Two Transcriptional Activators, CCAAT-Box-Binding Transcription ...

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Sep 2, 1986 - It will also be useful to compare the mechanism of induction of mammalian ... sulfate precipitate from 60 g of HeLa cells was chromato- graphed ...
MOLECULAR AND CELLULAR BIOLOGY, Mar. 1987, p. 1129-1138

Vol. 7, No. 3

0270-7306/87/031129-l10$02.00/0 Copyright C 1987, American Society for Microbiology

Two Transcriptional Activators, CCAAT-Box-Binding Transcription Factor and Heat Shock Transcription Factor, Interact with a Human hsp7O Gene Promoter WILLIAM D. MORGAN,' GREGG T. WILLIAMS,2 RICHARD I. MORIMOTO, JOHN GREENE,3 ROBERT E. KINGSTON,3 AND ROBERT TJIAN1* Department of Biochemistry, University of California, Berkeley, California 947201; Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 602012; and Department of Molecular Biology, Massachusetts General Hospital, and Department of Genetics, Harvard Medical School, Boston, Massachusetts 021143 Received 2 September 1986/Accepted 10 December 1986

We characterized the activity of a human hsp7O gene promoter by in vitro transcription. Analysis of 5' deletion and substitution mutants in HeLa nuclear extracts showed that the basal activity of the promoter depends primarily on a CCAAT-box sequence located at -65. A protein factor, CCAAT-box-binding transcription factor (CTF), was isolated from HeLa nuclear extracts and shown to be responsible for stimulation of transcription in a reconstituted in vitro system. DNase I footprinting revealed that CTF interacts with two CCAAT-box elements located at -65 and -147 of the human hsp7O promoter. An additional binding activity, heat shock transcription factor (HSTF), which interacted with the heat shock element, was also identified in HeLa extract fractions. This demonstrates that the promoter of this human hsp7O gene interacts with at least two positive transcriptional activators, CTF, which is required for CCAAT-box-dependent transcription as in other promoters such as those of globin and herpes simplex virus thymidine kinase genes, and HSTF, which is involved in heat inducibility.

Promoters recognized by RNA polymerase II require cis-acting regulatory elements both for constitutive activity and for modulation of transcription by various regulatory processes characteristic of different genes. These elements include sequences near the start of transcription, such as the TATA box, various upstream sequences in the region from approximately -50 to -200, and enhancer sequences that may be located at much greater distances either 5' or 3' to the start site. The mechanisms by which these elements act to control gene expression at the level of transcription are of great interest. One valuable approach to this problem is the identification and purification of sequence-specific DNAbinding proteins that interact with promoter and enhancer elements (9, 25). One regulatory system that has attracted attention is heat shock induction (23). The response of organisms to abnormally high temperature or to a variety of other metabolic stresses involves a rapid and dramatic increase in the rate of transcription initiation of several specific genes encoding heat shock proteins. Control also occurs at the level of mRNA splicing and translation in some, but not all, instances. This heat shock or stress response is very general, and perhaps universal, among eucaryotic organisms. Some of the heat shock gene products, such as the 70-kilodalton family, show remarkable amino acid sequence conservation (23). Perhaps more surprisingly, a 14-base-pair (bp) DNA sequence element, originally identified upstream of several Drosophila heat shock genes, also occurs with a high degree of homology in organisms as distantly related as yeasts and humans (23, 28). In addition, the Drosophila hsp70 gene promoter is functional and responsive to heat shock induction in mammalian cells (28, 29). *

The hsp70 gene family is of particular interest because hsp70 is the most abundant and generally conserved heat shock protein. This multicopy gene family includes both heat-inducible genes and noninducible cognates. A human gene coding for a heat shock-inducible 70-kilodalton member of this family has been cloned and sequenced, and its regulation in vivo has been investigated (36). These studies have permitted identification of at least two separate promoter domains (38). One, located between -107 and -68, contains the sequences required for heat shock induction (including at least one match to the 14-bp heat shock response element [HSE] consensus sequence). Another domain, located downstream of -68, is sufficient for basal promoter activity. This proximal domain is also subject to control by a number of additional regulatory processes. These include stimulation by nuclear oncogenes (such as the adenovirus ElA gene product and simain virus 40 and polyomavirus large T antigens), induction by serum growth factors, and cell cycle-dependent transcription (17, 18, 21, 22, 26, 37, 39; K. L. Milarski and R. I. Morimoto, Proc. Natl. Acad. Sci. USA, in press). Analysis of the human hsp70 promoter provides an important opportunity to investigate and compare the function of this inducible system with analogous ones in mammalian cells, such as metal regulation of metallothionein genes (5, 19, 20, 30, 33) or various hormone-regulated genes. It will also be useful to compare the mechanism of induction of mammalian heat shock genes to the function of the heat shock system in other organisms as diverse as yeasts and Drosophila melanogaster. To complement the in vivo mutational analysis already reported, it is important to characterize the sequencespecific DNA-binding proteins that recognize the cis-acting promoter elements, to correlate their binding sites with the effects of mutations, and to examine the relationship be-

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tween specific DNA-protein interactions and in vitro transcriptional activity. To this end, we identified and isolated proteins from HeLa nuclear extracts that interact with multiple distinct binding sites in the human hsp70 promoter region and are responsible for activating transcription in vitro. They include factors that are unique to heat shockinducible genes and factors common to other viral and cellular promoters. We report here the initial characterization of some of these binding sites and in particular the role of the positive transcriptional activator CCAAT-box-binding transcription factor (CTF) that acts within the proximal promoter domain. MATERIALS AND METHODS Abbreviations. hsp70, 70-kilodalton major heat shock protein; HSTF, heat shock transcription factor; CTF, CCAATbox-binding transcription factor; NF-I, nuclear factor I (DNA replication stimulatory factor); HSE, heat shock response element; SRE, serum response element; HSV-TK, herpes simplex virus thymidine kinase. Plasmids. Two series of hsp70 promoter plasmid constructions were used. One series was derived from pHsm-CAT, a pUC13 derivative containing the human hsp70 promoter sequence from -1100 to +150 fused to the bacterial chloramphenicol acetyltransferase (CAT) gene (38). 5' Bal 31 deletions of pHsm-CAT to -133, -68, and -47, as described previously, were used. Two additional derivatives were made from the -133 5' deletion of pHsm-CAT. A 5-bp substitution mutant, OS57-63, was prepared by synthetic oligonucleotide-directed in vitro mutagenesis. The sequence of OS57-63 is shown in Fig. 1. A small internal deletion within the transcribed sequence for use as a pseudo-wildtype control template in transcription assays was constructed by cutting the -133 5' deletion of pHsm-CAT at the SmaI linker between the hsp70 and CAT sequences, partial digestion with Bal 31, and ligation. The resulting plasmid, AS-8, has a unidirectional 9-bp deletion from the SmaI site extending into the CAT untranslated leader sequence. Another series of 5' deletion mutants was derived from a construction containing a 2.0-kilobase HindIII-BamHI partial digest fragment from -188 of the hsp7O promoter sequence to + 1634 of the CAT gene (derived from pHsm-CAT) inserted into the SphI site of pBR-N/B (13). 5' Bal 31 deletions fusing the hsp70 promoter to the vector at the BglII site were prepared as described by Haltiner et al. (13). The sequences of these deletion mutants are shown in Fig. 1. Plasmid DNA was prepared as described by Maniatis et al. (24) by alkaline hydrolysis and purified on CsCl gradients containing ethidium bromide. Preparation and fractionation of HeLa nuclear extracts. Extracts were prepared by 0.42 M KCl extraction of nuclei from actively growing HeLa cells (5 x 105 to 7 x 105 cells per ml) essentially as described by Dignam et al. (6). The nuclear extract was then precipitated with ammonium sulfate (0.38 g/ml of extract). Extract for use in transcription assays was desalted on a Bio-Gel (Bio-Rad Laboratories) P-10 column equilibrated with TM buffer containing 0.1 M KCl; TM buffer is 50 mM Tris hydrochloride (pH 7.9)-12.5 mM MgCI2-1 mM EDTA-1 mM dithiothreitol-20% glycerol. Fractionation of the nuclear extract was performed by a protocol similar to that used for Spl (3). The ammonium sulfate precipitate from 60 g of HeLa cells was chromatographed on a 750-ml Sephacryl S-300 column equilibrated with TM buffer containing 0.1 M KCl, and fractions eluting at a volume of 1.2 to 1.4 relative to the void volume peak

MOL. CELL. BIOL.

were pooled, passed through a 10-ml DEAE-Sepharose CL-6B column in TM buffer containing 0.1 M KCl, and loaded directly onto a 6-ml heparin-agarose column. The heparin-agarose column was eluted with steps of TM buffer containing 0.2, 0.3, and 0.5 M KCl. The 0.3 M KCl fraction (H.3 fraction), which contained the specific transcriptional stimulatory activity, had 7.5 mg of protein. Samples of the H.3 fraction containing 2.5 to 3.3 mg of protein were diluted to 50 mM KCl with TM buffer and loaded onto a 1-ml Pharmacia FPLC MonoS column. The MonoS column was eluted with a 20-ml linear gradient of 50 to 400 mM KCl in TM buffer. Sequence-specific DNA affinity chromatography with an ot-globin CCAAT-box sequence resin was performed as described elsewhere (K. A. Jones, J. T. Kadonaga, P. J. Rosenfeld, T. J. Kelly, and R. T. Tjian, Cell, in press) with a sample of the H.3 fraction. In vitro transcription assays. Buffer conditions for in vitro transcription assays were 25 mM Tris hydrochloride (pH 7.9)-6.25 mM MgCl2-0.5 mM EDTA-0.5 mM dithiothreitol-50 mM KCl-2% polyvinyl alcohol-10% glycerol-250 ,uM each ribonucleoside triphosphate. The amounts of DNA template and protein fractions are indicated in the figure legends. Reactions (50 ,ul) were initiated by the addition of DNA and nucleoside triphosphates to the protein fraction, and the mixtures were incubated at 30°C for 30 min. The reactions were terminated, RNA transcripts were purified, primer extension reactions were performed as described by Jones et al. (15), and primer extension products were electrophoresed on 6% polyacrylamide gels. A 24-residue synthetic deoxynucleotide that hybridizes to a sequence within the CAT gene was used as a primer (25 fmol per assay). The major products of the in vitro reactions corresponded to the correct in vivo start position (data not shown). Reconstituted in vitro transcription reactions were performed with partially purified HeLa RNA polymerase II prepared as described by Dynan and Tjian (8). General transcription factors (Sp2) were prepared as described by Dynan and Tjian (8) except that the Sephacryl S-300 column was pooled selectively for Sp2 transcriptional activity with the adenovirus major late promoter. This procedure avoids contamination of Sp2 with transcription factors eluting earlier on the S-300 column. DNase I footprinting assays. Reactions for DNase I footprinting assays were performed as described by Jones et al. (15) with the amounts of DNA probe, protein fraction, and nonspecific carrier DNA indicated in the figure legends. Wild-type probes were prepared from the 420-bp BamHINcoI fragment of pHsm-CAT or pH2.8 (36) and 5' end labeled at either site with [y-32P]ATP and T4 polynucleotide kinase. Probes of the -133 5' deletion mutant of pHsm-CAT and the OS57-63 substitution mutant were prepared from the HindIl-BamHI fragment 5' end labeled at the HindlIl sites (-135). Probes from the 5' deletions made in the pBR-N/B construct were cut at the BamHI site in the hsp7o promoter and the NdeI site within the vector sequence and 5' end labeled at the BamHI site. DNA probes for footprinting the HSE were prepared from DNA grown in an Escherichia coli strain deficient in DNA cytosine methylase activity. Methylation of bases within the DNA cytosine methylase recognition sequence CC(A/T)GG, one of which occurs within the heat shock element consensus, decreased binding affinity in footprint assays (data not shown). RESULTS Transcriptional activity in nuclear extracts. We examined in vitro transcription of the human hsp7O gene to determine

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