©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of the Promoter Region of the Human Apurinic Endonuclease Gene (APE) (*)

(Received for publication, November 1, 1994; and in revised form, December 15, 1994)

Lynn Harrison Antony Gian Ascione David M. Wilson III (§) Bruce Demple (¶)

From the Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Apurinic/apyrimidinic (AP) sites are mutagenic and block DNA synthesis in vitro. Repair of AP sites is initiated by AP endonucleases that cleave just 5` to the damage. We linked a 4.1-kilobase pair HindIII DNA fragment from the region upstream of the human AP endonuclease gene (APE) to the chloramphenicol acetyltransferase (CAT) gene. Deletions generated constructs containing 1.9 kilobase pairs to 50 base pairs (bp) of the APE upstream region. Transient transfection studies in HeLa cells established that the basal APE promoter is contained within a 500-bp fragment. The major transcriptional start site in HeLa, hepatoma (HepG2), and myeloid leukemic (K562) cells was mapped to a cluster of sites 130 bp downstream of a putative ``CCAAT box,'' 130 bp 5` of the first splice junction in APE. Deletion of 5` sequences to within 10 bp of the CCAAT box reduced the CAT activity by only about half, and removal of the CCAAT box region left a residual promoter activity 9%. Deletion to 31 bp upstream of the transcriptional start site abolished APE promoter activity. DNA sequence analysis revealed potential transcription factor recognition sites in the APE promoter. Gel mobility-shift assays showed that both human upstream factor and Sp1 can bind their respective sites in the APE promoter. However, DNase I footprinting using HeLa nuclear extract showed that the binding of Sp1 and upstream factor is blocked by the binding of other proteins to the nearby CCAAT box region.


INTRODUCTION

Apurinic endonucleases initiate the repair of apurinic/apyrimidinic (AP) (^1)sites, of which perhaps thousands per day are introduced into the human genome by spontaneous base hydrolysis and reactions with oxygen radicals and other cellular metabolites (Lindahl, 1993). If left unrepaired, the bypass of AP sites during DNA replication can result in mutations and loss of genetic integrity (Loeb and Preston, 1986). For example, yeast strains deficient in Apn1 (the major AP endonuclease of Saccharomyces cerevisiae) have a substantially elevated frequency of spontaneous mutations (Ramotar et al., 1991). The extra mutations arising in these repair-deficient yeast strains include all classes of single-base pair substitutions, but most dramatically transversions prompted by the loss of purines (Kunz et al., 1994).

Molecular studies of prokaryotic and eukaryotic AP endonucleases have revealed two families of proteins: those related to Escherichia coli endonuclease IV and yeast Apn1, or those related to E. coli exonuclease III and the major human AP endonuclease, Ape (Demple and Harrison, 1994). Both enzyme families are ``class II'' AP endonucleases, which hydrolyze the phosphodiester bond on the 5` of AP sites (Levin and Demple, 1990) to allow excision and repair DNA synthesis (Demple and Harrison, 1994). These AP endonucleases also remove 3`-phosphoglycolate esters and 3`-phosphates from oxidative strand breaks (Chen et al., 1991; Demple et al., 1986; Henner et al., 1983; Johnson and Demple, 1988; Winters et al., 1992, 1994). Although E. coli exonuclease III has a robust 3`-repair diesterase function (Demple et al., 1986), this is a minor activity of the homologous human Ape protein (Chen et al., 1991). The cDNA (Demple et al., 1991; Robson and Hickson, 1991; Seki et al., 1992) and the gene (Harrison et al., 1992) encoding the major human AP endonuclease have been isolated. The APE gene has five exons, one untranslated, and four introns, and is contained within a 3-kb segment of DNA located on chromosome 14q at position 11.2-12 (Harrison et al., 1992). The same gene was identified in independent cloning efforts as HAP1 (Robson et al., 1992; Zhao et al., 1992) and as APEX (Akiyama et al., 1994), and assigned to the same chromosomal site. No disease related to DNA repair has been directly linked to this genome locus. However, the importance of this repair enzyme is suggested by the increased sensitivity to H(2)O(2) or alkylating agents of rat glioma cells expressing of an antisense APE transcript (Ono et al., 1994).

APE and its protein product were also isolated in a screen for an activity that restores DNA binding activity to oxidized c-Fos and c-Jun proteins in vitro (Xanthoudakis et al., 1992). This ``Ref1'' activity was also found for a larger, related protein from the plant Arabidopsis thaliana (Babiychuk et al., 1994). The in vitro Ref1 activity seems to reside in a short segment of Ape outside the region homologous to exonuclease III and independent of the AP endonuclease function (Walker et al., 1993; Xanthoudakis et al., 1994).

Hypoxic conditions in colon cancer cells increased APE expression (Yao et al., 1994). Expression of APE is also transcriptionally modulated during regeneration of the epithelium after physical injury. (^2)We have therefore attempted to identify the cellular components that regulate expression of the APE gene. The work presented here identifies promoter elements and DNA-binding activities that mediate basal APE expression in cultured cells.


MATERIALS AND METHODS

Cell Culture

HeLa-S3 cells were grown in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Inc.) supplemented with 10% bovine calf serum. K562 and HepG2 cells were grown in RPMI 1640 supplemented with 10% heat-inactivated horse serum and modified Eagle's medium with Earle's salts (Life Technologies, Inc.) supplemented with 10% fetal calf serum, respectively. Treatment of cells was carried out at 37 °C in tissue culture flasks for treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), dimethyl sulfoxide (Me(2)SO), paraquat, or dexamethasone. HeLa cells were grown in roller bottle cultures for treatment with bleomycin sulfate or heat shock.

Chemical Agents

TPA (Life Technologies, Inc.) was dissolved in Me(2)SO (Sigma) at a concentration of 1.62 mM and stored at -20 °C. Bleomycin sulfate (Sigma) was dissolved in double distilled water at 5 mg/ml and stored at -20 °C. Paraquat (methyl viologen; Sigma) was prepared in double distilled water as a 50 mM stock and used immediately. Dexamethasone was prepared at 20 µg/ml in 2% (v/v) ethanol in DMEM supplemented with 10% bovine calf serum.

Protein and RNA Analysis

Total cellular RNA was isolated using a procedure described by Chomczynski and Sacchi(1987). For Northern blot analysis, 10 µg of RNA was subjected to electrophoresis in a 1% agarose gel containing 0.5 M formaldehyde. The RNA was transferred to positively charged nylon membranes (Boehringer Mannheim) by capillary action and incubated at 42 °C with P-labeled, heat-denatured APE cDNA, according to standard procedures (Sambrook et al., 1989).

To determine the level of Ape activity, cells were harvested after washing with phosphate-buffered saline (PBS) and extracts prepared according to Chen et al.(1991). After treatment of HeLa cells with bleomycin sulfate or heat shock, S100 extracts were used to partially purify Ape using DE52 and Bio-Rex 70 chromatography, as described previously (Chen et al., 1991). Assays for 3`-repair diesterase were carried out according to Chen et al.(1990).

Transcription Start Site

To identify the transcriptional start site for APE, an oligonucleotide complementary to the 3` portion of the first exon (5`-CGAGATCTGCCCTCCAGCCAATT-3`; Operon Technologies, Inc., Alameda, CA) was end-labeled using [-P]ATP and T4 polynucleotide kinase (Sambrook et al., 1989). The kinase was heatinactivated at 90 °C for 2 min and the end-labeled primer stored at -20 °C until required. For primer extension reactions, 5 µg of total RNA and 12.5 pmol of end-labeled primer were mixed and denatured by heating to 70 °C for 10 min in a final volume of 12.5 µl. Samples were placed on ice for 1 min before resuspension in reaction buffer for avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI), or Superscript II from Life Technologies, Inc. The samples were warmed to 42 °C for 2 min, and 1 µl of reverse transcriptase was added to the mixture. Synthesis of cDNA was performed at 42 °C for 30 min, and the samples were separated on a 6% polyacrylamide gel.

Production of Promoter Constructs

Plasmid pLHBS1 (Harrison et al., 1992) was digested with HindIII, and the 4.1-kb APE promoter fragment (Fig. 1B) ligated into the HindIII site of pCATBASIC (Promega) in both orientations to produce plasmids pCB1 (antisense) and pCB2 (sense). Plasmid pAPE2 (Harrison et al., 1992) was digested with SmaI and the 5.4-kb human genomic fragment containing the APE gene ligated into the SmaI site of pBluescript-SK to produce pLHBS3. After digestion of pLHBS3 with SmaI and NruI, a 2-kb APE promoter fragment was isolated and ligated into the SalI site of pCATBASIC to produce pCB9. Digestion of pCB9 with SphI and SpeI and re-ligation removed a 1-kb fragment to produce pCB10. Digestion of pCB9 with PstI and re-ligation removed a 1.5-kb fragment to produce pCB11. Plasmid pCB9 was digested with SphI, together with DraIII, BssHII, or AvaI to produce pCB18, pCB19, and pCB23, respectively. To produce pCB21, pCB9 was digested with HindIII to remove 1.95 kb of the upstream region of APE and re-ligated. Digestion of pCB11 with HindIII and PstI allowed the isolation of a 500-bp fragment that was then ligated into the SalI site of pCATBASIC to produce pCB22. To produce pCB26, pCB27, pCB29, and pCB30, pCB22 was digested with SacII, digested with Bal-31 nuclease for 1-1.5 min and re-ligated. pCB15 was isolated following AvaI digestion of pCB10 to remove a 184-bp segment of the APE promoter, and re-ligation. Plasmid pCB22 was digested with DraIII together with BssHII or BanII to produce pCB33 and pCB34, respectively. Plasmid pCB20 was produced by the removal of a 43-bp fragment from pCB11 by digestion with BssHII and BanII. All the plasmids were confirmed by DNA sequencing.


Figure 1: Physical structure of the APE promoter. A, the 14-kb fragment encompassing the APE gene showing the restriction sites for XhoI (X), SmaI (S), HindIII (H), PstI (P), SacI (S1), and BamHI (B). B, the 4.1-kb HindIII fragment, which contains 65 bp of the first untranslated exon (). C, the region between the PstI and NruI sites, indicating potential recognition sites for Sp1, glucocorticoid receptor (GR), USF, and AP1. A potential CCAAT box is also situated in this region.



Transient Transfection

The amounts of DNA used for transfection corresponded to the number of molecules in 15 µg of pCB1 or pCB2. The total amount of test plasmid DNA in each transfection was made up to 15 µg by the addition of pCATBASIC DNA and then mixed with 10 µg of pSVbetagal (Promega) in 200 µl of PBS. 5 times 10^6 HeLa-S3 cells were washed in PBS and resuspended in the DNA solution. The cells were electroporated at 240 V, 200 ohms, and 960 microfarads, resuspended in 30 ml of DMEM and 10% bovine calf serum, and placed in two 100-mm Petri dishes. After 48 h at 37 °C, the cells were harvested, resuspended in 200 µl of 0.25 M Tris-HCl (pH 8), and extracted by three freeze-thaw cycles. After centrifugation for 15 min at 14,000 rpm and 4 °C, the cell-free extracts were stored at -80 °C. beta-Galactosidase assays were carried out according to Sambrook et al.(1989). The extracts were then heated at 60 °C for 10 min to inactivate mammalian acetylases and centrifuged at 4 °C. The resulting supernatants were assayed for chloramphenicol acetyltransferase (CAT) activity (Promega Technical Bulletin 084).

Mobility-shift Analysis

Digestion of pCB18 or pCB19 with HindIII released APE promoter fragments of 208 and 165 bp, respectively, while AvaI digestion of pCB11 or pCB20 produced fragments of 191 and 148 bp, respectively. The isolated fragments were end-labeled by incubation with Klenow DNA polymerase (New England Biolabs, Inc., Beverly, MA), 167 µM each of unlabeled dNTPs, and 50 µCi of [alpha-P]dGTP or [alpha-P]dCTP at room temperature for 15 min. End-labeled DNA (0.5-4 fmol) was incubated with HeLa nuclear extract (prepared according to Dignam et al., 1983) or with 50-150 ng of purified recombinant human Sp1 protein (Promega) in 10 µl of binding buffer (4% glycerol (v/v), 1 mM MgCl(2), 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 50 µg/ml poly(dI-dC)) for 20 min at room temperature. For reactions with purified Sp1, 1 µg of bovine serum albumin (molecular biology grade; Boehringer Mannheim) was added to the binding reaction. In some cases a synthetic double-stranded oligonucleotide, containing a consensus Sp1 recognition site, was incubated with the protein for 5 min at room temperature prior to the addition of the labeled probe. Where indicated, polyclonal antiserum against the USF transcription factor (a generous gift from Dr. M. Sawadogo, Houston, TX) (^3)was added after 5 min to a final dilution of 10 to 10 and the incubation continued for another 15 min. After electrophoresis at 100 V in a 4% polyacrylamide gel in 0.5 times TBE buffer (Sambrook et al., 1989) and drying, the gels were autoradiographed.

DNase I Footprinting Studies

Plasmid pCB29 was digested with AvaI and end-labeled as for the protein binding probes. The DNA was then precipitated with ethanol, resuspended in HaeII reaction buffer, and digested with HaeII. After polyacrylamide electrophoresis, the gel containing the end-labeled 239-bp fragment was incubated in 0.5 ml of elution buffer (0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA, 0.1% SDS) for 12 h. The DNA was precipitated with ethanol and resuspended in 30 µl 10 mM Tris-HCl, pH 8.0, 1 mM EDTA. End-labeled probe (4 fmol) was incubated with 0-30 µg of HeLa nuclear extract or 0-50 ng of recombinant human Sp1 protein, in 10-µl binding reactions (poly(dI-dC) omitted from Sp1 reactions). After 20 min at room temperature, 40 µl of binding buffer and 50 µl of 5 mM CaCl(2), 10 mM MgCl(2) were added. RQ1 RNase-free DNase (Promega) was diluted in cold 10 mM Tris-HCl (pH 8) to 0.05 units/µl, and 1-3 µl added to the reaction. After 75 s, 90 µl of stop solution (200 mM NaCl, 30 mM EDTA, 1% SDS, 100 µg/ml yeast tRNA) was added. Protein was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and the DNA precipitated with ethanol. After washing with 70% ethanol and drying, the DNA pellet was resuspended in 4 µl of loading solution (0.1 M NaOH:formamide (1:2, v/v), 0.1% xylene cyanol, 0.1% bromphenol blue), heated to 95 °C for 2 min, and subjected to electrophoresis in a denaturing 6% polyacrylamide gel.


RESULTS

Significant regulation of the APE gene in response to DNA-damaging agents has not been reported. In our hands, the amount of Ape protein or APE mRNA was not altered after treatment of HeLa cells with various DNA damaging agents: bleomycin sulfate, paraquat (a free radical generator), or heat shock (summarized in Table 1). However, our own recent work indicates possible transcriptional regulation of APE during epithelial wound healing,^2 and the recently reported hypoxic response of APE (Yao et al., 1994) shows that APE transcription is regulated under some circumstances. We therefore analyzed the functional elements of the APE promoter.



Analysis of the 2-kb region upstream of APE (data not shown) for possible regulatory sequences (using the GenBank transcription factor data base) revealed several potential recognition sites for transcription factors within the 600 bp upstream of the start point of APE cDNA (GenBank accession no. M99703). These sites included potential binding sequences for Sp1, glucocorticord receptor, c-Myc-like proteins (Faisst and Meyer, 1992) such as upstream factor (USF; Sawadogo et al., 1988; Gregor et al., 1990), and AP1 (Fig. 1). In order to determine whether the AP1 or glucocorticoid receptor binding sites were biologically functional in the APE promoter, K562 or HeLa cells were treated with a phorbol ester (TPA) or dexamethasone. Although Northern analyses demonstrated strong induction of the 2.4-kb c-Fos mRNA after a 6-h TPA treatment, the level of the 1.5-kb APE message was not detectably altered during a 24-h exposure to TPA (Table 1). The APE mRNA also did not vary significantly following dexamethasone treatment (Table 1). Despite the response of APE to hypoxic conditions, incubation of K562 cells with the hydroxyl radical scavenger dimethyl sulfoxide (1.2% final concentration) did not change APE transcription (Table 1).

Transcriptional Start Sites

For understanding the significance of the transcription factor binding sites, the transcriptional start site of APE was determined. Total RNA was isolated from three different cell types: K562 myeloid leukemic cells, HepG2 derived from hepatoma cells, and HeLa cells derived from a cervical carcinoma, and analyzed by primer extension. Although multiple start sites were found in all the cell types, three major start sites were identified consistently. These sites were clustered within a 7-bp region, 130 bp downstream of the putative CCAAT box in all three cell types (Fig. 2). Such clusters of multiple start sites are common for genes lacking a TATA box (e.g. phenylalanine hydroxylase, Konecki et al.(1992); cystic fibrosis gene, Yoshimura et al.(1991); macrophage colony stimulating factor receptor gene, Yue et al.(1993)). The transcriptional start sites reported for the human lymphoblastoid cell line WIL2-NS (Zhao et al., 1992) and for HeLa cells (Robson et al., 1992), located approximately 75 bp upstream of the sites observed here, were not detected in our experiments.


Figure 2: Transcription start sites for APE. A, primer extension was used to identify the transcriptional start sites of the APE mRNA in total RNA isolated from HeLa, HepG2, and K562 cells. Yeast tRNA was used as a negative control. The template DNA sequence is shown with the start site of the longest transcript indicated by +1. B, sequence of the region around the transcription start sites (coding strand). The three major, consistent start sites are underlined. The first major start site (+1) is 128 bp downstream of the putative CCAAT box (shown in bold). The intervening region also contains two consensus recognition sequences (CACGTG; shown in bold) for USF/Myc-like protein.



Identification of the APE Promoter Region

To identify functional regions of the APE promoter, a series of DNA fragments upstream of the APE coding region was ligated into pCATBASIC, which contains the reporter gene encoding CAT. The resulting series of reporter plasmids was analyzed in transient transfection experiments in HeLa cells. Transfection of pCATBASIC alone resulted in negligible CAT activity in the cell extracts.

A 500-bp insert (in pCB22) had equivalent basal promoter activity to a 4-kb insert (in pCB2, Fig. 3A). Ligation of the 4-kb insert in the reverse orientation (pCB1) or deletion of 775 bp 5` to the transcriptional start site (pCB17) resulted in negligible CAT activity. No significant difference was detected between pCB22 and pCB11 (the latter plasmid containing a putative recognition sequence for AP1), which suggests that the segment downstream of the transcription start sites (+65 to +118) is not necessary for basal promoter activity.



Figure 3: Reporter gene constructs for the APE promoter. Individual plasmids (see text for construction) were transfected into HeLa cells together with pSVbetagal. After 48 h cells were harvested, extracted and assayed for beta-galactosidase and CAT activities. The ratio of CAT activity to beta-galactosidase was calculated and is expressed as a percentage of the ratio obtained for pCB22 in the same experiment. Each plasmid was transfected at least four independent times, using two different passages of HeLa cells. Symbols are as for Fig. 1. A, activity of promoter segments of 0.5-4.1 kb. B, activity of promoter segments deleted from -462 to -138. C, activity of promoter segments deleted from -138 to +65. D, effect of internal deletions on APE promoter activity.



Deletion of the region between -462 and -412, which contains a putative Sp1 recognition site, also did not alter basal promoter activity (Fig. 3B). In order to detect a significant decrease in basal promoter activity, it was necessary to delete the 5` terminus of the genomic insert to -138 (in pCB18), which expressed approximately half the CAT activity of pCB22 (Fig. 3B). The end point in pCB18 lies only 10 bp upstream of the CCAAT box. The region between -173 (pCB29) and -138 (pCB18) contains two overlapping Sp1 consensus recognition sequences (Fig. 4A) that may contribute to basal expression. As this paper was being finished, a report (Akiyama et al., 1994) appeared that confirms the results of Fig. 3(A and B).


Figure 4: DNA probes for protein binding studies with the APE promoter. A, the sequence shown between the AvaI sites was essential for APE basal promoter activity. Two overlapping Sp1 sites, three USF/Myc-like consensus sequences, and a CCAAT box-like sequence are shown in bold. Regions 1 and 2, which are underlined, are the sections of DNA protected by purified recombinant human Sp1 protein and HeLa nuclear extract, respectively (see Fig. 7). B, structures of probes I-IV used in protein binding studies. The hatchedboxes indicate the first exon of APE, and the numbering is relative to the first transcription start site (+1).




Figure 7: Nuclear protein-binding sites in the coding strand of the APE promoter. A, Sp1 binding studies. A 3`-labeled probe (containing the -173 to -26 region of the APE promoter) was incubated with the indicated amount of purified Sp1 protein and digested with 0.05, 0.10, or 0.15 units of RQ1 RNase-free DNase. The protected region shown corresponds to bp -169 to -148 of the APE promoter, which contains two overlapping Sp1 consensus recognition sites (see Fig. 4A). B, HeLa nuclear protein binding studies. The same probe as in A incubated with the indicated amount of HeLa nuclear extract and digested with 0.05, 0.10, or 0.15 units of RQ1 RNase-free DNase. The protected region shown corresponds to bp -130 to -105 of the APE promoter and encompasses the CCAAT box. A further region -141 to -131 also shows an alteration in the DNase I digestion pattern.



The deletion experiments showed that the basal APE promoter for HeLa cells is contained in a relatively small region of DNA. Therefore additional reporter gene constructs were prepared to determine the minimum region required for full promoter activity of APE. Little or no CAT activity was expressed with inserts of 53 or 87 bp of the APE upstream region (in pCB21 and pCB23, respectively, Fig. 3C). However, a fragment lacking the putative CCAAT box (-95 to +118) conferred 80% promoter activity relative to pCB22 in four independent transfections (Fig. 3C). Addition of the CCAAT box segment to pCB19 actually decreased the promoter activity consistently 2-fold. It seemed possible that the residual fragment in pCB19 might not accurately represent the APE promoter. Therefore, various internal deletions of pCB22, pCB11 and pCB10 were prepared (Fig. 3D).

All promoter activity was lost by deleting a segment between -210 and -25 (yielding pCB15), which contains the putative CCAAT box, the Sp1 site and a 64-bp segment that conferred promoter activity in pCB19 (Fig. 3C). Promoter activity was reduced 11-fold by deleting the CCAAT box and the Sp1 site (yielding pCB33). When another 43 bp were deleted from pCB22 (to generate pCB34), negligible amounts of CAT activity were expressed (Fig. 3D). This confirmed that promoter activity could be conferred by the region -98 to -55 within a larger genomic fragment. To test the role of this sequence in the context of the CCAAT box, plasmid pCB20 was constructed (Fig. 3D). Six independent transfections of each of the constructs shown in Fig. 3D, using three different passages of HeLa cells, consistently showed pCB20 to express higher levels of CAT activity than pCB22. These results can be contrasted with those for pCB18 and pCB19, which indicated significant promoter activity for the region downstream of the CCAAT box (Fig. 3C).

DNA Binding Studies

The functional significance of the putative protein binding sites in the APE promoter was assessed in a series of protein-DNA binding experiments. For this purpose four different APE promoter fragments (Fig. 4) were isolated by restriction digestion of plasmids pCB19 (probe I), pCB18 (probe II), pCB11 (probe III), and pCB20 (probe IV). After labeling and incubation with HeLa nuclear extracts, gel electrophoresis resolved distinct protein-DNA complexes (Fig. 5A). Probe I yielded two complexes, one of which electrophoresed with a similar mobility to the single complex observed with probe II (Fig. 5). Probes I and II both contained the 43-bp region that conferred promoter activity in pCB19 (Fig. 3C), but probe II contained the CCAAT box in addition (Fig. 4). Probes III and IV also yielded single protein-DNA complexes that contained a significant fraction of the total DNA at the highest protein levels (Fig. 5A). The complexes with probes II, III, and IV were not eliminated by increasing in the binding reactions the concentration of poly(dI-dC) up to 200 µg/ml (Fig. 5B). In contrast, such competition was effective in reducing the amounts of protein-DNA complexes containing probe I (Fig. 5B).


Figure 5: APE promoter binding by HeLa nuclear extracts. A, effect of increasing amounts of nuclear extract. Probes I-IV were labeled (see text) and incubated with 0, 1, 2, and 3 µg (probes II-IV) or 0, 1, and 3 µg (probe I) of HeLa nuclear extract in 50 µg/ml poly(dI-dC), for 20 min at room temperature. After separation in a 4% polyacrylamide gel, complexes were visualized by autoradiography. B, effect of poly(dI-dC). Probes I-III were labeled and, except for the first lane of each set, incubated with 3 µg of HeLa nuclear extract for 20 min at room temperature, in the presence of 50, 100, or 200 µg/ml poly(dI-dC). Labeled probe IV was incubated with 3 µg of extract (except for the first lane of this set) in 50 or 200 µg/ml poly(dI-dC).



In order to determine the contribution of USF and of Sp1 protein to the formation of these complexes, additional binding studies were carried out. The 64-bp region that conferred promoter activity in pCB19 (absent from pCB23; Fig. 3C), contains two consensus binding sites (CACGTG) for c-Myc-like proteins (Prendergast and Ziff, 1991), such as the human upstream factor (USF, Gregor et al., 1990; Fig. 4A). One site (TCACGTGA) is the sequence recognized by the major late transcription factor of adenovirus, of which USF is the human counterpart (Sawadogo et al., 1985; Carthew et al., 1985; Miyamoto et al., 1985). To test for the binding of this protein, we employed specific antibodies.^3 Labeled probe I was incubated with HeLa nuclear extract and polyclonal anti-USF antiserum added to the reactions. Of the two complexes formed, one (the upper complex; Fig. 6A) decreased in intensity with increasing antibody concentration as a series of complexes of slower mobility appeared (Fig. 6A). Neither the second complex formed with probe I nor those formed with probes III and IV were affected by the anti-USF antibody (Fig. 6A). Although it is possible that the binding of other proteins to probe III may have masked the crucial epitopes in bound USF, the formation of a similar mobility complex with probe IV (lacking the USF site; Fig. 4) argues against this interpretation.


Figure 6: USF and Sp1 binding to the APE promoter. A, ``supershift'' by anti-USF antibodies. Probes I, III, and IV were labeled and incubated with 3 µg of HeLa nuclear extract at 50 µg/ml poly(dI-dC) for 5 min at room temperature. Polyclonal anti-USF antiserum was then added to yield a final dilution of 10, 5 times 10, 2.75 times 10, and 10 for probe I, or 5 times 10 and 10 for probes III and IV. Probe alone is indicated by ``-''. After another 15 min, protein-DNA complexes were analyzed as described in the legend to Fig. 5. No complexes were observed with any probe incubated with the antiserum alone (data not shown). B, binding of purified Sp1. Labeled probe III was incubated with 0, 50, and 150 ng of purified recombinant human Sp1 protein in 50 µg/ml poly(dI-dC) and 100 µg/ml bovine serum albumin, for 20 min at room temperature. In some incubations with 100 ng of Sp1 protein, an unlabeled double-stranded oligonucleotide containing the consensus Sp1 recognition site (Promega) was added (0.0175 or 1.75 pmol; rightmostlanes).



Incubation of pure Sp1 protein with probe III yielded two complexes, one of which was less intense and had slower mobility (Fig. 6B). Since this Sp1 site consists of two overlapping recognition sites, the upper band in Fig. 6B may correspond to the binding of a second Sp1 molecule to this second site. The binding by pure Sp1 was competed by a double-stranded synthetic oligonucleotide containing the Sp1 consensus sequence; a 20-fold molar excess diminished probe III binding significantly, and a 2000-fold excess eliminated it (Fig. 6B). Such competition was not observed for binding of HeLa nuclear extract protein(s) to probe III, although those extracts contain Sp1 that binds the synthetic oligonucleotide (data not shown).

Pure Sp1 formed a distinct footprint spanning the base pairs -169 to -148 in the APE promoter, as revealed in DNase protection experiments (Fig. 7A; for sequence see Fig. 4A). However, such a distinct footprint was not observed after incubation with HeLa nuclear extract (Fig. 7B), which instead had strongest binding activity for a 40-bp region containing the CCAAT box (Fig. 7B; for the sequence, see Fig. 4A). A partial protection of the USF site and adjacent 3` sequences may also occur (Fig. 7B). Addition of 50 ng of Sp1 protein to 30 µg of nuclear extract still did not result in a distinct Sp1 footprint (data not shown). Thus, although both USF and Sp1 are available in our nuclear extract, and the respective DNA sites are capable of binding these proteins, the binding of one or more other nuclear proteins over the CCAAT box region prevents USF and Sp1 from efficiently binding their DNA targets.


DISCUSSION

The APE gene product is expressed constitutively at relatively high levels in the nuclei (Demple et al., 1991) of transformed cells such as HeLa cells, Chinese hamster ovary cells, or HPB-ALL T-lymphoblasts (7 times 10^6 molecules/cell; Chen et al., 1991). Conversely, attempts to modulate the expression of this probable DNA repair protein by DNA-damaging treatments have yielded consistently negative results (Table 1). Such experiments have not been reported for untransformed human fibroblasts, in which the Ape levels are 10-20 times lower (Chen et al., 1991). A rather small upstream region (140 bp) of APE seems to be required for the high basal expression in HeLa cells, as shown here by deletion analysis of the APE promoter linked to a reporter gene.

Within this basal APE promoter lie several potential regulatory sites. Surprisingly, deletion of the putative CCAAT box of the APE promoter failed to obliterate transcriptional activity (in pCB19 and pCB33). This expression could result from promoter elements that remain silent when the CCAAT box is present, as may be the case with the USF-binding site (see below).

It is not known whether the altered constructs direct the use of the same transcriptional starts employed for basal expression by the intact APE promoter in HeLa, HepG2, or K562 cells. Like many TATA-less genes (Konecki, et al., 1992; Yoshimura et al., 1991; Yue at al., 1993), APE displays multiple transcription start sites, with those identified here clustered 130 bp upstream from the first splice junction (Harrison et al., 1992). Additional initiation sites for APE mRNA in HeLa cells were seen in some of our experiments (data not shown), and still other sites have been reported from other laboratories (Zhao et al., 1992; Robson et al., 1992; Akiyama et al., 1994). It remains to be seen whether these same sites are used during the induction of APE transcription in hypoxic cells (Yao et al., 1994) or possibly during epithelial regeneration.^2

Potential binding sites for transcription factors are present in the APE promoter (e.g. Sp1 and USF) and within the structural gene (e.g. AP1; Fig. 1). The AP1 site in exon 1 seems not to exert an effect in K562 cells, as suggested by the lack of response of APE transcription to TPA (Table 1). Similarly expression of the rat homolog of APE was not altered with c-fos induction in the hypothalamus after light exposure (Rivkees and Kelley, 1994).

Sp1 sites located -420 bp and -168 bp 5` of APE may make modest contributions to the basal expression, although at least one of these sites was evidently not strongly bound by Sp1 in HeLa nuclear extracts, even though the site 30 bp 5` of the CCAAT box is bound by pure Sp1 protein in vitro. The nuclear extracts also contained functional USF protein that could bind a cognate site in a DNA fragment lacking the CCAAT box, as judged by the ``supershifting'' effect of polyclonal anti-USF antiserum. Some binding was detected overlapping the USF site (40 bp 3` to the CCAAT box) with HeLa nuclear extracts and a hypersensitive site is situated at -69, just 3` to this region (Fig. 7B, and data not shown), but the lack of ``supershifting'' by anti-USF antiserum suggests that this binding does not involve USF itself.

The CCAAT-containing fragments exhibited strong and specific binding of nuclear protein(s) and the protection of a 40-bp region, including the CCAAT box itself. It seems possible that the latter binding represents recognition by basal transcription factor(s) in HeLa extracts, such as CP1 (Chodosh et al., 1988), NF-Y (Dorn et al., 1987), and CP2 or NF-1 (Chodosh et al., 1988). If so, such binding evidently precludes interaction with either USF or Sp1 at their nearby sites. In the absence of the CCAAT box, USF can bind its site and may contribute to the promoter activity of pCB19 and pCB33 (Sawadogo and Roeder, 1985; Sawadogo et al., 1988; Roy et al., 1991).

The APE promoter bears some features in common with so-called ``housekeeping'' genes: lack of a TATA box, multiple transcription start sites (Konecki et al., 1992; Yoshimura et al., 1991; Yue et al., 1993), and similar expression in a variety of tissues (^4)(Akiyama et al., 1994). Such widespread expression suggests the action of transcription factors present in many cell types and active under a variety of conditions. Nonetheless, APE transcription does increase in response to hypoxia in cultured carcinoma cells (Yao et al., 1994) and may be modulated during epithelial wound healing.^2 It will be of interest to determine whether and how the protein binding sites observed here and the functional regions of the APE promoter might be employed during these regulated responses.


FOOTNOTES

*
This work was supported in part by American Cancer Society Grant CN-86 (to B. D.) and National Institutes of Health Grant GM40000 (to B. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of an National Research Service Award from the United States Public Health Service.

To whom correspondence should be addressed.

(^1)
The abbreviations used are: AP, apurinic/apyrimidinic; kb, kilobase pair(s); bp, base pair(s); DMEM, Dulbecco's modified Eagle's medium; TPA, 12-O-tetradecanoylphorbol-13-acetate; PBS, phosphate-buffered saline; USF, upstream factor.

(^2)
L. Harrison, T. Galonopoulos, A. G. Ascione, H. N. Antoniades, and B. Demple, manuscript in preparation.

(^3)
M. Sawadogo, personal communication.

(^4)
L. Hughes-Davies, T. Galonopoulos, L. Harrison, M. Maxwell, H. N. Antoniades, and B. Demple, submitted for publication.


ACKNOWLEDGEMENTS

We thank members of our laboratory for helpful discussions, and S. Phelan and Dr. K. Call for RNA samples from TPA-treated K562 cells. We are grateful to Dr. M. Sawadogo for the generous gift of anti-USF antiserum.


REFERENCES

  1. Akiyama, K., Seki, S., Oshida, T. & Yoshida, M. (1994) Biochim. Biophys. Acta 1219, 15-25 [Medline] [Order article via Infotrieve]
  2. Babiychuk, E., Kushnir, S., Van Montagu, M. & Inze, D. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3299-3303 [Abstract]
  3. Carthew, R. W., Chodosh, L. A. & Sharp, P. A. (1985) Cell 43, 439-448 [Medline] [Order article via Infotrieve]
  4. Chen, D. S., Herman, T. & Demple, B. (1991) Nucleic Acids Res. 19, 5907-5914 [Abstract]
  5. Chodosh, L. A., Baldwin, A. S., Carthew, R. W. & Sharp, P. A. (1988) Cell 53, 11-24 [Medline] [Order article via Infotrieve]
  6. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  7. Demple, B. & Harrison, L. (1994) Annu. Rev. Biochem. 63, 915-948 [CrossRef][Medline] [Order article via Infotrieve]
  8. Demple, B., Johnson, A. W. & Fung, D. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7731-7735 [Abstract]
  9. Demple, B., Herman, T. & Chen, D. S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 11450-11454 [Abstract]
  10. Dignam, J. P., Lebowitz, R. M. & Roeder, R. G. (1983) Nucleic Acids Res. 11, 1475-1489 [Abstract]
  11. Dorn, A., Bollekens, J., Staub, A., Benoist, C. & Mathis, D. (1987) Cell 50, 863-872 [Medline] [Order article via Infotrieve]
  12. Faisst, S. & Meyer, S. (1992) Nucleic Acids Res. 20, 3-26 [Medline] [Order article via Infotrieve]
  13. Gregor, P. D., Sawadogo, M. & Roeder, R. G. (1990) Genes & Dev. 4, 1730-1740
  14. Harrison, L., Ascione, A. G., Menninger, J. C., Ward, D. C. & Demple, B. (1992) Hum. Mol. Genet. 1, 677-680 [Abstract]
  15. Henner, W. D., Rodriguez, L. O., Hecht, S. M. & Haseltine, W. A. (1983) J. Biol. Chem. 258, 711-713 [Abstract/Free Full Text]
  16. Johnson, A. W. & Demple, B. (1988) J. Biol. Chem. 263, 18009-18016 [Abstract/Free Full Text]
  17. Konecki, D. S., Wang, Y., Trefz, F. K., Lichter-Konecki, U. & Woo, S. L. C. (1992) Biochem. 31, 8363-8368 [Medline] [Order article via Infotrieve]
  18. Kunz, B. A., Henson, E. S., Roche, H., Ramotar, D., Nunoshiba, T. & Demple, B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8165-8169 [Abstract]
  19. Levin, J. D. & Demple, B. (1990) Nucleic Acids Res. 18, 5069-5075 [Abstract]
  20. Lindahl, T. (1993) Nature 362, 709-715 [CrossRef][Medline] [Order article via Infotrieve]
  21. Loeb, L. A. & Preston, B. (1986) Annu. Rev. Genet. 20, 201-230 [CrossRef][Medline] [Order article via Infotrieve]
  22. Miyamoto, N. G., Moncollin, V., Egly, J. M. & Chambon, P. (1985) EMBO J. 4, 3563-3570 [Abstract]
  23. Ono, Y., Furuta, T., Ohmoto, T., Akiyama, K. & Seki, S. (1994) Mutat. Res. 315, 55-63 [Medline] [Order article via Infotrieve]
  24. Prendergast, G. C. & Ziff, E. B. (1991) Science 251, 186-189 [Medline] [Order article via Infotrieve]
  25. Ramotar, D., Popoff, S. C., Gralla, E. B. & Demple, B. (1991) Mol. Cell Biol. 11, 4537-4544 [Medline] [Order article via Infotrieve]
  26. Rivkees, S. A. & Kelley, M. R. (1994) Brain Res. 666, 137-142 [Medline] [Order article via Infotrieve]
  27. Robson, C. N. & Hickson, I. D. (1991) Nucleic Acids Res. 19, 5519-5523 [Abstract]
  28. Robson, C. N., Hochhauser, D., Craig, R., Rack, K., Buckle, V. J. & Hickson, I. D. (1992) Nucleic Acids Res. 20, 4417-4421 [Abstract]
  29. Roy, A. L., Meisterernst, M., Pognonec, P. & Roeder, R. G. (1991) Nature 354, 245-248 [CrossRef][Medline] [Order article via Infotrieve]
  30. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  31. Sawadogo, M. & Roeder, R. G. (1985) Cell 43, 165-175 [Medline] [Order article via Infotrieve]
  32. Sawadogo, M., Van Dyke, M. W., Gregor, P. D. & Roeder, R. G. (1988) J. Biol. Chem. 263, 11985-11993 [Abstract/Free Full Text]
  33. Seki, S., Hatsushika, M., Watanabe, S., Akiyama, K., Nagao, K. & Tsutsui, K. (1992) Biochim. Biophys. Acta 1131, 287-299 [Medline] [Order article via Infotrieve]
  34. Walker, L. J., Robson, C. N., Black, E., Gillespie, D. & Hickson, I. D. (1993) Mol. Cell Biol. 13, 5370-5376 [Abstract]
  35. Winters, T. A., Henner, W. D., Russell, P. S., McCullough, A. & Jorgensen, T. J. (1994) Nucleic Acids Res. 22, 1866-1873 [Abstract]
  36. Winters, T. A., Weinfeld, M. & Jorgensen, T. J. (1992) Nucleic Acids Res. 20, 2573-2580 [Abstract]
  37. Xanthoudakis, S., Miao, G., Wang, F., Pan, Y.-C. E. & Curran, T. (1992) EMBO J. 11, 3323-3335 [Abstract]
  38. Xanthoudakis, S., Miao, G. G. & Curran, T. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 23-27 [Abstract]
  39. Yao, K.-S., Xanthoudakis, S., Curran, T. & O'Dwyer, P. (1994) Mol. Cell Biol. 14, 5997-6003 [Abstract]
  40. Yoshimura, K., Nakamura, H., Trapnell, B. C., Dalemans, W., Pavirani, A., Lecocq, J.-P. & Crystal, R. G. (1991) J. Biol. Chem. 266, 9140-9144 [Abstract/Free Full Text]
  41. Yue, X., Favot, P., Dunn, T. L., Cassady, A. I. & Hume, D. A. (1993) Mol. Cell Biol. 13, 3191-3201 [Abstract]
  42. Zhao, B., Grandy, D. K., Hagerup, J. M., Magenis, R. E., Smith, L., Chauhan, B. C. & Henner, W. D. (1992) Nucleic Acids Res. 20, 4097-4098 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.