The Role of an Inverted CCAAT Element in Transcriptional Activation of the Human DNA Topoisomerase IIα Gene by Heat Shock*

Expression of the DNA topoisomerase IIα (topoIIα) gene is highly sensitive to various environmental stimuli including heat shock. The amount of topoIIα mRNA was increased 1.5–3-fold 6–24 h after exposure of T24 human urinary bladder cancer cells to heat shock stress at 43 °C for 1 h. The effect of heat shock on the transcriptional activity of the human topoIIα gene promoter was investigated by transient transfection of T24 cells with luciferase reporter plasmids containing various lengths of the promoter sequence. The transcriptional activity of the full-length promoter (nucleotides (nt) −295 to +85) and of three deletion constructs (nt −197 to +85, −154 to +85, and −74 to +85) was increased ∼3-fold 24 h after heat shock stress. In contrast, the transcriptional activity of the minimal promoter (nt −20 to +85), which lacks the first inverted CCAAT element (ICE1), the GC box, and the heat shock element located between nt −74 and −21, was not increased by heat shock. Furthermore, the transcriptional activity of promoter constructs containing mutations in the GC box or heat shock element, but not that of a construct containing mutations in ICE1, was significantly increased by heat shock. Electrophoretic mobility shift assays revealed reduced binding of a nuclear factor to an oligonucleotide containing ICE1 when nuclear extracts were derived from cells cultured for 3–24 h after heat shock. No such change in factor binding was apparent with an oligonucleotide containing the heat shock element of the topoIIα gene promoter. Finally, in vivo footprint analysis of the topoIIα gene promoter revealed that two G residues of ICE1 that were protected in control cells became sensitive to dimethyl sulfate modification after heat shock. These results suggest that transcriptional activation of the topoIIα gene by heat shock requires the release of a negative regulatory factor from ICE1.

DNA topoisomerases are essential enzymes that participate in the segregation of newly replicated chromosome pairs, in chromosome condensation, and in modification of the super-helical content of DNA (1)(2)(3). Human topoisomerase II (topoII) 1 functions as a homodimer by cleaving and opening one DNA duplex, passing a second duplex through the opening, and then resealing the break (4 -6). Two topoII isoforms have been identified in mammals: 170-kDa topoII␣ and 180-kDa topoII␤ (7). Although both enzymes are closely related in structure, they differ in important biochemical and pharmacological properties, including sensitivity to topoII-targeting drugs, cellular localization, and regulation by the cell cycle (8). Whereas the amount of topoII␤ remains relatively constant throughout the cell cycle, topoII␣ expression is coupled to the cell cycle (9,10). topoII␣ is of particular importance because of its association with DNA replication, mitosis, and cell proliferation.
Expression of topoII␣ is highly susceptible to environmental stimuli, and such regulation is thought to be mediated at both the transcriptional and post-transcriptional levels. The promoter region of the topoII␣ gene contains various regulatory elements, including five inverted CCAAT elements (ICEs), one GC box, and one heat shock element (HSE) (11). Exposure of human colon cancer cells to glucosamine induces down-regulation of topoII␣, resulting in the development of resistance to the topoII␣-targeting epipodophyllotoxin, etoposide (12). Development of resistance to such topoII␣-targeting agents is often associated with down-regulation of topoII␣ in various mammalian cell lines (13,14). In one etoposide-resistant cell line derived from human head and neck cancer KB cells (15,16), the transcription factor Sp3 was implicated in the down-regulation of topoII␣ (17).
Introduction of the wild-type p53 tumor suppressor gene into murine cells results in reduced expression of the topoII␣ gene, and this effect appears to be mediated by one of the ICEs in the topoII␣ gene promoter (18). Apoptosis induced by adenovirus E1A protein in human KB cells is associated with a marked decrease in the amount of topoII␣ that is due to accelerated degradation of topoII␣ by the ubiquitin proteolysis pathway (19,20). The amount of topoII␣ mRNA in late S phase is ϳ15 times that during the G 1 phase of the cell cycle in human HeLa cells, apparently because of increased mRNA stability in S phase (10). These observations indicate that topoII␣ expression is regulated by multiple mechanisms that operate at the levels of transcription, mRNA stability, and protein degradation.
Heat shock stress also affects the abundance of topoII␣ mRNA in mammalian cells. Exposure of human head and neck or colon cancer cells to high nonpermissive temperatures results in an increase in expression of the topoII␣ gene, apparent 6 -12 h later, and consequent sensitization to the cytotoxic effect of etoposide (21,22). The same heat shock stress markedly increases the abundance of the heat shock protein HSP70 and induces a transient decrease in the amount of topoII␣ mRNA and protein immediately after exposure to hyperthermia (10,22,23). Whereas this early effect of heat shock stress on topoII␣ expression appears to be mediated by increased degradation of topoII␣ mRNA (10), the later up-regulation of topoII␣ gene expression appears to be due to transcriptional activation (22). We have now investigated which elements in the 5Ј-flanking region of the human topoII␣ gene are responsible for the heat shock-induced activation of transcription.

EXPERIMENTAL PROCEDURES
Materials-Restriction enzymes and other nucleic acid-modifying enzymes and reagents were obtained from Promega (Madison, WI), Life Technologies, Inc., or Takara Shuzo (Kyoto, Japan), unless indicated otherwise. Both [␣-32 P]dCTP and [␥-32 P]ATP were from NEN Life Science Products. Human topoI cDNA was kindly provided by T. Andoh (Sohka University, Tokyo, Japan), and human topoII␣ cDNA (pBS-hTOP2) was provided by J. C. Wang (Harvard University, Boston, MA). Human HSP70 cDNA was kindly given by R. T. N. Tjian (University of California, Berkeley, CA). All cDNA fragments were separated from vector DNA by agarose gel electrophoresis and labeled by random primer DNA synthesis.
Cell Culture and Heat Shock Conditions-The T24 cell line, established from human transitional cell carcinoma of the urinary bladder (24), was cultured at 37°C under a humidified atmosphere of 5% CO 2 in Eagle's minimal essential medium (Nissui Seiyaku, Tokyo) supplemented with 10% newborn calf serum (Sera-Lab, Sussex, United Kingdom), 1 mg/ml Bacto-peptone (Difco), 0.292 mg/ml L-glutamine, 100 units/ml penicillin, and 100 g/ml kanamycin. For heat shock, culture plates were sealed with paraffin film and immersed in a water bath at 43°C for 1 h.
Northern Blot Analysis-Northern blot analysis was performed as described previously (17). Briefly, total RNA was extracted from T24 cells with the use of guanidine isothiocyanate (25), subjected (15 g/ lane) to electrophoresis on a 1% agarose gel containing formaldehyde, and transferred to a Hybond N ϩ membrane (Amersham International, Buckinghamshire, United Kingdom). The membranes were exposed to 32 P-labeled cDNA probes for 18 h and washed twice at 42°C in 2ϫ SSC containing 0.1% SDS and twice at 42°C in 0.2ϫ SSC containing 0.1% SDS. Radioactivity was detected with a Fujix BAS 2000 image analyzer (Fuji Film, Tokyo).
Construction of topoII␣ Plasmids-We used the polymerase chain reaction (PCR) to clone the human topoII␣ gene promoter (nt Ϫ295 to ϩ85, relative to the major transcription start site) as described previously (17). The 3Ј-end of all inserts was nt ϩ85, 10 base pairs upstream of the translation initiation site. For the construction of other deletion constructs, HindIII fragments (nt Ϫ295 to ϩ85) of the pTII␣Ϫ295 plasmid were digested with BfaI (pTII␣Ϫ197), ScrFI (pTII␣Ϫ154), HphI (pTII␣Ϫ74), and SacI (pTII␣Ϫ20). The digestion products were blunt-ended with the Klenow fragment of DNA polymerase I, ligated to HindIII linkers, and cloned into the HindIII site of the pGL2-Basic vector (Promega).
Site-directed mutagenesis of ICE1, the GC box, and the HSE in pTII␣Ϫ295 was performed by a PCR-based method. The promoter sequences were amplified first with Pfu polymerase (Stratagene, La Jolla, CA), the 3Ј-primer ϩ85 (5Ј-CGGTCGTGAAGGGGCTCAAG-3Ј), and 5Јprimers that introduce specific mutations into the target elements: m5 (5Ј-CAGGGAAAAACTGGTCTGCTTCGGGCGGGCTAAAGGAAGGT-TCAAGTGGAGCT-3Ј) for mutation of ICE1, m6 (5Ј-CAGGGATTGGC-TGGTCTGCTTCAAAAAAGCTAAAGGAAGGTTCAAGTGGAGCT-3Ј) for mutation of the GC box, and m7 (5Ј-CAGGGATTGGCTGGTCTGCT-TCGGGCGGGCTAAAGAAAGGAAAAAATGGAGCT-3Ј) for mutation of the HSE (mutated nucleotides are underlined). A second PCR was then performed with Taq polymerase, the first PCR products, and the 5Ј-primer Ϫ295 (corresponding to the normal promoter sequence with a 5Ј-end at nt Ϫ295). The second PCR products were digested with Hin-dIII and ligated into pGL2-Basic. The mutations introduced into these clones were confirmed by DNA sequencing.
Transient Transfection-T24 cells (1 ϫ 10 5 ) were transferred to 60-mm dishes, incubated at 37°C for 48 h, and transfected with lucif-erase plasmid DNA (2.5 g) by calcium phosphate precipitation as described previously (26). Four hours after transfection, the cells were washed, incubated at 37°C for 24 h in fresh medium, and exposed to 43°C for 1 h. The treated cells were then harvested immediately (0 h) or after further incubation at 37°C for 1, 6, 12, or 24 h for determination of luciferase activity.
Luciferase Assays-Cells were lysed in 200 l of 25 mM Tris phosphate buffer (pH 7.5) containing 1% Triton X-100 and subjected to centrifugation at 14,000 ϫ g for 15 s. The resulting supernatants were assayed for luciferase activity with the use of a Picagene kit (Toyoinki, Tokyo); light intensity was measured for 15 s with a luminometer (Model TD-20/20, Promega). Cells were cotransfected with pSV2-␤-GAL as a control for transfection efficiency, and ␤-galactosidase activity was measured with an Aurora GAL-XE kit (ICN, Costa Mesa, CA).
Isolation of Stable Transfectants-T24 cells (5 ϫ 10 5 ) were transfected with a luciferase reporter vector containing the topoII␣ gene promoter (pTII␣Ϫ295; 10 g) and pRSV-neo (0.5 g) with the use of Trans-it reagent (PanVera, Madison, WI). After 8 h, the medium was replaced, and the cells were incubated for 24 h. The cells were then incubated in selection medium containing G418 (0.8 mg/ml; Life Technologies, Inc.), and growing colonies (20 -30/10 6 cells) were cloned, expanded, and tested for luciferase activity.
PCR-Unless indicated otherwise, PCR was performed in a final volume of 100 l containing 1 ng of template DNA, a 100 pM concentration of each oligonucleotide primer, a 200 M concentration of each deoxynucleotide triphosphate, 2.5 units of Taq DNA polymerase, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl 2 , and 0.01% (w/v) gelatin. Amplification was carried out in a DNA thermal cycler (Perkin-Elmer) for 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 1 min, and polymerization at 72°C for 2 min.
Preparation of Nuclear Extracts-Nuclear extracts were prepared as described previously (17). Briefly, T24 cells (4 ϫ 10 7 ), subjected or not to heat shock at 43°C for 1 h, were collected by exposure to trypsin; resuspended in 200 l of an ice-cold solution containing 10 mM Hepes-NaOH (pH 7.9), 10 mM KCl, 0.75 mM spermidine, 0.15 mM spermine, 0.2 mM EDTA, 0.2 mM EGTA, 0.5 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride; and incubated on ice for 15 min. The cells were then lysed by passing 10 times through a 25-gauge needle attached to a 1-ml syringe, and the lysate was centrifuged for 40 s in a microcentrifuge. The resulting nuclear pellet was resuspended in 100 l of an ice-cold solution containing 20 mM Hepes-NaOH (pH 7.9), 0.4 M NaCl, 0.75 mM spermidine, 0.15 mM spermine, 0.2 mM EDTA, 0.2 mM EGTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 25% (v/v) glycerol; incubated for 30 min on ice with frequent gentle mixing; and then centrifuged for 20 min at 4°C in a microcentrifuge to remove insoluble material. The resulting supernatant (nuclear extract) was stored at Ϫ70°C, and its protein concentration was determined with a protein assay kit (Bio-Rad).
Electrophoretic Mobility Shift Assay (EMSA)-EMSAs were performed as described previously (29). Briefly, 6 g of nuclear extract protein were incubated for 30 min at room temperature in a final volume of 20 l containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM MgCl 2 , 1 mM EDTA, 8% glycerol, 1 mM dithiothreitol, 0.1 g of poly(dI-dC), and 1 ϫ 10 4 cpm of 32 P-labeled oligonucleotide probe (1 ng) in the absence or presence of various competitors. The reaction mixtures were then applied to a nondenaturing 5% polyacrylamide gel and separated by electrophoresis at 100 V for 3 h in a buffer containing 50 mM Tris, 380 mM glycine, and 2 mM EDTA. The gel was exposed to x-ray film with intensifying screens. The following oligonucleotides were used for EM-SAs: topo-ICE1 (5Ј-GAGTCAGGGATTGG CTGGTCTGCTTCGGGC-3Ј, nt Ϫ77 to Ϫ48 of the topoII␣ gene), topo-HSE (5Ј-GGGCTAAAGG AAG-GTTCAAGTGGAGCTCTC-3Ј, nt Ϫ47 to Ϫ18 of the topoII␣ gene), and HSP70-HSE (5Ј-GA AACCCCTGGAATATTCCCGACC-3Ј, nt Ϫ114 to Ϫ91 of the human HSP70 gene). For supershift assays, 2 g of antibodies to heat shock factor HSF1 or HSF2 (30) were incubated with nuclear extract for 30 min at room temperature before addition of 32 P-labeled oligonucleotide probe.

Effects of Heat Shock
Stress on the Abundance of topoI, topoII␣, and HSP70 mRNAs-Consistent with our previous observations with human head and neck or colorectal cancer cells (22,23), Northern blot analysis revealed that exposure of T24 cells to 43°C for 1 h resulted in an initial small decrease in the amount of topoII␣ mRNA, which was followed by an increase in transcript abundance that was maximal (ϳ3-fold) at 24 h (Fig. 1). The amount of HSP70 mRNA was increased immediately after heat treatment, reaching a maximum (ϳ18fold induction) at 1 h. In contrast, the amount of topoI mRNA was not affected by heat stress. The HSP70 and topoII␣ genes thus showed characteristics of immediate-early and late genes, respectively, in response to heat shock.
Basal Transcriptional Activity of the topoII␣ Gene Promoter-We measured the basal transcriptional activity of the to-poII␣ gene promoter in T24 cells transiently transfected with various luciferase reporter plasmids (Fig. 2). Maximal luciferase activity was obtained with the reporter construct with the pTII␣Ϫ295 insert, which contains four ICEs, the GC box, and the HSE between nt Ϫ295 and ϩ85 of the topoII␣ gene.
Stepwise deletion of ICE3, ICE2, and the combination of ICE1, GC box, and HSE from the 5Ј-end of the promoter resulted in marked -fold decreases in luciferase activity, in general agreement with previous results (11).
Effect of Heat Shock Stress on the Transcriptional Activity of the topoII␣ Gene Promoter-Exposure at 43°C for 1 h of T24 cells transiently transfected with the reporter construct containing pTII␣Ϫ295 resulted in an initial ϳ80% decrease in luciferase activity, followed by an increase that was maximal (3-fold) 24 h after heat treatment (Fig. 3). This experiment was repeated with two T24 cell lines stably transfected with the pTII␣Ϫ295 luciferase construct. Again, luciferase activity was decreased immediately after heat treatment, but then showed a time-dependent increase that was maximal (3-4-fold) after 24 h (data not shown).
To identify the promoter sequences responsible for conferring sensitivity to heat shock, we measured luciferase activity 24 h after exposure to 43°C for 1 h of T24 cells transiently transfected with various topoII␣ gene promoter constructs (Fig.  4). Heat shock increased luciferase activity ϳ3-fold in cells transfected with pTII␣Ϫ295, pTII␣Ϫ197, pTII␣Ϫ154, or pTII␣Ϫ74, but did not increase luciferase activity in cells transfected with pTII␣Ϫ20. The promoter sequence between nt Ϫ74 and Ϫ20, which contains ICE1, the GC box, and the HSE, thus appears to mediate transcriptional activation by heat shock.
Effects of Mutations in the topoII␣ Gene Promoter on Heat Shock Sensitivity-The roles of ICE1, the GC box, and the HSE FIG. 1. Effects of heat shock stress on the abundance of topoI, topoII␣, and HSP70 mRNAs. A, T24 cells were plated, incubated at 37°C for 24 h, and then exposed to 43°C for 1 h. Total RNA was isolated from the cells either immediately (0 h) or after further incubation at 37°C for 1, 6, 12, 24, or 36 h and subjected to Northern blot analysis with 32 P-labeled topoI, topoII␣, HSP70, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probes. Lane C corresponds to RNA isolated from control cells not subjected to heat treatment. B, data in A were subjected to image analysis, and the amounts of topoI (f), topo II␣ (E), and HSP70 (q) mRNAs at the various times were normalized by the amount of glyceraldehyde-3-phosphate dehydrogenase mRNA and are expressed relative to the value for control (C) cells. Data are representative of two similar experiments.

FIG. 2. Basal transcriptional activity of topoII␣ promoter constructs in T24 cells.
Plasmids containing various lengths of the human topoII␣ gene promoter upstream of the luciferase gene were constructed as described under "Experimental Procedures." The nucleotide positions indicated are relative to the major start site of transcription (16), shown by arrows. The four ICEs, GC box, and HSE in pTII␣Ϫ295 are indicated. T24 cells were subjected to transient transfection with 2.5 g of luciferase reporter plasmid and 0.5 g of pSV2-␤-GAL by calcium phosphate precipitation. Four hours after transfection, the cells were washed, incubated in fresh medium for 48 h at 37°C, and subjected to determination of luciferase activity. Data were corrected for differences in transfection efficiency on the basis of ␤-galactosidase activity, are expressed as a percentage of the corrected luciferase activity of cells transfected with pTII␣Ϫ295, and are means of triplicate determinations from a representative experiment.
FIG. 3. Effect of heat shock on the transcriptional activity of the topoII␣ gene promoter. T24 cells were transiently transfected with the luciferase reporter plasmid containing pTII␣Ϫ295 and with pSV2-␤-GAL. Four hours after transfection, the cells were washed, incubated in fresh medium for 24 h at 37°C, and exposed to 43°C for 1 h. The cells were then harvested immediately (0 h) or after further incubation at 37°C for 1, 6, 12, or 24 h and assayed for luciferase activity. Data were normalized to ␤-galactosidase activity, are expressed as a percentage of the corrected luciferase activity of control (C) non-heat-treated cells, and are means Ϯ S.D. of three independent experiments. in heat induction of topoII␣ gene promoter activity were investigated in T24 cells transiently transfected with luciferase reporter plasmids containing promoter sequences with specific mutations in these elements: GGATTGGCT in ICE1 was converted to GGAAAAACT (pTII␣Ϫ295m5), GGGCGGG in the GC box to AAAAAAG (pTII␣Ϫ295m6), and GGAAGGTTCAAGTG in the HSE to GAAAGGAAAAAATG (pTII␣Ϫ295m7) (Fig. 5A). The pTII␣Ϫ295m5 construct showed increased basal transcriptional activity, but luciferase activity was not increased further by heat shock (Fig. 5B). In contrast, heat shock increased the transcriptional activities of pTII␣Ϫ295m6 and pTII␣Ϫ295m7 ϳ3-fold; the transcriptional activities of these two plasmids were ϳ30 and 10%, respectively, of that of the wild-type plasmid. Thus, a factor that binds to ICE1 might negatively regulate basal promoter activity, and ICE1 appears to play a key role in heat-induced activation of the topoII␣ gene promoter. Whereas the GC box and HSE appear to contribute to basal promoter activity, they do not appear to be directly responsible for heat-induced promoter activation.
EMSA Analysis-We next investigated the effects of heat shock on the ICE (Y-box) binding proteins and HSFs with the use of EMSAs. A marked decrease in Y-box binding activity was apparent 3, 6, 12, and 24 h after heat shock (Fig. 6A). Formation of the complex was inhibited in the presence of either excess unlabeled oligonucleotide.
EMSAs performed with a typical HSE derived from the human HSP70.1 gene revealed the absence of a retarded signal in untreated cells (Fig. 6B). A retarded complex was detected with nuclear extracts of cells prepared immediately (0 h) after heat treatment; formation of this complex was inhibited in the presence of excess unlabeled oligonucleotide, and the complex was "supershifted" in the presence of antibodies to HSF1, but not in the presence of antibodies to HSF2. With the HSE of the topoII␣ gene as probe, a retarded complex was observed with nuclear extracts prepared from untreated cells and from cells after heat shock (Fig. 6C). However, the amount of this retarded complex was not affected by heat stress. Formation of this complex was inhibited by excess unlabeled oligonucleotide, but was not affected by antibodies to HSF1 or HSF2.
In Vivo Genomic Footprint Analysis-We examined the effects of heat shock on the dimethyl sulfate methylation patterns in the promoter region of the topoII␣ gene by in vivo genomic footprint analysis. Both G Ϫ64 and G Ϫ65 in ICE1 were protected in untreated cells, but protection was markedly reduced 3, 6, and 24 h after heat shock (Fig. 7). Methylation patterns of the GC box, HSE, and other elements in the topoII␣ promoter region (nt Ϫ295 to ϩ85) were not substantially affected by heat shock stress (data not shown). DISCUSSION We have previously shown that expression of the topoII␣ gene is increased 6 -24 h after exposure of human head and neck or colorectal cancer cells to heat shock stress (22,23). In the present study, we have shown that heat stress also induced activation of topoII␣ gene expression in human urinary bladder cancer cells. This heat-induced up-regulation of topoII␣ gene expression appeared to be mediated through an ICE or Y-box located between nt Ϫ74 and Ϫ21 on the basis of the following results. (i) The luciferase activity of T24 cells transfected with reporter constructs containing pTII␣Ϫ295, pTII␣Ϫ197, pTII␣Ϫ154, or pTII␣Ϫ74 was increased ϳ3-fold by heat shock FIG. 4. Identification of the promoter elements responsible for transcriptional activation of the topoII␣ gene by heat shock. T24 cells were transiently transfected with luciferase reporter plasmids containing the indicated topoII␣ gene promoter constructs and with pSV2-␤-GAL. Four hours after transfection, the cells were washed, incubated in fresh medium at 37°C for 24 h, exposed to 43°C for 1 h, and then incubated at 37°C for an additional 24 h. Luciferase activity was assayed and normalized to ␤-galactosidase activity. Data are expressed as a percentage of the corrected luciferase activity of the corresponding transfected cells not subjected to heat treatment and are means Ϯ S.D. of three independent experiments.

FIG. 5. Effects of mutations in the topoII␣ gene promoter on heat induction of transcriptional activity.
A, shown are sequences (nt Ϫ70 to Ϫ21) of the promoter constructs with mutations in ICE1 (pTII␣Ϫ295m5), the GC box (pTII␣Ϫ295m6), or the HSE (pTII␣Ϫ 295m7). Mutation sites are underlined. B, T24 cells were transiently transfected with the mutant constructs, subjected (closed bars) or not (open bars) to heat treatment, and assayed for luciferase activity as described in the legend to Fig. 4. Data were normalized to ␤-galactosidase activity, are expressed as a percentage of the corrected luciferase activity of nonheat-treated cells transfected with pTII␣Ϫ295, and are means Ϯ S.D. of three independent experiments. *, p Ͻ 0.01. stress, whereas that of cells transfected with a construct containing pTII␣Ϫ20 was not increased by heat treatment. (ii) Introduction of mutations into ICE1 of the topoII␣ gene promoter virtually eliminated the heat shock-induced increase in transcriptional activity, whereas mutation of the GC box or HSE had no such effect. (iii) EMSA analysis with nuclear extracts revealed a marked decrease in ICE1-binding activity 3-24 h after heat shock, consistent with the time course of the heat shock-induced increase in promoter activity, whereas HSE-binding activity was not affected by heat stress. (iv) In vivo genomic footprint analysis revealed a specific change in the methylation pattern of ICE1 induced by heat shock stress.
Members of the ICE-binding (YB-1) family of proteins are expressed in a wide range of cell types and function as important regulators of growth-associated and other genes (31)(32)(33)(34). The expression of genes encoding the epidermal growth factor receptor (35), proliferating cell nuclear antigen (36), DNA polymerase ␣ (37), and thymidine kinase (38) is regulated in a positive manner by ICEs. In contrast, such elements mediate down-regulation of the expression of genes encoding serum albumin, estrogen-dependent very low density lipoprotein apolipoprotein II, aldolase B, and class II major histocompatibility complex (39 -41). In the present study, deletion of nt Ϫ197 to Ϫ155, with contain ICE3, reduced basal promoter activity to about half of that apparent with the topoII␣ gene promoter constructs pTII␣Ϫ295 and pTII␣Ϫ197. Further deletion of nt Ϫ154 to Ϫ75, containing ICE2, and of nt Ϫ74 to Ϫ21, containing ICE1, reduced basal promoter activity to ϳ10 and 2%, respectively, of that apparent with pTII␣Ϫ295. Consecutive deletion of the five ICEs from the topoII␣ gene promoter was also previously shown to reduce basal promoter activity in a stepwise manner (11,18). Thus, the ICEs in the promoter of the human topoII␣ gene appear to play an important role in basal transcriptional activity.
Introduction of point mutations into ICE1 of the topoII␣ gene promoter alleviated the inhibition of topoII␣ gene expression by wild-type p53 (18). Fraser et al. (42) showed that the topoII␣ gene promoter is activated at an early stage during monocytic differentiation of human leukemia cells induced by phorbol ester or sodium butyrate and that this sodium butyrate-dependent up-regulation of topoII␣ gene expression is mediated by the promoter region between nt Ϫ90 and ϩ90, which contains ICE1. In contrast, inhibition of topoII␣ gene promoter activity in confluence-arrested cells appears to be mediated through interaction of the CCAAT-binding factor CBF/NF-Y with ICE2 (43).
In the present study, deletion or mutation of ICE1 in the topoII␣ gene promoter prevented the heat shock-induced increase in transcriptional activity. Moreover, both EMSA and in vivo genomic footprint analysis indicated that nuclear ICE1binding activity was decreased after heat shock stress. These observations indicate that ICE1 negatively regulates the human topoII␣ gene and that heat shock stress reverses this effect, possibly by inducing the dissociation of negative regulatory factors from ICE1. The Y-box binding protein YB-1 has been shown to inhibit interferon ␥-induced activation of class II major histocompatibility complex genes (41). In contrast, activation of the human MDR1 gene in response to heat shock, DNA-damaging anticancer agents, or ultraviolet light is mediated by interaction of a Y-box binding protein with an ICE in the promoter of this gene (29, 44 -47). Expression of YB-1 is also increased in response to genotoxic stress, suggesting that the promoter of the YB-1 gene itself is also sensitive to cytotoxic environmental stimuli (32,48). ICEs thus appear to mediate either negative or positive regulation of specific genes in response to exogenous stimuli. Brandt et al. (21) recently showed that c-Myb activated the human topoII␣ gene promoter via a Myb-binding site at nt Ϫ16 to Ϫ11 in human leukemia cells. In the present study, the basal promoter activity of pTII␣Ϫ20 was only 1.6% of that of pTII␣Ϫ295, and heat shock did not increase the transcriptional activity of this construct. It is thus unlikely that the Myb-binding site at Ϫ16 to Ϫ11 plays an important role in the heat activation of promoter activity of the topoII␣ gene.
Heat shock induces the expression of heat shock-related genes in mammalian cells, and this activation is mediated by HSFs (49 -53). HSFs bind to HSEs, which consist of contiguous arrays of the pentanucleotide motif 5Ј-NGAAN-3Ј present in FIG. 6. EMSA analysis of the effects of heat shock on the binding activity of proteins that interact with the topoII␣ gene promoter. A, EMSAs were performed with 32 P-labeled topo-ICE1 oligonucleotide as probe, and nuclear extracts were prepared from untreated control (C) cells or from heat-treated (43°C for 1 h) cells after incubation for the indicated times at 37°C. The effect of a 100-fold excess of unlabeled topo-ICE1 oligonucleotide as a competitor (Competitor) is shown. Arrowheads indicate specific retarded complex (S), nonspecific complex (NS), and free labeled probe (Free). B, EMSAs were performed with 32 P-labeled HSP70-HSE oligonucleotide as probe, and nuclear extracts were prepared from untreated control cells or from heat-treated cells after incubation for the indicated times at 37°C. The effects of a 100-fold excess of unlabeled HSP70-HSE oligonucleotide (Competitor) and of antibodies to HSF1 (anti-HSF1) or HSF2 (anti-HSF2) are shown. Arrowheads indicate specific complexes (S), specific supershifted complex (SS), nonspecific complex (NS), and free probe (Free). C, EMSAs were performed with 32 P-labeled topo-HSE oligonucleotide as probe, and nuclear extracts were prepared from control cells or from heat-treated cells after incubation for the indicated times at 37°C. The effects of a 100-fold excess of unlabeled topo-HSE (Competitor) and of antibodies to HSF1 (anti-HSF1) or HSF2 (anti-HSF2) are shown. Arrowheads indicate specific complexes (S), nonspecific complex (NS), and free probe (Free).
alternating orientations in the promoter regions of heat shock genes. Most heat-inducible genes, including HSP genes, contain an HSE consisting of four or more pentanucleotide motifs and respond to heat treatment within 1 h concomitant with marked fluctuations in nuclear HSF content (27,30,54,55). Our data confirm that HSF1, but not HSF2, binds to the HSE of the human HSP70 gene immediately after heat shock. However, the HSE of the topoII␣ gene consists of only two pentanucleotide motifs, and heat shock-induced transcriptional activation of the topoII␣ gene was not apparent until 6 -24 h after heat treatment. Furthermore, no increase in the binding of nuclear factors to the HSE of the topoII␣ gene after heat treatment was apparent by EMSA or in vivo footprint analysis. It is thus unlikely that the HSE in the topoII␣ gene promoter is responsible for the heat-induced activation of this gene. FIG. 7. In vivo footprint analysis of the topoII␣ gene promoter. A, untreated control (C) T24 cells or cells that had been incubated at 43°C for 1 h and then at 37°C for 3, 6, or 24 h were exposed to 0.05% dimethyl sulfate as indicated. Genomic DNA was isolated, and the promoter region of the topoII␣ gene was subjected to footprint analysis. The lane labeled Naked represents protein-free DNA that was methylated in vitro. The sequence of nt Ϫ70 to Ϫ25 of the topoII␣ gene promoter and the positions of G Ϫ65 and G Ϫ64 in ICE1, the GC box, and the HSE are indicated. B, data in A were subjected to image analysis, and the radioactivities of G Ϫ65 and G Ϫ64 at the various times were normalized to the radioactivity of G Ϫ56 and are expressed as a percentage of the value for naked DNA. Data are representative of two similar experiments.