Eur. J. Biochem. 188,231 -237 (1990)

0FEBS 1990

Interaction of upstream stimulatory factor with the human heme oxygenase gene promoter Michihiko SATO’, Shinobu ISHIZAWA’, Tadashi YOSHIDA’ and Shigeki SHIBAHARA’ Department of Molecular and Pathological Biochemistry, Yamagata University School of Medicine, Yamagata, Japan

’ Department of Applied Physiology, Tohoku University School of Medicine, Sendai, Japan Received July 31/November 6, 1989) - EJB 89 0951

Upstream stimulatory factor (USF), originally identified in HeLa cells, interacts with the upstream promoter sequence of adenovirus 2 major late promoter (Ad2MLP) and activates its transcription. USF is present in uninfected HeLa cells and appears to be involved in the transcription of cellular genes related to stress. Recently, we have proposed that the rat heme oxygenase gene, newly identified heat-shock protein gene, is regulated at least in partly by a rat homolog of USF [Sato, M., Fukushi, Y., Ishizawa, S., Okinaga, S., Muller, R. M. & Shibahara, S. (1989) J . Biol. Chem. 264, 10251 - 102601. We therefore confirm that the heme oxygenase gene is expressed in HeLa cells and its expression is increased by cadmium, suggesting that human heme oxygenase is a stress protein similar to the metallothioneins. Using partially purified USF from HeLa cells, we show that USF binds to the human heme oxygenase gene promoter and stimulates its cell-free transcription. The cis-acting element, identified as CACGTGACCCG, is located 34 bp upstream from the transcription initiation site, and contains the core sequence of the upstream promoter sequence of Ad2MLP. We propose that USF contributes to the transcription of the human heme oxygenase gene. Heme oxygenase, a rate-limiting enzyme of heme catabolism, cleaves heme to form biliverdin, which is subsequently reduced to bilirubin by biliverdin reductase in mammals [1, 21. Heme oxygenase activity is strongly induced by its substrate heme [2 - 41 and by various non-heme substances (reviewed in [5]). Recently, we have shown that rat heme oxygenase is transcriptionally induced by heat shock [6,7]. However, the heme-mediated induction of heme oxygenase is of particular significance, since this phenomenon is observed in all cell lines examined of various species including man, monkey, pig, mouse and rat [8]. Hemin treatment has been shown to result in increased amounts of heme oxygenase protein [9, 101 and mRNA [9, 111. In contrast, the evidence available indicates that only rat heme oxygenase is a heat-shock protein [7, 8, 121. Recently, it has been shown that hemin as well as cadmium act at the transcriptional level to induce heme oxygenase in mouse hepatoma cells [13]. We have partially purified a nuclear protein from rat glioma cells, designated heme oxygenase transcription factor, that interacts with the promoter region (- 51 to - 35) of the rat heme oxygenase gene and activates its transcription [14]. The core sequence of the heme-oxygenase-transcription-factor-binding site is identical to the sequence of CCACGTGAC found in the upstream promoter sequence of the adenovirus 2 major late promoter (Ad2MLP) [15-171, that is bound by upstream stimulatory factor (USF, [IS]) or Correspondence to S. Shibahara, Department of Applied Physiology, Tohoku University School of Medicine, Sendai, Japan 980. Abbreviations. USF, upstream stimulatory factor; Ad2MLP, adenovirus 2 major late promoter; DMEM, Dulbecco’s minimal essential medium. Enzyme. Heme oxygenase (EC 1.14.99.3).

major late transcription factor [17] originally identified in HeLa cells. We have therefore provided several lines of evidence that heme oxygenase transcription factor is a rat homolog of USF [14]. It is of interest that USF is involved in the basal levels of expression of cellular genes related to stress: mouse metallothionein I gene [19], rat y-fibrinogen gene [20] and possibly rat heme oxygenase gene [14]. The cis-acting elements of these genes contain a CNCGTGAC motif identical to the core sequence of the upstream promoter sequence of Ad2MLP [14]. Like heme oxygenase [13], metallothionein genes are transcriptionally induced by heavy metals, while fibrinogens are induced during acute phase response to inflammation, infection and tissue injury. In contrast to rat heme oxygenase, the human enzyme is not a heat-shock protein [12, 211, but is a stress protein induced by cadmium [22] or exposure to ultraviolet light [23]. It has been suggested that heme oxygenase is involved in the defense mechanisms against oxidation stress, since bilirubin produced locally may work as a physiological antioxidant [24]. It is therefore of significance in understanding the mechanisms by which human heme oxygenase gene expression is regulated. In this study, using USF partially purified from HeLa cells, we provide evidence that USF is involved in the transcription of the human heme oxygenase gene. MATERIALS AND METHODS

Materials HeLa cell lysates were obtained from Bethesda Research Laboratories. Restriction cndonucleases were purchased from Bethesda Research Laboratories, Pharmacia LKB Biotechnology, Boehringer Mannheim and New England BioLab; DNA polymerase (Klenow fragment), DNase I, DNase-free

232 bovine serum albumin and S1 nuclease from Boehringer Mannheim; T4 polynucleotide kinase from Pharmacia LKB Biotechnology; [ Y - ~ ~ P ] Aand T P [ U - ~ ~ P I ~ from C T PAmersham PLC, England. Other reagents used were of analytical grade commercially available. Partial purification of USFjrom HeLa cells Nuclear extracts were prepared from HeLa cells according to Dignam et al. 1251. The extracted proteins were precipitated by the addition of saturated ammonium sulfate solution, pH 7.0, at a final concentration of 50%, and dissolved in 20 mM Hepes, pH 7.9, containing 0.1 M KCl, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol and 15% glycerol, followed by dialysis against the same buffer overnight. After centrifugation, the resulting supernatant was subjected to Sephadex G25 column chromatographyequilibrated with buffer A (50 mM Tris/HCl, pH 7.9, 12.5 mM MgC12, 1 mM EDTA, 1 mM dithiothreitol and 20% glycerol) containing 0.1 M KCI [26]. The protein fraction was subsequently applied to a heparin-agarose column equilibrated with buffer A containing 0.1 M KCl, followed by extensive washing with buffer A containing 0.2 M KC1. The proteins bound to the heparin-agarose were then eluted with buffer A containing 0.3 M KC1 (USF fraction) [17] and used for cell-free transcription as well as for binding assays. Induction of heme oxygenase in HeLa cells HeLa cells were cultivated in Dulbecco’s minimal essential medium (DMEM) containing 10% fetal calf serum. About 4 x lo7 cells/l5-cm-diameter dish were incubated at 37°C in serum-free DMEM containing 5 pM hemin or 5 pM cadmium chloride. For heat treatment, cells were incubated at 42°C in serum-free DMEM. After incubation for 5 h, the microsomes were prepared from two dishes of cells, arid used for the determination of heme oxygenase activity [7, 91. One unit of the enzyme was defined as the amount catalyzing the formation of l nmol bilirubin/min. For RNA preparation, HeLa cells were treated for 3 h under the conditions described above. Total RNA was prepared from treated cells and analyzed as described previously [ l l , 211. The hybridization probe used was the XhoIIXbaI fragment (- 64 to 923) derived from the human heme oxygenase cDNA, pHHOl [21].

DNase I,footprint analysis The 5’-labelled DNA fragments (10 - 20 fmol, 20 000 30 000 cpm) were incubated with USF fraction (0 - 30 pg protein) in 20 pl 20 mM Hepes, pH 7.9, 50 mM KC1, 1 mM dithiothreitol, 2 mM MgC12, 5% glycerol and 5 pg poly(d1dC) for 20 min at room temperature, and partially digested with 5 pl DNase I (5-10 pg DNase I in 20 mM MgC12 and 10 mM CaC12)for an additional minute at room temperature. Digestion was stopped by the addition of 7Spl 12.5 mM EDTA, followed by extraction with phenol/CHC13 (9: 1, by vol.). Products were separated on an 8% polyacrylamide sequencing gel [27]. Gel-shift assays Gel-qhift assays [28] were performed in a volume of 20 p1 contai I ~ I I I Z5’-end-labelled synthetic oligonucleotides (2 5 fmol. I 0 ’ cpm), 1.5 pg poly(d1-dC), USF fraction (0.5 - 1 pl, 1-2 pg protein), 20 mM Hepes, pH 7.9, 40 mM NaCl, 4%

Ficol and 6 mM MgC12. The mixture was incubated for 15 min at room temperature, then half of the sample (10 pl) was loaded onto a 4% polyacrylamide gel in 6.25 mM Tris/ Borate, pH 8.3 and 0.125 mM EDTA. The gel was electrophoresed for 90 - 120 min at 10 V/cm. Cell-Jree transcription of the human heme oxjigenuse gene The subcloned plasmid, harbouring the 5‘-flanking region of about 1.4 kb, exons 1, 2, and 3, and a part of intron 3 of the human heme oxygenase gene [12], was transcribed using HeLa cell lysates according to the instructions of the supplier. The products were identified by S1 nuclease mapping analysis. The S1 probe was the PstI/HinfI fragment (nucleotide residues -283 to intron 1) end-labelled at the HinfI site, which is located 221 bp downstream from the transcription initiation site [12].

RESULTS AND DISCUSSION Partial purification of USF Recently, we have proposed that a rat homolog of USF is required for the basal levels of the rat heme oxygenase gene expression [14]. The cis-acting element of the rat heme oxygenase gene (-48 to -37; Fig. 1B) is almost identical to the nucleotide residues (- 45 to - 34) of the human heme oxygenase gene [12], suggesting that the same nuclear protein, possibly USF, may interact with this element of the human heme oxygenase gene. In order to investigate whether USF is indeed involved in the expression of the human heme oxygenase gene, we partially purified USF from HeLa cells, which was shown to bind to the upstream promoter sequence of Ad2MLP (Fig. 1A). The XhoI/PvuII fragment containing the upstream promoter sequence, derived from the pSmaF 1291, was incubated with the USF fraction, and subjected to footprint analysis, which indicated that a HeLa nuclear protein in the USF fraction does indeed bind to the upstream promoter sequence of Ad2MLP (Fig. 1A, lanes 1 - 3). The interaction of USF with the upstream promoter sequence was specifically inhibited if synthetic elements of the rat or human heme oxygenase genes containing a core sequence of the upstream promoter sequence (underlined in Fig. 1B) [19] were included in the binding reactions (Fig. l A , lanes 5 and 6). In contrast, the addition of a unspecific competitor, AP-1-binding element 1301, caused no apparent inhibition of protein binding (Fig. 1A, lane 4). These results confirm the quality of our USF fraction and suggest that the heme oxygenase gene may share a common nuclear protein, USF, with the Ad2MLP. Regulation ojheme oxygenase activity in HeLa cells USF is present in uninfected HeLa cells, and it appears to be required for the basal levels of expression of cellular genes related to stress, such as metallothionein I gene [19] and y-fibrinogen gene [20]. Consequently, we analyzed the regulation of the heme oxygenase gene expression in HeLa cells (Fig. 2). Heme oxygenase activity and mRNA levels are increased following treatment with hemin or cadmium, although basal levels of heme oxygenase gene expression are low in untreated HeLa cells (Fig. 2). In contrast, heat shock failed to induce heme oxygenase activity (Fig. 2A, column 2) and mRNA levels (Fig. 2B, lane 2) in HeLa cells, which is consis-

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1234 5 6 7 8 Fig. 1. Binding of USF to adenovirus 2 major lute promoter. (A) DNase I footprint analysis. The XhoI/PvuII fragment (-260 to 33) containing the upstream promoter sequence of Ad2MLP, end-labelled at the PvuIT site, was incubated with USF fraction (0-2Opg protein indicated at the top of the lanes). A protected region is shown on the left side of the gel. Competitors used were synthetic AP-1-binding element (see Fig. 5A), heme oxygenase transcription factor-binding element of the rat heme oxygenase gene (RHO shown in B) [14], and the element of the human heme oxygenase gene (HHO shown in Fig. 5A, c). About 500-fold excess of these synthetic elements was included in binding reactions (lanes 4 - 6). (B) Homologous nucleotide sequences of the protein-binding regions of three promoters, Ad2MLP, human heme oxygenase gene (HHO), and rat heme oxygenase gene (RHO). The core sequence is underlined (see Fig. 5A)

28 S18S-

1 2 3 4 Fig. 2. Induction of heme oxygenase in HeLa cells. (A) Enzyme activity. HeLa cells were incubated for 5h at 37°C (column 1) or at 42°C (column 2) or with cadmium at 37°C (column 3) or with hemin at 37 "C (column 4). (B) mRNA levels. The relative changes in the amount of hybridizable heme oxygenase mRNA were determined by Northern blot analysis. Each lane contained 8 pg total RNA prepared from HeLa cells, treated for 3 h under the conditions indicated at the top of the lanes. The size markers used were human rRNA

tent with our previous observations [8, 12, 211. Since human heme oxygenase is a stress protein induced by cadmium (Fig. 2) or by exposure to ultraviolet light [23], the human heme oxygenase gene could be a good candidate for the stressrelated genes, whose expression is maintained by USF.

Binding of U S F to the human and rat heme oxygenase gene promoters Using USF fraction, we analyzed the 5'-flanking region of about 1.4 kb of the human heme oxygenase gene for protein-

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Fig. 3. Specific binding of a nuclear protein to both the human and rat heme oxygenase gene promoters. (A) Identification of a protein-binding region of the human heme oxygenase gene. The ApaI/AvaI fragment (-98 to 20) of the human heme oxygenase gene, end-labelled at the AvaI site, was incubated with USF fraction (0-20 pg protein indicated at the top of the lanes). The digests were analyzed on a sequencing gel. Chemical sequence ladders of the same DNA fragment, A G and C + T, were included as size markers. The sequence of the protected region is indicated at the left side of the lanes ( the sequence shown is for a non-coding strand). (B) Gel-shift assays. USF fraction was incubated with end-labelled synthetic human element, shown in Fig. SA, c (lanes 1 and 2). The PvuII/XhoI fragment of the human heme oxygenase gene (- 59 to 20) was incubated with rat heme oxygenase transcription factor (lanes 3 and 4). Binding reactions were performed in the presence of a 400-fold excess of the human element (lanes 2 and 4). Open and filled arrow heads represent DNA’protein complexes and unbound DNA, respectively. (C) Specific binding of USF to the rat heme oxygenase gene promoter. The NdeIiAvaII fragment of the rat heme oxygenase gene (-137 to 125), end-labelled at the NdeI site, was incubated with USF fraction (0-20 pg protein indicated at the top of the lanes). Synthetic elements (500-fold excess) were included in the binding reaction for the specificity of this analysis: RHO, proteinbinding element of the rat heme oxygenase gene; HHO, protein-binding element of the human heme oxygenase gene. Size markers shown were end-labelled pUC8 DNA fragments generated by digestion with HpaII (lane M). (D) Nucleotide sequence of the 5’-flanking region of the human heme oxygenase gene. Nucleotide residues are numbered in the 5’ - 3’ direction, beginning with the transcription initiation site. Exon 1 is indicated by thick solid lines. A transcription initiation site is marked with an asterisk. A TATA-like sequence is indicated by overline and underline. Several potential binding sites for different transcription factors are indicated as follows: Sp-I , double underline; AP-1, underline. A protein-binding region shown in A is indicated by an open box

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binding activity, and found that only its promoter region was bound by USF (see Fig. 4A). Namely, DNase I footprint analysis revealed that the sequence CTGGCCCACGTGACCCGC (nucleotide residues - 50 to - 33 of the human heme oxygenase gene) was bound by a protein (Fig. 3A and D). Consequently, the oligonucleotide, CCACGTGACCCGC

(-45 to - 33; shown in Fig. 5 A, c) was synthesized and shown to be bound by USF (Fig. 3 B, lane l), while an excess of the same element prevented its binding (Fig. 3 B, lane 2). Unspecific competitors, AP-1-binding or GCN4-binding elements 114, 30, 311 (shown in Fig. 5A) failed to prevent the formation of protein. DNA complexes (data not shown).

235

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These results indicate that USF specifically binds to the region (-45 to - 33) of the human heme oxygenase gene. We then examined whether rat heme oxygenase transcription factor binds to the human heme oxygenase gene promoter using gel-shift assays. The PvuII/XhoI fragment (- 59 to 20) of the human heme oxygenase gene was specifically bound by rat heme oxygenase transcription factor, since an excess of synthetic human element completely prevented protein binding (Fig. 3 B, lanes 3 and 4), supporting the hypothesis that heme oxygenase transcription factor is a rat homolog of USF. On the other hand, USF of HeLa cells specifically binds to the rat heme oxygenase gene promoter, since footprint analysis revealed one protected region between the size markers 78 bp and 109 bp (Fig. 3C), which corresponds to the cis-acting element of the rat heme oxygenase gene [14]. This binding was inhibited by the presence of a synthetic human or rat element, but was not inhibited by unspecific competitors (Ap-1-binding or GCN4-binding elements). These results indicate that a common nuclear protein, USF, is able to bind to both human and rat heme oxygenase gene promoters.

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Identification of the core sequence ofthe human heme oxygenase gene promoter required f o r protein binding

Since the protein-binding element of the human heme oxygenase gene also contains TGACCC motif similar to the core sequence previously identified as binding sites of AP-1 [30] or GCN4 [31], we examined whether synthetic binding elements for AP-1 or GCN4 were bound by USF. No proteins bound to these oligonucleotides of AP-1 nor GCN4 consensus sequences (Fig. 5B, lanes 1 -4), while USF bound to the element of the human heme oxygenase gene (Fig. 5 B, lanes 5 - 6). Additional signals may represent complexes with a monomeric form of USF (lane 5). To identify the minimal sequence of the human heme oxygenase gene promoter required for protein binding, we examined the binding activity of three synthetic oligonucleotides (Fig. 5A, d, e and f). The C residues at positions -45 and - 33 were not indispensable for the protein binding itself (Fig. 5B, lanes 7 and 9), since the oligonucleotide lacking these two C residues (Fig. 5 A, d) is bound by USF. In contrast, when the C residue at -44 is

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Stimulation of cell-free transcription of the human heme oxygenase gene by USF

We analyzed the effects of USF on the transcription of the human heme oxygenase gene in HeLa cell lysates. The template DNA, harbouring the 5‘-flanking region of about 1.4 kb (Fig. 4A), contains only one functional binding site for USF (see Fig. 3A). In this series of experiments, we reduced the amount of template DNA (4 pg/ml) in order to detect the stimulation of the transcription of the heme oxygenase gene. The addition of USF apparently increased transcripts of the human heme oxygenase gene by 10-fold (Fig. 4B, lanes 46). In contrast, no stimulation was observed when the plasmid pSV2CAT [32] was transcribed in vitro (Fig. 4B, lanes 1-3) under the same conditions. Moreover, the core sequence of CACGTGAC (see Fig. 1B) was shown to be crucial for the binding of USF as well as for the stimulation of transcription of Ad2MLP [33], metallothionein I gene [19], y-fibrinogen gene [20] and the rat heme oxygenase gene [14]. Considering all these observations, we propose that USF specifically stimulates the in vitro transcription of the human heme oxygenase gene.

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190 147 Fig. 4. Activation of the in vitro transcription of the human heme oxygenase gene by USF. (A) Schematic representation of the template DNA. The template DNA, SpHHOS, harbours the EcoRI/EcoRI fragment of about 8 kb in the EcoRI site of plasmid pUC8. Promoter elements are indicated as follows: protein-binding region, closed box; TATA-like sequence, stippled box. Exons are shown in open boxes. (B) Cell-free transcription. Uncut plasmids pSV2CAT(6 pg/ml) and SpHHOS (4pg/ml) were transcribed in HeLa cell lysates with (lanes 2 and 5 ) or without (lanes 1 and 4) USF fraction (22 pg protein). Transcripts in the presence of cc-amanitin are shown in lanes 3 and 6. The S1 probe used was the AccIIHinff fragment labelled at the Hinff site, derived from pSV2CAT (1 140 nucleotides; expected size of transcripts, 660 nucleotides), and the PstIIHinfI fragment (nucleotide residue -283 to intron 1) derived from SpHHOS. The products were analyzed on a 5% polyacrylamide/7M urea gel. The size markers were end-labelled dXl74 replicative form DNA fragments generated by digestion with Hue111 (MI) and end-labelled pUC8 DNA fragments generated by digestion with HpaII (M2); these are given in base pairs

changed to T (Fig. 5A, e and f), USF is not able to bind to such a mutated element. Moreover, functional analysis of the rat heme oxygenase gene promoter indicates that GTGA (- 44 to 43) of the core sequence shown in Fig. 1 B is required for protein binding [14], whereas TCGA (- 39 to - 36) is not indispensable for protein binding and cell-free transcription [ 141. Therefore, the minimal sequence required for binding of the USF is identical to the core sequence of the upstream promoter sequence (see Fig. 1B) [19], corresponding to CACGTGAC (- 44 to - 37) of the human heme oxygenase gene. The C residue at position -44 of the human heme oxygenase gene is equivalent to the C residue at position -60 of Ad2MLP (see Fig. 1 B) [16]. Double point mutations at po~

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Fig. 5. Identification of the core sequence of the protein-binding site of the human heme oxygenuse gene. (A) The synthetic oligonucleotides used are shown. Large letters represent the nucleotide sequence of the AP-I-binding element (a), GCN4-binding element (b) and the human heme oxygenase gene promoter (c-f), lower case letters represent mutated nucleotides. (B) Gel-shift assay. USF fraction ( 5 Fg protein) was incubated with end-labelled oligonucleotides shown in A (2 x lo4 cpm), and the mixtures were separatcd on 4% polyacrylamide gel (lanes 1, 3, 5, 7, 9 and 11). Bovine serum albumin was used instead of USF in lanes 2,4, 6, 8, 10, and 12. The open triangle ( A ) represents frcc oligonucleotides and the filled triangle (A)indicates bound oligonucleotides

sitions -62 and -60 of Ad2MLP were shown to reduce its in vitro transcription [15], whereas a point mutation at position - 61 caused no effects [16]. These functional analyses indicate that the C residue at position -60 of Ad2MLP is indispensable for efficient transcription from its promoter, which is consistent with the result that the C residue at position -44 of the human heme oxygenase gene is crucial for protein binding (Fig. 5). We therefore propose that USF interacts with the human heme oxygenase gene promoter and is responsible for the stimulation of its transcription. Available evidence suggests that USF itself is not directly involved in the induction of stress-related genes, such as metallothionein I gene [19], and rat heme oxygenase gene [14], and that USF is required for the basal levels of expression of these genes. Biochemical characterization and molecular cloning of USF will allow us to understand the mechanisms by which these stress-related genes are regulated. This study was supported in part by Grants-in-Aid to S.S. for Special Project Cancer-Bioscience from the Ministry of Education, Science and Culture of Japan and for scientific research from the Naito Foundation.

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Interaction of upstream stimulatory factor with the human heme oxygenase gene promoter.

Upstream stimulatory factor (USF), originally identified in HeLa cells, interacts with the upstream promoter sequence of adenovirus 2 major late promo...
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