Cytochrome c Oxidase Subunit VIIa Liver IsoformCHARACTERIZATION AND IDENTIFICATION OF PROMOTER ELEMENTS IN THE BOVINE GENE*

(Received for publication, July 21, 1995, and in revised form, November 15, 1995)

R. Sathiagana Seelan‡, Lekha Gopalakrishnan§, Richard C. Scarpulla§,and Lawrence I. Grossman‡¶

From the ‡Center for Molecular Medicine and Genetics, Wayne State University School of Medicine,Detroit, Michigan 48201 and the §Department of Cell and Molecular Biology,Northwestern University Medical School, Chicago, Illinois 60611

Cytochrome c oxidase subunit VIIa is specified by twonuclear genes, one (COX7AH) producing a heart/muscle-specific isoform and the other (COX7AL) a form ex-pressed in all tissues. We have isolated both genes toexamine their transcriptional regulation. Here, we char-acterize the core promoter of COX7AL and show that a92-base pair region flanking the 5*-end promotes most ofthe activity of this gene. The 92-bp basal promoter con-tains sites for the nuclear respiratory factors NRF-1 andNRF-2, which have been shown to contribute to the tran-scription of a number of nuclear genes involved in mi-tochondrial respiratory activity, and also at least fourSp1 motifs. We show that both the NRF-1 and NRF-2binding sites are functional in COX7AL and present ev-idence suggesting that interaction between the NRF-1site and an upstream element contributes to expression.

Cytochrome c oxidase (COX),1 the rate-limiting component ofthe electron transport chain, catalyzes the transfer of electronsfrom reduced cytochrome c to molecular oxygen; this processhelps generate the proton gradient that fuels ATP synthesis.Although mammalian COX consists of 13 subunits, 3 encodedby mitochondrial DNA and the rest by the nuclear genome, thecatalytic functions reside in the mitochondrially encoded sub-units I and II. Attention has recently focused on elucidatingmore clearly the role of the nuclear-encoded subunits, whichhave been presumed to play a role in regulation and assemblyof COX (1–5). Among the nuclear-encoded subunits, most mam-mals have three (VIa, VIIa, and VIII) that exist as isoforms, anL (liver) isoform, which is ubiquitously expressed, and an H(heart) isoform, expressed primarily in adult heart and skeletalmuscles (6–8). The fetus, by contrast, contains the L isoform asthe dominant contractile muscle form (9–12) and switches tothe H isoform during development.The detailed roles of these nuclear subunits still remain

unclear. Several subunits, however, including isoforms, havebeen linked to function. Subunit VIa appears to sense adeninenucleotide concentrations and thereby modulates COX activity(13–16); furthermore, regulation of the bovine enzyme has beenshown to be isoform-specific (14, 16). However, the mechanism

and metabolic role are still matters of debate. Yeast subunit V(homologous to mammalian subunit IV) has a pair of isoforms(Va and Vb) that are preferentially expressed in a high or lowO2 environment, respectively (17, 18). The subunit V isoformshave been shown to modulate holoenzyme activity by alteringits kinetic properties, such as turnover number (19), by chang-ing the environment at the binuclear reaction center (20). Sub-unit VIb is required for assembly of a fully active yeast enzymebut is not required thereafter (21); when subunit VIb is selec-tively removed from the mammalian holoenzyme, COX activityis increased, suggesting that VIb has a suppressor-like function(22). A role for mammalian subunit IV in proton pumping,possibly by mediating access of protons into the transmem-brane proton channel, has been inferred from limited trypsindigestion experiments of COX (23).Both COX function and synthesis, therefore, may involve

responses to regulatory signals. One way the nuclear genescould be regulated is through common signals that reside intheir DNA sequence. The characterization of several genes ofcomplexes I to V of the respiratory chain has elucidated anumber of candidate signals: (i) NRF-1, a positive activator oftranscription found to have a role in at least two COX genes(rat COX6C and mouse COX5B). It appears to be a key factor incoordinating respiratory metabolism with other biosyntheticand degradative pathways (24, 25); (ii) NRF-2, an ets-relatedmultisubunit activator that recognizes a GGAA motif. It hasbinding sites in the mouse and rat COX4 and mouse COX5Bgenes (26, 27) and is also known as the GA-binding protein (28);(iii) the OXBOX, a tissue-specific element that promotes theexpression of genes in heart/skeletal muscles (29). The OXBOXfactor is found only in myogenic cells and acts in concert withanother element, often overlapping the OXBOX, known as theREBOX. The REBOX element apparently binds to a ubiquitousfactor and is modulated by the redox state, pH, and thyroidhormone levels (30); and (iv) an enhancer-like element found inthe ATP synthase b-subunit, cytochrome c1, and pyruvate de-hydrogenase E1a subunit genes (31). Thus far, only the NRF-1and NRF-2 motifs have been shown to be functionally associ-ated with COX genes.To understand the detailed regulation of COX isoforms in

various tissues, we have isolated the bovine genes for thesubunit VIIa isoforms, including their 59-flanking regions (32,33). In this initial communication, we characterize the firsttissue-specific COX gene promoter. We describe the basal pro-moter of the bovine COX7AL isoform gene and identify regu-latory elements and transcription factors that appear involvedin its expression. Specifically, we report that both NRF-1 andNRF-2 participate in COX7AL expression and that NRF-1 mayneed to interact with an upstream factor for maximal activity.

* This work was supported by National Institutes of Health GrantsGM 48517 (to L. I. G.) and GM 32525 (to R. C. S.). The costs of publi-cation of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.¶ To whom correspondence should be addressed. Tel.: 313-577-5326;

Fax: 313-577-5218; E-mail: LG@cmb.biosci.wayne.edu.1 The abbreviations used are: COX, cytochrome c oxidase; CAT, chlor-

amphenicol acetyltransferase; bp, base pair(s); PAGE, polyacrylamidegel electrophoresis; EMSA, electrophoretic mobility shift assay; PCR,polymerase chain reaction; ds, double stranded.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 4, Issue of January 26, pp. 2112–2120, 1996© 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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EXPERIMENTAL PROCEDURES

Materials—CAT enzyme-linked immunosorbent assay kit and chlo-rophenol red b-D-galactopyranoside were from Boehringer Mannheim;Klenow polymerase and T4 polynucleotide kinase were from Promega;pCH110 was obtained from Pharmacia Biotech Inc.; [g-32P]ATP and[a-32P]dCTP (each 3000 Ci/mmol) were from Dupont NEN; media andsera for transfection analysis were from Life Technologies, Inc.; oligo-nucleotides were synthesized by Integrated DNA Technologies, Inc.(Coralville, IA) or Center for Molecular Biology (Wayne State Univer-sity); pRSV CAT (34) was from Dr. J. Moshier, and pGKO CAT (35) wasfrom Dr. G. Kumar, both at Wayne State University. All other chemi-cals were from Sigma or Fisher.Construction of CAT Deletion Plasmids—Various 59-flanking regions

of the COX7AL gene beginning upstream of the transcriptional startsite (11) (32) and extending to the non-coding portion of the first exon(ThaI or DdeI) (Fig. 1) were cloned into the multiple cloning site ofpGKO CAT. A 1.5-kilobase HincII-ThaI fragment (21442 to 152), a569-bp PstI-ThaI fragment (2517 to 152), a 427-bp HincII-ThaI frag-ment (2375 to 152), a 168-bp XmaIII-DdeI fragment (298 to 170), anda 58-bp HaeIII-ThaI fragment (26 to 152) were isolated, filled in withKlenow polymerase, and cloned into pGKO CAT to generate, respec-tively, HincII(A)-CAT, PstI-CAT, HincII(B)-CAT, XmaIII-CAT, andHaeIII-CAT. Inserts were identified by restriction enzyme analyses andDNA sequencing, and plasmid DNA was purified on a preparative scalevia two CsCl gradients.Construction of Hae43-CAT, Hae46-CAT, and PCR59-CAT—The

92-bp region between the XmaIII and the HaeIII sites (Fig. 1) wasfurther analyzed as follows. Digestion with HaeIII results in two prod-ucts, a 59 43-bp and a 39 46-bp fragment (Fig. 2B). Each fragment wascloned intoHaeIII-CAT (Fig. 1) to generateHae43-CAT andHae46-CAT(Fig. 2B). A 59-bp PCR fragment was also generated using primers thatspan the internal HaeIII site (Fig. 2B) and similarly cloned to generatePCR59-CAT. The HaeIII-CAT vector contains the transcriptional startsite of COX7AL and has negligible CAT activity.Cell Transfection and CAT Analysis—HeLa cells (8 3 105) grown in

Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serumwere seeded into each 100-mm dish and transfected by the calciumphosphate method (36) using 30–35 mg of the test plasmid and 4 mg ofa b-galactosidase control plasmid, pCH110. Cell extracts were preparedby freeze-thawing (36). Half of the cell extract was removed and frozenfor b-galactosidase assay. The rest was incubated at 65 °C for 5 min(37), centrifuged, and the supernatant frozen for CAT activity analyses.Each transfection, done in duplicate or triplicate, was repeated at leastthree times. CAT activity of each sample was determined in duplicateon 2–4 ml of the extract by using the CAT enzyme-linked immunosorb-ent assay kit and normalized to b-galactosidase activity. b-Galactosid-ase activity was determined in duplicate in a 100-ml reaction using 2–5ml of the extract and chlorophenol red b-D-galactopyranoside in Hepesbuffer, according to the manufacturer’s protocol. The normalized CATactivity of the test samples was expressed relative to the largest CATconstruct or to XmaIII-CAT, as indicated.Site-directed Mutagenesis—The 92-bp promoter region between the

XmaIII and HaeIII sites was subjected to single- and double-site mu-tation analyses, using overlapping oligonucleotides to reconstruct the92-bp fragment (25). For the wild-type sequence, ten oligonucleotidesspanning both strands of the 92-bp region were synthesized, purifiedthrough PAGE (36), and assembled as follows. 100 ng each of all but theterminal oligonucleotides were mixed and phosphorylated with T4polynucleotide kinase and ATP. The kinased mixture was precipitatedwith ethanol and ligated after addition of 100 ng each of the terminaloligonucleotides. The ligation mixture after ethanol precipitation wasrepaired with Klenow polymerase, and the products were resolved byPAGE (15%). The 92-bp reassembled promoter fragment was isolated,cloned into HaeIII-CAT, and verified by sequencing. Single-site anddouble-site mutations were introduced by substituting the appropriatecomplementary mutant oligonucleotides (see “Results”) into theassembly.DNase I Footprinting and Methylation Interference Analysis—Bind-

ing reactions for DNase I were carried out with 20 fmol of an end-labeled DNA fragment (200-bp XbaI-HindIII) derived from a constructcontaining the wild-type 92-bp promoter (298 to 26) in TM buffer (25mM Tris at pH 7.9, 6.25 mMMgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol,10% glycerol) containing 50 mM KCl. Approximately 40 ng of purifiedrecombinant NRF-1 (26) and 200 ng of NRF-2 (38) (containing equimo-lar amounts of bacterially expressed human a and b2 subunits) wereincubated separately with labeled DNA along with 2% polyvinyl alcoholand 100 mg of bovine serum albumin. Following a 15-min incubation at

room temperature, the samples were adjusted to 5 mM MgCl2, 2.5 mM

CaCl2 and treated with 2.5 ng of DNase I for 1 min. Cleaved DNA wasextracted with phenol/chloroform/isoamyl alcohol, ethanol precipitated,and analyzed on a 6% polyacrylamide-urea sequencing gel.Methylation interference was performed as described (39) on the

200-bp wild-type promoter fragment described above. Following diges-tion with either XbaI or HindIII, the DNA was 39-end labeled withKlenow enzyme. After a second digestion with either XbaI or HindIII,the resulting 200-bp fragments were gel purified and electroeluted.Each fragment (1 3 106 cpm) was treated with dimethyl sulfate for 5min at room temperature as described (40). Binding reactions contained5 3 105 cpm of methylated fragment and either 40 ng of recombinantNRF-1 or 200 ng of NRF-2. Following electrophoresis, the wet gel wasautoradiographed for 2 h at 4 °C, and the free and protein-bound bandswere excised and electroeluted. The electroeluted DNA was extractedonce with phenol/chloroform, ethanol precipitated with 10 mg of Esch-erichia coli tRNA, and cleaved at methylated guanosine residues bytreatment with 1 M piperidine. Cleavage products were electrophoresedon a 6% polyacrylamide-urea sequencing gel.Electrophoretic Mobility Shift Assays (EMSA)—Mobility shift assays

were carried out essentially as described (39). Duplex DNA for EMSAwas assembled from complementary oligonucleotides or their mutantforms by annealing in TE buffer containing 150 mM NaCl after heatingto 65 °C and cooling slowly to room temperature. The mixture wasprecipitated from ethanol and filled in with Klenow polymerase, andthe duplex was isolated from 15% PAGE. DNA was end labeled, andEMSA was performed using 5–20 fmol of DNA and 25 mg of HeLaextract, prepared as described (41), in each binding reaction. For com-petition, the indicated molar excesses of unlabeled duplex oligonucleo-tides were preincubated with the extract for 10 min at room tempera-ture and for an additional 10 min with the labeled probe.Complementary oligonucleotides from adjacent regions of the promoterwere similarly annealed and used as nonspecific DNA. Gel shifts wereresolved on 5% PAGE (58:1), run in 0.5 3 TBE (39) in the cold, andautoradiographed. Purified recombinant proteins used were NRF-1 (20ng/reaction) and NRF-2 (a plus b2) (50 ng each/reaction).Antibody Supershift Assays—Reactions were carried out as described

for EMSA, using 15 mg of HeLa extract and 20 ng of purified recombi-nant NRF-1. After incubation for 15 min at room temperature, 1 ml ofundiluted goat anti-NRF-1 antibody was added and incubated for anadditional 15 min. Samples were electrophoresed as for EMSA butusing 4% gels.

RESULTS

Characterization of the Upstream Regulatory Regionof COX7AL

CAT constructs harboring 6 bp to 1.5 kilobase pairs ofCOX7AL upstream sequence were analyzed for promoter activ-ity in HeLa cells (Fig. 1). Significant activity (18% relative topRSV CAT or 56% relative to the longest construct) was re-tained when the upstream sequence was deleted to only 98 bpof flanking DNA (XmaIII-CAT). Further deletion to only 6 bp(HaeIII-CAT) abolished most of this activity, suggesting thatthe basal promoter region of COX7AL is localized within a92-bp XmaIII-HaeIII (298 to 26) region. This region containsan NRF-1 site showing 10/12 homology with the consensussequence and two potential NRF-2 sites (designated A and B,respectively, from the 59-end) that have a GGAA purine core asa recognition motif (Fig. 2A). Based on nucleotides that flankthis motif, only the A site appears to be an ideal ets binding site(28). The region also contains a number of Sp1 motifs. At leastone is located in the distal (d) portion of the fragment andseveral in the more proximal (p) highly G-C rich region. Two ofthese motifs have the core GGGCGG sequence (42) and aredesignated dSp1 and pSp1 (Fig. 2A). dSp1 has a 9/10 matchwith an extended Sp1 consensus sequence (42), and pSp1 has a7/10 match. A perfect match with the Sp1 consensus compiledby Faisst and Meyer (43) is found a few nucleotides upstream ofpSp1 and is designated as pSp19. A fourth motif with a singlemismatch overlaps an internal HaeIII site (Fig. 2A) and will bereferred to as pSp199.The activities of fragments containing additional upstream

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sequences (Fig. 1) are 1.6–1.8-fold higher than that of the 92-bpfragment, suggesting that additional positive elements are lo-cated upstream of the basal promoter region. At least one ofthese elements is located between the HincII (2375) and

XmaIII sites (61% stimulation relative to the 92-bp fragment)and possibly another between the PstI and HincII (2375) sites(84% relative to the 92-bp fragment). The HaeIII-CAT con-struct, which contains only 6 bp of flanking DNA and 52 bp of

FIG. 1. Characterization of the upstream regulatory region of COX7AL. 6 bp to ;1.5 kilobase pairs of COX7AL upstream region werecloned in pGKO CAT vector and assayed for promoter activity in HeLa cells. A restriction map of the upstream region is shown on top with thearrow indicating the major transcriptional start site at 11. The nt positions of the fragments used to make these constructs are indicated on eachline. The CAT activity (%) of each construct is expressed relative to HcII(A)-CAT, set at 100%. The name of each construct is shown on the left. Theempty CAT vector, pGKO CAT, has a relative activity of 5%.

FIG. 2. A, Sequence of COX7AL containing the basal promoter region and various regulatory motifs. The basal promoter region extends from 298(XmaIII) to 26 (HaeIII) of the COX7AL gene. Various regulatory motifs (boxed or bracketed horizontally) and restriction enzyme sites (underlined)used to make some of the CAT constructs are indicated. The four NRF-2 sites are indicated as A, B, C, and D from the 59-end. dSp1 refers to thedistal Sp1 site and pSp1, pSp19, and pSp199 refer to the proximal Sp1 site cluster. The arrow denotes the major transcriptional start site (11). B,CAT activity of subfragments derived from the 92-bp XmaIII-HaeIII basal promoter region. The XmaIII-CAT construct (bold) was digested at theinternal HaeIII site to generate the Hae46-CAT and the Hae43-CAT subclones. Horizontal arrows indicate the PCR primers used to amplify afragment containing the internalHaeIII site. The transcriptional start site is depicted by the bent arrow. The CAT activity of each construct (6S.D.)relative to XmaIII-CAT (100%) is indicated on the right. Dashes indicate deleted regions.

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the untranslated region of the first exon, has only 5% theactivity of the largest CAT construct.

Characterization of the COX7AL Basal Promoter

The 92-bp basal promoter region was further analyzed byassessing the activity of constructs containing subfragments(Fig. 2B). The HaeIII digestion products and the PCR productas described under “Experimental Procedures” were individu-ally cloned in theHaeIII-CAT vector (Fig. 1). Each of the clonedHaeIII products accounted for only 14–19% of the CAT activityof the intact 92-bp fragment (Fig. 2B). Thus, either NRF-1 doesnot contribute significantly to promoter activity or the lowactivity of the NRF-1 site-containing fragment (Hae46-CAT) isdue to the absence of upstream sequences. By contrast, the59-bp PCR fragment (PCR59-CAT), which lacks the NRF-1 site(Fig. 2B), retained 65% of the CAT activity. Although thisresult suggests that the basal promoter is located within a59-bp region that spans the proximal overlapping Sp1 motifs, itis not in its original spatial context with respect to the tran-scriptional start site. To examine the roles of the various siteswithin a normal promoter context, we performed site-directedmutagenesis.

Analysis of the Basal Promoter by Site-directedMutagenesis

Mutant complementary oligonucleotides were synthesized inwhich the core recognition motifs were altered. Mutations wereintroduced at the NRF-1 (GCGCTTGCGC to GtaaTTGaat),dSp1, pSp1, and pSp199 sites. For dSp1 and pSp1, the corerecognition sequence of GGGCGG was altered to atGCGG and

GGGatG, respectively. In addition, for pSp199 the HaeIII sitecontained within this motif was altered (GGCC to ataC), sincethis site was observed to be critical for promoter function bydeletion analysis (Fig. 2B). The basal promoter was then as-sembled from these oligonucleotides harboring one or two mu-tant sites and transfected into HeLa cells. For single-site mu-tants, a pronounced decrease in activity occurred when theNRF-1 (27%), the dSp1 (45%), or the pSp199 (26%) site wasmutated (Fig. 3). A mutation in pSp1, however, showed anuncharacteristic increase (112%). These results were confirmedwith double-site mutants.In general, double mutants showed a steep drop in activity.

However, if the pSp1 site was included, the decrease was moremodest compared to a single-site mutation of the other memberof the pair. The basis for the apparent stimulation by a muta-tion at the pSp1 site is not presently understood. The doublemutations at pSp199- NRF-1 (12%) and at the dSp1-pSp199 sites(12%) show the lowest activities; comparison with the singlesite mutants suggests that the latter sites act independentlywhereas the former sites may interact.

Analysis of DNA-Protein Interactions by DNase IFootprinting and Methyl Interference Assays

The results of transfection experiments with site-directedmutants point to the NRF-1 site as a major determinant of COXpromoter function. To investigate whether NRF-1 displays spe-cific binding to this site, DNase I footprinting was performedusing purified recombinant NRF-1 protein. A clear footprintdisplaying the characteristic enhanced cleavage pattern at theboundaries (Fig. 4, lane 3) was obtained in the region of the

FIG. 3. Promoter analysis by site-directed mutagenesis. The top line indicates the XmaIII-CAT vector with the various regulatory motifs.Mutated sites are indicated below the figure (✖) and summarized to the left of each line. The top four lines indicate single-site mutations, and thebottom six lines indicate double-site mutations. Normalized CAT activity of these constructs relative to a wild-type 92-bp reassembled sequence(100%) is indicated on the right. pGKO is the empty CAT vector used to make the various constructs.

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NRF-1 site. Point mutations within the NRF-1 site that re-sulted in a loss of promoter activity (Fig. 3) also eliminatedprotein binding to the site (data not shown). A similar analysisof the promoter fragment with purified recombinant NRF-2showed the presence of two NRF-2 footprints in the region ofthe A and C/D sites (Fig. 4, lane 8, top and bottom, respective-ly). Furthermore, the inclusion of a 100-fold excess of oligonu-cleotide containing NRF-1 (RC4 2172/2147) or NRF-2 (RCO4113/136) binding sites (44) in the footprinting reactions estab-lished the specificity of the interaction of the proteins to theirrespective sites (Fig. 4, lanes 4, 5, 9, and 10).The pattern of protein-DNA contacts was determined by

methylation interference analysis. Radiolabeled promoter frag-ments containing intact NRF-1 and NRF-2 sites were methyl-ated and incubated with purified protein as described under“Experimental Procedures.” Analysis of the piperidine cleavageproducts showed the guanine contacts at the NRF-1 site (Fig. 5,left) to be consistent with those described previously (39). Anal-ysis of the guanine contacts made with NRF-2 indicates bind-ing of protein to the A and C sites (Fig. 5, right), localized to theadjacent guanine nucleotides of the core GGAA motif as previ-ously observed (45). Interestingly, the A and C sites are theonly ones preceded by the nucleotide A or C, which is favoredfor the binding of Ets proteins (28).

Analysis of DNA-Protein Interactions by MobilityShift Assays

DNase I footprinting reveals that NRF-1 binds to its motif inCOX7AL and that NRF-2 binds to the A and C/D sites but notto the B site (Fig. 4). To confirm these results, EMSA wasperformed on double-stranded 21–24-bp oligonucleotides con-taining these motifs.DNA-Protein Interactions at the NRF-1 Site—NRF-1 binds to

a 21-bp DNA (ds NRF-1) containing a consensus NRF-1 site ofthe COX7AL promoter (Fig. 6B). This binding (top band) iscompeted by a 500-fold molar excess of the specific unlabeledfragment but not by an equivalent molar excess of a 21-bpfragment mutated at the NRF-1 site (ds NRF-1mut) or by anonspecific 23-bp fragment (ds NS). Further confirmation thatthis band represents NRF-1 binding comes from using purifiedrecombinant NRF-1 on ds NRF-1 and ds NRF-1mut; whereas ashift was seen with the former, none was observed with thelatter (data not shown). Additional evidence was obtained byantibody supershifts. The binding of NRF-1 from HeLa nuclearextract (Fig. 6A, lane 1, upper band) is supershifted whenantibody against NRF-1 is used (lane 2). The position of theduplex after incubation with purified recombinant NRF-1 (lane3) and its supershifting (lane 4) is also shown. It is clear fromthese results that the upper band represents a specific

FIG. 4. Competition DNase I footprinting of NRF-1 and NRF-2sites in the COX7AL promoter. Promoter fragments were 39-endlabeled with Klenow enzyme on the non-coding strand and subjected toDNase I footprinting in the absence (lanes 2 and 7) or presence ofpurified recombinant NRF-1 (lanes 3–5) or NRF-2 (lanes 8–10). A 100-fold molar excess of synthetic oligonucleotides containing rat cyto-chrome c (RC4) promoter sequences from 2172 to 2147 (lanes 4 and 10)or rat cytochrome c oxidase (RCO4) promoter sequences from 113 to136 (lanes 5 and 9) were included in the binding reactions as indicated.The vertical lines adjacent to the autoradiographs indicate the regionsprotected from DNase I digestion. The protected NRF-2 regions at thetop and bottom refer to the A and C/D sites, respectively. G, G-specificsequencing reaction.

FIG. 5. Methylation interference footprinting of NRF-1 andNRF-2 sites. COX7AL promoter fragments were 39-end labeled withKlenow enzyme on either upper or lower strands relative to the tran-scriptional orientation and methylated with dimethyl sulfate. Methyl-ated fragments were incubated with purified NRF-1 or NRF-2 andelectrophoresed on a 4% non-denaturing preparative acrylamide gel.DNA from free (F) and protein-bound (B) bands was eluted, cleavedwith piperidine, and electrophoresed on a 6% urea-acrylamide gel.Arrowheads and filled circles indicate guanine nucleotides that com-pletely inhibited protein binding when methylated, and the underlinedsequences indicate regions protected in DNase I footprint analysis.Methylation interference at the NRF-2A site is indicated on the upperstrand and at the C site on the lower strand.

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DNAzNRF-1 complex. The typically diffuse supershifted com-plexes obtained with the crude extract may result from otherproteins interacting with antibody. These observations suggestthat the NRF-1 motif in COX7AL is functional and corroboratethe results of footprinting analyses (Fig. 4).The lower band in these figures probably represents NRF-2

binding at the B site, which is present in the ds NRF-1 and dsNRF-1mut duplexes. The binding of NRF-2 to the B site was notseen by DNase I footprinting (Fig. 4). Since footprinting wasdone on a larger fragment, containing all of the NRF-2 sites (A,B, and C/D), and EMSAwas performed on a 21-bp fragment, webelieve the footprinting data to be more consistent with nativeNRF-2 interactions. We thus conclude that NRF-2 does notinteract at the B site.DNA-Protein Interactions at the NRF-2A Site—The interac-

tions at the NRF-2A site were examined by EMSA (Fig. 7A),utilizing the following duplexes: ds NRF-2A, containing theNRF-2A site and the pSp1 site; ds NRF-2A*, in which the pSp1site has been altered; and a 21-bp nonspecific oligonucleotidespanning an adjacent upstream region (ds NS). The most prom-inent band in these lanes represents NRF-2 binding. This con-clusion is derived from the observation that purified NRF-2 (aand b2 subunits) forms a complex with ds NRF-2A that issimilar in mobility to a complex formed with HeLa nuclearextract (lanes 2 and 4). This interaction is partly competed at a500-fold molar excess of specific unlabeled DNA (ds NRF-2A ords NRF-2A*), containing an intact NRF-2A site (lanes 6 and 8).The nonspecific DNA fragment (ds NS) is unable to compete forthis binding at equimolar levels (lanes 9 and 10).Purified human Sp1 binds to ds NRF-2A (lane 3), forming a

faint complex that is not evident in lanes containing totalnuclear extract from HeLa cells. Since the pSp1 site is fused tothe 59 end of NRF-2A, it is possible that binding to the Sp1motif is blocked by the relatively higher affinity of NRF-2 to thefused A site. The apparent weak Sp1 complex formation maynot be surprising given that the pSp1 motif has only a 7/10match with the extended consensus (42).

DNA-Protein Interactions at the NRF-2C/D Sites—Both pu-rified NRF-2 and the HeLa extract form identical complexeswith a duplex DNA containing the tandem C/D sites (Fig. 7B,lanes 2 and 3). The complex formed is readily competed at a500-fold molar excess of the specific unlabeled DNA (lane 4) butnot by a nonspecific DNA (lanes 6 and 7). The NRF-2 complexformed at the tandem C/D sites (ds NRF-2C/D) differs from theNRF-2 complex formed at the A site (Fig. 7A) in two ways: thiscomplex is larger, and it is readily competed at a 500-fold molarexcess of specific competitor.

DISCUSSION

COX7AL, the first COX isoform gene to our knowledge ana-lyzed for promoter function, has a TATA-less promoter locatedat a CpG island (32). We have mapped the basal promoter to a92-bp segment immediately upstream of the major transcrip-tional start site. It contains functional NRF-1 and NRF-2 (siteA) motifs as well as a tandem pair of functional NRF-2 sites(C/D) located a few nucleotides downstream of the transcrip-tional start site (Fig. 2A). In addition, at least four Sp1 sites arelocated at the distal and proximal regions. Further deletion ofthe 92-bp region defines a 59-bp core region that includes allthe proximal Sp1 sites and contains up to 65% of the CATactivity. We observed a significant loss of activity (;74%) wheneither the pSp199 or the NRF-1 site was altered (Fig. 3). Indeed,a double mutant for these sites showed one of the lowest activ-ities, suggesting that these two sites are important forCOX7AL regulation. These mutations, however, did not totallyabolish promoter activity, suggesting that other factor(s) alsoparticipate in promoter function. This residual activity could bedue, at least in part, to NRF-2 binding to the tandem motifs,since these were intact in all of the constructs discussed.The proximal Sp1 region includes three Sp1 motifs (pSp199,

pSp19, and pSp1) of which pSp199, which spans the HaeIII site,appears to be the most important. Deletion of fragments up-stream of the HaeIII site, or mutating the HaeIII site in thecontext of an intact 92-bp promoter, drastically reduces CAT

FIG. 6. A, antibody supershift analysisof NRF-1 binding to the COX7AL pro-moter. The duplex oligonucleotide dsNRF-1 was incubated with HeLa extract(lane 1), HeLa extract and NRF-1 anti-body (lane 2), purified recombinantNRF-1 (lane 3), and purified recombinantNRF-1 and NRF-1 antibody (lane 4). B,specificity of NRF-1 binding to theCOX7AL promoter. The duplex oligonu-cleotide, ds NRF-1, was incubated withHeLa extract (lane 1) and HeLa extractand competitor DNAs (lanes 2–7). Lanes 2and 3, specific competition with dsNRF-1; lanes 4 and 5, competition with dsNRF-1mut; lanes 6 and 7, competition witha nonspecific DNA. Competitors wereused at 100- and 500-fold molar excess ofunlabeled DNA. The top band in theselanes represents NRF-1 binding. Se-quences of the various duplexes areshown at the bottom of the figure.

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activity. Although we presume that Sp1 binds to this motifbased on its G-C richness and its resemblance to the Sp1consensus, a similar element (CGGCCCC) found in the etsdomain binding region of the human mitochondrial ATP syn-thase b-subunit gene promoter (46) does not appear to form aprotein-DNA complex with Sp1. Hence, the nature of the factorbinding to pSp199 needs to be elucidated. At the distal site(dSp1), a mutation decreased activity to 45% relative to the92-bp fragment, and a double mutation at the dSp1 and pSp199sites abolished 88% of the basal promoter activity. At the pSp1site, however, there was a surprising enhancement of activityto 112% (Fig. 3). This effect was also noted in double mutants.A possible explanation for this could be the observation that thepSp1 site is fused to the 59-end of NRF-2A (Fig. 2A). Theaffinity of NRF-2 to the A site may thus mask accessibility tothe adjacent Sp1 site. Any competition between Sp1 and NRF-2to bind to the fused site should, therefore, be relieved when thepSp1 motif is altered. This hypothesis is supported by thefollowing observations: (i) NRF-2 clearly footprints at the A site(Fig. 4), spanning the GGAA motif and extending into the pSp1site (Fig. 5, bottom), and (ii) DNA-protein interactions at thefused site (Fig. 7A) indicate the presence of a single prominentNRF-2 complex but no Sp1 interaction.The importance of NRF-1 to promoter function judged by

site-directed mutagenesis seems to be at variance with thedeletion analysis data (Hae46-CAT; Fig. 2B), where a fragmentharboring the NRF-1 site was found to be insufficient for pro-moter activity. These observations can be reconciled if NRF-1were to interact with upstream regions (Fig. 8). Thus, in thedeletion construct, the lower promoter activity may result be-

cause NRF-1, although able to bind to its target motif, is unableto interact with factor(s) binding upstream. This model, whichemphasizes the importance of promoter context in NRF-1 func-tion (25), is supported by the site-directed mutagenesis data, inwhich a mutation at the NRF-1 site abolishes 73% of thepromoter activity.Studies of the murine COX4 and COX5B promoters (26, 27)

reveal that NRF-2 has a 10–20-fold greater affinity for tandemthan monomeric sites. Similarly, the pair of tandem NRF-2sites (C/D) located downstream in the untranslated region ofthe first exon of COX7AL appears to form a stable high affinitycomplex with NRF-2 (Fig. 7B). Moreover, strong binding isobserved when purified NRF2 a and b2 subunits are added to aduplex containing the tandem sites (Fig. 7B), whereas a com-plex at the single site (Fig. 7A) was detectable only on pro-longed exposure, suggesting that tandem sites are preferredsubstrates for NRF-2. Alternatively, the weak complex forma-tion at the A site with purified NRF-2 could suggest thatanother member of the Ets family has a preferred affinity forthis site. Interestingly, the tandem NRF-2 motifs in mouseCOX4 are also located in the untranslated region of the firstexon and have features resembling an initiator element, sincemutating either motif of the ets pair appears to determine thestart site of transcription (27).A more detailed examination of the tandem sites indicates

that they are not equivalent. Methyl interference analysisclearly indicates protein contact between NRF-2 and site C butnot site D. This agrees well with the consensus for ets bindingmotifs (28); the C site is in a favorable binding context whereasthe D site is not. Since DNase I footprinting and EMSA suggest

FIG. 7. A, NRF-2 interaction at the A site of the COX7AL promoter. The duplex oligonucleotide, ds NRF-2A, was used. Lane 1, free probe; lane2, HeLa extract; lane 3, purified human Sp1; lane 4, purified recombinant NRF-2a and b2 subunits. Lanes 5–10 contain HeLa extract and labeledds NRF-2A and depict competition with specific DNA (ds NRF-2A, lanes 5 and 6), specific DNA mutated at the pSp1 site (ds NRF-2A*, lanes 7 and8), and a nonspecific DNA (ds NS, lanes 9 and 10). Competitors were used in a 100- and 500-fold molar excess of the unlabeled DNA fragment. TheNRF-2 band is indicated on the right. The binding of purified Sp1 and NRF-2 to ds NRF-2A can be visualized only upon prolonged exposure. B,NRF-2 interaction at the tandem C/D sites. A duplex oligonucleotide containing the tandem C and D sites (ds NRF-2 C/D) was used. Lane 1, freeprobe; lane 2, HeLa extract; lane 3, purified recombinant NRF-2 subunits a and b2; lanes 4 and 5, competition with specific DNA; lanes 6 and 7,competition with nonspecific DNA. The competitors were used in a 500- and 1000-fold molar excess of the unlabeled fragment. The sequences ofthe duplexes used are shown at the bottom of the figure.

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that a protein complex is formed over both sites, these results,taken together, imply that the NRF-2a subunit recognizes andbinds to the C site and utilizes the weaker D site to form astable heterotetrameric complex. A model that summarizes thefactors interacting at COX7AL is shown in Fig. 8.There is a notable parallelism among the few COX promoters

analyzed thus far (26, 27, 45, and this study). Sp1 interactionappears to be an intrinsic component of the COX4, COX5B, andCOX7AL promoters (27, 47–49). Multiple, functional NRF-2motifs are also present in all three genes. NRF-2 is a memberof the ets family of proteins, and ets proteins have been found toregulate genes involved in development, growth control, andcell transformation (50). It is possible that other members ofthe ets family recognize the same target sequence and modu-late gene expression, as has been observed for the human ATPsynthase b subunit gene promoter, which is regulatable byNRF-2, Ets-1, and Ets-2 (46). Finally, NRF-1 interaction ap-pears to be present for murine COX5B (26), rat COX6C (39),and bovine COX7AL (this study). NRF-1 sites have been foundin a number of genes involved in mitochondrial biogenesis andfunction (25, 39) and in constitutively expressed housekeepinggenes, suggesting a mechanism whereby an external environ-ment or physiological stimulus could coordinately modulateboth nuclear and mitochondrial genomes via common nuclearsignals (24). There appear to be differences, however, in therelative contribution of NRF-1 and NRF-2 to promoter activityin genes containing both motifs. In COX4 and COX5B, pro-moter function is mainly dependent on the NRF-2 motifs (26,27, 45, 47), whereas for the human mitochondrial transcriptionfactor A gene, NRF-1 seems to be the major determinant (44).For COX7AL, NRF-1 appears to be the more important factorsince mutating this site abolishes 73% of the promoter activity;however, this conclusion should await site-directed point mu-tations introduced into the various ets motifs in COX7AL.Apart from Sp1, NRF-1, and NRF-2, a fourth component inCOX promoter regulation appears to be NF-E1 or YY1, whichconstitutes a significant component of the basal promoter ma-chinery of the mouse COX5B gene (48).In addition to transcriptional regulation by NRF-1 and

NRF-2, COX7AL is regulated post-transcriptionally throughthe requirement for a protein (COLBP) bound to the 39-un-translated region of the L message (51, 52). Some of the majorquestions that remain are as follows. Do these regulatory ele-ments respond to the energy status of the cell? If so, how? Whyare these elements present only in a subset of the genes encod-ing respiratory chain proteins? How are L isoform genes tran-scriptionally silenced in differentiated muscle cells? Furtherwork will be needed to define the nature and location of thesignals that mediate these events.

Acknowledgments—We thank Joseph V. Virbasius for helpful discus-sions, Narayan G. Avadhani for a preprint in advance of publication,and Wayne D. Lancaster and Margaret I. Lomax for comments on themanuscript.

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GrossmanR. Sathiagana Seelan, Lekha Gopalakrishnan, Richard C. Scarpulla and Lawrence I.IDENTIFICATION OF PROMOTER ELEMENTS IN THE BOVINE GENE

Oxidase Subunit VIIa Liver Isoform: CHARACTERIZATION ANDcCytochrome

doi: 10.1074/jbc.271.4.21121996, 271:2112-2120.J. Biol. Chem. 

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