(Received for publication, June 22, 1995; and in revised form, August 23, 1995)
From the
In order to investigate the mechanism(s) governing the striated muscle-specific expression of cytochrome c oxidase VIaH we have characterized the murine gene and analyzed its transcriptional regulatory elements in skeletal myogenic cell lines. The gene is single copy, spans 689 base pairs (bp), and is comprised of three exons. The 5`-ends of transcripts from the gene are heterogeneous, but the most abundant transcript includes a 5`-untranslated region of 30 nucleotides. When fused to the luciferase reporter gene, the 3.5-kilobase 5`-flanking region of the gene directed the expression of the heterologous protein selectively in differentiated Sol8 cells and transgenic mice, recapitulating the pattern of expression of the endogenous gene. Deletion analysis identified a 300-bp fragment sufficient to direct the myotube-specific expression of luciferase in Sol8 cells. The region lacks an apparent TATA element, and sequence motifs predicted to bind NRF-1, NRF-2, ox-box, or PPAR factors known to regulate other nuclear genes encoding mitochondrial proteins are not evident. Mutational analysis, however, identified two cis-elements necessary for the high level expression of the reporter protein: a MEF2 consensus element at -90 to -81 bp and an E-box element at -147 to -142 bp. Additional E-box motifs at closely located positions were mutated without loss of transcriptional activity. The dependence of transcriptional activation of cytochrome c oxidase VIaH on cis-elements similar to those found in contractile protein genes suggests that the striated muscle-specific expression is coregulated by mechanisms that control the lineage-specific expression of several contractile and cytosolic proteins.
Cytochrome c oxidase (COX) ()is the terminal
enzyme of the electron transport chain(1) . It catalyzes the
coupled reactions of electron transfer from ferocytochrome c to water (2) and proton translocation across the inner
mitochondrial membrane (eukaryotes) or the cytoplasmic membrane
(prokaryotes)(3, 4, 5) . Energy conserved in
the form of an electrochemical gradient across the membrane provides
the driving force for the ATP synthase to phosphorylate
ADP(6, 7) . Not surprisingly, alterations of the
activity of cytochrome c oxidase can exert significant control
over the flux of aerobic ATP production(8) .
Eukaryotic COX is a multicomponent enzyme consisting of 12 polypeptides in Saccharomyces cerevisiae(9) and 13 polypeptides in mammals(10) . The three largest subunits (I, II, and III) of the eukaryotic enzyme are encoded by mitochondrial genes(11, 12) , and the remainder (IV, Va, Vb, VIa, VIb, VIc, VIIa, VIIb, VIIc, and VIII (nomenclature of Kadenbach et al.(10) ) are encoded by nuclear genes(11, 13) . The isolation of catalytically active cytochrome c oxidase from Paracoccus denitrificans(14, 15, 16, 17) and the finding that it comprises only three subunits that show significant amino acid homology to subunits I, II, and III of the eukaryotic protein (18, 19) have led to the view that these subunits constitute the catalytic core of the eukaryotic enzyme(2, 20, 21) . The functions of the 9 or 10 nuclear encoded subunits in the eukaryotic cytochrome c oxidase are less well understood. Gene disruption studies in S. cerevisiae(22, 23, 24, 25, 26, 27, 28, 29) indicate that most of these are essential components for the assembly or maintenance of a functional protein. However, a null mutation in the gene encoding subunit VIII (homologous to mammalian subunit VIIc) reduces cellular respiration and cytochrome c oxidase activity to 80% of the wild type levels(30) . In addition, a yeast strain with a null mutation in the gene encoding subunit VIa (homologous to mammalian subunit VIa) exhibited altered responsiveness to ATP and potassium phosphate(31, 32) . Thus, subunits VIII and VIa may function to modulate enzyme activity in response to changes in metabolic conditions. It is uncertain whether conclusions about subunit function derived from gene disruption experiments in yeast can be extended to mammals (21, 25) since there are no known corresponding mutations in mammals.
In mammals, there are species differences in the occurrence of tissue-specific subunit isoforms, designated H (for heart) and L (for liver). The H form of subunits VIa, VIIa, and VIII is expressed only in heart and skeletal muscle tissue, whereas the L form is expressed in most tissues(33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) . Low levels of VIIaH and VIIIH are also present in smooth muscle and brown fat tissue(41, 50, 51) . The observations that COX isolated from heart and liver have different kinetic properties and respond differently to adenine nucleotides have led to the proposal that tissue-specific subunits function to regulate holoenzyme activity to meet tissue energy demands(52) . For example, intraliposomal ADP stimulated the activity of reconstituted cytochrome c oxidase from bovine heart but not that from liver(53, 54) . In addition, preincubation of the heart COX with a monoclonal antibody against subunit VIaH abolished the stimulatory effect of ADP (54) , indicating that COXVIaH may be required for mediating the tissue-specific allosteric effect of ADP on the heart enzyme.
Although the tissue and developmental specific expression of COXVIaH has been described, and the genes encoding this subunit isoform in bovine and rat have been isolated(55, 56) , no report has been published analyzing the regulatory mechanism(s) of its expression. While studies of the mechanisms of the lineage-specific expression of cytosolic and contractile proteins in striated muscle have progressed rapidly in recent years(57) , our understanding of the lineage-dependent expression of mitochondrial proteins is still at a rudimentary stage. Elucidation of the mechanisms of muscle-specific expression of COXVIaH, a regulatory subunit of a key mitochondrial enzyme, will expand the current understanding of the regulatory mechanism(s) governing the differentiation of specialized oxidative myotubes. In the present study, we have cloned and characterized the gene encoding murine COXVIaH and analyzed its transcriptional regulatory elements in murine skeletal muscle cell lines. The results indicate that the skeletal myotube-specific expression of COXVIaH is, in large part, regulated by the same myogenic factors that control the muscle-specific expression of contractile proteins. No regulatory elements essential for the transcription of genes encoding ubiquitously expressed mitochondrial proteins can be identified by sequence comparisons in the functional COXVIaH promoter region. This evidence and the developmental switch that occurs soon after birth suggest that there must be concerted mechanisms employed by specialized oxidative myotubes to coordinate COXVIaH expression with other mitochondrial proteins.
Figure 1: Organization of the gene encoding murine COXVIaH. A, partial restriction map of the 11-kb KpnI/SalI fragment encoding COXVIaH. E1, E2, and E3 denote the positions of the three exons. B, schematic structure of the gene encoding COXVIaH. Presentation is based on bidirectional sequence analysis of a DNA fragment spanning nucleotides -699 to 1212 relative to the transcriptional start site. Open boxes and solid lines represent exons and introns, respectively. The schematic is proportional to actual length in base pairs. The direction of transcription and the start site are represented by an arrow. The positions of the polyadenylation site and the translational start site are indicated by numbers in parentheses.
Genomic fragments covering nucleotides -699 to +1212 of the COXVIaH gene were bidirectionally sequenced. As illustrated in Fig. 1B and Fig. 2, the gene spans 689 bp and comprises three exons separated by two small introns. All of the exon-intron boundaries conform to consensus splice junction rules. The immediate 5`-region of the gene lacks both TATA and CAAT boxes.
Figure 2: Nucleotide sequence of the gene encoding COXVIaH. The nucleotide sequence of the entire gene as well as 682 bp of 5`-flanking region are shown. Capital letters represent exon sequences. Lowercase letters denote intron sequences. The translational start and stop codons are shown in boldface type. The deduced amino acid sequence of COXVIaH is shown below the nucleotide sequence.
Figure 3: Mapping the 5`-end of COXVIaH transcripts with RNase protection assay and 5`-RACE. A, RNase protection analysis with an antisense riboprobe spanning from -728 to +61 (relative to the translational start site). The riboprobe was hybridized to 30 µg of total RNA from the indicated sources, digested, and analyzed as described under ``Experimental Procedures.'' The positions and sizes (bp) of size standards are marked on the left. B, rapid amplification of 5`-cDNA ends (5`-RACE). Poly(A) RNA from mouse heart was reverse-transcribed as described under ``Experimental Procedures.'' After gel purification, the PCR products were cloned and sequenced. The 5`-sequence of the COXVIaH gene is shown on the first line. Capital letters represent the sequence of the first exon as derived from the RNase protection assay. Dashes indicate confirmed sequences from the PCR products. The translational ATG is indicated.
Figure 4:
Northern blot analysis of the steady state
levels of COXVIaH transcript in mouse cell lines. Total RNA was
isolated from various mouse cell lines at different growth stages as
indicated, size-fractionated on a formaldehyde-agarose gel, and
transferred to a nylon membrane as described under ``Experimental
Procedures.'' The blot was hybridized with a P-labeled COXVIaH cDNA. The positions and sizes (kb) of
RNA size standards are indicated on the left. In lanes
K, L, and M, 12.2, 16.4, and 13.4 µg of
total RNA were loaded, respectively. All of the other lanes contained 25 µg of total RNA. The blot was stripped and
reprobed with a labeled cDNA for the glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) gene (bottom of figure) to control for RNA
loading.
Figure 5: Deletion analysis of the expression of COXVIaH5`-luciferase gene constructs in mouse cell lines. Left, schematic illustration of the promoter-reporter fusion constructs. Various DNA fragments from the COXVIaH 5`-flanking region were fused directionally to the cDNA of firefly luciferase (open boxes). Numbers indicate the positions of the start and end points of the fragment relative to the transcriptional start site. Right, luciferase activity in Sol8 and 10T1/2 cells transfected with the promoter-reporter fusion constructs. Transfection, preparation of cell extracts, and enzymatic assay were performed as described under ``Experimental Procedures.'' Luciferase activity is expressed as chemiluminescence counts over that of background after correction for transfection efficiency. Solid and hatched bars represent data from Sol8 myoblasts and myotubes, respectively. Open and dotted bars represent data from 10T1/2 fibroblasts harvested at time windows parallel to that of Sol8 myoblasts and myotubes, respectively. Data are the means ± S.E. from three to seven separate experiments.
Figure 7: Expression of COXVIaH-luciferase in transgenic mice. Extracts were prepared from specified organs from a transgene negative mouse littermate (solid bar) and two transgene positive mice (from two founders, hatched bar and dotted bar) and assayed for luciferase activity as described under ``Experimental Procedures.'' Data are expressed as chemiluminescence counts per mg of protein.
Deletion of COXVIaH 5`-sequence from nucleotide -3500 to -283 slightly increased luciferase activity in Sol8 myotubes to 1250-fold over background but significantly reduced luciferase activity in Sol8 myoblasts and 10T1/2 fibroblasts to and of that found in Sol8 myotubes, respectively. This suggests there may be nonspecific transcriptional enhancer elements within this region. Further 5`-deletion from nucleotide -283 to -144 disrupted a consensus E-box sequence (CAGCTG) at -147 to -142 and resulted in a 9-12-fold decrease of luciferase activity in Sol8 myotubes as well as in Sol8 myoblasts and 10T1/2 fibroblasts, suggesting the existence of a positive element in this region, which may be regulated by a factor(s) common to myoblasts, myotubes, and fibroblasts. A 3`-deletion from nucleotide +17 to -23 of a fragment spanning nucleotides -698 to +17 of the COXVIaH gene resulted in a 3-fold decrease of luciferase activity in Sol8 myotubes, Sol8 myoblasts, and 10T1/2 fibroblasts. Further 3`-deletion of this fragment from nucleotide -23 to -142 caused a further 5-fold decrease of luciferase activity in Sol8 myotubes but had no effect on luciferase activity in Sol8 myoblasts and 10T1/2 fibroblasts, suggesting the existence of a positive element in this region, which is regulated by myotube specific factors. Together, the deletion analysis located a 300-bp fragment spanning nucleotide -283 to +17 of the COXVIaH gene that is sufficient to direct the high level, myotube-specific expression of luciferase in Sol8 cells.
Figure 6: Mutational analysis of COXVIaH promoter. A, nucleotide sequence of the 300-bp fragment of the COXVIaH gene sufficient to direct the myocyte-specific expression of luciferase in Sol8 cells. Nucleotides are numbered relative to the transcriptional start site. Potential regulatory elements are indicated by boldface letters. The name of each element is given on top of the sequence. E-1 to E-3 denotes E boxex 1-3. The corresponding mutations introduced at these sites are illustrated at the bottom. B, luciferase activity in Sol8 and 10T1/2 cells transfected with the promoter-reporter fusion construct with various mutations in the promoter region. Transfection, enzymatic assays, and data presentation are the same as in the Fig. 4legend. Solid and hatched bars represent data from Sol8 myoblasts and myotubes, respectively. Open bars represent data from 10T1/2 fibroblasts harvested at a time window parallel to that of Sol8 myoblasts. Inset, a blow-up view of the result from 10T1/2 cells to illustrate the effect of the E-1mut in the fibroblast line. Data are the means ± S.E. from three to seven separate experiments.
Remarkable insights into the regulatory mechanisms of skeletal muscle differentiation have been gained through the isolation of the basic helix-loop-helix (bHLH) myogenic determination factors and the MEF2 family of muscle-specific transcriptional factors. The MEF2 proteins are encoded by four separate genes, each of which undergoes alternative splicing. They bind to an A/T-rich motif found in many muscle genes and activate their transcription(65, 69, 70, 71, 72, 73) . Most studies on the mechanisms of tissue-specific transcription in striated muscle have focused on proteins associated with the contractile apparatus, since these are the obvious unique components in the muscle lineage. More recently, it has been documented that muscle-specific isoforms are present for several mitochondrial proteins coupled to oxidative phosphorylation(33, 74) , underscoring that in addition to contractile proteins, striated muscle must also acquire unique proteins associated with mitochondria for its special need in aerobic ATP production. This is particularly true for oxidative fibers. The 5`-flanking regions of genes encoding several of the muscle-specific isoforms of mitochondrial proteins contain E-box as well as MEF2 elements(55, 56, 74) ; however, the functional significance of these elements in the transcriptional regulation of these genes remains largely undefined.
The present study was undertaken to analyze the structure and transcriptional regulation of the gene encoding COXVIaH, a muscle-specific subunit isoform of cytochrome c oxidase. Characterization of the murine gene encoding COXVIaH revealed an exon-intron structure similar to that found in the genes from bovine and rat. The 5`-ends of transcripts from the murine gene are heterogeneous, with the majority of transcripts having a 30-bp 5`-untranslated region. This contrasts with the 181- and 203-bp 5`-untranslated regions reported for transcripts from the bovine and rat genes, respectively(55, 56) . Whether this reflects true species differences, or whether the discrepancy is due to different interpretations of primer extension experiments is unclear. In contrast to bovine, but similar to rat, the 5`-region of the murine gene lacks both TATA and CAAT boxes. Thus, the proximal region of the murine COXVIaH promoter has structural features resembling those of housekeeping genes (75) but not those of regulated and tissue-specific genes. The TATA-less feature and the ability of the COXVIaH promoter to direct skeletal muscle specific transcription in transgenic mice (Fig. 7) thus represents an unusual case of muscle-specific transcriptional regulation in the absence of a consensus TATA element.
Using transient transfection, we have located a 300-bp 5`-flanking region of the COXVIaH gene, which is sufficient to direct high level, skeletal myotube-specific expression of a heterologous protein in Sol8 cells in a pattern similar to the expression of the endogenous gene. Sequence analysis revealed three potential E-boxes and an MEF2 element clustered at nucleotide -147 to -60 of this region. Mutation in the MEF2 element diminished luciferase activity in differentiated myotubes to levels found in myoblasts, and the effect was restricted to myotubes. Thus, interaction of this site with a myotube-specific factor is essential for myotube-specific transcriptional activation. There is compelling evidence to suggest this factor is MEF2, since MEF2 binding is rapidly induced following downshift of myoblasts to form myotubes, and presumptive MEF2 sites undergo DNase footprinting only upon conversion to myotubes(72) . Finally, our data show that conversion of this site to a site incapable of binding MEF2 completely abolishes the myotube-specific transactivation of the luciferase reporter cassettes (but not myoblasts). It follows that direct interaction with the skeletal muscle-specific bHLH proteins alone can not account for the myotube specificity of transcription from the COXVIaH promoter, since in the absence of the MEF2 site, the three E-boxes are unable to confer myotube-specific transcription. Mutations in the proximal two E-boxes at nucleotides -75 and -60 of the COXVIaH promoter did not promote large decreases of luciferase activity in myotubes, indicating that as individual elements they are likely dispensable. Mutation in the distal E-box at nucleotide -147 resulted in a dramatic decrease in luciferase activity. This occurred in the presence of the two downstream E-boxes, indicating that either the location or additional sequences outside the canonical E-box motif distinguished the distal E-box from the two upstream ones. The parallel decrease in luciferase activity in 10T1/2 fibroblasts caused by the same mutation, however, argues against muscle-specific bHLH factors interacting with this site since they are known to be absent in 10T1/2 cells. Although not strictly excluded, it is unlikely that the parallel effects caused by this mutation in myoblasts, myotubes, and fibroblasts are due to disruption of interactions of this site with distinct factors in different cell types. A plausible explanation is that this E-box is interacting with ubiquitously present factors to enhance transcription from the COXVIaH promoter.
Taken together, the present study establishes the functional significance of the MEF2 and distal E-box sites in the skeletal muscle-specific transcription of COXVIaH. The dependence on common cis-elements in the transcriptional activation of both contractile proteins (69, 70, 71) and COXVIaH suggests that the striated muscle-specific expression of these two classes of molecules is coregulated by common mechanisms. Additionally, since COXVIaH is required for the tissue-specific allosteric effect of ADP on bovine heart cytochrome c oxidase(54) , such coregulation may be of selective advantage for the oxidative muscle lineage in signaling the energy demand of the contractile apparatus to the mitochondria.
As an isoform of a cytochrome c oxidase subunit, the
expression of COXVIaH must be regulated in concert with other
mitochondrial proteins. Coordinated up-regulation of the expression of
mitochondrially encoded and nuclear encoded mitochondrial proteins has
been documented in rabbit skeletal muscle after sustained contraction
via nerve stimulation (76, 77) and in patients
suffering from mitochondrial myopathy(78, 79) . Recent in vitro experiments identified several cis-elements
and trans-factors essential for the transcription of nuclear
encoded mitochondrial proteins. Two sequence motifs with the consensus
of YGCGCAYGCGCR and MGGAAG have been identified in several ubiquitously
expressed nuclear encoded mitochondrial proteins. Their cognate binding
factors, nuclear respiratory factors 1 and 2, have been isolated. The
functional significance of these cis-elements and trans-factors in the transcription of several nuclear encoded
mitochondrial proteins including the mitochondrial transcription factor
A has been demonstrated in HeLa
cells(80, 81, 82) . Promoter analysis in
skeletal muscle myogenic cell lines, on the other hand, has identified
the oxbox and rebox cis-elements essential for the
transcription of the genes encoding the muscle-specific adenine
nucleotide translocase and the -subunit of ATP
synthase(83, 84, 85) . While these studies
suggest that transcriptional regulation is an important mechanism in
coordinating mitochondrial biogenesis, the physiological significance
of these cis-elements and trans-factors in
coordinated regulation of the expression of mitochondrial proteins has
not been established. A search of the 300-bp COXVIaH promoter for NRF-1
and NRF-2 as well as the oxbox and rebox sequence motifs revealed no
such sequences, suggesting that the coordination of the expression of
COXVIaH with other mitochondrial proteins may be regulated by different
but concerted mechanisms. The identification of promoter elements
sufficient to direct expression of a reporter gene in striated muscle
in transgenic animals and the availability of animal models to induce
coordinated up-regulation of the expression of mitochondrial proteins,
nonetheless, will facilitate further experimentation aimed at the
elucidation of the mechanisms coordinating the expression of COXVIaH
during mitochondrial biogenesis.
Additional studies will be required to ascertain if these same elements are required for transcriptional regulation in cardiac myocytes. The GATA site present in the proximal region is an obvious candidate for this regulation, but the MEF2 and E-box motifs may also be indispensable. Finally, although not specifically addressed by this study, the question as to the physiological role of this subunit in the regulation of oxidative phosphorylation in striated muscle remains to be defined in mammals. Targeted mutagenesis of this gene in murine embryonic stem cells and creation of mutant mice that lack this subunit may provide insight into the functional significance of COXVIaH in oxidative myocytes.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U34801[GenBank].