Cell, Vol. 61,

1217-1224,

June 29, 1990,Copyright 0 1990 by Cell Press

A Specific Member of the ATF Transcription Factor Family Can Mediate Transcription Activation by the Adenovirus Ela Protein Fang Liu and Michael R. Green Department of Biochemistry and Molecular Biology Harvard University Cambridge, Massachusetts 02138

Summary The adenovirus Ela protein stimulates transcription of viral early genes. Recent experiments indicate that Ela contains a transcriptional activating region, which functions when directed to a promoter. Because Ela is not a sequence-specific DNA binding protein, how it targets to viral promoters has been a question. Several of the viral early promoters contain one or more binding sites for ATFs, a family of cellular transcription factors. Here we show that Ela can function through a specific ATF protein, designated ATF-2. We provide evidence that Ela interacts with a discrete region of promoter-bound ATF-2, thereby positioning the Ela activating region at a viral promoter.

adenovirus early promoters? One possibility is that Ela interacts through its promoter binding region with a cellular protein(s) that is already bound to DNA. Several of the adenovirus early promoters contain binding sites for activating transcription factors (ATFs), a family of cellular transcription factors that possess homologous DNA binding domains (Hai et al., 1989). A number of studies have implicated the involvement of an ATF protein in the Ela transcriptional response (for review see Flint and Shenk, 1990). For example, the ATF sites in the adenovirus E4 promoter are required for Ela responsiveness, and an E4 DNA fragment containing two ATF sites can confer Ela inducibility onto a heterologous gene (Gilardi and Perricaudet, 1984, 1986; Lee and Green, 1987). We have recently isolated multiple, independent ATF cDNA clones (Hai et al., 1989). Here we show that the protein product of one of these ATF cDNA clones, designated ATF-2, can mediate transcription activation by Ela. We provide evidence that ATF-2 functions to recruit Ela to the promoter. Results

Introduction The 289-amino-acid adenovirus Ela protein is both a potent activator of viral gene transcription and an oncoprotein. Three regions, designated regions 1, 2, and 3, are highly conserved among Ela proteins from different adenoviruses and are responsible for the various activities of Ela (for review see Moran and Mathews, 1987). Regions 1 and 2 are required for cellular transformation (Lillie et al., 1986, 1987; Moran et al., 1986; Zerler et al., 1986; Schneider et al., 1987; Subramanian et al., 1988; Whyte et al., 1988a), whereas region 3 is necessary and sufficient for transcriptional activation of viral early genes (for review see Flint and Shenk, 1990). Recent protein fusion experiments have shown that the 46 amino acids that constitute region 3 contain two sep arable activities (Lillie and Green, 1989). The N-terminal portion of region 3 contains a transcriptional activating region, functionally analogous to that of a typical cellular activator (for reviews see Ptashne, 1988; Mitchell and Tjian, 1989). For example, the Ela activating region can substitute for the acidic activating region of the yeast activator GAL4. Conversely, mutations in the Ela activating region are compensated for by addition of a heterologous acidic activating region. The C-terminal portion of region 3 contains a “promoter binding” function, required to direct Ela to its natural targets, the adenovirus early promoters. Fulnction of both the transcriptional activating and promoter binding regions depends upon a central portion of region 3 that includes a metal binding site (Culp et al., 1988; Lillie and Green, 1989; K. J. Martin, J. W. Lillie, and M. R. G., submitted). Ela is not a sequence-specific DNA binding protein (Ferguson et al., 1985). How then does Ela target to the

An Experimental Strategy for Determining Whether a Protein Can Support an Ela Transcription Response The presence of endogenous ATFs in mammalian tissue culture cells prevents us from assaying the function of ATF cDNA clones directly. To circumvent this problem we fused different ATF cDNA clones, or control genes, to the GAL4 DNA binding domain, GAL4(1-147) (see Ptashne, 1988). We then tested in a cotransfection assay the ability of each GAL4 fusion protein to support Ela-mediated transcriptional activation using reporter genes that either contained or lacked GAL4 binding sites. We imagined that if a GAL4 fusion protein binds Ela, it will recruit Ela to a promoter bearing GAL4 binding sites (designated the GAL4 reporter). This will position the Ela activating region at the promoter, and transcription will be stimulated (Figure 1). A GAL4-RB Fusion Protein Can Support Ela-Mediated Transcription Activation To test this experimental design, we constructed a control GAL4 fusion containing a protein known to bind Ela. Previous studies have shown that Ela can interact with plOS-RB, the protein product of the retinoblastoma susceptibility gene (Whyte et al., 1988b). We analyzed the ability of a GAL4-FIB fusion protein to support an Ela response in the cotransfection assay diagrammed in Figure 1. Figure 2 shows that the GAL4 reporter is not activated upon cotransfection of plasmids expressing either GAL4(1147) GAL4-RB, or Ela. Nor is the GAL4 reporter activated following cotransfection of plasmids expressing GAL4(1147) and Ela. However, if the reporter contains GAL4 binding sites, transcription activation occurs when both GAL4RB and Ela are present. Thus, in this assay transcription

Cell 1218

+Ela

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Figure I. Experimental

Design

A protein that can bind Ela (white) is shown fused to the GAL4 DNA binding domain, GALC (1-147). Ela can thus be recruited to a promoter containing GAL4 binding sites, enabling the Ela transcriptional activating region (stippled oval) to stimulate transcription.

GAL4 Sites

activation requires a promoter that contains GAL4 binding sites and cotransfection of plasmids directing the synthesis of GAL4-RB and Ela. A GAL4-ATF-2 Fusion Protein Can Support Ela-Mediated Transcription Activation Figure 3A shows that when ATF-2, one of our previously isolated ATF cDNA clones (Hai et al., 1989) is fused to GAL4(1-147), the resulting fusion protein supports an Ela transcription response. (ATF-2 has been independently isolated by another group and designated CRE-BPl; Maekawaet al., 1989.) By itself, GAL4-ATF-2 fails to stimulate transcription from the GAL4 reporter, suggesting that

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Acti-

CHO cells were cotransfected with plasmids directing the synthesis of GAL4 derivatives (2 ug of DNA), Ela (4 trg of DNA), and a CAT reporter (4 ug of DNA) as indicated. The structure of the reporters, the GAL4 derivatives, and Ela are diagrammed below the autoradiogram. The reporters are G5ElSCAT and ElSCAT (Lillie and Green, 1989). GAL4(1-147) is expressed from the plasmid pSG424 (Sadowski and Ptashne, 1989). Ad5 Ela is expressed from the plasmid pSVEla (Smith et al., 1985).

it lacks an activating region. As with GAL4-RB, activation requires the presence of both GAL4-ATF-2 and Ela, as well as a promoter bearing GAL4 binding sites. Figure 38 shows that GAL4-ATF-2 can also support Ela-mediated transcription activation from the adenovirus E4 promoter if GAL4 binding sites have been added. As an additional test of ATF-2 function, we performed cotransfection experiments in human 293 cells. Human 293 cells contain high levels of endogenous Ela, which enables adenovirus early promoters to be efficiently expressed (see Berk, 1988). We reasoned that in 293 cells a GAL4 reporter could be activated by cotransfection of a plasmid directing the synthesis of ATF-2. The endogenous Ela in 293 cells would be recruited to the promoter by interaction with bound GAL4-ATF-2. Figure 3C shows that, as expected, in 293 cells activation depends only upon the presence of GAL4 binding sites in the reporter and expression of GAL4-ATF-2. ATF-2 Can Bind to the ATF Sites in the Adenovirus E4 Promoter The adenovirus E4 promoter contains a TATA box and at least three upstream ATF binding sites. These E4 ATF sites are essential for Ela inducibility (Gilardi and Perricaudet, 1984, 1986; Lee and Green, 1987). As a further test for the role of ATF-2 in E4 transcription, we asked whether ATF-2 can bind to the E4 ATF sites in vivo, as evidenced by increased E4 transcription. Since ATF-2 lacks an activating region (Figure 3) we constructed an ATF2-VP16 fusion protein by adding to ATF-2 the potent VP18 acidic activating region (Triezenberg et al., 1988; Sadowski et al., 1988). Figure 4 shows that ATF-2-VP16 significantly increases transcription from the E4 promoter, but not from an E4 promoter deletion mutant that lacks ATF binding sites. We conclude that ATF-2 can bind to the E4 ATF sites in vivo. Other GAL4 Derivatives Fail to Support an Ela Response The ability of GAL4 derivatives, such as GAL4-RB and GAL4-ATF-2, to support an Ela transcription response is highly specific (Figure 5). In particular, a full-length ATF-1,

ATF-2 Mediates 1219

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I Figure 4. ATF-2 Can Bind to ATF Sites within the E4 Promoter In Vivo

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CHO cells were cotransfected with a pECE expression plasmid directing the synthesis of an activator (1 pg of DNA) and a CAT reporter (1 ug of DNA). The structures of the reporters and ATF-2 derivatives are diagrammed below the autoradiogram. -

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Fusion Protein Can Mediate Transcription

(A]I As in Figure 2 except that GAM-ATF-2 is used instead of GAL4RB. GAL4-ATF-2 is expressed from pSG424-ATF-2 plasmid, with ATF2 amino acids l-505 fused in-frame to GAL4(1-147) in the pSG424 velctor. (B), As in (A) except that G5E4A-38CAT and E4A-38CAT were used as reporters. Their structures are shown below the autoradiogram. (C]I The reporter (5 ug of DNA) and a plasmid directing the synthesis of the indicated GAL4 derivative (5 pg of DNA) were cotransfected into 29.3 cells using the calcium-phosphate procedure. GlElBCAT and ElBCAT (Lillie and Green, 1989) were used as reporters. The structures of the reporters are indicated below the autoradiogram. Both

another member of the ATF family (Hai et al., 1989) does not support an Ela response in our assay. (GAL4-ATF-1 and GAL4-ATF-2 are expressed at comparable levels; Figure 8B, lanes 1 and 2.) In addition, a number of previously characterized GAL4 derivatives (Ptashne, 1988 and reference therein) do not mediate activation by Ela. These GAL4 derivatives contain acidic activating regions of various strengths and therefore stimulate transcription from the GAL4 reporter, but this level of transcription is not significantly augmented by addition of Ela. The N-Terminal Region of ATF-2 Is Required to Support Ela Responsiveness To identify the region of ATF-2 required to support an Ela response, we determined the activity of several GAL4ATF-2 derivatives. As summarized in Figure 8A, progressive deletion of the first 143 amino acids of ATF-2 destroys its ability to support Ela inducibility. For example, GAL4ATF-2 (350-505), which contains the complete ATF-2 DNA binding domain, does not support an Ela response. Figure 8B indicates that the various GAL4-ATF-2 derivatives are expressed at comparable levels; the minor variations in levels are not sufficient to account for their differences in activity. We conclude that the N-terminal portion of ATF2 (amino acids l-143) which is separate from the DNA binding domain, is required to support an Ela response. Effect of Ela Mutations on Ela-Mediated Activation by GAL4-RB and GALA-ATF-2 To test whether activation requires interaction between Ela and the DNA-bound GAL4 derivative, we analyzed four previously characterized Ela mutants (Figure 7, bottom). Mutant 936 changes amino acid 126 in region 2 from Glu to Gly. Ela-936 is transformation defective, but acti-

GAL4(1-147) and GAL4-ATF-2 are expressed Rous sarcoma virus long terminal repeat.

under the control of the

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5 GAL4 Sites

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Figure 5. Failure of Other GAL4 Fusion Proteins to Support an Ela Response CHO cells were cotransfected with plasmids directing the synthesis of GAL4 derivatives (2 wg), Ela(4 Kg), and the G5ElBCAT reporter (4 ug). GAL4-ATF-1 and GAL4-ATF-2 are full-length ATF-1 and ATF-2 cDNA clones fused in-frame to GAL4(1-147) in the pSG424 vector, respectively. GAL4-WT is the wild-type GAL4 protein, amino acids 1-881, expressed from pSG4; GAL4-B17 is an acidic activating region from the Escherichia coli genome fused to GAL4(1-147) and is expressed from pSGB17 (Sadowski et al., 1988). GAL4-I is GAL4 amino acids l-238, containing the GAL4 activating region I (148-238) cloned in pECE; GAL4-I+II is GAL4 amino acids l-238 and 768-881, containing GAL4 activating regions I (148-238) and II (768-881) cloned in pECE; GALC AH encodes an acidic 15-amino-acid sequence that can form a putative amphipathic helix fused in-frame to GAL4(1-147) in pSG424 (gifts of Ivan Sadowski). In this experiment, the incubation times of the CAT assays were varied so that all the reactions were kept in the linear range.

vates transcription of adenovirus early promoters as effectively as wild-type Ela (Lillie et al., 1988). The transformation deficiency of Ela-936 is presumably due to its inability to interact with plO!XB, which binds to Ela regions 1 and 2 (Whyte et al., 1989). Figure 7 shows that Ela-936 fails to activate transcription in the presence of GAL4-FIB, but can activate transcription in the presence of GAL4-ATF-2. Likewise, in-frame deletions of region 1 or region 2 also eliminate activation in the presence of GAL4-FIB but do not affect the response supported by GAL4-ATF-2 (data not shown). The second mutation, 1098 (amino acid 180 Gly to Asp), destroy8 the promoter binding activity of Ela region 3 (Lillie and Green, 1989). Ela-1098 can activate transcription in the presence of GAL4-FIB but not in the presence of GAL4-ATF-2. These results are expected since the regions of Ela that interact with ~105RB, regions 1 and 2, lie outside of region 3. Finally, Ela mutants A148-152 and 171AM74A, which destroy the activating region of Ela (Lillie and Green, 1989; K. J. Martin, J. W. Lillie, and M. R. G., submitted), fail to activate transcription in the presence of either GAL4-RB or GAL4ATF-2. On the basis of these results we conclude that activation requires that Ela contains an activating region and is able to interact with the promoter-bound protein. Additional Evidence for Interaction between Ela and ATF-2 As further evidence for an interaction between Ela and ATF-2, Figure 8 shows that the functions of these two pro-

teins can be switched. ElaAN149, an Ela derivative lacking its activating region but maintaining its promoter binding activity, was fused to the GAL4 DNA binding domain. Thus, by itself the GAL4-ElaAN149 protein fails to stimulate transcription. ATF-2-VP16, which alone cannot bind to the GAL4 reporter, also does not activate transcription. However, cotransfection of GAL4-ElaAN149 and ATF2-VP16 expressing plasmids results in activation of the GAL4 reporter. (The VP16 activating region, by itself, cannot stimulate transcription; data not shown.) As expected, ATF-2, which lacks an activating region, fails to stimulate transcription. In this experiment, therefore, DNA-bound Ela recruits ATF-2-VP16 to the promoter, thereby enabling the acidic VP16 activating region to stimulate transcription. In essence, ATF-P-VP16 functions as the fransactivator. Discussion Our previous study using GAL4-Ela fusion proteins provided evidence that Ela region 3 contains both a transcriptional activating function and a promoter binding activity (Lillie and Green, 1989). The result8 presented here performed with the wild-type 289-amino-acid Ela protein strengthen and extend these previous conclusions. First, we show that when an appropriate protein is tethered to the promoter, the wild-type 289-amino-acid Ela protein can stimulate transcription. Second, we show that transcriptional activation is impaired by various mutations within Ela that interfere with its ability to associate with the particular DNA-bound protein. Third, we identify a specific ATF protein, ATF-2, that can mediate an Ela response and can bind to the ATF sites within a natural Ela target, the adenovirus E4 promoter. It is often stated that Ela is a “promiscuous” activator, stimulating transcription from diverse promoters (see Berk, 1986; Flint and Shenk, 1990). How can we rationalize our finding that a specific ATF protein mediates an Ela response with this notion? We suggest several possibilities. First, the N-terminal ATF-2 protein motif responsible for Ela inducibility may be present in other cellular DNA binding proteins. For example, acidic activating regions are present in a wide variety of otherwise dissimilar cellular transcription activators (for reviews see Ptashne, 1988; Mitchell and Tjian, 1989). Furthermore, Eta’s promiscuity may result from its association with protein motifs other than that present in ATF-2. Second, we have described one way in which Ela may be recruited to the promoter: through interaction with a bound ATF protein. Our results do not exclude other mechanisms for recruitment of Ela. For example, it has been reported that Ela can bind to DNA nonspecifically (Chatterjee et al., 1988), which may explain why some promoters that lack ATF sites are Ela inducible. Third, our studies have specifically analyzed transcription activation by Ela region 3. The Ela inducibility of some promoters is by a region 3-independent mechanism (Simon et al., 1987; Zerler et al., 1987; KaddurahDaouk et al., 1990). Perhaps some of the Ela-inducible promoters that lack ATF sites are not activated by a region 3-dependent mechanism. Finally, the extent of Ela’s

ATF-2 Mediates 1221

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Figure 6. Deletion Analysis of ATF-2 (A) Transcriptional activity. The ATF-2 N-terminal deletion mutants illustrated were fused to GAL4(1-147). Some of the protein motifs within ATF-2 are indicated. CHO cells in a 100 m m plate were cotransfected with plasmids directing the synthesis of GAL4 derivates (2 pg of DNA), Ela (4 pg of DNA), and the G5ElBCAT reporter (4 pg of DNA). CAT activity was quantitated by scintillation counting and indicated on the right. (6) lmmunoblot analysis. Proteins from one-third of the transfected cells in (A) were fractionated on a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose filter. GAL4 derivatives were detected by incubation with an anti-GAL4(1-147) antibody, followed by incubation

with [1125]proteinA. The ATF-2 amino acids present in the GAL4ATF-2 derivative are indicated above each lane.

promiscuity is not clear. The low levels of transcription in the absence of Ela has made accurately quantitating Ela

inducibility difficult. In our experiments Ela is not acting promiscuously: the presence of GAL4 sites has an enormous effect on the level of transcription in the presence of Ela. Furthermore, a relatively small deletion of ATF-2 destroys the ability of GAL4-ATF-2 to support an Ela response. Ela Functions by Interacting with Cellular Proteins In addition to transcription activation, Ela has a number of other activities, which are related to its ability to function as an oncoprotein. These transformation-related activities are carried out by highly conserved regions 1 and 2. Previous studies have identified a number of cellular polypeptides with which Ela interacts (Yee and Branton, 1985; Harlow et al., 1988). Several of these cellular proteins, such as ~105RB, directly interact with Ela regions 1 and 2; these interactions are important for Ela’s ability to transform cells (Whyte et al., 1989). Here we provide evidence that there is a region 3-specific interaction between Ela and ATF-2. At present, however, we cannot rule out that the interaction between Ela and ATF-2 is not direct, but occ:urs, for example, through additional cellular proteins. In any case, all Ela activities appear to be carried out through associations with cellular proteins.

Other Viral Transcription Activators It is characteristic for animal viruses to encode proteins that facilitate expression of viral genes. Several of these viral transcription activators appear to work in a manner similar to Ela. For example, the HSV-1 VP18 protein (Sadowski et al., 1988), hepatitis I3 pX protein (Seto et al., 1990), pseudorabies immediate-early protein (K. J. Martin, J. W. Lillie, and M. R. G., unpublished data), and HTLV-1 tax protein (J. W. Lillie, unpublished data) can be shown to contain transcriptional activating regions when fused to heterologous DNA binding domains. Moreover, none of these viral proteins are sequence-specific DNA binding proteins and are apparently directed to the promoter by other mechanisms. For example, the HSV-1 VP16 protein targets to viral promoters by direct interaction with Ott-1, a cellular factor that binds to viral DNA elements (Gerster and Roeder, 1988; Stern et al., 1989); the pseudorabies immediate-early protein binds DNA promiscuously (Cromlish et al., 1989); and the HTLV-1 tax and hepatitis I3 proteins appear to function through ATF and AP-2, respectively (Park et al., 1988; Seto et al., 1990). The ATF Family of Cellular Transcription Factors In addition to their presence in adenovirus promoters, ATF binding sites are found in a wide variety of other viral and cellular promoters (Lin and Green, 1988 and reference

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therein). Although all ATF proteins can bind to ATF sites (Hai et al., 1989) we provide evidence that different ATF proteins are functionally distinguishable. For example, ATF-2 but not ATF-1 can support an Ela transcription response. Correspondingly, the N-terminal region of ATF-2, which is required for Ela responsiveness, is absent from ATF-1. Our preliminary results indicate that ATF-2 cannot efficiently support a CAMP-inducible transcription response, whereas CREB, another ATF protein, can (Hoeffler et al., 1988; Gonzalez et al., 1989; Gonzalez and Montminy, 1989). The failure of ATF-2 to support efficiently CAMP inducibility is presumably because the region of CREB implicated in this response is absent from ATF-2. Thus, the various members of the ATF family bind to similar DNA sequences but have different transcriptional effector functions. Accordingly, the only significant amino acid similarity among these proteins lies within their DNA

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The 13s mANA and its product, the 289-amino-acid Ela protein, are schematically represented. 1,2, and 3 represent the three highly conserved regions of the Ela protein. The positions of the Ela mutations are indicated. The Ela derivative used is indicated above each lane. pH3G936 and pH3GlOQE is in the background of pH3G-13s Ela, which encodes the 289-amino-acid Ela protein and a portion of an El6 gene product (Lillie et al., 1986). In lane 1 pH3G-13s was used. A148-152 deletes five internal amino acids from region 3, and 171A/174A changes the cysteines at amino acids 171 and 174 to alanines (K. J. Martin, J. W. Lillie, and M. R. G., submitted). A148-152 and 17tA/174A are in the background of the Ela 13s cDNA (Svensson et al., 1983). In lane 4 the Ela 13s cDNA plasmid was used.

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(A) Schematic diagram. Notice that the stippled oval representing the transcriptional activating region of Ela is absent. (6) CAT assay. CHO cells were cotransfected with plasmids directing the synthesis of GAL4-ElaAN149 (1 ug of DNA), an ATF-2 derivative (1 ug of DNA), and the reporter (3 ug of DNA) as indicated. GAL4ElaAN149 contains Ela amino acids 150-223 fused in-frame to GAL4* (1-147) (Lillie and Green, 1989).

binding regions (Hai et al., 1989). The differential functions of ATF proteins may explain the presence of ATF binding sites in various promoters that are regulated by different physiological inducers. The API/c-Jun transcription factor family is related to the ATF family (Hai et al., 1989). Recent experiments indicate that different Jun proteins also are functionally distinguishable (Chiu et al., 1989; Schtitte et al., 1989). Function of ATF-2 Our results raise questions concerning the normal cellular role of ATF-2. It seems reasonable to assume that ATF-2 is involved in transcriptional activation. However, our protein fusion experiments indicate that ATF-2 lacks a constitutive activating region, How then is ATF-2 converted into an activator? The difficulty in answering this question is that we currently do not know the conditions under which ATF-2 functions as an activator, nor the cellular genes upon which ATF-2 acts. Nonetheless, we offer two possibilities. First, ATF-2 may be converted into an activator through a covalent modification(s). In fact, there is evidence that the activation potential of CREB is regulated by phosphorylation (Yamamoto et al., 1988; Gonzalez and Montminy, 1989). Second, a cellular factor may bind to

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Ela Transcription

Activation

ATF-2 and supply the transcriptional activating function. This putative cellular factor would be the cellular counterpart of the viral Ela protein. Experimental Procedures Isolation of Full-Length ATF-2 and AT&l cDNA Clones ATF-2 was initially isolated as a partial cDNA encoding amino acids 95-505 (Hai et al., 1989). The 5’ end of ATF-2 cDNA was obtained by the polymerase chain reaction. The first strand cDNA was synthesized using 15 pg of cytoplasmic RNA from MG63 cells and a 3’ primer (5’GAGGGGATAAATCTAGAGGC-3’). The amplification was performed with this 3’ primer and a 5’ primer (5’-TGTGATAAGTTATTCAACTT~37. The sequence of the 5’ end primer was based on the 5’ untranslated region of the published CRE-BP1 sequence (Maekawa et al., 1969), which is identical to ATF-2 except for two amino acid differences (Hai et al.. 1989). A full-length ATF-1 cDNA was isolated by screening an MG63 human osteosarcoma cDNA library (a gift of Chen-Ming Fan and Tom Maniatis) with a uniformly labeled RNA probe derived from the previously described partial ATF-1 cDNA clone (Hai et al., 1989). The fulllength ATF-1 cDNA clone encodes a 273-amino-acid protein, and the 5’ untranslated region is preceded by two TGA stop codons. The complete nucleotide sequence of ATF-1 will be presented elsewhere. Constructions pSG424-ATF-2(1-505), pSG424-ATF-2(34-505), pSG424-ATF-2(109505), pSG424-ATF-2(144-505), pSG424-ATF-2(196-505). and pSG424-ATF-2(350-505) were constructed by inserting the appropriate ATF-2 DNA fragment in-frame to the GAL4(1-147) sequence in the vector pSG424. pRSV424 directs the synthesis of GAL4(1-147) from the RSV promoter. pRSV424-ATF-2 contains a DNA fragment encoding A‘TF-2 amino acids l-505 inserted in-frame to the GAL4(1-147) sequence in pRSV424. The junctions of all GAL4 fusion proteins were confirmed by DNA sequencing. pATF-2 contains a DNA fragment encoding ATF-2 amino acids l-505 in pECE (Ellis et al., 1986). pATF-P-VP16 contains the DNA fragment coding for ATF-2 (I-502) fused in-frame to VPl6(413-490) in the pECE vector. pE4CAT contains E4 DNA sequences from -240 to +38 inserted upstream of the CAT gene (Lillie and Green, 1989). pE4A-38CAT contains E4 DNA sequences from -38 to +38 inserted upstream of the CAT gene. pG5E4A-38CAT contains five GAL4 binding sites inserted upstream of the E4 sequences in pE4A-38CAT. Transfections Chinese hamster ovary (CHO-DUKX) cells were grown in alpha minimal essential medium with nucleotides plus 10% fetal bovine serum and were split I:8 24 hr prior to transfection. DNA was introduced into cells by the the DEAE-dextran technique (Cat0 et al., 1986). The concentration of DEAE-dextran was 250 Kg/ml. In a particular experiment the total amount of DNA in each transfection was identical. After 12 hr, cells were treated with dimethyl sulfoxide. followed by addition of choloroquine for 2.5 hr. Forty-eight hours post-dimethyl sulfoxide shock, cells were collected and assayed for CAT activity as described (Gorman et al., 1982). Human 293 cells were maintained in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum and were split I:6 20 hr prior to transfection. Cells were refed 4 hr before addition of the calciumphosphate precipitate (Wigler et al.. 1978). The cells were incubated with calcium-phosphate precipitate for 18 hr; 36 hr following removal of the precipitate, the cells were harvested and CAT activity assayed. Acknowledgments We gratefully acknowledge James W. Lillie for the Ela mutants before publication, William J. Coukos for the GAL4-RB fusion plasmid, ChenMing Fan and Tom Maniatis for the MG63 cDNA library, Ivan Sadowski for the anti-GAL4(1-147) antibody and various GAL4 derivative plasmids, Ed Harlow for the Ela region 1 and region 2 deletion mutants, and Michael F. Carey for helpful suggestions. We thank members of tha Green and Ptashne laboratories for critical reading of the manu-

script. This work was supported by grants from the National Institutes of Health to M. R. G. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adverfisement” in accordance with 16 USC. Section 1734 solely to indicate this fact. Received March 14, 1990; revised April 19, 1990. References Berk, A. J. (1986). Adenovirus Annu. Rev. Genet. 20, 45-79.

promoters

and EIA transactivation.

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