MOLECULAR AND CELLULAR BIOLOGY, June 1992, p. 2599-2605 0270-7306/92/062599-07$02.00/0

Vol. 12, No. 6

Copyright ©) 1992, American Society for Microbiology

The hsp7O Gene CCAAT-Binding Factor Mediates Transcriptional Activation by the Adenovirus Ela Protein LYNETTE S. Y. LUM, STEPHANIE HSU, MICHAEL VAEWHONGS, AND BARBARA WU* Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208 Received 16 December 1991/Accepted 12 March 1992

Expression of the human hsp70 gene is cell cycle regulated and is inducible by both serum and the adenovirus Ela protein (K. Milarski and R. Morimoto, Proc. NatI. Acad. Sci. USA 83:9517-9521, 1986; M. C. Simon, K. Kitchener, H.-T. Kao, E. Hickey, L. Weber, R. Voellmy, N. Heintz, and J. R. Nevins, Mol. Cell. Biol. 7:2884-2890, 1987; B. Wu, H. Hurst, N. Jones, and R. Morimoto, Mol. Cell. Biol. 6:2994-2999, 1986; B. Wu and R. Morimoto, Proc. Natl. Acad. Sci. USA 82:6070-6074, 1985). This regulated expression is predominantly controlled by the CCAAT element at position -70 relative to the transcriptional initiation site (G. Williams, T. McClanahan, and R. Morimoto, Mol. Cell. Biol. 9:2574-2587, 1989; B. Wu, H. Hurst, N. Jones, and R. Morimoto, Mol. Cell. Biol. 6:2994-2999, 1986). A corresponding CCAAT-binding factor (CBF) of 999 amino acids has recently been cloned and shown to stimulate transcription selectively from the hsp70 promoter in a CCAAT element-dependent manner (L. Lum, L. Sultzman, R. Kaufman, D. Linzer, and B. Wu, Mol. Cell. Biol. 10:6709-6717, 1990). We report here that the first 192 residues of CBF, when fused to the DNA-binding domain of the heterologous activator GAL-4, are necessary and sufficient to mediate Eladependent transcriptional activation. Ela and CBF exhibit complex formation in vitro, suggesting that an in vivo interaction between these proteins may be relevant to the well-characterized Ela-induced transcriptional activation of the hsp70 promoter. The specificity and rate of transcriptional initiation by RNA polymerase II are controlled by sequence-specific DNA-binding transcription factors (for a review, see reference 14). These DNA-bound factors appear to function by interacting with specific target proteins that in turn interface with the general transcriptional apparatus (17, 18). Viral trans-activators that are themselves unable to bind DNA, such as adenovirus Ela, could potentially gain promoter specificity by interacting with sequence-specific DNA-binding transcription factors (10, 23). The human hsp7O promoter is transcriptionally activated by adenovirus Ela (21, 29), a 289-amino-acid protein that is both a potent transcriptional activator of specific viral and cellular genes and an oncoprotein (for reviews, see references 3 and 15). This regulated expression is predominantly controlled by the basal promoter containing sequences upstream from position -70 relative to the transcriptional initiation site (5, 28). While no single mutation within the basal promoter abolishes the response to Ela, the CCAAT and TATA elements play prominent roles. A fragment from -100 to -44, containing the CCAAT element and adjacent sequences, confers Ela responsiveness on a heterologous promoter (28). In the context of this chimeric promoter, the CCAAT element is essential for detectable basal and Elainduced expression (28). The importance of the TATA element is demonstrated by the loss of Ela responsiveness when the hsp70 TATA element is replaced by the simian virus 40 TATA element (20). While the hsp70 TATA element plays an important role in the context of the hsp70 promoter, it is not sufficient to confer Ela inducibility on heterologous promoters (1). These data argue for the interplay between the CCAAT and TATA elements for proper Ela-induced

In our effort to understand the molecular mechanisms governing the serum-stimulated (31), cell cycle-regulated (13), and Ela-induced transcription of the hsp70 gene, we have recently isolated a cDNA encoding a CCAAT-binding factor (CBF) that binds to the CCAAT element of the hsp70 promoter in vitro and selectively stimulates transcription from the hsp70 promoter in vivo (12). We report the identification of two regions of the CBF polypeptide, residues 98 to 192 and 438 to 534, that are required for transcriptional stimulation of the hsp7O promoter in COS cells. When appended to the DNA-binding domain of yeast activator GAL4, only one region, residues 1 to 192, is required to mediate Ela-dependent transcriptional stimulation from a synthetic promoter containing five GAL4-binding sites in CHO cells. An association of Ela and CBF residues 1 to 192 can be detected in vitro. We suggest that Ela-induced transcription from the hsp70 promoter in vivo is achieved in part by the interaction of Ela and CBF.

MATERIALS AND METHODS Bacterial expression. pGEX-CBF(1-192) was constructed in plasmid pGEX2T (22) and encodes the fusion protein GST-CBF(1-192), containing residues 1 to 192 of CBF. Induction and purification of GST and GST-CBF(1-192) were performed as previously described (12) with the exception that the proteins were not eluted from the glutathioneagarose beads. Expression constructs and transfection. COS and CHO cell transfections (2, 8) and chloramphenicol acetyltransferase (CAT) assays (4) were performed as previously described. Radioactivity was measured by phosphor image (Molecular Dynamics) analysis, and enzymatic activity was calculated as the percentage of ['4C]chloramphenicol converted to acetylated forms. The hsp70-CAT reporter gene contains the hsp70 pro-

expression.

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moter and 5' untranslated sequences from -100 to +150 fused to a bacterial CAT gene; this construct has been described elsewhere (30). The G5CAT reporter gene contains five GAL4-binding sites inserted upstream of the Elb TATA element, and the Ela expression plasmid pCE has been previously described (6, 10). Mt-CBF and Mt-CXM encode CBF residues 1 to 999 and 1 to 710, respectively (12). Mt-CSSP, which has a carboxyterminal deletion, encodes CBF residues 1 to 192 and was constructed by inserting the 583-bp PstI-SspI fragment of Mt-CXM into the Mt vector. Mt-CBFA1, which has an internal deletion, encodes an in-frame deletion derived by removing the internal 741-bp SspI fragment of Mt-CBF. Mt-CBFAH3, which has an amino-terminal deletion, encodes CBF residues 98 to 999 and was constructed by replacing the 304-bp PstI-HindIII fragment of Mt-CBF with an oligonucleotide adaptor containing a methionine initiator codon. Mt-CBFA1/R, a double-deletion construct derived from Mt-CBFAH3 by removing the downstream 1.5-kb EcoRI fragment, encodes CBF residues 98 to 534. MtCBFAH/A1/R, a triple-deletion construct derived from MtCBFAH/R by removing the internal 741-bp SspI fragment, encodes CBF residues 98 to 192 and 438 to 534. GAL-CBF encodes full-length CBF inserted downstream of GAL residues 1 to 147 in the vector pSG424 (8). GALCXM encodes a protein with a carboxy-terminal deletion and was constructed by inserting the coding region from Mt-CXM into pSG424. GAL-CXR encodes a protein with a carboxy-terminal deletion and was constructed by inserting the coding region of Mt-CXR into pSG424. GAL-CBF-AH3 encodes a protein with an amino-terminal deletion and was constructed by transferring the 2.9-kb HindIII-XbaI fragment of Mt-CBF into pSG424. GAL-CBF-HAP encodes a protein with an internal deletion and was constructed by replacing the 1.9-kb HindIII-PstI fragment of GAL-CBF with an in-frame oligonucleotide adaptor. GAL-CBF-AX and -AR encode proteins with amino-terminal deletions and were constructed by ligating the 1.5-kb EcoRI-XbaI or the 2.0-kb XhoI-XbaI fragment of Mt-CBF into pSG424. GAL-CBF-A1 encodes a protein with an internal deletion and was constructed by deleting the 741-bp SspI fragment from GALCBF. GAL-CSSP encodes a protein with a carboxy-terminal deletion and was constructed by deleting the 741-bp SspI and the 1.8-kb SspI-XbaI fragments of GAL-CBF. All constructs were confirmed by DNA sequencing. The expression of each construct was monitored by both immunoprecipitation of pulse-labeled samples and immunoblot assays of unlabeled samples with either anti-CBF (12) or anti-GAL4 (a gift from Doug Last) antisera. In vitro transcription-translation and binding assays. Ela 13S and 12S cRNAs were synthesized from plasmids Sp13S and Sp12S (19) and translated in rabbit reticulocyte lysates (Promega) by following the manufacturer's instructions. After in vitro translation in the presence of [35S]methionine and [35S]cysteine (trans-label, ICN), 4 ,ul of each translation reaction mixture was diluted with 100 ,ul of MJ buffer (25 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid]-KOH, pH 7.8, 5 mM MgCl2, 0.1 mM ZnC12, 0.1 mM EDTA, 2 mM dithiothreitol, 10 mg of bovine serum albumin per ml, 110 mM NaCl) and applied to affinity beads containing 5 ,ug of GST, GST-CBF(1-192), or GST-Topoisomerase (a gift from A. Mondragon). The slurry was incubated at 4°C for 2 h, and the beads were repeatedly washed with MJ buffer supplemented with 0.1% Nonidet P-40. Bound material was eluted by boiling the beads in sodium dodecyl sulfate

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FIG. 1. Two regions of the CBF polypeptide are needed to stimulate transcription from the hsp70 promoter. (A) Enzymatic CAT activity expressed from the hsp70-CAT reporter gene in COS cells cotransfected with the following constructs (see diagram in panel B): lane 1, Mt vector; lane 2, Mt-CBF; lane 3, Mt-CXM; lane 4, Mt-CSSP; lane 5, Mt-CBF-A1; lane 6, Mt-CBFAH3; lane 7, Mt-CBFAH/R; and lane 8, Mt-CBFAH/A1/R. (B) Schematic representation of CBF and derivatives. Hatched bars indicate residues retained in each construct. Numbers correspond to amino acid residue positions. Relative activities are averages of duplicate

experiments.

(SDS)-sample buffer and then was subjected to SDS-polyacrylamide gel electrophoresis (PAGE). RESULTS Regions of the CBF polypeptide required to stimulate transcription from the hsp7O promoter in COS cells. We have previously shown that CBF stimulates transcription from a cotransfected hsp70-CAT reporter gene in COS monkey cells (12). A series of CBF cDNA deletions were constructed to identify the regions of the CBF polypeptide that are essential for stimulating transcription from the hsp70 promoter. A construct with a carboxy-terminal deletion to residue 710 (CXM; Fig. 1A, lane 3) was able to stimulate transcription from the hsp70-CAT reporter gene in transfected COS cells. As measured by enzymatic CAT activity, CXM resulted in a 6-fold stimulation, compared with an

hsp70 CBF MEDIATES TRANSCRIPTIONAL ACTIVATION BY Ela

VOL. 12, 1992 B. COS cells

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FIG. 2. Cotransfected CBF or Ela exhibits differential transcriptional activation capabilities on the hsp70 promoter in a cell linedependent manner. Enzymatic CAT activity expressed from the hsp70-CAT reporter gene in CHO (A) and COS (B) cells cotransfected with the indicated constructs.

11-fold stimulation with CBF. The loss of transcriptional stimulation was observed when the carboxy-terminal deletion was extended to residue 192 (CSSP; Fig. 1A, lane 4). Since this construct results in an activity that is less than twofold greater than that seen with the vector alone, it is likely due to variability of the assay conditions rather than a significant contribution to transcription. A construct with an amino-terminal deletion of 97 residues (CBFAH3; Fig. 1A, lane 6), as well as one with an internal deletion of residues 192 to 438 (CBFA1; Fig. 1A, lane 5), remained capable of stimulating transcription from the hsp70 promoter. These data suggest that three regions of the CBF polypeptide, amino acid residues 1 to 97, 193 to 437, and 711 to 999, are dispensable for stimulating transcription from the hsp70 promoter. This prediction was confirmed and the localization was refined by making a construct with a double deletion (CBFAH/R; Fig. 1A, lane 7) and one with a triple deletion (CBFAH/A1/R; Fig. 1A, lane 8) of these nonessential regions. These mutants define two essential regions as lying within residues 98 to 192 and 438 to 534. The assay depicted in Fig. 1 requires both DNA-binding and transcriptional activation functions. Loss of activity can result from loss of either function. If DNA-binding and transcriptional activation functions reside on separable domains of CBF, then it is likely that each of the two essential regions defined will correspond to one of these functions. Localization of the transcriptional activation function of CBF is addressed below. Sequence comparisons of these regions did not reveal any striking similarity to DNA-binding or trans-activation domains of known transcription factors. Cell line-dependent activity of CBF and Ela affecting transcription from the hsp7O promoter. Unlike transfections into COS cells (Fig. 2B, lanes 3 and 4), cotransfection of CBF and hsp70-CAT into CHO cells does not result in increased hsp70 promoter activity (Fig. 2A, lanes 3 and 4). On the contrary, increasing the amount of transfected CBF results in the repression of hsp70 promoter activity (data not shown). Conversely, cotransfection of Ela and hsp7o-CAT results in stimulation of hsp70 promoter activity in CHO cells (Fig. 2A, lanes 1 and 2) but not in COS cells (Fig. 2B, lanes 1 and 2). Since Ela remains capable of promoting transcription from the adenovirus E3 promoter in COS cells (data not shown), the trans-activator activity of Ela is functional in COS cells. The implications of these observations are discussed below. Because of the restricted pheno-

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types, subsequent experiments on Ela trans-activation were conducted with CHO cells. CBF mediates Ela-dependent transcriptional stimulation. Previous studies of the hsp7o promoter concluded that CCAAT and TATA elements play an important role in Ela responsiveness (20, 24, 28). We reasoned that CBF could serve to recruit Ela to the hsp70 promoter, since transfer of hsp7O promoter sequences from -70 to -40 (containing the CCAAT element) onto a heterologous promoter confers Ela induction (28). We sought to determine whether CBF mediates Ela-induced trans-activation in CHO cells. To distinguish between the endogenous Ela-responsive activity and transfected CBF, synthetic activators composed of the DNA-binding domain of yeast GAL4 fused to various regions of CBF were constructed. The activities of these GAL-CBF fusions were monitored by their ability to stimulate transcription from G5CAT (10), a reporter gene containing five GAL4-binding sites. Ela alone affords a slight (twofold) stimulation of G5CAT (Fig. 3A, lanes 1 and 2). As expected, since transfected CBF is unable to stimulate transcription of hsp-CAT in CHO cells (Fig. 2B), GAL-CBF is also unable to promote transcription of G5CAT in the cell background (Fig. 3A, lane 3). However, combining Ela and GAL-CBF results in a 15-fold enhancement of transcription of G5CAT (Fig. 3A, lane 4). The synergy is specific, as Ela does not enhance the transcriptional activity of GAL4 (Fig. 3A, lanes 5 and 6). A series of deletions of the CBF moiety in GAL-CBF were constructed to determine the region of CBF required to mediate Ela-dependent transcriptional stimulation. The Ela-dependent stimulation is sensitive to amino-terminal deletions of the CBF moiety in GAL-CBF. For example, a deletion of 97 residues (GAL-CBF-AH3; Fig. 3B, lane 5) retains only one-third of the activity of GAL-CBF. Further deletions of 362 residues (GAL-CBF-AX; Fig. 3B, lane 7) and 534 residues (GAL-CBF-AR; Fig. 3B, lane 9) result in the complete loss of activity. In contrast, carboxy-terminal deletions to residue 710 (GAL-CXM; Fig. 3B, lane 3) or to residue 534 (GAL-CXR; Fig. 3B, lane 4) result in a 2.5- or 6.4-fold increase in activity, respectively, compared with GAL-CBF. Internal deletions further defined the extent of the amino terminus of CBF required for Ela trans-activation. A deletion of residues 98 to 746 (GAL-CBF-HAP; Fig. 3B, lane 6) retains only one-fifth of the activity of GAL-CBF, while a deletion of residues 193 to 437 (GAL-CBF-A1; Fig. 3B, lane 8) retains activity comparable to that of GAL-CBF. Since approximately equivalent levels of the polypeptides encoded by each construct are detected in transfected CHO cells by immunoblot assays with an anti-GAL4 antiserum (data not shown), it is not readily apparent why some constructs have more activity than GAL-CBF. The deletion analyses suggest that while residues 1 to 97 are necessary, they are not sufficient to mediate Ela-activated transcription, and that the first 192 residues are sufficient to mediate Ela-activated transcription (GALCSSP; Fig. 3B, lane 10). As shown in Fig. 4 (lanes 1 to 3), neither GAL-CSSP, which contains CBF residues 1 to 192, nor Ela alone promotes transcription of the G5CAT reporter gene. Together, they provide greater-than-50-fold stimulation. This implies an important, but not necessarily exclusive, role for residues 1 to 192 of CBF in Ela-activated transcription of the endogenous hsp70 gene. To elicit transcriptional stimulation of the hsp70 promoter, Ela may exert effects on other transcription factors and may require additional domains of the CBF polypeptide that were not addressed in the GAL-CBF assays. The Ela-responsive do-

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CBF in COS cells is not as sensitive to deletion of residues 1 to 97 (Fig. 1). Ela associates with CBF residues 1 to 192 in vitro. Viral trans-activators that are themselves unable to bind DNA, such as adenovirus Ela, could theoretically gain promoter specificity by interacting with sequence-specific DNA-binding transcription factors (10, 11, 23). We investigated the possibility that Ela and CBF form a complex in vitro by using bacterially produced glutathione-S-transferase (GST) and a GST-CBF(1-192) fusion protein (22) as affinity reagents. The adenovirus Ela gene encodes alternatively spliced 13S and 12S mRNAs whose protein products have distinct biological activities (3, 15). The ability of Ela to induce GAL-CSSP (containing CBF residues 1 to 192)directed transcription of G5CAT in vivo resides predominantly with the 13S product, as the 12S product confers only a modest twofold effect (Fig. 4A). This parallels the effects observed on the hsp70 promoter in HeLa cells (29) and CHO cells (data not shown). In vitro-transcribed and -translated Ela 12S and 13S polypeptides (Fig. 4B, lanes 1 and 4) were fractionated on GST-agarose or on GST-CBF(1-192)-agarose, and the bound fractions were examined by SDS-PAGE. The amount of 12S polypeptides retained by GST-CBF(1192)-agarose is at best twice that retained by GST-agarose alone (Fig. 4B, lanes 2 and 3). In contrast, 13S polypeptides were preferentially retained by GST-CBF(1-192)-agarose (Fig. 4B, lanes 5 and 6). Polypeptides unrelated to Ela, such as human hsp70 (Fig. 4C, lanes 4 through 6), are not retained by GST-CBF(1-192). Conversely, Ela is not retained by GST-fusion matrices composed of other polypeptides unrelated to CBF, such as bacterial topoisomerase I (Fig. 4C, lanes 7 through 9). Since Ela is the only radiolabeled polypeptide in this assay, we cannot discriminate between direct or indirect interaction of Ela and CBF residues 1 to 192. Factors present in the reticulocyte lysates may have contributed to the formation of the observed complex.

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main of CBF in CHO cells (residues 1 to 192) overlaps with a region of CBF that is essential for CBF activity in COS cells (Fig. 1). However, unlike GAL-CBF-mediated Ela trans-activation in CHO cells, the transcriptional activity of

DISCUSSION Model of CBF function. The cell type-dependent activity of CBF (Fig. 2) leads us to propose the following model for CBF function (Fig. 5). CBF, while able to bind to the hsp70 promoter, is unable to stimulate transcription in the absence of a coactivator, whose function is mimicked by Ela. This coactivator is predicted to be in excess in COS cells and limiting in CHO cells. The corollary prediction is that CBF is in excess in CHO cells and is limited in COS cells. Hence, transfection of CBF into COS cells results in the formation of more transcriptionally competent CBF-coactivator complexes and results in the observed increase in hsp70 promoter activity. Transfection of CBF into CHO cells does not increase the level of transcriptionally competent complexes but may instead result in decreased hsp70 promoter activity by competing with such complexes for binding to the hsp7O promoter. An alternative model is that CBF is transcriptionally competent, but that this activity is masked by a repressor. The function of Ela could then be to nullify the activity of the repressor. In this scenario, the repressor is present in CHO cells but not in COS cells. While the data do not clearly distinguish between these two models, we favor the former because of the following observations. Titration experiments with CBF led to repression of hsp70 promoter activity in CHO cells, and supplying both Ela and CBF does not stimulate the hsp70 promoter to a level beyond that accomplished by Ela alone in CHO cells or by CBF alone in COS cells. These observations are also compatible with the exist-

nsp70 CBF MEDIATES TRANSCRIPTIONAL ACTIVATION BY Ela

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FIG. 4. Ela stimulates GAL-CBF(1-192)-directed transcription of G5CAT in vivo and associates with CBF in vitro. (A) Enzymatic CAT activity expressed from the G5CAT reporter gene in CHO cells cotransfected with GAL-CSSP, encoding CBF residues 1 to 192 fused to the DNA-binding domain of GAL4 (see schematic in Fig. 3C), and the indicated Ela constructs. (B) The indicated in vitrotranslated polypeptides were either subjected directly to SDSPAGE (2 ,ul; lanes 1 and 4) or fractionated (4 ,ul) on GST-glutathione agarose (lanes 2 and 5) or GST-CBF(1-192)-glutathione agarose (lanes 3 and 6). The bound fraction was subjected to SDS-PAGE. Radioactivity was measured by phosphor image analysis. (C) In vitro-translated Ela 13S or hsp70 polypeptides were subjected directly to SDS-PAGE (lanes 1, 4, and 7) or fractionated on GST, GST-CBF(1-192), or GST-Topoisomerase (GST-Topo) (containing an N-terminal fragment of bacterial toposimerase I) affinity matrices as indicated.

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formation of primary cells in cooperation with ras) appear to be facilitated by protein-protein interactions. Previous studies have detected a number of cellular polypeptides that stably interact with Ela (7, 32). Some of these have recently been identified. p105 is the retinoblastoma gene product Rb (26), and p60 is cyclin A (16). Correlative data suggest that these interactions have biological consequences. For example, the interaction between Ela and pl05Rb is thought to be important for transformation because mutations of Ela that are transformation defective are also unable to bind p105-Rb (27). Transcriptional activation of the early viral E4 promoter by Ela is thought to involve an interaction of Ela and ATF-2 (11), although no physical data have been shown. Here, we present evidence that CBF-mediated Ela transcriptional activation is achieved in part by the interaction between Ela and CBF. Like the Ela-TFIID interaction (9), the Ela-CBF interaction is easily disrupted by detergents (0.1% SDS [data not shown]), which may explain why it was not detected in previous searches for cellular proteins that stably interact with Ela. The region of CBF necessary for mediating Ela transcriptional activation in vivo is also necessary for interacting with Ela in vitro. Both ATF2- and CBF-mediated Ela transcriptional stimulation are sensitive to mutations in conserved region 3 of the Ela polypeptide. Coincidentally, the region of ATF2 required for Ela induc-

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of a repressor that competes with the coactivator by occupying the coactivator-binding domain on the CBF polypeptide. Ela trans-activation of the hsp7O gene. Ela has a multitude of activities (for a review, see reference 15), two of which (i.e., transcriptional activation of specific genes and trans-

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ibility is also amino terminal, although a comparison of the CBF and ATF2 Ela-inducible regions did not reveal any striking sequence similarity. Our data suggest that by protein-protein interaction at residues 1 to 192, CBF recruits Ela to the target promoter; the transcriptional activation function is then provided by Ela. Lillie and Green (10) have shown that DNA-bound Ela can stimulate transcription, suggesting that proximity to a promoter allows Ela to function as a transcriptional activator. We proposed that the observed Ela-CBF interaction may play a fundamental role in targeting Ela to the hsp70 promoter. The in vitro binding results correlate with the in vivo activities observed with 13S and 12S: in vivo, 13S is the more potent transcriptional activator (Fig. 4A), and it binds more efficiently to CBF residues 1 to 192 in vitro (Fig. 4B). The reported stimulation of the hsp7O promoter by 12S (21) may therefore have a mechanism similar to that of 13S but with reduced efficiency in the promoter-targeting and/or transcriptional activation functions. This is supported by recent observations that Ela and TFIID interact in vitro and, moreover, that this interaction is also greater for 13S than 12S (9). It has been proposed that for Ela to accomplish in vivo trans-activation, it must interact both with the promoter-specific transcription factor and with components of the initiation complex (10, 11, 25). Thus, in vitro binding of CBF residues 1 to 192 with Ela, while necessary, is not sufficient for in vivo trans-activation by Ela. A construct with a point mutation at residue 150 of the 13S-encoded polypeptide (25) retained in vitro binding activity to CBF but was defective in in vivo trans-activation activity (data not shown). This mutation may result in the loss of the ability to interact with TFIID, since mutations of Ela at residues 148 and 151 severely diminish in vitro binding to TFIID (9). It will be most important to examine a series of mutations within conserved region 3 for their abilities to interact with CBF in vitro and to promote GAL-CBF-directed transcription in vivo. ACKNOWLEDGMENTS We thank Mark Ptashne, Michael Green, and Doug Last for providing GAL4 and GSCAT plasmids and anti-GAL4 antiserum, Nic Jones for providing Ela plasmids, and A. Mondragon for providing GST-Topoisomerase. We thank Doug Engel, Bob Holmgren, Nic Jones, Dan Linzer, and Alfonso Mondragon for encouragement, discussion, and critical reading of the manuscript. This work was supported by funds from the NIH, the Illinois Cancer Council, the Harris Fund, the Burroughs Wellcome Fund, and the Northwestern University Alumnae Board. REFERENCES 1. Agoff, N., D. Linzer, and B. Wu. Unpublished data. 2. Bonthron, D., R. Handin, R. Kaufman, L. Wasley, E. Orr, L. Mitsock, B. Ewenstein, J. Loscalzo, D. Ginsberg, and S. Orkin. 1986. Structure of pre-pro-von Willebrand factor and its expression in heterologous cells. Nature (London) 324:270-274. 3. Flint, J., and T. Shenk. 1990. Adenovirus Ela protein paradigm viral activator. Annu. Rev. Genet. 23:141-161. 4. Gorman, C., L. Moffat, and B. Howard. 1982. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2:1044-1051. 5. Greene, J., Z. Larin, I. Taylor, H. Prentice, K. Gwinn, and R. Kingston. 1987. Multiple basal elements of a human hsp70 promoter function differently in human and rodent cell lines. Mol. Cell. Biol. 7:3646-3655. 6. Haley, K. P., J. Overhauser, L. E. Babiss, H. S. Ginsberg, and N. C. Jones. 1984. Transformation properties of type-5 adenovirus mutants that differentially express the Ela gene products.

Proc. Natl. Acad. Sci. USA 81:5734-5738. 7. Harlow, E., P. Whyte, B. R. J. Franza, and C. Schley. 1986. Association of adenovirus early-region 1A proteins with cellular polypeptides. Mol. Cell. Biol. 6:1579-1589. 8. Kakidani, H., and M. Ptashne. 1988. GAL4 activates gene expression in mammalian cells. Cell 52:161-167. 9. Lee, W. S., C. C. Kao, G. 0. Bryant, X. Liu, and A. J. Berk. 1991. Adenovirus Ela activation domain binds the basic repeat in the TATA box transcription factor. Cell 67:365-376. 10. Lillie, J., and M. Green. 1989. Transcriptional activation by the adenovirus Ela protein. Nature (London) 338:39-44. 11. Liu, F., and M. Green. 1990. A specific member of the ATF transcription factor family can mediate transcription activation by the adenovirus Ela protein. Cell 61:1217-1224. 12. Lum, L., L. Sultzman, R. Kaufman, D. Linzer, and B. Wu. 1990. A cloned human CCAAT-box-binding factor stimulates tran-

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