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(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) ( 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
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 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].
Volume 270,
Number 44,
Issue of November 3, 1995 pp. 26433-26440
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)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) .
Isolation and Characterization of the Murine COXVIaH
Gene
A partial cDNA of murine COXVIaH spanning nucleotides
78-306 (provided by Dr. Bill Parsons) was used to screen a 129/Sv
mouse genomic library (Stratagene); four independent clones were
isolated. An 11-kb KpnI/SalI and a 9-kb XbaI
fragment encompassing the entire cDNA sequence were subcloned into
pSP72 (Stratagene) and characterized by restriction digestion and
Southern hybridization analyses using standard procedures (58) . Bidirectional sequencing of genomic fragments covering
nucleotides -698 to 1212 relative to the transcriptional start
site of the gene was performed with the dideoxynucleotide chain
termination method using a commercial kit (Sequenase v2, Stratagene).
Southern Blot Hybridization
Genomic DNA was
isolated from J1 embryonic stem cells, digested with various
restriction enzymes, size-fractionated on a 0.8% agarose gel, and
transferred to nylon membranes (GeneScreen, DuPont NEN)(58) .
The membrane was probed with P-labeled DNA synthesized
using the Klenow fragment of DNA polymerase I. Hybridization was
performed in a 50% formamide containing solution at 42 °C
overnight. After sequential washes in 2
SSC, 1% SDS at room
temperature for 30 min and 0.2 SSC, 1% SDS at 68 °C for 1
h, the membranes were subjected to autoradiography.Northern Blot Hybridization, Ribonuclease Protection
Assay, and Rapid Amplification of 5` cDNA Ends
(5`-RACE)
Total RNA was isolated from various mouse
organs or cultured cells using the methods of Glisin et al.(59) and Ullrich et al.(60) . For
Northern blot analysis, the RNA was size-fractionated on a 1.1%
formaldehyde agarose gel, transferred to a nylon membrane, and probed
with P-labeled DNA synthesized using the partial cDNA of
COXVIaH as template. Conditions for hybridization and posthybridization
washes were the same as those described for Southern analysis. The
ribonuclease protection assay was performed essentially as
described(58) . A 789-bp HindIII/NcoI genomic
fragment spanning nucleotides -728 to +61 relative to the
translational starting site was subcloned and used as the template to
direct the synthesis of a
P-labeled antisense riboprobe.
The probe was hybridized to 30 µg of total RNA isolated from
various mouse organs in a solution containing 80% formamide at 50
°C overnight. Following RNase H and T1 digestion, protected
fragments were fractionated on a 6% denaturing polyacrylamide gel, and
protected bands were visualized following autoradiography. 5`-RACE was
performed by the method described by Frohman(61) . Briefly,
poly(A)
RNA from mouse heart was reverse-transcribed
using an antisense oligonucleotide homologous to nucleotides +184
to +203 relative to the translational start site as the initial
primer. Homopolymeric dC was added to the 3`-end of the first strand
cDNA using terminal deoxynucleotidyl transferase in the presence of
dCTP. The dC-tailed cDNA of COXVIaH was amplified with PCR using a
poly-dG containing oligonucleotide as the 5`-primer and an antisense
oligonucleotide homologous to base pair +80 to +103 of
COXVIaH cDNA as the 3`-primer. Following gel purification, the PCR
products were subcloned into Bluescript II SK (Stratagene) and
sequenced.
Plasmid Constructions
The promoterless plasmid,
pBST-Luc (kindly provided by Dr. R. Bassel-Duby) served as the vector
backbone for the luciferase expression constructs. In this study, the
unique HindIII site of pBST-Luc was blunted and converted to
an XhoI site by linker addition. DNA fragments of the
5`-region of COXVIaH gene of varying lengths were generated via
convenient restriction enzyme sites and subcloning into pBS-SK
(Stratagene): pBSC6aHC was constructed by inserting a HindIII/blunted-MaeI fragment containing nucleotides
-698 to +17 of the COXVIaH gene into the HindIII/HincII sites; a HindIII fragment
containing nucleotide -1290 to -699 of the gene was
inserted into the HindIII site of pBSC6aHC to generate
pBSC6aHB; pBSC6aHA was constructed by subcloning an XbaI/HindIII fragment containing nucleotide
-3500 to -1291 of the gene into the XbaI/HindIII sites of pBSC6aHC and subsequent
insertion of the HindIII fragment containing nucleotide
-1290 to -699 of the gene into the HindIII site;
pBSC6aHD was generated by removing a HindIII/BglII
fragment spanning nucleotide -698 to -284 of the gene from
pBSC6aHC, blunting the ends and religating the plasmid; pBSC6aHF was
constructed by insertion of a HindIII/TaqI fragment
containing nucleotides -698 to -23 of the gene between the HindIII/AccI sites of pBS SK; a BamHI/PvuII fragment from pBSC6aHC containing
nucleotide -698 to -142 of the gene was subcloned into the BamHI/PvuII sites of pSP72 to generate pSP72C6aHG.
C6aHA, -B, -C, -D, -F, and -G fragments were then directionally cloned
into the BamHI/XhoI sites of pBST-luc between the
upstream SV40 poly(A) capture cassette (positioned to reduce
nonspecific background luciferase activity) and the cDNA of luciferase.
Construction of pBSC6aHE was accomplished by subcloning a PvuII/XhoI fragment from pBSC6aHC (-144 to
+17 of the gene) into the SmaI/XhoI sites of
pBST-Luc. Mutations of potential transcriptional regulatory elements in
C6aHD were generated by oligonucleotide directed mutagenesis using
sequential PCR as described(62) . Two oligonucleotides, with
the desired mutation in an overlapping region antisense to each other,
were paired with either T3 or T7 primers to amplify two fragments of
C6aHD overlapping each other at the mutational region using pBSC6aHD as
the template. The entire C6aHD bearing the desired mutation was then
generated by annealing the two overlapping fragments together, followed
by PCR amplification using the two universal primers. After digestion
with BamHI and XhoI, the mutated C6aHD region was
subcloned into pBST-luc as described. The identity of each
promoter-reporter construct was confirmed by bidirectional DNA sequence
analysis.Cell Culture and Transfection
Myoblasts (Sol8 and
C2C12) and fibroblasts (10T1/2 and NIH3T3) were grown in growth medium
(Dulbecco's modified Eagle's medium with high glucose
supplemented with 20% (Sol8) or 10% (all others) fetal bovine serum,
200 units/ml penicillin and 20 µg/ml streptomycin). Myotubes were
derived from myoblasts by growing subconfluent cultures (80%
confluent) in differentiation medium (same as growth medium except 2%
horse serum was substituted for the 20% fetal bovine serum).
Transfection was performed by trypsinization and electroporation of
approximately 10
cells in 0.5 ml of growth medium
containing 80 µg of sonicated salmon sperm DNA, 25 µg of test
DNA, and 25 µg of cytomegalovirus-
-galactosidase expression
vector (as an internal control of transfection efficiency).
Electroporation was conducted with a Bio-Rad Gene Pulser using field
strengths of 750 (Sol8) or 700 (10T1/2) volts/cm and a capacitance of
960 microfarads. Cells were immediately plated on 6-cm (myotubes) or
10-cm (myoblasts) gelatin-coated plates in growth medium containing 5
mM butyric acid. The medium was replaced with butyrate-free
medium 12-15 h later. Between 30 and 40 h after transfection,
Sol8 myoblast extracts were harvested while Sol8 myotubes were refed
with differentiation medium. Sol8 myotube extracts were then harvested
92-110 h after transfection. One experimental group of 10T1/2
cells was harvested 30-40 h after transfection, and the other was
maintained in growth medium and harvested 92-110 h after
transfection.Enzyme and Protein Assays
Cell extracts were
prepared and assayed for luciferase activity and -galactosidase
activity as described(63) . Luciferase activity was determined
by measuring the amplitude of the peak chemiluminescence with a
Berthold Biolumat LB 9500 C luminometer. Background luciferase activity
was assessed in extracts from parallel cultures transfected with the
promoterless plasmid pBSTLuc. -galactosidase activity was
determined spectrophotometrically after incubation of the extracts with o-nitrophenyl--D-galactopyranoside. Luciferase
activity in cell extracts transfected with various fusion constructs
was expressed as chemiluminescence counts over that of background after
correction for transfection efficiency with the -galactosidase
activity. Protein concentration was measured colorimetrically with the
Bio-Rad reagent (Bio-Rad). All assays were linear with respect to
protein concentration.Transgenic Animal Production
Transgenic mice were
generated by standard methods (64) using the KpnI/Not fragment of the promoter-reporter construct
pBST-LucC6aHA that was free of plasmid sequences. The presence of the
transgene in F1 progeny was determined by Southern analysis of tail DNA
using a 1-kb EcoRI fragment from the luciferase cDNA.
Luciferase activity in various organs from transgene-positive mice was
determined essentially as described (63) with the following
modifications: organs were excised and quickly frozen in liquid
nitrogen and then homogenized in a solution containing 70 mM
KH
PO
(pH 7.8), 2.1 mM MgCl, 0.7 mM dithiothreitol, 55.6 mM
Tris-HCl, 0.25 mg/ml soybean trypsin inhibitor (Sigma), 5 µg/ml
aprotonin (Sigma), and 0.1% Nonidet P-40. Following centrifugation at
12,000 rpm at 4 °C for 10 min, 50 µl of the supernatant was
immediately mixed with 250 µl of assay solution, and activity was
determined by measuring peak chemiluminescence during a 10-s interval
after injection of 100 µl of 5 mM luciferin.
Cloning and Structural Characterization of the COXVIaH
Gene
Four independent phage clones were isolated by
screening an 129/Sv mouse genomic library with a partial cDNA of murine
COXVIaH. Two overlapping fragments encompassing the entire gene of
COXVIaH were subcloned and characterized by restriction digestion and
Southern hybridization analysis. A partial restriction map of the 11-kb KpnI/SalI fragment is shown in Fig. 1A.
The restriction pattern of the genomic clones was then compared with
that of the endogenous gene obtained by Southern blot analysis of
genomic DNA from J1 embryonic stem cells. A DNA probe containing
nucleotides -283 to +91 relative to the transcriptional
start site of the COXVIaH gene hybridized to single restriction
fragments in genomic DNAs digested with a variety of restriction
enzymes, indicating that the gene was single copy and did not represent
a pseudogene (as is common for several nuclear encoded mitochondrial
proteins). The identical restriction digestion patterns between the
cloned gene and the endogenous DNA isolated from tail biopsy samples
verified that the structure of the gene was not altered during the
cloning process (data not shown).
Mapping the 5`-End of COXVIaH Transcripts
The
5`-end of COXVIaH transcripts was determined by RNase protection assay
as well as rapid amplification of 5` cDNA ends (5`-RACE). The results
are shown in Fig. 3. Total RNA from hearts and skeletal muscles
of mice at various developmental ages protected multiple fragments of a
riboprobe antisense to nucleotides -728 to +61 of the
COXVIaH gene. The sizes of these fragments ranged from 72 to 154 bp,
with the dominant fragment clustered at 91 bp (Fig. 3A). In order to exclude the possibility that
there were upstream untranslated exons beyond the range of the
riboprobe used in the RNase protection assay, upstream sequences of
COXVIaH transcripts were determined from 12 independent subclones of
the 5`-region of COXVIaH cDNA obtained from mouse heart poly(A) RNA by
5`-RACE, a procedure for amplification of nucleic acid sequences from
an mRNA template between a defined internal site and unknown sequences
at the 5`-end. Consistent with data from the RNase protection assay,
the 5`-ends of COXVIaH cDNAs are heterogeneous, located at nucleotide
-64 to +5 relative to the translational start site, and the
majority of the cDNAs have their 5`-ends clustered at nucleotide
-30 (Fig. 3B). Taken together, the results
indicate that the 5`-ends of COXVIaH mRNAs are heterogeneous, with the
predominant transcript having a 30-bp 5`-untranslated region. This site
was thus designated +1 with respect to transcript initiation.
Expression of COXVIaH in Murine Cell Lines
The
availability of tissue culture cell lines that undergo differentiation
to fully functional myotubes has allowed investigations into the
mechanisms that govern expression of some lineage-specific muscle
genes. To determine if the tissue-specific expression of COXVIaH in
striated muscle can be modeled in murine myogenic cell lines during
differentiation, steady state levels of COXVIaH transcript were
examined in the skeletal muscle cell lines Sol8 and
CC at the myoblast and myotube stages. For
comparison, two fibroblast lines, 10T1/2 and NIH3T3, were also
examined. Total RNA isolated from these cell lines, before and after
the induction of myogenic differentiation by downshift into serum-free
media, was probed with
P-labeled DNA prepared with a
partial cDNA of COXVIaH. As shown in Fig. 4, COXVIaH mRNA was
not detected in 10T1/2, NIH3T3 fibroblasts, Sol8, or
C
C myoblasts. Abundant COXVIaH mRNA, however,
was present in Sol8 and C
C myotubes 2 days
after the induction of differentiation of confluent cultures of
myoblasts in a serum-depleted medium. No COXVIaH mRNA was detected in
10T1/2 and NIH3T3 fibroblast cultures depleted of serum. Thus, the
expression of COXVIaH in skeletal muscle cell lines was dramatically
up-regulated during the transition from myoblasts to myotubes. The
up-regulation of COXVIaH expression during myotube formation thus
provided a suitable in vitro system to examine the mechanisms
controlling the lineage-dependent expression of COXVIaH in skeletal
muscle.
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.
Regulatory Element Analysis of the COXVIaH
5`-Region
To locate the proximal promoter region of the COXVIaH
gene and identify cis-elements essential for mediating its
skeletal myotube-specific expression, fragments of 5`-flanking
sequences of the gene with various deletions were fused to the cDNA of
firefly luciferase. Promoter activity of these fragments was assessed
by transient transfection of these promoter-reporter constructs into
Sol8 and 10T1/2 cells followed by assays for luciferase activity in
cell extracts before and after myogenic differentiation. The results
are shown in Fig. 5. The region containing nucleotides
-3500 to +17 of the COXVIaH gene directed the expression of
the heterologous protein in Sol8 myotubes about 900-fold over the
background. In comparison, the same construct was 5- and 9-fold less
active in Sol8 myoblasts and 10T1/2 fibroblasts, respectively. The
relatively high transcriptional activity of the 3500-bp region of the
COXVIaH gene in Sol8 myoblasts and 10T1/2 fibroblasts was surprising,
and may be due to nonspecific transcriptional initiation when present
as episomal DNA. This notion is supported by the observation that when
this promoter-reporter construct was microinjected into mouse oocytes
to generate transgenic mice, it was capable of directing the
tissue-specific expression of luciferase into the heart and skeletal
muscle in a pattern virtually identical to the endogenous gene (Fig. 7).
Regulatory Element Analysis of the COXVIaH
Promoter
The nucleotide sequence of the 300-bp COXVIaH promoter
is presented in Fig. 6A. Several enhancer elements are present
in this region that might be involved in its tissue-specific
expression. Three E-box elements (CANNTG), which function as
muscle-specific enhancers in the regulation of the expression of
several muscle-specific proteins through their interaction with the
MyoD family of transcription factors (57) , are present
beginning at nucleotides -147, -75, and -60. A single
MEF2 element, another muscle-specific enhancer sequence(65) ,
is present beginning at nucleotide -90. Finally, a single GATA
element (WGATAR), which may function as an enhancer in the regulation
of the lineage-specific gene expressions in T-cells(66) ,
erythroid cells (67) , and cardiac myocytes(68) , is
present beginning at nucleotide -105. In order to determine the
functional significance of these potential regulatory elements in the
transcriptional regulation of COXVIaH, mutant constructs of the 300-bp
COXVIaH promoter containing nucleotide substitutions in each of the
three E-boxes or the MEF2 element or both (at their respective sites in
the wild type promoter) were transfected into Sol8 and 10T1/2 cells.
The effects of the mutations on the promoter activity were assessed by
assaying luciferase activity in cell extracts. Mutation in the MEF2
element to a site incapable of DNA binding (65) completely
abolished the expression of luciferase in myotubes to the level found
in myoblasts (Fig. 6B) but had no effect on luciferase
activity in Sol8 myoblasts and 10T1/2 fibroblasts. This indicates that
this element is essential for the myogenic differentiation-dependent
expression of COXVIaH in skeletal myotubes. Mutation in the E-box at
nucleotide -147 reduced the expression of luciferase in myotubes
to 20% of the wild type level. This effect, however, was not
myotube-specific since the same mutation also resulted in a
10-20-fold reduction of the expression of the reporter gene in
Sol8 myoblasts and 10T1/2 fibroblasts. Mutation in the E-box at
nucleotide -60 decreased luciferase activity in myotubes,
myoblasts, and fibroblasts to 65% of the wild type level, whereas
mutation in the E-box at nucleotide -75 did not affect the
expression of luciferase in any cell type.
Analysis of the COXVIaH Promoter in Transgenic
Mice
In order to confirm that the 5`-flanking region of the
COXVIaH gene functions as a striated muscle-specific promoter/enhancer
element in vivo, the DNA construct from nucleotides
-3500 to +17 fused to luciferase was used to generate
transgenic mice. Three separate lines of mice were generated. One line
failed to transmit the transgene to subsequent progeny, precluding
further analysis as of this writing. Two lines transmitted the
transgene in a Mendelian fashion and were available for analysis. Of
these, only one line (Fig. 6) showed expression. Luciferase
activity was detected in heart and skeletal muscle and at a low level
in brain tissue. In contrast, no detectable luciferase activity was
present in the liver, kidney, or stomach from this line. Although
preliminary, these data suggest that all the information necessary for
the striated muscle-specific expression of COXVIaH is present on this
3.5-kb 5`-flank. A detailed analysis of these promoter elements in
transgenic animals is in progress and will be reported separately.
-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.
)
We sincerely appreciate the expert technical
assistance of George Albright and Willie Young and the collective
efforts of the Moreadith Laboratory. We thank R. Sanders Williams for a
critical review of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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