(Received for publication, December 16, 1996, and in revised form, January 31, 1997)
From the Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan 48201
Cytochrome c oxidase (COX) subunit VIIc is one of the nuclear encoded subunits of the 13-subunit holoenzyme that carries out the terminal step in the electron transport chain. We have isolated the gene for this subunit, previously shown to be ubiquitously expressed from a single copy gene in the genome, and show that 167 base pairs of DNA surrounding the transcriptional start site contain the minimal promoter of this gene. This basal promoter contains two YY1 sites and at least one site for NRF-2, which show binding to their cognate factors. Mutation of both YY1 sites eliminates most of the promoter activity. Mutation at the upstream YY1 site significantly reduces the efficiency of transcript initiation at the major start site and thus plays the dominant role in COX7C regulation. COX7C is, thus, the second nuclear gene of COX that is regulated by YY1, suggesting that it is a third common factor, along with NRF-1 and NRF-2, to be associated with COX gene regulation.
Mammalian cytochrome c oxidase (COX)1 is a 13-subunit protein complex located on the inner mitochondrial membrane that catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen and participates in the translocation of protons across the membrane. Proton translocation generates an electrochemical gradient that drives the synthesis of ATP. The complex consists of three primarily catalytic subunits encoded by mitochondrial DNA and 10 polypeptides encoded in the nucleus that are suggested to function in regulation and/or assembly (1-3). Three of the nuclear-encoded subunits (VIa, VIIa, and VIII) exist as a pair of isoforms (4-6) in most mammalian species: a liver (L) form that is present in most tissues and a heart/muscle (H) form specific for adult cardiac and skeletal muscle.
Several COX nuclear genes, including those of isoforms, have now been isolated and characterized and provide a means of assessing their role in function and regulation; in addition, defects in the expression of genes for COX nuclear subunits are likely to cause human disease. A detailed analysis of the basal promoters of three ubiquitously expressed COX genes thus far available (COX4, COX5B, and COX7AL) reveal that they have regulatory sites in common (7-12). COX4, COX5B, and COX7AL contain multiple, functional binding sites for uclear espiratory actor-2 (NRF-2 (Ref. 8); also called GABP, GA-binding protein). COX5B and COX7AL also harbor a functional site for NRF-1 (13, 14), which binds to a number of housekeeping genes; a functional site has also been detected in COX6C, although the promoter of this gene has not been extensively analyzed (15). Binding sites for the ubiquitous factor Sp1 are found in the basal promoters of COX4, COX5B, and COX7AL. Finally, the multifunctional factor Yin-Yang-1 (YY1) has only been found in COX5B, where it acts by initiating basal transcription. None of the above elements, however, is found in the basal promoter of COX6AH, the only H isoform COX promoter so far characterized (16). The basal promoter of this gene is dependent on muscle-specific elements such as the E-box (MyoD binding site) and MEF-2 (myocyte-specific enhancer-binding factor-2) binding sites.
To further elucidate the mechanisms of COX regulation and expression, we have characterized the bovine gene encoding the 63-amino acid precursor of subunit VIIc (COX7C), a gene encoding a subunit without isoforms and thus expressed in all tissues. We present here the sequence of the COX7C gene and identify the transcription factors that regulate its expression. We show that its basal promoter is regulated by two YY1 sites, one of which is required for efficient initiation of transcription from the major start site. COX7C is the second COX nuclear gene in which YY1 plays a role in promoter function, suggesting that YY1 may be a third common factor, along with NRF-1 and NRF-2, involved in COX gene regulation.
Ribonuclease A was obtained from
Sigma; Ribonuclease T1 was from Life Technologies,
Inc.; T3 and T7 RNA polymerases and avian myeloblastosis virus reverse
transcriptase were from Promega Corp. [-32P]ATP (3000 Ci/mmol), [
-32P]CTP (800 Ci/mmol), and
[
-35S]dATP (1500 Ci/mmol) were from DuPont NEN. pCH110
was from Pharmacia Biotech Inc. The CAT enzyme-linked immunosorbent
assay kit, chlorophenol red
-D-galactopyranoside, and
yeast tRNA were purchased from Boehringer Mannheim. The Sequenase
Version 2.0 DNA sequencing kit was from U. S. Biochemicals, and the
ribonuclease protection assay kit (RPA IITM) was from Ambion.
Oligonucleotide primer synthesis was by the Center for Molecular
Medicine and Genetics (Wayne State University) or Random Hill
Biosciences (Ramona, CA). All other chemicals were from Fisher,
Sigma, or Life Technologies, Inc.
HeLa (2-CCL), HeLa S3 (CCL-2.2), and C2C12 mouse myoblasts (1772-CRL) were obtained from American Type Culture Collection. Media and supplements were obtained from Life Technologies, Inc. HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) containing penicillin (40 units/ml) and streptomycin (40 µg/ml). HeLa S3 suspension cultures were grown in a similar medium containing 5% FBS. C2C12 myoblasts were cultured in DMEM supplemented with 15% FBS, 0.5% chick embryo extract, and gentamicin (50 µg/ml). Myotubes were obtained by growing myoblasts in dishes coated with calf skin collagen (Calbiochem) and inducing them to differentiate in DMEM plus 10% horse serum and gentamicin. Transfection conditions are described below.
Characterization ofBGL-2J was isolated from a
bovine genomic EMBL-3 Sp6/T7 library (17). The phage was purified
through two rounds of screening (18) using labeled COX7C
cDNA (19) as probe and the phage DNA purified (20). Restriction
fragments of the genomic (phage) DNA that were positive upon Southern
analysis with COX7C cDNA probe were subcloned into
pBluescript KS(
) vector (Stratagene) and sequenced (21).
Primer extension was carried out as
described previously (22), using total RNA isolated from adult bovine
heart tissues and a 23-nt antisense primer corresponding to nt 183-161
of the first exon (Fig. 3). The primer-extended products were resolved on 6% polyacrylamide, 6 M urea gels and autoradiographed
for 1-2 weeks at 70 °C. A pBluescript KS(
) plasmid sequenced
with the universal forward primer served as a DNA size marker.
Ribonuclease Protection Assay
A 395-bp
SacII-HincII fragment comprising 121 bp of the
first intron, at least 131 bp of the first exon, as deduced from the cDNA (19), and an additional contiguous region of 143 bp at the 5
end, was cloned at the SacII-HincII site of
pBluescript KS(
) and used for nuclease protection with 40 µg of
total RNA as described previously (23).
EcoRI, PstI, or SacII sites located upstream of the transcriptional start site and a HpaII site located within the non-coding portion of the first exon were used to isolate fragments that were cloned into pGKOCAT (24) to generate 1.8-kb EcoRI-7cCAT, 1.5-kb PstI-7cCAT, and 93-bp SacII-7cCAT. Recombinant plasmids were isolated, verified by sequencing, and purified through two CsCl gradients.
HeLa cells (8 × 105/ml) were grown, transfected, and the normalized CAT activity determined as described (9). For each CAT construct 3-4 dishes were used and each experiment repeated at least four times. C2C12 myoblasts (2 × 105 cells) and myotubes (5 × 105 cells) were similarly transfected. The medium was replenished 17-20 h after transfection. Myoblasts were allowed to grow for another 48 h; to generate myotubes the medium was switched to 10% horse serum 24 h after replenishment and cells allowed to grow for an additional 48 h before harvesting, as described for HeLa cells.
Electrophoretic Mobility Shift Assays (EMSA)The 167-bp
SacII-HpaII fragment (93 to +74) containing the
putative basal promoter was end-labeled with [
-32P]ATP
and T4 polynucleotide kinase and used for EMSA. A typical binding
reaction (25) in a 20-µl final volume contained 5-10 fmol of the
labeled fragment, 2 µg of poly(dI-dC), 20 mM Hepes (pH
7.9), 1 mM MgCl2, 4% Ficoll, 0.5 mM dithiothreitol, 50 mM KCl, and protein. The
latter consisted of HeLa (S3) extract (26) (7 µg of protein),
purified histidine-tagged YY1 (600-700 ng), or purified NRF-2 (45 ng
each of subunits
and
1). For competition assays, the
binding reactions were preincubated with a 100- and 1000-fold molar
excess of the unlabeled fragment for 30 min on ice prior to incubation
with the labeled probe for an additional 20 min. Samples were
electrophoresed on 4% PAGE in 0.25 × TBE (1 × TBE = 90 mM Tris borate, 2 mM EDTA, pH 8.0) for
3 h in the cold and autoradiographed. Complementary
oligonucleotides 21-32 bases long spanning the
SacII-HpaII fragment (Fig. 5) were annealed in TE
buffer plus 150 mM NaCl and the duplex DNA was recovered from PAGE (22), end-labeled with [
-32P]ATP, and used
in EMSA. Electrophoresis was as before but for 2 h.
Site-directed Mutagenesis of the YY1 Motifs in COX7C
The
167-bp basal promoter fragment was resynthesized from a set of 6 overlapping oligonucleotides spanning both strands as described (27).
Appropriate mutant or wild-type (WT) complementary oligonucleotides
were used to create the desired upstream (U), downstream (D), and
upstream-downstream (UD) YY1 mutant, or WT constructs. Mutations were created by introducing the sequence AAAGGG
for the YY1 core recognition motif NCCATN. For YY1-D, the introduced changes did not alter the overlapping NRF-2 site (Fig. 5).
Briefly, the oligonucleotides were phosphorylated, annealed, and
ligated; the 167-bp fragment recovered by PAGE was cloned into the
SmaI site of pGKOCAT vector. After transformation into Escherichia coli DH5 cells, recombinants harboring the
desired inserts were identified by restriction enzyme digestion and
sequencing.
Total RNA from C2C12 myoblasts,
myotubes, and HeLa cells transfected with the WT basal promoter, as
well as those harboring the various YY1 mutations, was subjected to a
ribonuclease protection assay. Two antisense probes were generated by
transcription from the T3 promoter using appropriately digested
pBluescript plasmid containing the WT 167-bp basal promoter fragment
(WT probe) or one with a mutant YY1-D site
(D-probe) (Fig. 9B). The WT probe was used to
detect transcripts arising from transfected WT and YY1-U
mutant basal promoter CAT constructs and the D-probe was used to detect transcripts arising from the YY1-D and
YY1-UD mutated constructs. Both the probes and the
transcripts arising from the transfected plasmids include a common
62-bp vector sequence (Fig. 9B) that serves to differentiate
these transcripts from any endogenous ones that may be protected by the
probe. Both probes will protect all transcripts initiating from the +1
site to the beginning of the CAT gene. Yeast tRNA and mock-transfected
cells were used as a control for each probe. Individual transcripts
were quantitated by densitometric scanning with a Molecular Dynamics
PhosphorImager. The results from the ribonuclease protection assays
were confirmed by primer-extension analysis (22) using an antisense
primer specific to the 5-untranslated region of the CAT gene
(5
-TAGCTCCTGAAAATCTCGCC) (data not shown).
The
COX7C genomic region from BGL-2J (see "Experimental
Procedures") was completely sequenced on both strands (Fig.
1A). The first exon (185 nt) encodes the
complete 16-amino acid presequence and the first 9 amino acids of the
mature protein; it also contains a 5
-non-coding region of 110 nt. The
second exon (129 bp) encodes the remaining 38 amino acids of the mature
protein and 15 nt of the 3
-non-coding region, whereas the third
encodes 218 nt of the non-coding region. All the intron/exon junctions
follow the GT-AG rule (28); the first and second introns are 838 and
915 bp, respectively.
The gene is located at a CpG island (29), based on the distribution of CpG residues. Of the 52 CpGs that span the sequenced gene (Fig. 1C), 94% are found near the transcriptional start site and the first intron; by contrast, the distribution of GpC residues appears to be fairly uniform (Fig. 1C).
The transcriptional start site of the COX7C gene was
determined by primer-extension and nuclease protection analyses. A
23-nt antisense primer that anneals to the first exon, beginning 2 nt 5 (nt 183-161; Fig. 3) of the first intron, was used to reverse transcribe total bovine RNA. A major primer-extended product of 183 nt
was obtained, suggesting that the first exon is 185 nt long. This
product is clustered along with at least two minor products that all
lie within 8-10 nt of each other (but not clearly evident in Fig.
2A). This result was consistent with a
ribonuclease protection assay. In the latter, a cloned 395-bp fragment,
extending from the SacII site (nt
93) to the
HincII site (nt +306), containing at least 131 bp of the
first exon as deduced from the cDNA, and an additional 143 bp of
5
-upstream sequence, was radioactively transcribed to yield sense and
antisense transcripts and each was hybridized to total bovine RNA. Only
the antisense transcript probe yielded protected fragments (Fig.
2B, lane CO-AS), the largest of which was 180 nt.
At least three protected bands (including the 180-nt band) were seen,
consistent with the primer extension data. The major band
(arrow) observed by primer-extension (Fig. 2A)
was used to assign nt +1. These results indicate that the gene contains
heterogeneous start sites. The end of the third exon was determined by
comparison with a poly(A) tail-containing processed pseudogene
(
BCOX7c-1; Ref. 19) since the available bovine cDNAs
were truncated in this region. The gene has two potential poly(A)
addition signals, one matching the consensus AATAAA and another,
AATTAA, located 132 nt and 32 nt upstream of the poly(A) tail,
respectively (Fig. 3).
Analysis for the presence of known regulatory sites revealed two
putative YY1 motifs (30, 31) between 17 and +69, one immediately
flanking the transcriptional start site (referred to as the
YY1-pstream site) and the other in the
5
-untranslated region, 55 bp downstream of the transcriptional start
site (YY1-ownstream site) (Figs. 3 and 5). The
two sites match the reported consensus (Fig. 5) (32, 33). The presence
of YY1 motifs is especially interesting since a YY1 site has also been
found in COX5B, where it acts as an initiator (10). Another
notable motif present is for NRF-2 ((A/C)GGAA): two sites upstream of
the translational start site (at +69 to +65 and
158 to
154; Fig. 3)
and four sites in the first intron, including a pair of tandem motifs
spaced 4 bp apart. Six more motifs can be found if the above consensus is extended to include a GGAT core: two in the upstream region (at
111 to
115 and
60 to
64), one at the start site (+6 to +2), and
three in the first intron. There are no canonical TATA or CCAAT boxes
or binding sites for NRF-1. There are also no GC-boxes (Sp1 binding
sites) with the core sequence GGGCGG (34), but several that show
partial homology are present. A perfect match to the Sp1 consensus
((G/T)(G/A)GGC(G/T)(G/A)(G/A)(G/T); Ref. 35) is present between +579
and +587 in the first intron and two more, with a single mismatch
(8/9), are located between +73 and +65 in the 5
-untranslated region
and +691 and +683 in the first intron.
Three CAT constructs were
made in pGKOCAT vector with 1.8 kb, 1.5 kb, and 93 bp of flanking DNA
(Fig. 4). These were transfected into HeLa and
C2C12 cells, and their normalized CAT activity
was compared with the largest CAT construct, EcoRI-7c CAT.
In HeLa cells, significant activity (117 ± 26%) is retained with
the smallest CAT construct used: SacII-7cCAT, which contains
only 93 bp of 5-flanking DNA and 74 bp of the 5
-untranslated region
(Fig. 5). Hence, the putative basal promoter must reside
within the 167-bp fragment defined by SacII and
HpaII ends. This fragment contains two putative sites for
YY1 and at least a single site for NRF-2. Similarly, in
C2C12 cells, the SacII-7cCAT
construct is as active as the largest construct both in myotubes
(113 ± 8%) and myoblasts (138 ± 17%) (Fig. 4).
Binding of Transcription Factors to the Core Promoter
We used
EMSA to examine the ability of YY1 to bind to the basal promoter both
with HeLa extracts (data not shown) and purified YY1 (Fig.
6). Increasing amounts of YY1 (0.1-600 ng) produced a
distinct shift (Complex A) at 10 ng that progressively increased in
intensity at 100 and 600 ng. A second shift (Complex B) of slower
mobility was observed at 600 ng, indicating the probable presence of
two YY1 binding sites in the basal promoter fragment, consistent with
the YY1-U and -D sites identified within this fragment. We speculate that the faster migrating band represents the
DNA fragment with one YY1 site bound and the slower one has both sites
bound. To confirm binding of YY1 to each of these sites, we annealed
two pairs of complementary oligonucleotides for EMSA, each harboring
one of these sites. The duplex oligonucleotide ds7c-6 contains the
YY1-D site, and ds7c-3.5 contains the YY1-U site
(Fig. 5). YY1 binds specifically to YY1-D (Fig.
7A), shown by competition with 100- and
1000-fold molar excesses of the specific (ds7c-6, lanes 3 and 4) but not nonspecific (lanes 5 and
6) DNA. Similarly, YY1 was able to bind specifically with
YY1-U (ds7c-3.5) (Fig. 7B). These results confirm
that the basal promoter fragment (93 to +74) has two functional
binding sites for YY1 and that these are the U and
D sites.
YY1-D overlaps an NRF-2 motif (Fig. 5). We examined whether NRF-2 could bind specifically to this complex regulatory site and found that it does (Fig. 7C), since the specific DNA (ds7c-6, lanes 3 and 4) but not a nonspecific DNA (ds7c-3, lanes 5 and 6) can effectively compete for this binding.
Mutational Analysis of YY1 SitesMutations were introduced at
YY1-U, -D, and at both sites (UD) in
the 167-bp basal promoter fragment. The effects of these mutations on
CAT activity were assessed in both undifferentiated and differentiated
C2C12 cells and in HeLa cells (Fig.
8). The mutations have similar effects in all three cell
types. YY1-U appears to be the relatively more important
site, since a mutation decreases activity by 68% in HeLa cells and
58% in myotubes, whereas a mutation in YY1-D decreases
activity by 46% and 39%, respectively. However, when both sites are
mutated, the CAT activity drops further (by a total of 79% and 71%,
respectively). Myoblasts show a similar pattern, but at more modest
levels. These decreases in promoter activity are significant in the
three cell types tested, as determined by a standardized t
test (36).
Transcriptional Analysis of the YY1 Mutations in Transfected Cells
The major transcriptional start site determined for the
wild-type transfected promoter (93 to + 74) in cultured cells is consistent with that obtained from mRNA isolated from tissue (data not shown). This suggests that the transfected CAT gene is driven by
the COX7C basal promoter and not by cryptic sequences. In
addition to the major start site, as already noted, a cluster of
heterogeneous transcripts beginning immediately downstream of this site
is seen. To examine if these initiation sites are affected by the two
YY1 mutations, we transfected HeLa cells with the WT and mutant
YY1-U, -D, and -UD basal promoter
constructs. Total RNAs from these cells were subjected to ribonuclease
protection assays with probes that would detect the major transcript
arising at the +1 site as well as the heterogeneous cluster of minor
transcripts initiating downstream. In Fig.
9A, the major start site (+1) (larger
arrow) and initiation sites for the three minor transcripts
(smaller arrows) in the WT-transfected promoter are shown.
The major transcript constitutes 47% of these transcripts by
densitometric analysis. This pattern is retained in the D
mutant, although the overall amount of transcripts is reduced.
Significantly, the major transcript in WT cells is reduced in both the
U and UD mutants (16 and 20%, respectively); one
of the downstream transcripts now constitutes the largest fraction
(32-33%, respectively). Overall, there is a net decrease in total
transcript levels in the U, D, and UD
mutants. Some of the decrease in the D lane is due to the
lesser amount of total RNA used (Fig. 9A,
legend). These observations hold true for both myoblasts and
myotubes (data not shown). Thus, the binding of YY1 to the U
site appears to correlate with initiating transcription from the +1
site.
The minimal promoter of COX7C can be localized to a
sequence that contains 93 bp of 5-flanking region and 74 bp of the
5
-untranslated region. This 167-bp region contains two sites for YY1
and at least one site for NRF-2. All three sites show binding to their
cognate factors (Fig. 10). In addition to
COX7C and COX5B (10), the nuclear encoded
-subunit of the mitochondrial F0F1 ATP
synthase is also regulated by YY1 (37). Thus, three subunits of the
respiratory chain are regulated by this factor, a multifunctional
zinc-finger protein, variously referred to as NF-E1 (31),
(38),
UCRBP (39), CF-1 (40), F-ACT 1 (41), and NMP-1 (42), that can activate,
repress, or initiate transcription. The presence of two or more binding
sites for YY1 and their location 3
to the transcriptional start site
have been frequently observed (32, 42-45).
COX7C expression appears to be more critically dependent on the binding of YY1 to the U site than it is to the D site. Mutating both sites together further reduces CAT activity, suggesting that the binding of YY1 to both sites is required for maximal promoter activity; furthermore, consideration of the activity associated with each mutant site compared with the double mutation suggests that the sites act independently. The titration of the basal promoter fragment with YY1 (Fig. 6) shows few molecules with two bound sites at lower YY1 concentrations, suggesting that one of the sites has a tighter association constant than the other; we presume it is the U site, based on the argument that bound YY1 must be present there to give the WT initiation pattern (Fig. 9A). The mutational data also suggests that YY1 binds to the U and D sites as an activator. The observation that mutating the YY1 sites affects promoter activity similarly in the three cell types tested is not surprising, since COX7C is ubiquitously expressed in all tissues, is not known to be differentially regulated, and fits with the general observation that ubiquitously expressed genes are activated by YY1 whereas highly regulated genes are repressed by it (32).
In transfected cells, transcription initiates at a cluster that includes the major start site at +1, consistent with that seen in tissues. Binding of YY1 to the U site appears to control the efficiency of initiation from the major start site, since a mutation at YY1-U decreases dramatically the synthesis of this transcript. A YY1-D mutation appears to have little or no effect on the relative proportion of WT transcripts but decreases their net amount. It is less clear how the interaction of YY1 to the D site affects promoter activity (but see below). We also note that the YY1 double mutation does not completely abolish CAT promoter activity. At least 20-45% of the CAT activity is still retained in these mutants, suggesting that the NRF-2 motif in the basal promoter could also contribute to promoter activity.
The close proximity of YY1-U to the transcriptional start site indicates that this binding site could function as an initiator, as seen for COX5B. YY1-U may therefore be involved in the formation or stability of the preinitiation complex, perhaps by interaction with the basal transcription machinery. The ability of YY1 to recruit Pol II to the initiation site by interaction with TFIIB, or TBP, has been demonstrated (46). Consistent with this view, we see that mutating the U site nearly abolishes initiation at the major start site, which accounts for nearly half of the transcripts arising from the WT promoter.
The effect of mutating the D site is less clear.
YY1-D overlaps an NRF-2 site. The core motif for YY1 (CCAT,
top strand) and NRF-2 (GGAA, bottom strand) are spaced only 3 nt apart.
However, the four zinc fingers of YY1 are presumed to interact with 12 nt of the bottom strand, with the core motif positioned at the center
(33). This indicates that the binding of one factor would sterically
hinder the binding of the other. As a repressor, YY1 is known to
overlap the DNA binding motifs of a wide range of activator proteins by
precluding their binding. For instance, overlapping binding sites have
been observed for YY1 and NF-B in the serum amyloid A1 gene promoter
(47), YY1 and serum response factor in the skeletal
-actin gene promoter (41), YY1 and MGF in the
-casein
promoter (48) and YY1 and GATA-1 in the
-globin gene promoter (49).
In COX7C, the significance of the YY1/NRF-2 overlap is not
clear, especially since NRF-2 is also a general activator of COX gene
expression (8). NRF-2, a member of the ets family of
proteins that regulate genes involved in development, growth control,
and cell transformation, has been found in the promoters of all
ubiquitously expressed COX nuclear genes so far characterized:
COX4 (7, 11, 12), COX5B (10), COX7AL
(9), and COX7C. It is conceivable that the binding of these
factors to the overlapping YY1/NRF-2 motif in COX7C is
dictated by the steady-state levels of YY1 and NRF-2 in different
tissues. YY1 levels are known to be decreased during the onset of the
myogenic program (41). Alternatively, YY1 binding at the D
site may serve to alter the topology of the promoter by DNA bending
(44), which would facilitate protein-protein interactions or enhance
protein binding to DNA. In the c-fos serum response element,
where the serum response factor and YY1 binding sites overlap, it has
been shown that both proteins can co-occupy this element, apparently facilitated by a YY1-induced bending of the serum response element DNA
to enhance binding of serum response factor (50). This possibility can
be extended to the YY1/NRF-2 motif of the COX7C promoter, and may explain why a mutation at the YY1-D site
decreases CAT activity. If a YY1-induced bend facilitates enhanced
binding of NRF-2 at the D site, it follows that a mutation
would decrease the kinetics of NRF-2 binding, thus explaining the
modest decrease in CAT activity.
COX7C (YY1/NRF-2), COX5B (YY1/NRF-1/NRF-2), COX7AL (NRF-1/NRF-2), and COX4 (NRF-2) are four ubiquitously expressed genes whose promoters have been analyzed in some detail. At least three factors appear to contribute to their expression. It is not clear why some of these are NRF-1-dependent and others YY1-dependent. NRF-2 appears to be a common factor in these genes and its interaction, if any, with YY1 and NRF-1 is not known. It is conceivable that some of these factors may sense the redox state of the cell and coordinate their interactions with other factors. A clearer picture will emerge only when the promoters of additional COX genes are characterized.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U58655[GenBank].
We thank Dr. Roderick A. Capaldi, University of Oregon, for providing adult bovine heart tissues; Dr. Nancy Bachman, University of Michigan, for the EMBL-3 Sp6/T7 bovine genomic library; Dr. Michael Atchison, University of Pennsylvania, for purified YY1; and Dr. Richard C. Scarpulla, Northwestern University, for purified NRF-2. We thank Drs. Scarpulla and Margaret I. Lomax, University of Michigan, for critical comments on the manuscript, and Dr. Gerard Tromp, Wayne State University, for assistance with statistical analysis.