©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Cytochrome c Oxidase Subunit VIIa Liver Isoform
CHARACTERIZATION 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 (1) Lekha Gopalakrishnan (2) Richard C. Scarpulla (2) Lawrence I. Grossman (1)(§)

From the  (1)Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan 48201 and the (2)Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Cytochrome c oxidase subunit VIIa is specified by two nuclear genes, one (COX7AH) producing a heart/muscle-specific isoform and the other (COX7AL) a form expressed in all tissues. We have isolated both genes to examine their transcriptional regulation. Here, we characterize the core promoter of COX7AL and show that a 92-base pair region flanking the 5`-end promotes most of the activity of this gene. The 92-bp basal promoter contains sites for the nuclear respiratory factors NRF-1 and NRF-2, which have been shown to contribute to the transcription of a number of nuclear genes involved in mitochondrial respiratory activity, and also at least four Sp1 motifs. We show that both the NRF-1 and NRF-2 binding sites are functional in COX7AL and present evidence suggesting that interaction between the NRF-1 site and an upstream element contributes to expression.


INTRODUCTION

Cytochrome c oxidase (COX), (^1)the rate-limiting component of the electron transport chain, catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen; this process helps generate the proton gradient that fuels ATP synthesis. Although mammalian COX consists of 13 subunits, 3 encoded by mitochondrial DNA and the rest by the nuclear genome, the catalytic functions reside in the mitochondrially encoded subunits I and II. Attention has recently focused on elucidating more clearly the role of the nuclear-encoded subunits, which have been presumed to play a role in regulation and assembly of COX(1, 2, 3, 4, 5) . Among the nuclear-encoded subunits, most mammals have three (VIa, VIIa, and VIII) that exist as isoforms, an L (liver) isoform, which is ubiquitously expressed, and an H (heart) isoform, expressed primarily in adult heart and skeletal muscles (6, 7, 8) . The fetus, by contrast, contains the L isoform as the dominant contractile muscle form (9, 10, 11, 12) and switches to the H isoform during development.

The detailed roles of these nuclear subunits still remain unclear. Several subunits, however, including isoforms, have been linked to function. Subunit VIa appears to sense adenine nucleotide concentrations and thereby modulates COX activity(13, 14, 15, 16) ; furthermore, regulation of the bovine enzyme has been shown 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 low O(2) environment, respectively (17, 18) . The subunit V isoforms have been shown to modulate holoenzyme activity by altering its kinetic properties, such as turnover number (19) , by changing the environment at the binuclear reaction center (20) . Subunit VIb is required for assembly of a fully active yeast enzyme but is not required thereafter(21) ; when subunit VIb is selectively removed from the mammalian holoenzyme, COX activity is 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 transmembrane proton channel, has been inferred from limited trypsin digestion experiments of COX(23) .

Both COX function and synthesis, therefore, may involve responses to regulatory signals. One way the nuclear genes could be regulated is through common signals that reside in their DNA sequence. The characterization of several genes of complexes I to V of the respiratory chain has elucidated a number of candidate signals: (i) NRF-1, a positive activator of transcription found to have a role in at least two COX genes (rat COX6C and mouse COX5B). It appears to be a key factor in coordinating respiratory metabolism with other biosynthetic and degradative pathways(24, 25) ; (ii) NRF-2, an ets-related multisubunit activator that recognizes a GGAA motif. It has binding sites in the mouse and rat COX4 and mouse COX5B genes (26, 27) and is also known as the GA-binding protein(28) ; (iii) the OXBOX, a tissue-specific element that promotes the expression of genes in heart/skeletal muscles(29) . The OXBOX factor is found only in myogenic cells and acts in concert with another element, often overlapping the OXBOX, known as the REBOX. The REBOX element apparently binds to a ubiquitous factor and is modulated by the redox state, pH, and thyroid hormone levels(30) ; and (iv) an enhancer-like element found in the ATP synthase beta-subunit, cytochrome c(1), and pyruvate dehydrogenase E1a subunit genes(31) . Thus far, only the NRF-1 and NRF-2 motifs have been shown to be functionally associated with COX genes.

To understand the detailed regulation of COX isoforms in various tissues, we have isolated the bovine genes for the subunit VIIa isoforms, including their 5`-flanking regions(32, 33) . In this initial communication, we characterize the first tissue-specific COX gene promoter. We describe the basal promoter of the bovine COX7AL isoform gene and identify regulatory elements and transcription factors that appear involved in its expression. Specifically, we report that both NRF-1 and NRF-2 participate in COX7AL expression and that NRF-1 may need to interact with an upstream factor for maximal activity.


EXPERIMENTAL PROCEDURES

Materials

CAT enzyme-linked immunosorbent assay kit and chlorophenol red beta-D-galactopyranoside were from Boehringer Mannheim; Klenow polymerase and T4 polynucleotide kinase were from Promega; pCH110 was obtained from Pharmacia Biotech Inc.; [-P]ATP and [alpha-P]dCTP (each 3000 Ci/mmol) were from Dupont NEN; media and sera for transfection analysis were from Life Technologies, Inc.; oligonucleotides were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA) or Center for Molecular Biology (Wayne State University); pRSV CAT (34) was from Dr. J. Moshier, and pGKO CAT (35) was from Dr. G. Kumar, both at Wayne State University. All other chemicals were from Sigma or Fisher.

Construction of CAT Deletion Plasmids

Various 5`-flanking regions of the COX7AL gene beginning upstream of the transcriptional start site (+1) (32) and extending to the non-coding portion of the first exon (ThaI or DdeI) (Fig. 1) were cloned into the multiple cloning site of pGKO CAT. A 1.5-kilobase HincII-ThaI fragment (-1442 to +52), a 569-bp PstI-ThaI fragment (-517 to +52), a 427-bp HincII-ThaI fragment (-375 to +52), a 168-bp XmaIII-DdeI fragment (-98 to +70), and a 58-bp HaeIII-ThaI fragment (-6 to +52) were isolated, filled in with Klenow polymerase, and cloned into pGKO CAT to generate, respectively, HincII(A)-CAT, PstI-CAT, HincII(B)-CAT, XmaIII-CAT, and HaeIII-CAT. Inserts were identified by restriction enzyme analyses and DNA sequencing, and plasmid DNA was purified on a preparative scale via two CsCl gradients.


Figure 1: Characterization of the upstream regulatory region of COX7AL. 6 bp to 1.5 kilobase pairs of COX7AL upstream region were cloned in pGKO CAT vector and assayed for promoter activity in HeLa cells. A restriction map of the upstream region is shown on top with the arrow indicating the major transcriptional start site at +1. The nt positions of the fragments used to make these constructs are indicated on each line. 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. The empty CAT vector, pGKO CAT, has a relative activity of 5%.



Construction of Hae43-CAT, Hae46-CAT, and PCR59-CAT

The 92-bp region between the XmaIII and the HaeIII sites (Fig. 1) was further analyzed as follows. Digestion with HaeIII results in two products, a 5` 43-bp and a 3` 46-bp fragment (Fig. 2B). Each fragment was cloned into HaeIII-CAT (Fig. 1) to generate Hae43-CAT and Hae46-CAT (Fig. 2B). A 59-bp PCR fragment was also generated using primers that span the internal HaeIII site (Fig. 2B) and similarly cloned to generate PCR59-CAT. The HaeIII-CAT vector contains the transcriptional start site of COX7AL and has negligible CAT activity.


Figure 2: A, Sequence of COX7AL containing the basal promoter region and various regulatory motifs. The basal promoter region extends from -98 (XmaIII) to -6 (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 5`-end. dSp1 refers to the distal Sp1 site and pSp1, pSp1`, and pSp1`` refer to the proximal Sp1 site cluster. The arrow denotes the major transcriptional start site (+1). B, CAT activity of subfragments derived from the 92-bp XmaIII-HaeIII basal promoter region. The XmaIII-CAT construct (bold) was digested at the internal HaeIII site to generate the Hae46-CAT and the Hae43-CAT subclones. Horizontal arrows indicate the PCR primers used to amplify a fragment containing the internal HaeIII site. The transcriptional start site is depicted by the bent arrow. The CAT activity of each construct (±S.D.) relative to XmaIII-CAT (100%) is indicated on the right. Dashes indicate deleted regions.



Cell Transfection and CAT Analysis

HeLa cells (8 times 10^5) grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum were seeded into each 100-mm dish and transfected by the calcium phosphate method (36) using 30-35 µg of the test plasmid and 4 µg of a beta-galactosidase control plasmid, pCH110. Cell extracts were prepared by freeze-thawing(36) . Half of the cell extract was removed and frozen for beta-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 least three times. CAT activity of each sample was determined in duplicate on 2-4 µl of the extract by using the CAT enzyme-linked immunosorbent assay kit and normalized to beta-galactosidase activity. beta-Galactosidase activity was determined in duplicate in a 100-µl reaction using 2-5 µl of the extract and chlorophenol red beta-D-galactopyranoside in Hepes buffer, according to the manufacturer's protocol. The normalized CAT activity of the test samples was expressed relative to the largest CAT construct 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 mutation analyses, using overlapping oligonucleotides to reconstruct the 92-bp fragment(25) . For the wild-type sequence, ten oligonucleotides spanning both strands of the 92-bp region were synthesized, purified through PAGE(36) , and assembled as follows. 100 ng each of all but the terminal oligonucleotides were mixed and phosphorylated with T4 polynucleotide kinase and ATP. The kinased mixture was precipitated with ethanol and ligated after addition of 100 ng each of the terminal oligonucleotides. The ligation mixture after ethanol precipitation was repaired with Klenow polymerase, and the products were resolved by PAGE (15%). The 92-bp reassembled promoter fragment was isolated, cloned into HaeIII-CAT, and verified by sequencing. Single-site and double-site mutations were introduced by substituting the appropriate complementary mutant oligonucleotides (see ``Results'') into the assembly.

DNase I Footprinting and Methylation Interference Analysis

Binding reactions for DNase I were carried out with 20 fmol of an end-labeled DNA fragment (200-bp XbaI-HindIII) derived from a construct containing the wild-type 92-bp promoter (-98 to -6) in TM buffer (25 mM Tris at pH 7.9, 6.25 mM MgCl(2), 0.5 mM EDTA, 0.5 mM dithiothreitol, 10% glycerol) containing 50 mM KCl. Approximately 40 ng of purified recombinant NRF-1 (26) and 200 ng of NRF-2 (38) (containing equimolar amounts of bacterially expressed human alpha and beta(2) subunits) were incubated separately with labeled DNA along with 2% polyvinyl alcohol and 100 µg of bovine serum albumin. Following a 15-min incubation at room temperature, the samples were adjusted to 5 mM MgCl(2), 2.5 mM CaCl(2) and treated with 2.5 ng of DNase I for 1 min. Cleaved DNA was extracted 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 digestion with either XbaI or HindIII, the DNA was 3`-end labeled with Klenow enzyme. After a second digestion with either XbaI or HindIII, the resulting 200-bp fragments were gel purified and electroeluted. Each fragment (1 times 10^6 cpm) was treated with dimethyl sulfate for 5 min at room temperature as described(40) . Binding reactions contained 5 times 10^5 cpm of methylated fragment and either 40 ng of recombinant NRF-1 or 200 ng of NRF-2. Following electrophoresis, the wet gel was autoradiographed for 2 h at 4 °C, and the free and protein-bound bands were excised and electroeluted. The electroeluted DNA was extracted once with phenol/chloroform, ethanol precipitated with 10 µg of Escherichia coli tRNA, and cleaved at methylated guanosine residues by treatment with 1 M piperidine. Cleavage products were electrophoresed on a 6% polyacrylamide-urea sequencing gel.

Electrophoretic Mobility Shift Assays (EMSA)

Mobility shift assays were carried out essentially as described(39) . Duplex DNA for EMSA was assembled from complementary oligonucleotides or their mutant forms by annealing in TE buffer containing 150 mM NaCl after heating to 65 °C and cooling slowly to room temperature. The mixture was precipitated from ethanol and filled in with Klenow polymerase, and the duplex was isolated from 15% PAGE. DNA was end labeled, and EMSA was performed using 5-20 fmol of DNA and 25 µg of HeLa extract, prepared as described(41) , in each binding reaction. For competition, the indicated molar excesses of unlabeled duplex oligonucleotides were preincubated with the extract for 10 min at room temperature and for an additional 10 min with the labeled probe. Complementary oligonucleotides from adjacent regions of the promoter were similarly annealed and used as nonspecific DNA. Gel shifts were resolved on 5% PAGE (58:1), run in 0.5 times TBE (39) in the cold, and autoradiographed. Purified recombinant proteins used were NRF-1 (20 ng/reaction) and NRF-2 (alpha plus beta(2)) (50 ng each/reaction).

Antibody Supershift Assays

Reactions were carried out as described for EMSA, using 15 µg of HeLa extract and 20 ng of purified recombinant NRF-1. After incubation for 15 min at room temperature, 1 µl of undiluted goat anti-NRF-1 antibody was added and incubated for an additional 15 min. Samples were electrophoresed as for EMSA but using 4% gels.


RESULTS

Characterization of the Upstream Regulatory Region of COX7AL

CAT constructs harboring 6 bp to 1.5 kilobase pairs of COX7AL upstream sequence were analyzed for promoter activity in HeLa cells (Fig. 1). Significant activity (18% relative to pRSV CAT or 56% relative to the longest construct) was retained when the upstream sequence was deleted to only 98 bp of flanking DNA (XmaIII-CAT). Further deletion to only 6 bp (HaeIII-CAT) abolished most of this activity, suggesting that the basal promoter region of COX7AL is localized within a 92-bp XmaIII-HaeIII (-98 to -6) region. This region contains an NRF-1 site showing 10/12 homology with the consensus sequence and two potential NRF-2 sites (designated A and B, respectively, from the 5`-end) that have a GGAA purine core as a recognition motif (Fig. 2A). Based on nucleotides that flank this motif, only the A site appears to be an ideal ets binding site(28) . The region also contains a number of Sp1 motifs. At least one is located in the distal (d) portion of the fragment and several in the more proximal (p) highly G-C rich region. Two of these motifs have the core GGGCGG sequence (42) and are designated dSp1 and pSp1 (Fig. 2A). dSp1 has a 9/10 match with an extended Sp1 consensus sequence(42) , and pSp1 has a 7/10 match. A perfect match with the Sp1 consensus compiled by Faisst and Meyer (43) is found a few nucleotides upstream of pSp1 and is designated as pSp1`. A fourth motif with a single mismatch overlaps an internal HaeIII site (Fig. 2A) and will be referred to as pSp1``.

The activities of fragments containing additional upstream sequences (Fig. 1) are 1.6-1.8-fold higher than that of the 92-bp fragment, suggesting that additional positive elements are located upstream of the basal promoter region. At least one of these elements is located between the HincII(-375) and XmaIII sites (61% stimulation relative to the 92-bp fragment) and possibly another between the PstI and HincII(-375) sites (84% relative to the 92-bp fragment). The HaeIII-CAT construct, which contains only 6 bp of flanking DNA and 52 bp of the untranslated region of the first exon, has only 5% the activity of the largest CAT construct.

Characterization of the COX7AL Basal Promoter

The 92-bp basal promoter region was further analyzed by assessing the activity of constructs containing subfragments (Fig. 2B). The HaeIII digestion products and the PCR product as described under ``Experimental Procedures'' were individually cloned in the HaeIII-CAT vector (Fig. 1). Each of the cloned HaeIII products accounted for only 14-19% of the CAT activity of the intact 92-bp fragment (Fig. 2B). Thus, either NRF-1 does not contribute significantly to promoter activity or the low activity of the NRF-1 site-containing fragment (Hae46-CAT) is due to the absence of upstream sequences. By contrast, the 59-bp PCR fragment (PCR59-CAT), which lacks the NRF-1 site (Fig. 2B), retained 65% of the CAT activity. Although this result suggests that the basal promoter is located within a 59-bp region that spans the proximal overlapping Sp1 motifs, it is not in its original spatial context with respect to the transcriptional start site. To examine the roles of the various sites within a normal promoter context, we performed site-directed mutagenesis.

Analysis of the Basal Promoter by Site-directed Mutagenesis

Mutant complementary oligonucleotides were synthesized in which the core recognition motifs were altered. Mutations were introduced at the NRF-1 (GCGCTTGCGC to GtaaTTGaat), dSp1, pSp1, and pSp1`` sites. For dSp1 and pSp1, the core recognition sequence of GGGCGG was altered to atGCGG and GGGatG, respectively. In addition, for pSp1`` the HaeIII site contained within this motif was altered (GGCC to ataC), since this site was observed to be critical for promoter function by deletion analysis (Fig. 2B). The basal promoter was then assembled from these oligonucleotides harboring one or two mutant sites and transfected into HeLa cells. For single-site mutants, a pronounced decrease in activity occurred when the NRF-1 (27%), the dSp1 (45%), or the pSp1`` (26%) site was mutated (Fig. 3). A mutation in pSp1, however, showed an uncharacteristic increase (112%). These results were confirmed with double-site mutants.


Figure 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 (&cjs3649;) and summarized to the left of each line. The top four lines indicate single-site mutations, and the bottom 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.



In general, double mutants showed a steep drop in activity. However, if the pSp1 site was included, the decrease was more modest compared to a single-site mutation of the other member of the pair. The basis for the apparent stimulation by a mutation at the pSp1 site is not presently understood. The double mutations at pSp1``- NRF-1 (12%) and at the dSp1-pSp1`` sites (12%) show the lowest activities; comparison with the single site mutants suggests that the latter sites act independently whereas the former sites may interact.

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

The results of transfection experiments with site-directed mutants point to the NRF-1 site as a major determinant of COX promoter function. To investigate whether NRF-1 displays specific binding to this site, DNase I footprinting was performed using purified recombinant NRF-1 protein. A clear footprint displaying the characteristic enhanced cleavage pattern at the boundaries (Fig. 4, lane 3) was obtained in the region of the NRF-1 site. Point mutations within the NRF-1 site that resulted in a loss of promoter activity (Fig. 3) also eliminated protein binding to the site (data not shown). A similar analysis of the promoter fragment with purified recombinant NRF-2 showed the presence of two NRF-2 footprints in the region of the A and C/D sites (Fig. 4, lane 8, top and bottom, respectively). Furthermore, the inclusion of a 100-fold excess of oligonucleotide containing NRF-1 (RC4 -172/-147) or NRF-2 (RCO4 +13/+36) binding sites (44) in the footprinting reactions established the specificity of the interaction of the proteins to their respective sites (Fig. 4, lanes 4, 5, 9, and 10).


Figure 4: Competition DNase I footprinting of NRF-1 and NRF-2 sites in the COX7AL promoter. Promoter fragments were 3`-end labeled with Klenow enzyme on the non-coding strand and subjected to DNase I footprinting in the absence (lanes 2 and 7) or presence of purified recombinant NRF-1 (lanes 3-5) or NRF-2 (lanes 8-10). A 100-fold molar excess of synthetic oligonucleotides containing rat cytochrome c (RC4) promoter sequences from -172 to -147 (lanes 4 and 10) or rat cytochrome c oxidase (RCO4) promoter sequences from +13 to +36 (lanes 5 and 9) were included in the binding reactions as indicated. The vertical lines adjacent to the autoradiographs indicate the regions protected from DNase I digestion. The protected NRF-2 regions at the top and bottom refer to the A and C/D sites, respectively. G, G-specific sequencing reaction.



The pattern of protein-DNA contacts was determined by methylation interference analysis. Radiolabeled promoter fragments containing intact NRF-1 and NRF-2 sites were methylated and incubated with purified protein as described under ``Experimental Procedures.'' Analysis of the piperidine cleavage products showed the guanine contacts at the NRF-1 site (Fig. 5, left) to be consistent with those described previously(39) . Analysis of the guanine contacts made with NRF-2 indicates binding of protein to the A and C sites (Fig. 5, right), localized to the adjacent guanine nucleotides of the core GGAA motif as previously observed(45) . Interestingly, the A and C sites are the only ones preceded by the nucleotide A or C, which is favored for the binding of Ets proteins (28) .


Figure 5: Methylation interference footprinting of NRF-1 and NRF-2 sites. COX7AL promoter fragments were 3`-end labeled with Klenow enzyme on either upper or lower strands relative to the transcriptional orientation and methylated with dimethyl sulfate. Methylated fragments were incubated with purified NRF-1 or NRF-2 and electrophoresed on a 4% non-denaturing preparative acrylamide gel. DNA from free (F) and protein-bound (B) bands was eluted, cleaved with piperidine, and electrophoresed on a 6% urea-acrylamide gel. Arrowheads and filled circles indicate guanine nucleotides that completely inhibited protein binding when methylated, and the underlined sequences indicate regions protected in DNase I footprint analysis. Methylation interference at the NRF-2A site is indicated on the upper strand and at the C site on the lower strand.



Analysis of DNA-Protein Interactions by Mobility Shift Assays

DNase I footprinting reveals that NRF-1 binds to its motif in COX7AL and that NRF-2 binds to the A and C/D sites but not to the B site (Fig. 4). To confirm these results, EMSA was performed on double-stranded 21-24-bp oligonucleotides containing 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 of the COX7AL promoter (Fig. 6B). This binding (top band) is competed by a 500-fold molar excess of the specific unlabeled fragment but not by an equivalent molar excess of a 21-bp fragment mutated at the NRF-1 site (ds NRF-1) or by a nonspecific 23-bp fragment (ds NS). Further confirmation that this band represents NRF-1 binding comes from using purified recombinant NRF-1 on ds NRF-1 and ds NRF-1; whereas a shift was seen with the former, none was observed with the latter (data not shown). Additional evidence was obtained by antibody supershifts. The binding of NRF-1 from HeLa nuclear extract (Fig. 6A, lane 1, upper band) is supershifted when antibody against NRF-1 is used (lane 2). The position of the duplex after incubation with purified recombinant NRF-1 (lane 3) and its supershifting (lane 4) is also shown. It is clear from these results that the upper band represents a specific DNAbulletNRF-1 complex. The typically diffuse supershifted complexes obtained with the crude extract may result from other proteins interacting with antibody. These observations suggest that the NRF-1 motif in COX7AL is functional and corroborate the results of footprinting analyses (Fig. 4).


Figure 6: A, antibody supershift analysis of NRF-1 binding to the COX7AL promoter. The duplex oligonucleotide ds NRF-1 was incubated with HeLa extract (lane 1), HeLa extract and NRF-1 antibody (lane 2), purified recombinant NRF-1 (lane 3), and purified recombinant NRF-1 and NRF-1 antibody (lane 4). B, specificity of NRF-1 binding to the COX7AL promoter. The duplex oligonucleotide, ds NRF-1, was incubated with HeLa extract (lane 1) and HeLa extract and competitor DNAs (lanes 2-7). Lanes 2 and 3, specific competition with ds NRF-1; lanes 4 and 5, competition with ds NRF-1; lanes 6 and 7, competition with a nonspecific DNA. Competitors were used at 100- and 500-fold molar excess of unlabeled DNA. The top band in these lanes represents NRF-1 binding. Sequences of the various duplexes are shown at the bottom of the figure.



The lower band in these figures probably represents NRF-2 binding at the B site, which is present in the ds NRF-1 and ds NRF-1 duplexes. The binding of NRF-2 to the B site was not seen by DNase I footprinting (Fig. 4). Since footprinting was done on a larger fragment, containing all of the NRF-2 sites (A, B, and C/D), and EMSA was performed on a 21-bp fragment, we believe the footprinting data to be more consistent with native NRF-2 interactions. We thus conclude that NRF-2 does not interact at the B site.

DNA-Protein Interactions at the NRF-2A Site

The interactions at the NRF-2A site were examined by EMSA (Fig. 7A), utilizing the following duplexes: ds NRF-2A, containing the NRF-2A site and the pSp1 site; ds NRF-2A*, in which the pSp1 site has been altered; and a 21-bp nonspecific oligonucleotide spanning an adjacent upstream region (ds NS). The most prominent band in these lanes represents NRF-2 binding. This conclusion is derived from the observation that purified NRF-2 (alpha and beta(2) subunits) forms a complex with ds NRF-2A that is similar in mobility to a complex formed with HeLa nuclear extract (lanes 2 and 4). This interaction is partly competed at a 500-fold molar excess of specific unlabeled DNA (ds NRF-2A or ds NRF-2A*), containing an intact NRF-2A site (lanes 6 and 8). The nonspecific DNA fragment (ds NS) is unable to compete for this binding at equimolar levels (lanes 9 and 10).


Figure 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; lane 2, HeLa extract; lane 3, purified human Sp1; lane 4, purified recombinant NRF-2alpha and beta(2) subunits. Lanes 5-10 contain HeLa extract and labeled ds 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 and 8), 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. The NRF-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, free probe; lane 2, HeLa extract; lane 3, purified recombinant NRF-2 subunits alpha and beta(2); 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 of the duplexes used are shown at the bottom of the figure.



Purified human Sp1 binds to ds NRF-2A (lane 3), forming a faint complex that is not evident in lanes containing total nuclear extract from HeLa cells. Since the pSp1 site is fused to the 5` end of NRF-2A, it is possible that binding to the Sp1 motif is blocked by the relatively higher affinity of NRF-2 to the fused A site. The apparent weak Sp1 complex formation may not be surprising given that the pSp1 motif has only a 7/10 match with the extended consensus(42) .

DNA-Protein Interactions at the NRF-2C/D Sites

Both purified NRF-2 and the HeLa extract form identical complexes with a duplex DNA containing the tandem C/D sites (Fig. 7B, lanes 2 and 3). The complex formed is readily competed at a 500-fold molar excess of the specific unlabeled DNA (lane 4) but not by a nonspecific DNA (lanes 6 and 7). The NRF-2 complex formed at the tandem C/D sites (ds NRF-2C/D) differs from the NRF-2 complex formed at the A site (Fig. 7A) in two ways: this complex is larger, and it is readily competed at a 500-fold molar excess of specific competitor.


DISCUSSION

COX7AL, the first COX isoform gene to our knowledge analyzed for promoter function, has a TATA-less promoter located at a CpG island(32) . We have mapped the basal promoter to a 92-bp segment immediately upstream of the major transcriptional start site. It contains functional NRF-1 and NRF-2 (site A) motifs as well as a tandem pair of functional NRF-2 sites (C/D) located a few nucleotides downstream of the transcriptional start site (Fig. 2A). In addition, at least four Sp1 sites are located at the distal and proximal regions. Further deletion of the 92-bp region defines a 59-bp core region that includes all the proximal Sp1 sites and contains up to 65% of the CAT activity. We observed a significant loss of activity (74%) when either the pSp1`` or the NRF-1 site was altered (Fig. 3). Indeed, a double mutant for these sites showed one of the lowest activities, suggesting that these two sites are important for COX7AL regulation. These mutations, however, did not totally abolish promoter activity, suggesting that other factor(s) also participate in promoter function. This residual activity could be due, 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 (pSp1``, pSp1`, and pSp1) of which pSp1``, which spans the HaeIII site, appears to be the most important. Deletion of fragments upstream of the HaeIII site, or mutating the HaeIII site in the context of an intact 92-bp promoter, drastically reduces CAT activity. Although we presume that Sp1 binds to this motif based on its G-C richness and its resemblance to the Sp1 consensus, a similar element (CGGCCCC) found in the ets domain binding region of the human mitochondrial ATP synthase beta-subunit gene promoter (46) does not appear to form a protein-DNA complex with Sp1. Hence, the nature of the factor binding to pSp1`` needs to be elucidated. At the distal site (dSp1), a mutation decreased activity to 45% relative to the 92-bp fragment, and a double mutation at the dSp1 and pSp1`` sites abolished 88% of the basal promoter activity. At the pSp1 site, however, there was a surprising enhancement of activity to 112% (Fig. 3). This effect was also noted in double mutants. A possible explanation for this could be the observation that the pSp1 site is fused to the 5`-end of NRF-2A (Fig. 2A). The affinity of NRF-2 to the A site may thus mask accessibility to the adjacent Sp1 site. Any competition between Sp1 and NRF-2 to bind to the fused site should, therefore, be relieved when the pSp1 motif is altered. This hypothesis is supported by the following observations: (i) NRF-2 clearly footprints at the A site (Fig. 4), spanning the GGAA motif and extending into the pSp1 site (Fig. 5, bottom), and (ii) DNA-protein interactions at the fused site (Fig. 7A) indicate the presence of a single prominent NRF-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 the deletion analysis data (Hae46-CAT; Fig. 2B), where a fragment harboring the NRF-1 site was found to be insufficient for promoter activity. These observations can be reconciled if NRF-1 were to interact with upstream regions (Fig. 8). Thus, in the deletion construct, the lower promoter activity may result because NRF-1, although able to bind to its target motif, is unable to interact with factor(s) binding upstream. This model, which emphasizes the importance of promoter context in NRF-1 function(25) , is supported by the site-directed mutagenesis data, in which a mutation at the NRF-1 site abolishes 73% of the promoter activity.


Figure 8: Schematic diagram depicting the various transcription factors that participate in COX7AL promoter function. Various motifs identified in the COX7AL promoter region are indicated below the line whereas the binding of factors is shown above. Shaded factors indicate confirmed binding as inferred from gel shifts and footprint analyses. The binding of Sp1 to the pSp1`/pSp1`` sites is speculative. &cjs3649; denotes lack of binding. The possible interaction of NRF-1 with upstream Sp1 sites is suggested from deletion and site-directed mutation analyses data.



Studies of the murine COX4 and COX5B promoters (26, 27) reveal that NRF-2 has a 10-20-fold greater affinity for tandem than monomeric sites. Similarly, the pair of tandem NRF-2 sites (C/D) located downstream in the untranslated region of the first exon of COX7AL appears to form a stable high affinity complex with NRF-2 (Fig. 7B). Moreover, strong binding is observed when purified NRF2 alpha and beta(2) subunits are added to a duplex containing the tandem sites (Fig. 7B), whereas a complex at the single site (Fig. 7A) was detectable only on prolonged exposure, suggesting that tandem sites are preferred substrates for NRF-2. Alternatively, the weak complex formation at the A site with purified NRF-2 could suggest that another member of the Ets family has a preferred affinity for this site. Interestingly, the tandem NRF-2 motifs in mouse COX4 are also located in the untranslated region of the first exon and have features resembling an initiator element, since mutating either motif of the ets pair appears to determine the start site of transcription(27) .

A more detailed examination of the tandem sites indicates that they are not equivalent. Methyl interference analysis clearly indicates protein contact between NRF-2 and site C but not site D. This agrees well with the consensus for ets binding motifs(28) ; the C site is in a favorable binding context whereas the D site is not. Since DNase I footprinting and EMSA suggest that a protein complex is formed over both sites, these results, taken together, imply that the NRF-2alpha subunit recognizes and binds to the C site and utilizes the weaker D site to form a stable heterotetrameric complex. A model that summarizes the factors 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 interaction appears to be an intrinsic component of the COX4, COX5B, and COX7AL promoters(27, 47, 48, 49) . Multiple, functional NRF-2 motifs are also present in all three genes. NRF-2 is a member of the ets family of proteins, and ets proteins have been found to regulate genes involved in development, growth control, and cell transformation(50) . It is possible that other members of the ets family recognize the same target sequence and modulate gene expression, as has been observed for the human ATP synthase beta subunit gene promoter, which is regulatable by NRF-2, Ets-1, and Ets-2 (46) . Finally, NRF-1 interaction appears to be present for murine COX5B(26) , rat COX6C(39) , and bovine COX7AL (this study). NRF-1 sites have been found in a number of genes involved in mitochondrial biogenesis and function (25, 39) and in constitutively expressed housekeeping genes, suggesting a mechanism whereby an external environment or physiological stimulus could coordinately modulate both nuclear and mitochondrial genomes via common nuclear signals(24) . There appear to be differences, however, in the relative contribution of NRF-1 and NRF-2 to promoter activity in genes containing both motifs. In COX4 and COX5B, promoter function is mainly dependent on the NRF-2 motifs(26, 27, 45, 47) , whereas for the human mitochondrial transcription factor A gene, NRF-1 seems to be the major determinant(44) . For COX7AL, NRF-1 appears to be the more important factor since mutating this site abolishes 73% of the promoter activity; however, this conclusion should await site-directed point mutations introduced into the various ets motifs in COX7AL. Apart from Sp1, NRF-1, and NRF-2, a fourth component in COX promoter regulation appears to be NF-E1 or YY1, which constitutes a significant component of the basal promoter machinery of the mouse COX5B gene(48) .

In addition to transcriptional regulation by NRF-1 and NRF-2, COX7AL is regulated post-transcriptionally through the requirement for a protein (COLBP) bound to the 3`-untranslated region of the L message(51, 52) . Some of the major questions that remain are as follows. Do these regulatory elements respond to the energy status of the cell? If so, how? Why are these elements present only in a subset of the genes encoding respiratory chain proteins? How are L isoform genes transcriptionally silenced in differentiated muscle cells? Further work will be needed to define the nature and location of the signals that mediate these events.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants GM 48517 (to L. I. G.) and GM 32525 (to R. C. S.). 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.

§
To whom correspondence should be addressed. Tel.: 313-577-5326; Fax: 313-577-5218; :LG{at}cmb.biosci.wayne.edu.

(^1)
The abbreviations used are: COX, cytochrome c oxidase; CAT, chloramphenicol acetyltransferase; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; EMSA, electrophoretic mobility shift assay; PCR, polymerase chain reaction; ds, double stranded.


ACKNOWLEDGEMENTS

-We thank Joseph V. Virbasius for helpful discussions, Narayan G. Avadhani for a preprint in advance of publication, and Wayne D. Lancaster and Margaret I. Lomax for comments on the manuscript.


REFERENCES

  1. Kadenbach, B., and Merle, P. (1981) FEBS Lett. 135, 1-11 [CrossRef][Medline] [Order article via Infotrieve]
  2. Kadenbach, B. (1986) J. Bioenerg. Biomembr. 18, 39-54 [Medline] [Order article via Infotrieve]
  3. Kadenbach, B., Kuhn-Nentwig, L., and Buge, U. (1987) Curr. Top. Bioenerg. 15, 113-161
  4. Capaldi, R. A., Takamiya, S., Zhang, Y. Z., Gonzalez-Halphen, D., and Yanamura, W. (1987) Curr. Top. Bioenerg. 15, 91-112
  5. Poyton, R. O., Trueblood, C. E., Wright, R. M., and Farrell, L. E. (1988) Ann. N. Y. Acad Sci. 550, 289-307 [Medline] [Order article via Infotrieve]
  6. Schlerf, A., Droste, M., Winter, M., and Kadenbach, B. (1988) EMBO J. 7, 2387-2391 [Abstract]
  7. Seelan, R. S., and Grossman, L. I. (1991) J. Biol. Chem. 266, 19752-19757 [Abstract/Free Full Text]
  8. Lightowlers, R., Ewart, G., Aggeler, R., Zhang, Y. Z., Calavetta, L., and Capaldi, R. A. (1990) J. Biol. Chem. 265, 2677-2681 [Abstract/Free Full Text]
  9. Ewart, G. D., Zhang, Y. Z., and Capaldi, R. A. (1991) FEBS Lett. 292, 79-84 [CrossRef][Medline] [Order article via Infotrieve]
  10. Bonne, G., Seibel, P., Possekel, S., Marsac, C., and Kadenbach, B. (1993) Eur. J. Biochem. 217, 1099-1107 [Abstract]
  11. Schillace, R., Preiss, T., Lightowlers, R. N., and Capaldi, R. A. (1994) Biochim. Biophys. Acta 1188, 391-397 [Medline] [Order article via Infotrieve]
  12. Grossman, L. I., Rosenthal, N. H., Akamatsu, M., and Erickson, R. P. (1995) Biochim. Biophys. Acta 1260, 361-364 [Medline] [Order article via Infotrieve]
  13. Taanman, J. W., and Capaldi, R. A. (1993) J. Biol. Chem. 268, 18754-18761 [Abstract/Free Full Text]
  14. Anthony, G., Reimann, A., and Kadenbach, B. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1652-1656 [Abstract]
  15. Taanman, J. W., Turina, P., and Capaldi, R. A. (1994) Biochemistry 33, 11833-11841 [Medline] [Order article via Infotrieve]
  16. Rohdich, F., and Kadenbach, B. (1993) Biochemistry 32, 8499-8503 [Medline] [Order article via Infotrieve]
  17. Trueblood, C. E., Wright, R. M., and Poyton, R. O. (1988) Mol. Cell. Biol. 8, 4537-4540 [Medline] [Order article via Infotrieve]
  18. Poyton, R. O., and Burke, P. V. (1992) Biochim. Biophys. Acta 1101, 252-256 [Medline] [Order article via Infotrieve]
  19. Waterland, R. A., Basu, A., Chance, B., and Poyton, R. O. (1991) J. Biol. Chem. 266, 4180-4186 [Abstract/Free Full Text]
  20. Allen, L. A., Zhao, X. J., Caughey, W., and Poyton, R. O. (1995) J. Biol. Chem. 270, 110-118 [Abstract/Free Full Text]
  21. LaMarche, A. E. P., Abate, M. I., Chan, S. H. P., and Trumpower, B. L. (1992) J. Biol. Chem. 267, 22473-22480 [Abstract/Free Full Text]
  22. Weishaupt, A., and Kadenbach, B. (1992) Biochemistry 31, 11477-11481 [Medline] [Order article via Infotrieve]
  23. Capitanio, N., Peccarisi, R., Capitanio, G., Villani, G., DeNitto, E., Scacco, S., and Papa, S. (1994) Biochemistry 33, 12521-12526 [Medline] [Order article via Infotrieve]
  24. Chau, C. M. A., Evans, M. J., and Scarpulla, R. C. (1992) J. Biol. Chem. 267, 6999-7006 [Abstract/Free Full Text]
  25. Virbasius, C. A., Virbasius, J. V., and Scarpulla, R. C. (1993) Genes & Dev. 7, 2431-2445
  26. Virbasius, J. V., Virbasius, C.-m. A., and Scarpulla, R. C. (1993) Genes & Dev. 7, 380-392
  27. Carter, R. S., and Avadhani, N. G. (1994) J. Biol. Chem. 269, 4381-4387 [Abstract/Free Full Text]
  28. Brown, T. A., and McKnight, S. L. (1992) Genes & Dev. 6, 2502-2512
  29. Li, K., Hodge, J. A., and Wallace, D. C. (1990) J. Biol. Chem. 265, 20585-20588 [Abstract/Free Full Text]
  30. Chung, A. B., Stepien, G., Haraguchi, Y., Li, K., and Wallace, D. C. (1992) J. Biol. Chem. 267, 21154-21161 [Abstract/Free Full Text]
  31. Tomura, H., Endo, H., Kagawa, Y., and Ohta, S. (1990) J. Biol. Chem. 265, 6525-6527 [Abstract/Free Full Text]
  32. Seelan, R. S., and Grossman, L. I. (1993) Genomics 18, 527-536 [Medline] [Order article via Infotrieve]
  33. Seelan, R. S., and Grossman, L. I. (1992) Biochemistry 31, 4696-4704 [Medline] [Order article via Infotrieve]
  34. Gorman, C. M., Merlino, G. T., Willingham, M. C., Pastan, I., and Howard, B. H. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6777-6781 [Abstract]
  35. Kumar, G. (1992) Gene (Amst.) 110, 101-103
  36. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, R. E., Seidman, J. G., Smith, J. A., and Struhl, K. (1989) Current Protocols In Molecular Biology , John Wiley & Sons, Inc., New York
  37. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  38. Gugneja, S., Virbasius, J. V., and Scarpulla, R. C. (1995) Mol. Cell. Biol. 15, 102-111 [Abstract]
  39. Evans, M. J., and Scarpulla, R. C. (1990) Genes & Dev. 4, 1023-1034
  40. Maxam, A., and Gilbert, W. (1980) Methods Enzymol. 65, 499-559 [Medline] [Order article via Infotrieve]
  41. Bothwell, A., Yancopoulos, G. D., and Alt, F. W. (1990) in Methods for Cloning and Analysis of Eukaryotic Genes , Jones and Bartlett Publishers, Boston
  42. Kadonaga, J. T., Jones, K. A., and Tjian, R. (1986) Trends Biochem. Sci. 11, 20-23 [CrossRef]
  43. Faisst, S., and Meyer, S. (1992) Nucleic Acids Res. 20, 3-26 [Medline] [Order article via Infotrieve]
  44. Virbasius, J. V., and Scarpulla, R. C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1309-1313 [Abstract]
  45. Virbasius, J. V., and Scarpulla, R. C. (1991) Mol. Cell. Biol. 11, 5631-5638 [Medline] [Order article via Infotrieve]
  46. Villena, J. A., Martin, I., Vinas, O., Cormand, B., Iglesias, R., Mampel, T., Giralt, M., and Villarroya, F. (1994) J. Biol. Chem. 269, 32649-32654 [Abstract/Free Full Text]
  47. Carter, R. S., Bhat, N. K., Basu, A., and Avadhani, N. G. (1992) J. Biol. Chem. 267, 23418-23426 [Abstract/Free Full Text]
  48. Basu, A., Park, K., Atchison, M. L., Carter, R. S., and Avadhani, N. G. (1993) J. Biol. Chem. 268, 4188-4196 [Abstract/Free Full Text]
  49. Hoshinaga, H., Amuro, N., Goto, Y., and Okazaki, T. (1994) J. Biochem. (Tokyo) 115, 194-201
  50. Wasylyk, B., Hahn, S. L., and Giovane, A. (1993) Eur. J. Biochem. 211, 7-18 [Abstract]
  51. Preiss, T., and Lightowlers, R. N. (1993) J. Biol. Chem. 268, 10659-10667 [Abstract/Free Full Text]
  52. Preiss, T., Chrzanowska-Lightowlers, Z. M. A., and Lightowlers, R. N. (1994) Biochim. Biophys. Acta 1221, 286-289 [Medline] [Order article via Infotrieve]

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