(Received for publication, July 21, 1995; and in revised form, November 15, 1995)
From the
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.
Cytochrome c oxidase (COX), ()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 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 -subunit, cytochrome c
, 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.
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%.
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.
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 10
cpm) was treated
with dimethyl sulfate for 5 min at room temperature as
described(40) . Binding reactions contained 5
10
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.
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.
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.
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.
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.
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-2 and
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
and
; 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) .
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
-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 and
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-2 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 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.