudhuri et al., 1990). The difference in E2F binding results from the as-

Cell 660

8

F

E2F,,

E2FL

n- + *.

-

+

-130kD -75kD

-

Figure

1. Heterogeneous

E2F Binding

Activity

in Extracts

of Various

39kD

Cell Lines

(A) Whole-ceil extracts were prepared from the indicated cell lines and assayed for E2F binding activity as described in Experimental Procedures. The position of the EPF-DNA complex previously described (Raychaudhuri et al., 1990) which involves the interaction of a single E2F factor with the DNA probe, is indicated ([E2F]m]). Each extract was assayed in the absence (-) or presence (+) of specific cold competitor DNA. (B) Methylation interference assay for sequence specificity of binding of L cell E2F complex. DNA-protein complexes were formed using the L cell extract and partially methylated probe DNA. The DNA probe used for this assay was mutated at the proximal recognition site and thus contained a single E2F recognition sequence. After separating the specific complex from the free DNA by gel electrophoresis, DNA bound in the complex (B) and free DNA(F) were isolated, cleaved with piperidine, and then analyzed in a 6% acrylamide sequencing gel. The two positions of methylation that interfere with binding are indicated by arrows, and the DNA sequence is depicted on the right. (C) UV cross-linking labels a 54 kd protein in the L cell complex. DNA-protein complexes were formed with either E2F purified from adenovirusinfected cells (EPFM) or the L cell extract (EPFL) using DNA probe substituted with bromodeoxyuridine. After UV irradiation and digestion with DNAase, the labeled proteins were analyzed by SDS-PAGE. The binding reactions were carried out in the absence (-) or presence (+) of specific cold comoetitor DNA. The oosition of a labeled 54 kd orotein. which is the size of purified E2F (Yee et al., 1969) that was not labeled in the presence of the specific competitor DNA is indicated by the arrow

sociation of a 19 kd E4 protein with E2F, allowing a cooperative interaction between two E2F molecules bound to the adjacent recognition sites. The observation that the E2F factor forms a complex with a viral protein in a lytic infection suggests the possibility that E2F may normally interact with cellular proteins. Previous assays of HeLa cell extracts have not afforded an indication of such an association. However, further analysis of E2F binding activity in extracts from a variety of other cell lines has revealed complexes distinct from that of the monomer E2F interaction observed with HeLa cell extracts (Figure 1A). In comparing extracts of a variety of cell lines, a considerable heterogeneity of complexes was detected on the E2F probe. In some cases, and most dramatically in mouse L cells, a single large complex was detected that exhibited a mobility similar to that of the E2F complex detected in adenovirus-infected HeLa cells. By several criteria, we judged that these complexes involved an E2F interaction. The formation of the complex was prevented by competition with an excess of the specific

sequence but not a nonspecific competitor. Furthermore, methylation interference assays of the large complex detected in the L cell extracts demonstrated that methylation of the same G residues that interfered with E2F binding (Yee et al., 1987) also interfered with binding of this slow migrating complex (Figure 16). Finally, affinity labeling of the DNA binding polypeptide in the L cell complex by UV cross-linking yielded a 54 kd labeled protein (Figure lC), identical in molecular size to previously characterized E2F (Yee et al., 1989). These results suggest that these complexes do involve an interaction via E2F. In addition, the fact that the UV cross-linking labels a protein of 54 kd in the L cell complex indicates that the slower migration of the complex is not due to direct binding of a considerably larger protein, but rather is due to additional components in the complex. We considered the possibility that the L cell E2F complex resulted from an alteration of E2F similar in nature to that brought about by the adenovirus E4 product, namely, a cooperative interaction of factors on adjacent promoter

ElA 661

Dissociates

Transcription

Factor

Complexes

B 0 5 10 20 SO 4s.‘. .

(min)

=%I---

Figure 2. The L Cell E2F Complex Manner to Form a Stable Complex

Does

Not Bind in a Cooperative

(A) DNA probe specificity, E2F binding was assayed with the L cell extract using E2 promoter probes that contain the normal two E2F sites (E2Fr + ,,), a mutated site I leaving only an intact site II (E2FlI), or a mutated site II leaving only the site I intact (E2Fr). (6) Off-rate measurements. The E2F complex was formed with the L cell extract and the normal two-site E2F probe. After incubation for 20 min, a large excess of cold competitor DNA was added and samples were removed at the indicated times. Each sample was immediately loaded onto a native gel that was already running.

sites (Hardy et al., 1989; Raychaudhuri et al., 1990). However, as shown in Figure 2A, this large complex was formed regardless of whether the probe contained a single E2F binding site or two adjacent binding sites, a result clearly distinct from that for E2F from adenovirus-infected ceils (Hardy et al., 1989; Raychaudhuri et al., 1990). Furthermore, this complex did not represent a stable interaction on the promoter as seen in a measurement of off rate (Figure 28). The L cell complex clearly exhibits a rapid off rate, whereas the complex formed with E2F from adenovirus-infected cells is stable for at least 80 min (Hardy et al., 1989; Raychaudhuri et al., 1990). We conclude that the slowly migrating complex found in extracts of L cells results from the interaction of a 54 kd E2F factor with a single recognition site. The fact that the complex migrates slowly in the native gel, at a position near to that for the complex from adenovirus-infected cells that contains two E2F molecules and the E4 19 kd protein, suggests that another protein or proteins are contained in the complex. DOC Dissociates E2F from the Large Complex A further indication that the slowly migrating complex detected in L cell extracts involved an interaction of E2F with another factor was provided by the experiment shown in Figure 3A. Treatment of the L cell extract with sodium

deoxycholate (DOC), a reagent known to disrupt protein-protein interactions (Baeuerle and Baltimore, 1988), eliminated the slowly migrating complex and yielded a complex typical of the interaction of a single E2F factor with the DNA. This was most dramatically shown with the L cell complex, since prior to DOC treatment a single complex was observed that was then converted to the smaller complex by DOC treatment. However, the same was true for the E2F-containing complexes from other cell extracts. In each case, the slowly migrating complexes were converted by DOC treatment to the complex typical of an E2F monomer interaction with the DNA. Analysis of E2F by glycerol gradient sedimentation provided additional evidence of an interaction between E2F and another factor. An L cell extract was sedimented in a glycerol gradient with or without prior treatment with DOC. Fractions of the gradient were then assayed for E2F binding activity as depicted in Figure 38. In the absence of DOC treatment, E2F binding activity was detected in a rapidly sedimenting form near the bottom of the gradient, cosedimenting with aldolase, which has a native molecular size of 180 kd. Furthermore, all of the binding activity was in the form of the slowly migrating complex as detected in the crude extract. Treatment of these fractions with DOC prior to the assay for E2F binding converted the activity to the more rapidly migrating complex, consistent with the results obtained in the crude extracts. In contrast, if the L cell extract was first treated with DOC prior to the glycerol gradient, the E2F binding activity sedimented near the top of the gradient, two fractions slower than bovine serum albumin, which has a native molecular size of 88 kd, and all of this E2F activity was in the form of the rapidly migrating gel complex. We therefore conclude that the vast majority of E2F in L cells is associated with another protein or proteins. This complex can nevertheless bind to DNA with the appropriate specificity, and E2F can be released from this interaction by treatment with DOC. The fact that the native molecular size suggested from the sedimentation analysis after DOC treatment agrees well with the molecular size of the E2F polypeptide (54 kd) (Yee et al., 1989; Figure 1C) argues that this binding activity is the result of a monomer interaction. ElA Proteins Can Dissociate the EPF-Containing Complex The results presented thus far demonstrate that the E2F transcription factor can be found in association with other protein(s) in the uninfected cell and that this complex can be disrupted with DOC, releasing free E2F. The experiment shown in Figure 8 suggests a functional relevance with respect to ElA-dependent rrans-activation. Although the experiments presented thus far indicate that L cells exhibit the most dramatic difference in the nature of the E2F complex, we have found that L cells are refractory to an adenovirus infection. The reasons for this are not clear at present. As an alternative approach, we have employed differentiated F9 cells for an infection since these cells also exhibit a slowly migrating E2F complex that can be dissociated with DOC, and we have previously shown that F9 cells can be infected by adenovirus (Imperiale and

Cdl 662

Figure 3. DOC Dissociates the EPF-Containing Complexes and Releases E2F

-E’%

.“A . ../ *

I + DOC

Nevins, 1984; Reichel et al., 1987). Infection of the differentiated F9 cells with the E4 mutant dl388 results in the disappearance of the large EPF-containing complex (Figure 4). There is also a substantial increase in the E2F monomer complex, more than can be accounted for by the loss of the large complex. We attribute this to the ability of ElA to stimulate E2F binding activity (Raychaudhuri et al., 1990). Infection of dF9 cells with wild-type Ad5 yields a large complex of similar mobility to that in the uninfected cell, but which is clearly a distinct complex as evidenced by dissociation measurements as well as the requirement for the two adjacent E2F recognition sequences in the probe (data not shown). This complex is the ECdependent stable complex previously described in adenovirus-infected HeLa cells (Hardy et al., 1989; Raychaudhuri et al., 1990). Thus, in the absence of E4 function (d1388 infection), adenovirus infection has the same effect on the EPF-containing complex as DOC treatment. In a wild-type infection in which E4 is produced, it appears that this released E2F is then able to form the typical stable complex. There are, of course, a number of early gene products produced in a dl388 infection. We have therefore used an in vitro assay to determine if ElA is actually involved in the dissociation of E2F from preexisting complexes. Our source of ElA protein in these experiments was in vitro translation products of a rabbit reticulocyte lysate programmed with RNA produced in vitro from an ElA 13s cDNA insert. As seen in Figure 5A, the addition of reticulocyte lysate alone or lysate programmed with a control RNA (brome mosaic virus) had no effect on the L cell E2F com-

(A) EPF-containing complexes were formed with the E2 probe and with extracts of L cells, differentiated F9 cells, and J556L cells that were either untreated (-) or treated (+) with DOC as described in Experimental Procedures. (6) Sedimenatation analysis of E2F binding activity in L cell extracts. L cell extracts, which were either untreated or treated with DOC, were fractionated by glycerol gradient sedimentation. After centrifugation, an aliquot of each fraction was assayed for E2F binding activity as described in Experimental Procedures. In addition, aliquots of the fractions from the control sample (no DOC treatment) were treated with DOC prior to the assay for E2F binding (bottom). The band present above the E2F complex in each lane is a probe artifact.

plex. In contrast, the addition of the lysate programmed with the ElA RNA completely eliminated the large complex and released free E2F that bound to the probe as a monomer. Although the result was most clearly seen in the L cell extract due to the absence of any free E2F, a similar result was found in each of the other cell extracts in that the addition of in vitro translated ElA protein dissociated the EPF-containing complexes (Figure 58). It thus appears that ElA possesses the ability to dissociate these EPF-containing complexes in a manner similar to the effects of DOC. ElA-Mediated Dissociation of E2F Allows Interaction of the 19 kd E4 Protein and Results in a Stimulation of Transcription Previous results from our laboratory and others have shown that the E2F factor found in a wild-type adenovirusinfected cell is capable of forming a stable complex on the E2 promoter as a result of an association with the 19 kd product of the viral E4 gene (Huang and Hearing, 1989; Neil1 et al., 1990). The importance of this action to E2 transcription is indicated by the observations that the E4 gene, and in particular the E4 open reading frame 8/7 that encodes the 19 kd protein, is an effective trans-activator of the E2 promoter (Goding et al., 1985; Reichel et al., 1989; Neil1 et al., 1990). As depicted in Figure 8A, the E4 19 kd protein, translated in vitro in a reticulocyte lysate, cannot associate with the L cell E2F-containing complex as indicated by the lack of an alteration in gel mobility, but more importantly by the fact that there is no increase in the stability of the E2F complex. Dissociation of the L cell com-

EIA Dissociates 663

Transcription

Factor

E2bei-

Complexes

mation of the stable E2F complex normally seen in virusinfected cells. Thus, these results demonstrate that the viral E4 protein cannot interact with the E2F factor when it is part of the complex found in the uninfected cell but it does so if ElA is present to liberate E2F. This result also demonstrates a distinct difference between the E2F complex containing the E4 protein and the E2F complex involving a cellular protein, since ElA clearly does not dissociate the E4-E2F complex. This result immediately suggests a transcriptional role for ElA in allowing the ECmediated stabilized binding of E2F That this is indeed of functional significance is indicated by the experiment shown in Figure 88, in which L cell extracts were used for in vitro transcription. Transcription was assayed using a promoter construct containing the two E2F sites from the E2 promoter fused upstream of the early SV40 promoter. Previous experiments have shown that the addition of the E2F sites in this construct confers ElA inducibility in in vivo assays and renders the promoter responsive to the addition of affinity-purified E2F in in vitro assays (Yee et al., 1989). We have now measured the transcriptional activity of this promoter in L cell extracts treated as in the experiment of Figure 8A. In the absence of any addition to the L cell extract, we could only measure low level transcription from the promoter (Figure 8B). The addition of E4 protein had no effect, and there was little if any stimulation of transcription upon the addition of ElA protein. However, the addition of both ElA and E4 proteins to the L cell extract, which results in the generation of E2F that can form a stable promoter complex as shown in Figure 8A, resulted in a 7.fold stimulation of transcription. Furthermore, as shown in Figure 8B (right), the stimulation of transcription by the addition of ElA and E4 proteins to the L cell extract required the presence of the

*E~FI,I

E%I-

Figure 4. Adenovirus Complex

Infection

Dissociates

the

EOF-Containing

Differentiated F9 cells were infected with dl312, dl366, or wild-type Ad5 as described in Experimental Procedures. Whole-cell extracts were prepared and assayed for E2F binding activity using a probe that contains two E2F binding sites. The position of the dF9-specific E2Fcontaining complex is indicated (E2Ft& as is the E2F complex resulting from binding a single E2F factor (E2Ftrt) and the stable complex formed as a result of cooperative binding of two E2F factors along with the E4 19 kd protein (E2F&

plex with ElA releases free stable complex. However, dissociated by the addition then the addition of the E4

A

E2F, which also forms an unif the L cell complex is first of the in vitro translated EiA, protein does promote the for-

B

Figure 5. ElA Protein Containing Complexes

n-&n* - + II,-.

-

+ 1

-

+ .

- + m*

-

+

(EIA)

Dissociates the E2F and Releases E2F

(A) E2F binding was measured in the L cell whole-cell extract alone (WCE) or after the addition of 1 pl of a 1:lO dilution of reticulocyte lysate programmed with brome mosaic virus RNA (RUBMV), reticulocyte lysate alone (RL), or reticulocyte lysate programmed with 13s ElA mRNA (RUElA). Binding was also assayed with each of the reticulocyte lysates in the absence of L cell extract. (6) E2F binding was assayed in extracts of the indicated cell lines in the absence (-) or presence (+) of reticulocyte @ate programmed with the 13s ElA mRNA.

Cdl 664

A

+E4

+ElA

mm+w

0

5 1020

0 5 1020

0

5 1020

0

5 10 20

(mid

* I’.

*

-+2?2)

E2FL

-E2F

.J,

(1)

-Primer-

Figure

6. Dissociation

of the L Cell Complex

by ElA Allows

the E4 19 kd Protein to Interact

with E2F and Form a Stable and Functional

Complex

(A) Gel retardation assays. E2F binding was assayed in the L cell extract alone or after the addition of the 19 kd E4 open reading frame 617 product, the 13s ElA product, or a combination of the E4 protein and the ElA protein, each synthesized in reticulocyte lysates. In each case, 1 rJ of a 15 dilution of the reticulocyte lysate was added. Dissociation rate measurements were carried out for each assay as described in Experimental Procedures and Figure 38. (6) In vitro transcription. Left: the EPF-SV template (pAlOCAT-E2F) described in Experimental Procedures was incubated in L cell extracts supplemented with reticulocyte lysates that were programmed with the indicated RNAs. After incubation as described in Experimental Procedures, RNA was isolated and analyzed by primer extension with a CAT-specific primer. The position of the expected 136 nucleotide primer extension product is indicated by the arrow. Right: the EPF-SV template and the SV promoter (pA1OCAT) (same early SV40 promoter lacking the E2F sites) were transcribed in the L cell extract with the addition of reticulocyte lysate programmed with brome mosaic virus RNA or ElA + E4 RNA.

E2F sites. The presence of the E2F sites had no effect on transcription in the control extract; we only observed a stimulation when the E2F sites were present as well as the addition of ElA and E4. It would therefore appear that the ability of ElA to dissociate E2F from the preexisting complex in the L cell extracts, then allowing the E4 19 kd protein to interact, is a functionally important event with respect to transcription activation. E2F Dissociation Is Independent of MA Domain 3 The “ElA gene” is actually a complex transcription unit that encodes at least five distinct mRNA species through alternative splicing (Nevins, 1989). The major products in a lytic infection or in transformed cells are proteins of 289 and 243 amino acids that are encoded by 13s and 12s mRNAs, respectively (Berk and Sharp, 1977). As shown in Figure 7A, the 12s ElA product, translated in the reticulocyte lysate, was as efficient as the 13s ElA product in

promoting the dissociation of E2F from the L cell complex. Furthermore, the dissociation mediated by either ElA protein allowed the E4-E2F interaction to form. We could find no significant difference in the ability of the two ElA proteins to promote the dissociation, suggesting that the ElA protein sequences common to the two proteins, namely conserved domains 1 and 2, were involved in this dissociation event. Indeed, as shown in Figure 78, it appears that domain 2 sequences are essential. A plasmid bearing a 12s ElA cDNA with a mutation at nucleotide position 928 (Moran et al., 1986) was transcribed and the RNA translated in vitro. In contrast to the wild-type 12s product, the 928 mutant was not capable of dissociating the E2F complex. SDS gel analysis of the in vitro translation products demonstrated that equivalent amounts of the wild-type 125 protein and the 928 mutant protein were produced (data not shown). From these results we conclude that ElA protein sequence in the 12s product, and specifically con-

ElA 665

Dissociates

Transcription

Factor

Complexes

A

E2F,-.

E2Flll--

B

-E2F121 E~FL+ =hj5

Figure 8. Transcriptional EIA Gene Product

Activation

Function

of the Adenovirus

12s

(A) HeLa cells or L cells were transfected with the pAlOCAT-EPF plasmid (Yee et al., 1989) together with a 13s EIA cDNA-expressing plasmid or a 12s EIA cDNA-expressing plasmid. CAT activity was measured as described in Experimental Procedures. (6) L cells were transfected with the pAlOCAT-EPF plasmid either alone or together with the wild-type 12s ElA-expressing plasmid or the 12s EIA plasmid bearing the 928 mutation. CAT activity was measured as described in Experimental Procedures. Figure 7. Dissociation of the L Cell Complex dependent of EIA Conserved Domain 3

Releases

E2F and Is In-

(A) E2F binding was assayed in the L cell extract in the absence or the presence of 1 pl of a I:30 dilution of reticulocyte extract programmed with either the 12s ElA mRNA or the 13s ElA mRNA and in the presence or absence of the E4 protein (1 ~1 of a 1:5 dilution). (6) E2F binding in the L cell extract before or after the addition of 1 d of a I:10 dilution of reticulocyte lysate programmed with wild-type 12s RNA or the 12s RNA containing the 928 mutation.

served domain 2 sequence, is required for the dissociation of the complexes containing the E2F transcription factor. The results presented thus far demonstrate that E2F can be released from cellular complexes through the action of the EIA protein. The functional significance to transcriptional control is indicated by the fact that the E2F protein is a transcription factor (Yee et al., 1989) and the direct demonstration of a stimulation of transcription in vitro. Although several previous studies have shown that the 243 amino acid 12s ElA product can fmns-activate transcription, including the E2 gene (Leff et al., 1984; Ferguson et al., 1985; Winberg and Shenk, 1984; Simon et al., 1987; Zerler et al., 1987), the results have been somewhat variable and the most responsive targets are often cellular genes such as that encoding PCNA (Zerler et al., 1987) and hsp70 (Simon et al., 1987). However, most fransactivation assays have used HeLa cells, and as we have noted previously, the majority of E2F in HeLa cells is apparently free of interactions with other factors. Thus, we would predict that the 12s ElA-dependent dissociating action that we describe here would be cell type dependent, having little contribution to trar?s-activation in HeLa cells. To address this issue directly, we have compared the ability of a 125 ElA cDNA and a 13s ElA cDNA to trans-

activate in either HeLa cells or L cells, employing the same promoter construct used for in vitro transcription (pAlOCAT-E2F) that measures E2F-dependent transcription (Yee et al., 1989). As shown in Figure 8A, whereas the 1% ElA product could efficiently stimulate transcription in HeLa cells, the 12s ElA product was inactive, a result similar to that seen in many previous experiments. In contrast, the same 12s ElA cDNA was clearly active when the Rafts-activation assay was performed in L cells, although the activity was reproducibly lower than that of the 13s ElA cDNA. Furthermore, this result was not limited to L cells but was also found in CVl cells and Vero cells and was not limited to this one construct but was also true for the intact E2 promoter (data not shown). Moreover, as shown in Figure 8B, the 12s ElA-mediated activation was dependent on domain 2 since the 928 mutation markedly reduced the ffans-activation. We therefore conclude that the 12s ElA product, involving domain 2, can indeed tmns-activate transcription through the E2F factor. Discussion We wish to emphasize what we believe to be two important issues deriving from these studies. First, the E2F transcription factor, which exists in a complex with the 19 kd E4 gene product in a virus-infected cell (Huang and Hearing, 1989; Neil1 et al., 1990), can be found in association with cellular factors in the uninfected cell. Furthermore, if the gel retardation mobilities are any indication, it appears that multiple interactions involving E2F can exist. Second, these associations can be disrupted by adenovirus ElA proteins, resulting in the liberation of a free, monomer E2F molecule. This ElA-mediated dissociation is of functional significance in allowing the interaction of the 19 kd E4 pro-

tein with E2F so as to generate a stable DNA complex that stimulates E2 transcription. Significance of Cellular, EPF-Containing Complexes We imagine two possible cellular roles for proteins in a complex with E2F. It has now become clear from recent experiments that the E2F transcription factor can interact with the viral 19 kd E4 gene product (Huang and Hearing, 1989; Neili et al., 1990), resulting in a complex that can bind cooperatively to adjacent promoter recognition sites (Hardy et al., 1989; Raychaudhuri et al., 1990). The cellular protein(s) in association with E2F could perform the same function in a cellular context. Clearly, when compared with the E2F-E4 interaction on the E2 promoter, the heteromeric E2F complex found in L cells did not form a stable complex on the E2 promoter. Nevertheless, it is also true that the E2 promoter is not the normal target for this complex, and it is quite possible that when assayed on the correct target promoter, this interaction could impart stability. Alternatively, it is also conceivable that this interaction provides a transcriptional activating function to E2F. Thus, although free E2F as isolated from uninfected HeLa cells where it exists as a monomer might stimulate transcription, it is also possible that the protein or proteins that are found in complexes with E2F might provide promoter specificity to the factor, not in the form of stable DNA interactions but by allowing specific contacts to be made with other transcription factors. Of course, it is also possible that the factor interacting with E2F blocks transcription function and that the complex is in fact nonfunctional. Role of ElA Proteins in the Dissociation of E2F Complexes The dissociation assays we describe here clearly demonstrate a function for the ElA proteins not previously recognized. We suggest that this action of ElA is important in redirecting the cellular transcriptional machinery. The E2F factor is released from interactions with cellular proteins to then allow its efficient use by the viral E2 promoter. It is also obvious that the relative importance of this event to viral infection will be a function of the host cell in terms of the extent to which E2F exists in a free state. For instance, we suspect that this activity plays a minor role in HeLa cells where very little of the E2F factor is found in complexes. indeed, various previous experiments have provided little evidence for a role for 12s ElA in transactivation of viral transcription in HeLa cells. Our results now clearly show that the magnitude of 12s Pansactivation, dependent on E2F, is a function of the cell type used for the assay and we believe that this is a function of the state of E2F. Of course, it is also true that adenovirus did not evolve in a HeLa cell, and we thus suggest that this activity of ElA, along with the 19 kd E4 protein, is important for efficient transcription in the normal host. Although our in vitro transcription assay gave no clear indication of an effect by ElA alone, we suspect this simply reflects the lesser sensitivity of the in vitro assay, since the in vivo transfection assay clearly demonstrated a stimulation by

the 12s ElA product alone. This result suggests that the release of E2F from the complex, without the accompanying interaction of the E4 protein, can be of functional significance. The subsequent addition of E4 would stabilize the interaction on the promoter, increasing the time the promoter is bound by factor, and thus further increase transcriptional activity. Although our assays have focused on the E2F factor, we do not exclude the possibility that other transcription factors are also targeted by ElA in this fashion. Previous experiments have suggested the involvement of multiple transcription factors in ElA trans-activation, including a TATAA-specific factor @Vu et al., 1987; Simon et al., 1988), the TFIIIC factor (Yoshinaga et al., 1988; Hoeffler et al., 1988), and an E4 promoter-specific factor (Raychaudhuri et al., 1987). It is thus a possibility that one or more of these factors is also complexed to cellular proteins and is a target for release by 12s ElA. In fact, previous assays of hsp70 induction by ElA, which requires the TATAA element, have shown that the 12s ElA product is nearly as efficient as the 135 ElA in activating transcription (Simon et al., 1987). Although it is tempting to suggest that there may be an interaction involving a TATAA factor, which is then dissociated by ElA, this is unfortunately difficult to address at the moment due to the lack of direct assays for TFIID binding. We also note that the human papillomavirus E7 protein and the large T antigen of SV40 have been shown to transactivate the adenovirus E2 promoter (Phelps et al., 1988; Loeken and Brady, 1989). T antigen and the E7 protein bear homology to ElA domains 1 and 2, sequences found in the 12s product, and there is no sequence homology to the 13Sspecific domain 3 sequences. Furthermore, recent experiments indicate that mutations in the region of E7 that is homologous with ElA domain 2 abolish E7 &ins-activation function (W. Phelps, personal communication). Thus, a variety of data clearly demonstrate the role for ElA 125 sequence in trans-activation, and the results in this paper provide a specific mechanism to account for this function. With respect to the issue of cell type specificity in 12s ElA trans-activation, it is also of interest that the cell extract we have assayed that exhibits the greatest amount of E2F in a free form is HeLa, a cell that expresses the human papillomavirus E7 protein. Clearly, the next step in this analysis is a determination of the ability of these other trans-activators to mediate a dissociation of E2F. A variety of past experiments have demonstrated a role for the 13S-specific domain 3 sequence in rrans-activation (Ricciardi et al., 1981; Montell et al., 1984; Glenn and Ricciardi, 1985; Moran et al., 1986; Liliie et al., 1986). Indeed, some assays have shown that the domain 3 sequence may also be sufficient for trans-activation (Liilie et al., 1987; Green et al., 1988). This result, together with past experiments and the data we present here that document a role for 125 ElA in Vans-activation, therefore suggests that ElA proteins contain at least two independently functioning units of trans-activation function. The results presented here define one activity that is specific for the 12s

:A$

Dissociates

Transcription

Factor

Complexes

EIA product. It is likely that the domain 3-specific event involves other types of transcription factor alterations. For instance, previous experiments have documented an increase, dependent on ElA function, in the level of active E2F factor that can bind to DNA (Raychaudhuri et al., 1990). Furthermore, adenovirus infection also leads to an increase in levels of the E4F transcription factor, again an ElA-dependent event (Raychaudhuri et al., 1987; Raychaudhuri et al., 1989; Rooney et al., submitted). Other experiments have suggested a role for phosphorylation in the control of E2F and E4F DNA binding activity (Raychaudhuri et al., 1989; Bagchi et al., 1989) as well as in changes in TFIIIC binding to DNA (Hoeffler et al., 1988). We believe that it is this function, a stimulation of phosphorylation, that ElA also participates in and that is dependent on the domain 3 sequence unique to the 13s product. In addition to this role, other experiments have suggested a functional role for the domain 3 sequence in providing a transcriptional activating domain to a promoter complex (Lillie and Green, 1989). However, despite these various observations, the exact target and mechanism for the domain 3-mediated activation remain unclear. Unfortunately, unlike the situation described here for the domain e-dependent event as well as recent experiments concerning the ECmediated alteration in E2F binding (Neil1 et al., 1990), an in vitro assay has not yielded a clear determination of the target or targets for domain 3-mediated activation. Finally, in considering the mechanism by which ElA may mediate the release of E2F from these complexes, another role of the ElA protein may be relevant. It is now well established that the EIA protein can be found complexed to a variety of cellular proteins in virus-infected and transformed cells (Harlow et al., 1986; Yee and Branton, 1985) one of which is the product of the retinoblastoma susceptibility gene, pRblO5 (Whyte et al., 1988). The ability of ElA to associate with Rb as well as most of the other proteins is dependent on sequences in conserved domain 2, and independent of sequences of conserved domain 3, the 13Sspecific sequence (Whyte et al., 1989). Furthermore, SV40 T antigen and the E7 gene product of human papillomavirus also interact with the Rb protein, dependent on sequences that are homologous to domain 2 of ElA (DeCaprio et al., 1988; Dyson et al., 1989a, 1989b; Mtinger et al,, 1989). Given the observation that a mutation in domain 2 abolished the ability of ElA to dissociate the E2F complexes, and given the fact that ElA does interact with cellular proteins via these sequences, it is tempting to speculate that this is accomplished through a direct interaction of ElA with the EPF-associated factor, which could be one of the various cellular proteins known to be complexed with EIA, resulting in a displacement of E2F. Of course, this is only speculation and firmer conclusions must await an extensive analysis of ElA mutants so as to clearly link or unlink this new activity of ElA with the ability of ElA to associate with these cellular proteins. Nevertheless, our results do now provide a biochemical activity of transcriptional importance that is dependent on the ElA protein sequence found in the 243 amino acid 12s prod-

uct, the ElA protein essential for cell transformation, and thus consideration of the mechanisms by which ElA alters cell growth and leads to an oncogenic state must take this into account.

Cells and Virus All cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum. F9 cells were induced to differentiate by the addition of retinoic acid and CAMP as described previously (Young et al., 19S9). Wild-type Ad5, the E4 mutant dl366, and the EIA mutant dl312 were grown and purified as described previously (Nevins et al., 1980). Preparation of Extracts Procedures for the preparation been described (Raychaudhuri

of whole-cell and nuclear extracts have et al., 1987; Kovesdi et al., 1986).

E2F Binding Assaya Procedures for the assay of E2F binding by gel retardation have been described previously (Yee et al., 1987). E2 promoter-containing probes employed for the binding assays contained one or both of the E2F sites and did not contain the ATF site. The probes are EcoRI-Hindlll fragments released from plasmids described by Loeken and Brady (1989) derived from the cloning of appropriate oligonucleotides into pUCl8. The one-site probe is the same as the two-site probe except that in addition to the alteration of the ATF binding site, sequences between -36 and -45 are also altered, destroying the proximal E2F binding site. DOC lbatmmt DOC treatment was carried out essentially as described (Baeuerle and Baltimore, 1988). Extracts were incubated at 4OC in the presence of 0.6% DOC for 20 min and then NP40 was added to a final concentration of 0.7%. UV Croaa-Linking The procedure for specific labeling of E2F by UV cross-linking to a s*P-labeled and bromodeoxyuridine-substituted probe has been described previously (Yee et al., 1989). Glycerol Gradient Sedlmentatlon Whole-cell extracts prepared from L cells were used. Control extracts and extracts treated with or without DOC and adjusted to 4% (v/v) glycerol using buffer containing no glycerol. Five hundred micrograms of extracts (5 mglml) was layered onto 4.2 ml 5%-25% (v/v) glycerol gradients, The gradients were centrifuged in a SW60 Ti rotor at 55,000 rpm for 18 hr. At the end of the run, the tubes were punctured and 0.2 ml fractions were collected from the bottom. Plasmlds Hindlll-Pstl DNA fragments of cDNAs encoding wild-type sequences of either the 12s or 13s gene products of ElA were cloned at the Hindlll-Pstl sites of pGEM1. To obtain the EIA 128-928 mutant, a BstXl fragment from the wild-type 12s cDNA clone was replaced by a BstXl fragment from EIA-928 (gift of E. Moran, Cold Spring Harbor Laboratory) that contained the point mutation at nucleotide position 928. EIA-expressing plasmids for the transfection experiments were constructed by subcloning Hindlll-BamHI fragments from the pGEM1 clones described above into the plasmid pBCl2/CMV/IL2 (Neil1 et al., 1990) from which IL2 cDNA insert was removed by Hindlll-BamHl digestion. The pGEM1 plasmid containing E4 6/7 open reading frame cDNA sequences was described previously (Neil1 et al., 1990). In Vltm lhnslatlon of ElA and E4 Pfotelns The plasmids (1 ug) were linearized at the EcoRl site, and capped RNA was made using SP6 polymerase following the procedure described previously (Gilmartin et al., 1986). FtNAs were translated in vitro using the reticulocyte lysate translation kit from Promega Biotechnology. [srS]methionine-labeled full-length translation products were assayed by SDS-polyacrylamide gel and autoradiography.

Cell 668

In Vitro lanscrlptlon Transcription reactions were carried out essentially as described previously (Yee et al., 1989) with the following modification. Five microliters of whole-cell extract (8 mglml) from L cells was used instead of HeLa nuclear extract as a source of transcription factors and polymerase II. Reticulocyte lysate (2 ul) programmed with brome mosaic virus RNA or SP6 RNA encoding the 13s ElA gene product or SP6 RNA corresponding to E4 6/7 open reading frame was added along with 250 ng of circular plasmid, either pAlOCAT-EPF or pAlOCAT (Yee et al., 1989). After preincubation in a 50 ul reaction volume for 15 min at room temperature, nucleotides were added (500 urn each) and incubation was continued for 60 min at 3oOC. RNA products were analyzed by primer extension assays as described previously (Yee et al., 1989). Transfectlon Assays Transfections were performed as described previously using DEAEdextran procedures (Cullen, 1987). Dishes (80 mm) at 70% confluency were transfscted with a total of 3 pg of DNA. pAlOCAT-EPF plasmid (1 ug) and 0.1 ug of ElA-expressing plasmids along with pUC18 DNA were used in these experiments. Cells were harvested for 48 hr posttransfection, and CAT assays were performed as described previously (Simon et al., 1988). Acknowledgments We thank Dr. Elizabeth Moran for the kind gift of the ElA-928 mutant. P R. and S. 8. were supported by the Howard Hughes Medical Institute. This work was supported by a grant from the National Institutes of Health (GM26785). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

February

8, 1990; revised

April 27, 1990.

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Note

Raychaudhuri, P., Bagchi, S., Neill, S., and Nevins, J. R. (1990). Activation of the E2F transcription factor in adenovirus-infected cells involves ElA-dependent stimulation of DNA-binding activity and induction of cooperative binding mediated by an E4 gene product. J. Virol. 64, 2702-2710. Reichel, R., Kovesdi, I., and Nevins, J. R. (1967). Developmental control of a promoter-specific factor that is also regulated by the ElA gene product. Cell 48, 501-518. Reichel, R., Kovesdi, I., and Nevins, J. R. (1988). Activation isting cellular factor as a basis for adenovirus EIA-mediated tion control. Proc. Natl. Acad. Sci. USA 85, 387-390.

of a preextranscrip-

Reichel, R., Neill, S. D., Kovesdi, I., Simon, M. C., Raychaudhuri, I?, and Nevins, J. R. (1989). The adenovirus E4 gene, in addition to the ElA gene, is important for tfans-activation of E2 transcription and for E2F activation. J. Virol. 63, 3643-3650. Ricciardi, R. P, Jones, R. L., Cepko, C. L., Sharp, F! A., and Roberts, B. E. (1981). Expression of early adenovirus genes requires a viral encoded acidic polypeptide. Proc. Natl. Acad. Sci. USA 78, 6121-6125. Simon, M. C., Kitchener, K., Kao, H. T., Hickey, E., Weber, L., Voellmy, R., Heintz, N., and Nevins, J. R. (1987). Selective induction of human heat shock gene transcription by the adenovirus ElA gene products, including the 12s ElA product. Mol. Cell. Biol. 7, 2884-2890. Simon, M. C., Fisch, T. M., Benecke, B. J., Nevins. J. R., and Heintz, N. (1988). Definition of multiple, functionally distinct TATA elements, one of which is a target in the hsp70 promoter for ElA regulation, Cell 52, 723-729. Simon, M. C., Rooney, R. J., Fische, T., Heintz, N., and Nevins, J. R. (1990). ElA-dependent activation of the c-fos promoter requires the TATAA sequence. Proc. Natl. Acad. Sci. USA 8Z 513-517. Weeks, D. L.. and Jones, N. C. (1983). ElA control is mediated by sequences 5’ to the transcriptional viral genes. Mol. Cell. Biol. 3, 1222-1234.

of gene expression starts of the early

Whyte, P., Buchkovich, J. J., Horowitz, J. M., Friend, S. H., Raybuck, M., Weinberg, R. A., and Harlow, E. (1988). Association between an oncogene and an anti-oncogene: the adenovirus ElA proteins bind to the retinoblastoma gene product. Nature, 334, 124-129. Whyte. P, Williamson, N. M., and Harlow, E. (1989). Cellular targets transformation by the adenovirus ElA proteins, Cell 56, 67-75.

for

Added

in Proof

The work referred to throughout as Rooney et al., submitted, can now be updated as Rooney, R. J., Raychaudhuri, t?. and Nevins. J. R. (1990). Two transcription factors that recognize the same site, E4F and ATF, can be distinguished both physically and functionally: a role for E4F in EIA trans-activation. Mol. Cell. Biol., in press.