Comparison of the promoters of the mouse (APEX) and human (APE) apurinic endonuclease genes

Mutation Research 385 Ž1997. 159–172

Accelerated publication

Comparison of the promoters of the mouse žAPEX / and human žAPE / apurinic endonuclease genes Lynn Harrison a , Antony Gian Ascione a , Yuichi Takiguchi b, David M. Wilson III a , David J. Chen b, Bruce Demple a,) a b

Department of Cancer Cell Biology, HarÕard School of Public Health, 665 Huntington AÕenue, Boston, MA 02115, USA DNA Damage and Repair Group, Life Sciences DiÕision, Los Alamos National Laboratory, Los Alamos, NM 87545, USA Accepted 6 October 1997

Abstract We investigated the minimal promoter of APEX, which encodes mouse apurinic DNA repair endonuclease. A 1.85-kb fragment with APEX upstream sequences and ; 290 bp of the transcribed region linked to a chloramphenicol acetyltransferase ŽCAT. reporter gene was assayed by transient transfection in NIH-3T3 cells. The minimal APEX promoter was comprised of ; 190 bp of upstream and ; 170 bp of transcribed DNA Žexon 1 and most of intron 1.. This ; 360-bp region contains two CCAAT boxes and other consensus protein binding sites, but no TATA box. Deletion of the 5X-most CCAAT box decreased activity ; 5-fold. The second CCAAT box Žsituated in exon 1. may play an independent role in APEX expression. Transcription start sites have been identified downstream of the second CCAAT box, and DNase I footprinting demonstrated NIH-3T3 nuclear proteins binding this region, including an Sp1 site located between the CCAAT boxes. Electrophoretic mobility-shift assays indicated binding by purified Sp1. Mouse proteins did not bind three myc-like ŽUSF. sites in the APEX promoter, in contrast to the APE promoter. The APEX and APE promoter had similar activity in Hela cells, but in mouse cells, the murine promoter had ; 5-fold higher activity than did the human promoter. Both the APEX and APE promoters exhibited bidirectional activity in their cognate cells. q 1997 Elsevier Science B.V. Keywords: DNA repair; Abasic site; Transcription; Development; Redox regulation

1. Introduction Apurinic ŽAP. endonucleases play a pivotal role in base excision repair by initiating the correction of AP sites generated spontaneously w1x or by the action of DNA glycosylases w2x. AP sites in vitro can block DNA synthesis and cause replication errors w3x. Endogenously generated AP sites are evidently mutaAbbreviations: AP, abasic or apurinicrapyrimidinic Corresponding author. Tel.: q1 Ž617. 432-3462; fax: q1 Ž617. 432-0377; e-mail: [email protected] )

genic: AP endonuclease-deficient yeast displays an elevated spontaneous mutation rate w4x, largely due to a dramatic increase in the production of AT ™ CG transversions w5x. There are two families of class II AP endonuclease proteins based on homology to either Escherichia coli exonuclease III or endonuclease IV Žreviewed in w2x.. These enzymes cleave the 5X-phosphodiester bonds of AP sites to initiate the excision process. The major mouse AP endonuclease, encoded by the APEX gene w6–8x, is homologous to exonuclease III Ž E. coli ., Rrp1 Ž Drosphila

0921-8777r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 9 2 1 - 8 7 7 7 Ž 9 7 . 0 0 0 5 3 - 0

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melanogaster ., Arp1 Ž Arabidosis thaliana., rApen Žrat., Bap1 Žbovine. and Ape Žhuman; also known as Hap1, Apex and Ref1. Žsee w9x.. Like the other class II AP endonucleases, APEX protein demonstrates 3X-repair diesterase, DNA 3X-phosphatase and 3X ™ 5X exonuclease activities w10x, although these activities occur at relatively low levels in the mammalian proteins w2,11,12x. The mammalian proteins have two distinct functional domains: the C-terminus contains the nuclease activities, while the N-terminus independently contains a redox-active cysteine that reduces some transcription factors in vitro Ž‘Ref1 activity’. w13,14x. Homozygous ‘knockout’ of APEX

r REF1 produces an embryonic-lethal phenotype, implying critical roles for at least one of the protein’s functions w15x. APE expression is inducible by hypoxia and reoxygenation of HeLa cells w16,17x. Expression of the porcine homolog of APE shows biphasic changes during skin regeneration after wounding w18x. In mice w19x or rats w20x, APEX expression also seems to vary during brain development and in the mature brain w21x. Human Ape protein has also been implicated in transcriptional regulation of the parathyroid hormone Ž PTH . gene in response to extracellular calcium w22x. Ape was proposed to interact with the

Fig. 1. Physical structure of the APEX gene. Two gene structures have been proposed for the APEX gene: one does not have an untranslated exon ŽA. w7x, while the other has an additional intron and hence an untranslated exon ŽB. w8x. The filled boxes represent exons. The genomic inserts in plasmids pSKB-Bs and pSKN-Bs are shown. C: comparison of published APEX gene sequences Ž Genome, upper case; Genbank accession no. D38077 w8x; or accession no. U12273 w7x. with the published APEX cDNA sequence Ž cDNA-2, lower case; accession no. D90374 w6x. and the sequence of the longest cloned cDNA from this work Ž cDNA-1, lower case.. Dashes indicate an intron absent from the cDNA.

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Ku proteins to form a negative calcium-responsive factor w23x that may also negatively regulate the APE gene itself w24x. The foregoing considerations and the increasingly intensive focus on genetic analysis of the APEX gene w15x point to the need for a detailed understanding of the structure and control of the APEX promoter and for a comparison to APE, the human homolog. A previous study delineated the fundamental characteristics of the promoter of the human gene w25x. These included a compact basal promoter, alternative transcription start sites, and actual or potential binding sites for transcription factors such as Sp1, USF or CCAAT-box factors w25x. The cloning of the mouse APEX gene w7,8x has now allowed molecular analysis of its promoter. Deletion analysis of constructs with the chloramphenicol acetyltransferase ŽCAT. reporter gene in cultured cells established a minimal APEX promoter with some features distinct from those of the human gene. In vitro protein binding experiments helped localize some functional elements of the mouse gene. 2. Materials and methods 2.1. Cell culture HeLa-S3 cells were grown in Dulbecco’s modified Eagle’s medium ŽDMEM; Mediatech. supplemented with 10% bovine calf serum, and K562 cells in RPMI 1640 ŽMediatech. supplemented with 10% heat-inactivated horse serum, at 378C and 5% CO 2 . Murine 3T3 cells were grown in DMEM, supplemented with 10% fetal calf serum, at 378C and 10% CO 2 . 2.2. Isolation of the mouse APEX cDNA Oligonucleotide primers Ž5X-CTGGTAACAGCCTATGTTC-3X , 5X-GTGATGGGACAGTGGTC-3X . were used to amplify a ; 400-bp fragment of the mouse APEX exon 5, by the polymerase chain reaction ŽPCR.. This fragment was 32 P-labeled in random priming reactions ŽLife Technologies, Inc.. with w a- 32 PxdCTP and used to probe ; 2 = 10 5 clones of a lZAPII mouse T-cell cDNA library Ža gift from Dr. L. Glimcher, Harvard School of Public Health. as described by technical literature from Stratagene. Sixteen positive clones were identified

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following autoradiography, of which 5 isolates were purified through tertiary screening. In vivo excision reactions were performed and the phagemid constructs were isolated as detailed by Stratagene using their Rapid Excision Kit. Phagemid DNA was sequenced to determine the 5X-termini of the APEX cDNAs. 2.3. Production of promoter constructs 2.3.1. Mouse promoter constructs Digestion with BamHI and Bsu36I, or with BamHI and NotI was used to isolate 1.85-kb and 0.6-kb fragments, respectively, from a previously cloned region of mouse genomic DNA carrying the mouse APEX gene w7x. Following treatment with Klenow DNA polymerase and addition of BamHI linkers to the Bsu36I 3X-termini, the BamHI–Bsu36I and NotI–Bsu36I DNA fragments were subcloned into the BamHI or NotI and BamHI sites of pBluescript II SKq to produce plasmids pSKB-Bs and pSKN-Bs, respectively ŽFig. 1.. Plasmid pSKN-Bs was digested with NotI and BamHI to isolate a 0.6-kb DNA fragment, which was ligated into the SalI site of pCATBASIC ŽPromega, Midaleton, WI. to produce pCBM1. Similarly, the 1.85-kb BamHI fragment isolated from pSKrB-Bs was ligated into the SalI site of pCATBASIC in both orientations to produce pCBM12 Žantisense. and pCBM14 Žsense.. After NotIrXbaI digestion of pCBM12, a 4.9-kb DNA fragment was retained that produced pCBM15 after re-ligation of the vector. SphI digestion of pCBM1, followed by KpnI, BanII or SacII digestion removed 393-, 295- and 134-bp fragments from pCBM1; following the generation of blunt termini using Klenow DNA polymerase, the plasmids were recircularized to produce pCBM2, pCBM18 and pCBM6, respectively. Similarly, XbaI digestion of pCBM1 combined with NruI, KpnI or BanII removed 217-, 230- and 337-bp fragments to generate, respectively, pCBM16, pCBM7 and pCBM8 upon re-ligation. AÕaI digestion of pCBM1 released a 470-bp fragment of APEX promoter DNA, which was ligated into the SalI site of pCATBASIC to produce pCBM10. Plasmids pCBM5 and pCBM17 were produced by the deletion of small segments of DNA from pCBM1 by limited digestion with Bal31 nuclease ŽNew England Biolabs, Beverly, MA.. Briefly, pCBM1 was linearized using DraIII or

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Fig. 2. Comparison of upstream sequences of the APE and APEX genes. The sequences y462 to q65 of APE and y309 to q290 of APEX were compared using Bestfit ŽGenetics Computer Group Package, University of Wisconsin.. The aligned ; 130-bp DNA fragments are 71% identical, and gaps are indicated by a dot. The putative CCAAT box no. 1 Žbold italics. is aligned in both promoters. Two of the three myc-like protein ŽUSF. binding sites Žbold. in the human promoter are aligned with sites in the mouse promoter. The mouse sequence has an Sp1 consensus binding site Žitalics. not present in this region of the human promoter. Transcriptional start sites reported within this region of the mouse Ž) w8x. and human genes Ža w28,29x. are marked. Other start sites have been identified downstream of this sequence for the human gene w25,29x. The numbering for the human sequence defines a downstream transcriptional start site as q1, and the underlined region of the human sequence binds nuclear proteins w25x.

BanII Žfor construction of pCBM5 and pCBM17, respectively., then treated for 60–90 s at 308C with one unit of Bal31 nuclease in the recommended buffer. The reaction was stopped by the addition of EGTA to a final concentration of 20 mM. Blunt termini were generated by treatment with Klenow DNA polymerase, and the DNA was recircularized. The resulting plasmid inserts were sequenced using the primer 5X-GATCTTGTTGGCGCTGC-3X ŽOperon Technologies, Alameda, CA.. 2.3.2. Human promoter constructs Digestion of pCB1 w25x with PstI, or with PstI and DraIII, followed by re-ligation, generated pCB36 and pCB37, respectively. A 2-kb APE promoter fragment was isolated by SmaI and NruI digestion of pLHBS3 w25x and ligated into the SalI site of pCATBASIC to produce pCB8. XbaI digestion of pCB8 combined with DraIII, SacII or SpeI diges-

tion, removed 1670-, 1370- and 1290-bp fragments from pCB8. Re-ligation of the resulting fragments produced plasmids pCB39, pCB40 and pCB38, respectively. The production of pCB22 was described previously w25x. The structures of all plasmids were confirmed by DNA sequencing. 2.4. Transient transfections For comparison of the mouse promoter constructs and the human construct, pCB22, the amounts of DNA used for transfection were normalized to the number of plasmid molecules in 10 mg of pCBM14. For comparison of the human constructs, the amounts of DNA corresponded to the number of molecules in 10 mg of pCB22. The total amount of test plasmid DNA in each transfection was brought to 10 mg by the addition of pCATBASIC DNA. The test DNA was mixed with 10 mg pCMVbgal Ža gift of Mr. L. Chen, Harvard School of Public Health. in 200 ml of

Fig. 3. Identification of the minimal APEX promoter. Reporter plasmids were cotransfected into NIH-3T3 cells with pCMVbgal, followed by assay for b-galactosidase and CAT activities. The ratio of these values was normalized to that obtained for pCBM1 in the same experiment and expressed as a percentage. The values are the means" SEM of at least 6 independent transfections. Potential binding sites for myc-like proteins ŽUSF., Sp1 and CCAAT box-binding proteins are indicated. The filled boxes represent exons and q1 the start site for transcription reported by w8x. A: activity of promoter segments between y1534 and q83. B: activity of promoter segments between q290 and y21.

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phosphate-buffered saline ŽPBS.. HeLa-S3 or K562 Ž5 = 10 6 cells. or 3T3 Ž; 3.5 = 10 6 cells. were washed in PBS and resuspended in the DNA solution. HeLa cells were electroporated at 240 V, 200 V and 960 mF, while 3T3 cells were electroporated at 260 V, and plated as previously described w25x. HeLa or 3T3 cells were grown for 48 h at 378C and cell extracts prepared as described in Harrison et al. w25x. K562 cells were electroporated at 260 V, 200 V and 960 mF and resuspended in 30 ml RPMI 1640 supplemented with 10% heat-inactivated horse serum. After ; 48 h at 378C, K562 cells were harvested by centrifugation at 1000 rpm for 5 min and washed with PBS. Cells were resuspended in 200 ml of 0.25 M Tris-HCl ŽpH 8. and extracted as described previously w25x. Cell-free extracts were stored at y808C. b-Galactosidase assays were carried out according to Sambrook et al. w26x. The extracts were then heated at 608C for 10 min to inactivate mammalian acetylases and centrifuged at 48C. The resulting supernatants were assayed for CAT activity Žsee Promega Technical Bulletin 084..

2.5. Mobility-shift analysis A 287-bp fragment was isolated following digestion of pCBM1 with SacII and NruI. This fragment was further digested with BfaI to produce a 274-bp fragment with a 3X recessed terminus, which was end-labeled by incubation at room temperature for 15 min with Klenow DNA polymerase ŽNew England Biolabs Inc., Beverly, MA. in a reaction with 50 mCi w a- 32 PxdTTP and 156 mM dATP and dGTP. After purification through a Sephadex G50 column, end-labeled DNA Ž5–10 fmol, containing ; 9000 cpm. was incubated with various amounts of 3T3 nuclear extract Ža gift from Dr. Anne Lane, Harvard Medical School. or 50 ng of purified recombinant Sp1 protein ŽPromega. in 10 ml of binding buffer Ž4% glycerol Žvrv. 1 mM MgCl 2 , 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 mM NaCl, 10 mM TrisHCl, pH 7.5, 50 mgrml polyŽdI-dC.. for 20 min at room temperature. Binding reactions with Sp1 also contained 1 mg of bovine serum albumin Žmolecular biology grade; Boehringer Mannheim.. Where speci-

Fig. 4. Comparison of the minimal APE and APEX promoters. Promoter activity assays were performed as described for Fig. 3, and each plasmid was transfected at least 6 independent times Žinto Hela or NIH-3T3 cells.. The filled boxes represent exons in APEX and the hatched box the first exon of APE. Other symbols are as described for Fig. 3.

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fied, unlabeled, synthetic double-stranded oligonucleotides Ž17.5 fmol to 1.75 pmol. containing the Sp1 or AP2 consensus binding sequences ŽPromega. were incubated with 3T3 nuclear extract for 5 min at room temperature prior to the addition of the labeled 274-bp fragment. Binding reactions were analyzed as described previously w25x. 2.6. DNase I footprinting studies Plasmid pCBM10 was digested with XbaI and incubated at room temperature for 25 min with Klenow DNA polymerase, 135 mM each of dGTP, dTTP and dATP and 50 mCi w a- 32 PxdCTP. The DNA was then precipitated with ethanol, resuspended in SacII reaction buffer, and digested with SacII. After heating at 658C for 20 min, the labeled DNA fragment was isolated as described previously w25x and resuspended in 40 ml 10 mM Tris-HCl, pH 8, 1 mM EDTA. End-labeled probe Ž; 10 fmol; ; 25 000 cpm. was incubated with 13 or 26 mg 3T3 nuclear extract or 50 ng recombinant Sp1 protein in 10-ml reactions in binding buffer Žexcept that polyŽdI-dC. was omitted from the Sp1 binding reactions.. After 20 min at room temperature, samples were digested with 0.03–0.13 unit of RQ1 RNasefree DNase ŽPromega. for 50 s at room temperature and analyzed as described previously w25x. 3. Results Two structures have been proposed for the mouse APEX gene ŽFig. 1A w7x; and Fig. 1B w8x., which differ by the presence of an untranslated exon. We cloned and sequenced four independent mouse APEX cDNAs from a T-cell library, each of which clearly contained the untranslated exon. The sequence of the longest cDNA isolated in our experiments ŽFig. 1C. is only 10 bp shorter than the predicted full-length cDNA of Akiyama et al. w8x. The gene structure depicted in Fig. 1B has therefore been used in the subsequent promoter diagrams. This result does not rule out an alternative splicing mode that would leave this first intron in place in some cell types. Note that the overall structure of the transcribed regions of the mouse and human AP endonuclease genes are the same: 5 exons Žone untranslated. and 4 introns, with virtually identical sizes and positions.

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Comparison of the sequences between y462 and q65 of APE Žhuman. and y309 and q290 of APEX Žmurine. using the Genetics Computer Group program Bestfit ŽFig. 2. revealed a high level of sequence identity between the mouse and human promoters w25x. These homologous sections of APE and APEX DNA contain a putative CCAAT box and potential binding sites for myc-like proteins; neither promoter contains an obvious TATA box. The y74 to q45 region of the mouse sequence differs from the human sequence in containing an additional CCAAT box element in the untranslated exon ŽFig. 2.. Akiyama et al. w8x identified multiple transcription starts ŽFig. 2., as previously found for the human APE gene w27–29x. Some of these could originate from the second CCAAT box. 3.1. Identification of the APEX promoter region To identify the functional elements of the mouse APEX promoter, a series of constructs was produced, with fragments from the upstream region of APEX ligated to the CAT gene in pCATBASIC Žsee Section 2: Materials and methods.. The constructs were analyzed for CAT activity after transient transfection into NIH-3T3 cells, with the results normalized to a co-transfected b-galactosidase control. Negligible CAT activity was detected in cell extracts if the parent pCATBASIC vector was used Ždata not shown.. A 1.85-kb fragment of DNA, containing ; 1.5 kb of upstream sequence Žin pCBM14., demonstrated promoter activity similar to that directed by a fragment containing only ; 190 bp of upstream sequence and 290 bp of the transcribed region ŽpCBM6; Fig. 3A.. Promoter activity was decreased ; 3-fold when the upstream CCAAT box Žno. 1. was deleted ŽpCBM18; Fig. 3A.. Deletion of the first Žuntranslated. exon further reduced APEX promoter activity ; 6-fold, which indicates that the CCAAT box Žno. 2. in the untranslated exon may be functional. Although ; 190 bp of upstream sequence was essential for basal promoter activity in our experiments, a region of ; 170 bp downstream of the transcriptional start site was also required. This segment of DNA contains the untranslated exon Žexon 1. and part of the first intron. Deletion in pCBM1 to the NruI site in the first intron reduced promoter activity to ; 33%. The ; 80 bp deleted in this way

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Fig. 5. Bidirectional promoter activity in APEX and APE. A: promoter activity of mouse sense ŽpCBM1. and antisense ŽpCBM12 and pCBM15. constructs transfected into NIH-3T3 Ž6 independent experiments.. B: promoter activity of human sense ŽpCB22. and antisense Žall others. constructs in HeLa or K562 cells Ž4 independent experiments except for pCB8 and pCB39, which were transfected three times..

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Fig. 6. APEX promoter binding by NIH-3T3 nuclear proteins. A: a 3 -labeled DNA fragment ŽD. containing the y189 to q90 region of the APEX promoter was incubated with 0, 0.7, 1.4 or 2.1 mg of 3T3 nuclear extract ŽNE. and 50 mgrml poly ŽdI-dC. Žleft 4 lanes., or with 1.4 mg nuclear extract and 50, 100 or 200 mgrml poly ŽdI-dC. Žright 3 lanes.. B: binding specificity of nuclear proteins. Nuclear extract Ž1 mg. was preincubated with 0, 17.5, 70 or 1750 fmol of oligonucleotide containing the consensus binding sequences for Sp1 Žlanes 2–5. or AP2 Žlanes 6–8.. The 3X-labeled APEX promoter DNA fragment ŽD. was then added. For lane 9, purified Sp1 was incubated with D in the absence of competitors.

Žcompare pCBM10 and pCBM16, Fig. 3B. is a GC-rich sequence containing an Sp1 site. However, DNase I footprinting of nuclear extracts with the SacII–Bsu36I fragment of pCBM1 did not indicate protein binding to this region Ždata not shown.. Despite this apparent lack of stable protein binding, further deletion of the downstream APEX sequence to the 3X end of exon 1 decreased promoter activity to ; 10%, and complete deletion of exon 1 eliminated significant promoter activity ŽpCBM7 and pCBM8 in Fig. 3B.. In summary, the basal APEX promoter is encoded in ; 360 bp, which includes

; 190 bp of upstream sequence, all of exon 1 and part of intron 1. 3.2. Comparison of the APE and APEX promoters In order to compare the activity of the murine and human AP endonuclease gene promoters, the activities of pCBM1 Žmouse. and pCB22 Žhuman. were measured in NIH-3T3 and HeLa cells. Both the promoters were active in the mouse and human cell lines ŽFig. 4., with comparable activities in HeLa cells. However, in the mouse cells, the activity of the human construct pCB22 was 5-fold lower than that

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of the mouse vector pCBM1. Consistent with Fig. 3A, deletion of CCAAT box 1 from pCBM1 Žproducing pCBM5. did not abolish the activity of the mouse promoter in mouse or human cells ŽFig. 4.. Deletion of both the CCAAT boxes from the mouse promoter Žto generate pCBM17. further diminished, but did not eliminate, transcription from the mouse promoter construct in both cell types ŽFig. 4.. It should be noted that the constructs with only a single CCAAT box Žmouse pCMB5 and human pCB22. had equivalent activities in 3T3 cells. This result may be due to the similar arrangement of transcription factor binding sites in the pCBM5 and pCB22 promoters: Sp1, USF, CCAAT box and USF ŽFig. 4.. 3.3. Are the bidirectional?

APEX

and

APE

promoters

Fragments of ; 2 kb of either the mouse promoter Žin pCBM12; Fig. 5A. or the human promoter Žin pCB8; Fig. 5B. placed in the antisense orientation did not exhibit promoter activity in the homologous cell types. However, shorter Ž500 bp. fragments of either promoter directed substantial transcriptional activity ŽFig. 5.. Deletion analysis of the APE Žhuman. promoter ŽFig. 5B. demonstrated that the putative antisense promoter lies between base pairs y140 and y471. This reverse promoter thus partially overlaps the forward promoter of the APE gene and may share regulatory elements with it. 3.4. DNA binding studies Protein-binding sites within the Žmurine. APEX promoter were identified by electrophoretic mobility-shift and DNase I footprinting assays. Using a 274-bp DNA fragment that includes 186 bp of

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APEX upstream sequence and 92 bp of the transcribed region Žsee below., incubation with increasing amounts of nuclear extract from NIH-3T3 cells produced multiple protein–DNA complexes ŽFig. 6A.. Of these, complexes II, IV, and V were observed in the presence of competitor polyŽdI-dC. up to 100 mgrml, while the amount of complex III Ža doublet in some experiments. was unchanged even in the presence of 200 mgrml polyŽdI-dC. ŽFig. 6A.. We tested whether the observed binding was due to interaction of specific transcription factors with the APEX promoter. Although antiserum against human upstream factor ŽUSF. recognizes the mouse protein ŽM. Sawadogo, personal communication., addition of the antiserum to binding reactions did not alter the pattern of complexes formed Ždata not shown.. However, Sp1 protein evidently does bind the APEX promoter: synthetic oligonucleotides containing the Sp1 consensus binding site competed effectively with the formation of complex V, and less effectively complex IV, by the mouse cell extract and led to formation of a doublet in the II complexes ŽFig. 6B.. These changes were not effected by an oligonucleotide with the AP2 consensus, which is not represented in the DNA fragment used in these studies. Purified recombinant Sp1 bound the APEX promoter with high affinity to produce a complex of slightly faster mobility than complexes V of the crude extract ŽFig. 6B.. Further information on the mouse proteins binding the APEX promoter was obtained from DNase I footprinting studies. NIH-3T3 nuclear extracts protected two regions including CCAAT boxes 1 and 2 and the Sp1 and myc-like DNA consensus binding sites ŽFig. 7A.. The sequences footprinted by the binding of murine proteins are summarized in Fig. 7B.

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Fig. 7. Nuclear protein-binding sites in the coding strand of the APEX promoter. A: DNase I footprinting. A 3 -labeled APEX promoter fragment Žcontaining base pairs y189 to q184. was incubated with 0, 13 or 26 mg NIH-3T3 nuclear extract protein 1 mg BSA, or 1 mg BSA and 50 ng pure Sp1 protein, followed by digestion with DNase I. The regions protected by nuclear extract proteins ŽNE. included CCAAT box nos. 1 ŽCa1. and 2 ŽCa2., one Sp1 ŽS., and two myc-like ŽM. consensus binding sequences. The region protected by purified Sp1 protein is indicated by the bracketed segments labeled Sp1. B: positions of binding sites determined by footprinting. The underlined DNA sequences Žy94 to q22 and q48 to q83. correspond to the regions protected by mouse nuclear extract proteins. The bracketed sequence y35 to q9 was the major region protected by purified Sp1 protein. The Sp1 consensus binding sequence is italicized, the myc-like sites are bold and underlined, and the CCAAT box sequences are bold, underlined and italicized.

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4. Discussion Previous characterization of the human AP endonuclease gene Ž APE . revealed multiple transcription start sites and a relatively compact functional promoter Ž; 240 bp; w25x. located in a CpG island w29x. The mouse APEX promoter, also situated within a CpG island w8x, shares with APE several consensus binding sites for transcription factors ŽCCAAT boxes, Sp1 sites and myc-like sites. and is also contained in a short segment of DNA Ž; 360 bp.. Unlike the APE promoter, however, the mouse promoter has two functional CCAAT boxes. Of these, our analysis indicates that the upstream site 1 is the major functional CCAAT box in APEX. Since deletion of site 1 did not eliminate promoter activity Žin pCBM18, Fig. 3A, and in pCBM5, Fig. 4. and CCAAT box 2 is bound by nuclear proteins, this second site is probably functional and may account for the transcription start sites identified downstream from exon 1 ŽFig. 2; w8x.. It will be of interest to determine whether CCAAT box 2 is used more intensively in other cell types than fibroblasts, or under different environmental conditions. Consensus binding sites for Sp1 and myc-like proteins are present in the APE and APEX basal promoters. However, USF, Ža c-myc-like protein., did not appear to bind in vitro to DNA fragments containing the USF sites from either the APEX promoter Žthis work. or the APE promoter w25x. In both promoters, these USF regions are bound by proteinŽs. present in nuclear extracts and appear to be essential elements of the promoters. The binding of these other proteins to the APE promoter seems to interfere with the binding of USF w25x. Other proteins may also interfere with binding by Sp1 protein. Purified Sp1 binds the human w25x and mouse promoters in vitro, but this is not always the case for Sp1 in nuclear extracts. For example, the CCAAT-proximal Sp1 site in APE Žsee pCB22; Fig. 4. was not footprinted detectably by proteins in human nuclear extracts w25x. In the APEX promoter, the Sp1 site located between the CCAAT boxes was bound by mouse nuclear proteins, and electrophoretic mobility-shift analysis suggested that Sp1 was involved. However, the DNase I footprint for nuclear proteins at this Sp1 site was different from that for purified Sp1 ŽFig. 7.. This difference may be due to binding by other proteins at adjacent se-

quences, which could also give rise to the multiple protein–DNA complexes seen in vitro ŽFig. 6.. There was no evidence for nuclear protein binding to other Sp1 sites in the APEX promoter, although deletion of the segment q98 to q184 Žsee pCBM10 and pCBM16 in Fig. 3B. decreased promoter activity ; 3-fold. Conceivably, the Sp1 site at q152 binds Sp1 more weakly than other sites and is not detected in vitro, even though Sp1 can be a limiting factor in gene expression in cultured cells w30x. Other protein binding sites in the transcribed region may contribute to APEX expression, as shown by the reduced activity measured when one such site was removed from pCBM16 to generate pCBM7 ŽFig. 3B.. Some evidence emerged recently indicating that the APE gene might be autoregulated in response to calcium. Initially, Ape protein was identified as a possible component of a Ca2q-dependent, negative modulator of the parathyroid hormone Ž PTH1. gene w22,23x. Prompted by these observations, Izumi et al. w24x located possible negative Ca2q-response elements in the APE promoter. These workers also described binding of Hela nuclear proteins to APE DNA fragments containing the Ca2q-response sites and indirectly implicated Ape protein in the binding complex. However, the possible effects of Ca2q on APE expression were not tested, and neither our own studies w25x nor those of Akiyama et al. w31x are consistent with the presence of a significant negative element in the 4 kb upstream of APE. Both the human Ž APE . and the mouse Ž APEX . upstream regions display bidirectional promoter activity in transfection assays. For the APE gene, the putative antisense promoter is distinct from the sense promoter and lies upstream. The APE and APEX promoters are similar to ‘house-keeping’ gene promoters, which are often TATA-less, compact segments with multiple transcription starts and GC-rich sequences w32–34x. Among these, both the mouse w35,36x and human w37x dhfr genes are joined to bidirectional promoters that also govern transcription of upstream mismatch repair genes. For MRP1, the gene upstream of human dhfr, GC boxes are essential for divergent transcription w37x. Both APE and APEX contain GC-rich elements appropriately situated Žy360 to y471, and y200 to y309, respectively. to drive bidirectional transcription. It will be of interest to test for bidirectional transcription from the APE and APEX promoters in vivo and to

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identify the transcription start sites and the genes lying upstream. Northern blot analysis showed that APE w31,38x and the rat homolog w20x are expressed significantly in diverse tissues. However, developmental regulation of APE in rat brain and testes w20x and in mouse brain w19x has been suggested. Furthermore, the porcine homolog of APE undergoes substantial biphasic changes in expression during epithelial healing from surgical wounds w18x, and the level of the Ape protein in rat hepatocytes is increased in animals treated with thioacetamide, which may induce oxidative DNA damage w39x. In cultured cells, APE is significantly induced following hypoxia and reoxygenation w16,17,40x, or exposure to asbestos ŽH. Fung and B.T. Mossman, personal communication.. The levels of APE mRNA are reduced substantially in HL-60 cells treated with retinoic acid or phorbol esters to induce apoptosis w41x, and during senescence of human fibroblasts in vitro ŽL. Harrison and T. Chang, unpublished data.. Determining the contribution of individual promoter elements and transcription factors to these complex variations constitutes an important goal. A recent study indicates that the APEX gene plays a critical role in mouse embryonic viability beyond day 5 w15x. The Ape protein has now been associated with three distinct activities: DNA repair w2x, protein binding to negative Ca2q-regulated elements w22–24x, and the Ref1 activity that restores DNA binding by diverse transcription factors w42x. Any or all of these functions might underlie the critical role played by Ape during development. Whichever activities are found to be important, the regulated expression of the APEX r APE gene clearly plays a key role, and further delineation of the underlying mechanisms is essential to understanding the biological role of this important protein.

Acknowledgements We thank our colleagues for helpful discussions. We are grateful to Dr. Anne Lane for generously supplying samples of 3T3 nuclear extracts. This work was supported by NIH grants to B.D. ŽGM40000. and D.J.C. ŽCA50519., and by the U.S. Department of Energy.

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