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Originally published In Press as doi:10.1074/jbc.M003024200 on August 4, 2000
J. Biol. Chem., Vol. 275, Issue 42, 33123-33133, October 20, 2000
Differential Regulation of the Catalytic and Accessory Subunit
Genes of Drosophila Mitochondrial DNA Polymerase*
Etienne
Lefai §¶,
Miguel A.
Fernández-Moreno§,
Anuradha
Alahari ,
Laurie S.
Kaguni, and
Rafael
Garesse**
From the Departamento de Bioquímica,
Instituto de Investigaciones Biomédicas "Alberto Sols"
CSIC-UAM, Facultad de Medicina, Universidad Autónoma de Madrid,
c/Arzobispo Morcillo 4, 28029 Madrid, Spain and the Department
of Biochemistry, Michigan State University,
East Lansing, Michigan 48824-1319
Received for publication, April 11, 2000, and in revised form, July 25, 2000
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ABSTRACT |
The developmental pattern of expression of the
genes encoding the catalytic ( ) and accessory ( ) subunits of
mitochondrial DNA polymerase (pol  ) has been examined in
Drosophila melanogaster. The steady-state level of pol
- mRNA increases during the first hours of development,
reaching its maximum value at the start of mtDNA replication in
Drosophila embryos. In contrast, the steady-state level of
pol - mRNA decreases as development proceeds and is low in
stages of active mtDNA replication. This difference in mRNA
abundance results at least in part from differences in the rates of
mRNA synthesis. The pol genes are located in a compact cluster
of five genes that contains three promoter regions (P1-P3). The P1
region directs divergent transcription of the pol - gene and the
adjacent rpII33 gene. P1 contains a DNA replication-related element (DRE) that is essential for pol - promoter activity, but
not for rpII33 promoter activity in Schneider's cells. A
second divergent promoter region (P2) controls the expression of the orc5 and sop2 genes. The P2 region contains two
DREs that are essential for orc5 promoter activity, but not
for sop2 promoter activity. The expression of the pol
- gene is directed by P3, a weak promoter that does not contain
DREs. Electrophoretic mobility shift experiments demonstrate that the
DRE-binding factor (DREF) regulatory protein binds to the DREs
in P1 and P2. DREF regulates the expression of several genes encoding
key factors involved in nuclear DNA replication. Its role in
controlling the expression of the pol - and orc5
genes establishes a common regulatory mechanism linking nuclear and
mitochondrial DNA replication. Overall, our results suggest that the
accessory subunit of mtDNA polymerase plays an important role in the
control of mtDNA replication in Drosophila.
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INTRODUCTION |
Animal mitochondrial DNAs, with few exceptions, encode 13 subunits
of the respiratory complexes I and III-V located in the inner
mitochondrial membrane. The remainder of the ~1000 mitochondrial proteins, including those involved in mtDNA replication and
transcription, are encoded in the nucleus, translated on
cytoplasmic ribosomes, and targeted by a complex import mechanism to
the mitochondrial compartment (1, 2).
The molecular mechanisms involved in mitochondrial proliferation and
maturation (i.e. mitochondrial biogenesis) during cell division, growth, and differentiation are poorly understood. During early embryonic stages, the mitochondria that are stored in the oocyte
are segregated into various cellular territories; as development proceeds, mitochondrial proliferation begins. When differentiation ceases, the cells contain the correct number of mitochondria with the
specific morphology and intracellular distribution to meet the highly
variable energetic requirements of the various tissues of the organism
(3, 4). How the mitochondrial mass, the extent of mitochondrial
differentiation, and the mtDNA copy number are controlled in a defined
cell type is not known.
In addition to this increase in mitochondrial biogenesis in response to
unidentified developmental cues, cells can also regulate the expression
of the genes encoding mitochondrial proteins in response to a variety
of physiological signals, including hormones, cyclic nucleotides,
contractile activity, and thermogenesis (5-8). Several DNA elements
recognized by regulatory proteins involved in the transcriptional
regulation of nuclearly encoded mitochondrial genes have been
identified in mammals, including Mts, REBOX/OXBOX, and GRBOX
elements (9-11). In addition, two transcription factors involved in
nucleo-mitochondrial communication, nuclear respiratory factors 1 and 2 (NRF1 1 and NRF2), have been
characterized during the last few years by Scarpulla and co-workers
(12-14). NRF1 activates the expression of several genes that encode
functional components of the oxidative phosphorylation pathway and key
elements of the transcriptional/replication machinery of mitochondria
such as Tfam (also known as mitochondrial transcription
factor A) and the RNase mitochondrial RNA processing (7).
Binding sites for NRF1 have been also identified in several genes
involved in cell proliferation and intermediary metabolism (12). Thus,
NRF1 likely plays an important role in the control of mammalian
mitochondrial biogenesis under a variety of conditions requiring high
energy supply.
Although mtDNA replication is required for eukaryotic cell
proliferation, the mechanisms that regulate this process are poorly understood. Recent studies have shown that the cytoskeleton plays an
active role in the mitochondrial dynamics of the cell (for a review,
see Ref. 15) and that the replication of the mitochondrial genome
occurs in the perinuclear space (16). The key enzyme in mtDNA
replication is DNA polymerase (pol ), which constitutes only
~1% of the total cellular DNA polymerase activity (17, 18). The pol
holoenzyme has been characterized extensively in
Drosophila (19); it is a heterodimer of a 125-kDa subunit containing DNA polymerase and 3'-5' exonuclease catalytic
activities (20) and a 35-kDa accessory subunit that increases the
catalytic efficiency of the holoenzyme and is likely involved in primer recognition and in enhancing processivity (21, 22). Both subunits have
also been identified in other systems, and their structures are
evolutionarily conserved. The catalytic subunit shares homology with
the family A DNA polymerases (20, 23, 24), whereas the accessory
subunit is related to aminoacyl-tRNA synthetases (22, 25). In
Drosophila, the nuclear genes and the corresponding cDNAs have been cloned (20, 26, 27) and map in the same genomic
region (Adh region, chromosome II) within a tight cluster of
five genes (28). Here we report on the transcriptional regulation of
the pol - and pol - genes. Whereas our data indicate that the catalytic subunit of pol is expressed constitutively at a low
level, the developmental pattern of expression of the pol - gene
suggests that the accessory subunit plays an important role in the
control of mtDNA replication. Moreover, the transcription factor
DREF, which activates the expression of genes encoding components of the nuclear replication machinery (29), is critical for
pol - promoter activity. This regulatory protein is also critical
for the activity of the mitochondrial single-stranded DNA-binding
protein (mtSSB) promoter (30), establishing firmly a molecular link
between nuclear and mitochondrial DNA replication.
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EXPERIMENTAL PROCEDURES |
RNA Extraction and Northern Analysis
Total RNAs from staged embryos; first-, second-, and
third-instar larvae; early and late pupae; and adults of
Drosophila melanogaster Oregon R were extracted using the
Trizol kit (Life Technologies, Inc.) according to the manufacturer's
directions. For Northern analysis, 30 µg of total RNA was
electrophoresed on 1.8 M formaldehyde and 1.2% agarose
gels, blotted on a Zeta Probe membrane (Bio-Rad), and probed in
ZAP buffer (0.25 M phosphate buffer, pH 7.2., 7% SDS) at 65 °C using various [-32P]dCTP-labeled
cDNA clones as probes. Filters were washed in 0.1% SDS and 0.1×
SSC at 65 °C and autoradiographed with intensifying screens at
 70 °C.
Nuclear Run-on Transcription Analysis
To obtain nuclei from Drosophila adults, flies (100 in a typical experiment) were homogenized in 1 ml of buffer containing 10 mM KCl, 15 mM HEPES (pH 7.6), 5 mM MgCl2, 0.35 M sucrose, 0.5 mM EGTA, 0.1 mM EDTA, 1 mM
dithiothreitol, and 10 mM phenylmethylsulfonyl fluoride.
The homogenate was filtered through Miracloth (Calbiochem) and
centrifuged at 400 × g for 5 min. The supernatant was
centrifuged at 700 × g for 10 min, and the nuclear
pellet was resuspended in buffer containing 50 mM Tris-HCl
(pH 8.3), 5 mM MgCl2, 0.1 mM EDTA,
and 40% glycerol. Embryos were dechorionated by sodium hypochloride
treatment and washed extensively before homogenization. Transcriptional
run-on reactions were performed essentially as described by Linial
et al. (31) using 150 µCi of [-32P]UTP
and ~107 nuclei. Radiolabeled RNA was purified by
phenol/chloroform extraction followed by precipitation with isopropyl
alcohol and hybridized in ZAP buffer at 65 °C to 5 µg of 18 S rDNA
and pol - and pol - cDNAs that were immobilized on
nylon filters using the Manifold II system (Schleicher & Schüll)
and denatured by NaOH treatment. Filters were washed in 0.1% SDS and
02 × SSC at 65 °C and autoradiographed with intensifying
screens at 70 °C.
Promoter Constructs
DNA fragments containing the promoter regions (P1-P3) of the
five genes were amplified by PCR using several sets of oligonucleotide primers (Table I). The resulting
parental DNA fragments were used as substrates for generation of
deletion constructs. After digestion with restriction endonucleases,
DNA fragments were excised from agarose gels and inserted into the pxp2
vector, which contains the luciferase gene as reporter. The nucleotide
sequences of the parental DNA fragments and each of the promoter
constructs were confirmed by DNA sequence analysis.
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Table I
Oligonucleotide primers used in this study
+1 represents the transcriptional start site of pol -,
orc5, and pol -, respectively. The XhoI
restriction sites included in the reverse oligonucleotides are
underlined. Their positions are according to +1.
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pol - Promoter (P3)--
Deletions of the parental DNA
fragment were generated as follows. EcoRV/XhoI
and XmnI/XhoI fragments were cloned into the pxp2
vector that was digested with SmaI/XhoI to
generate constructs 1710/+113 and 1197/+113 (where +1
corresponds to the transcriptional start site). The
XmnI/XhoI fragment was also digested with
DraI, and the XmnI/DraI fragment was
cloned into pxp2 that was digested with SmaI to generate
construct 1197/183. To obtain construct 407/+113, a 520-bp
BglII/XhoI fragment was cloned into pxp2 that was
digested with BglII/XhoI. To mutate the putative
NRF1-like binding site in P3, we used the oligonucleotide
5'-GCCAGCCTAGGTATTTTTGccCcCTccCcCGGCGG-3' (from positions
+42 to +8 in the template strand for transcription); the
lowercase letters represent the nucleotides that were changed from the
original sequence (5'-GCCAGCCTAGGTATTTTTGTGCGCTTGCGCGGCGG-3'), and
those that are underlined represent the mutated NRF1 site. After
several steps of subcloning, PCR, and additional subcloning, construct
407/+113mut was obtained. This plasmid is identical to its
parental construct, 407/+113, except for the six nucleotide changes within the NRF1-like site.
rpII33/pol - Promoter (P1)--
Deletions in the
parental PCR fragment were generated as follows.
HindIII/XhoI and PvuII/XhoI
fragments were cloned into the pxp2 vector that was digested with
HindIII/XhoI and SmaI/XhoI, respectively, to generate constructs 1806/+467 and 1319/+467. To obtain constructs 569/+467 and 212/+467, the parental DNA fragment was digested with PstI and then with
ClaI, rendered blunt-ended with T4 DNA polymerase, and cut
with XhoI. Purified fragments were then ligated into the
pxp2 vector that was digested with SmaI/XhoI. A
797-bp DNA fragment from positions 326 to +473 was also amplified by
PCR using two oligonucleotides as primers: the same reverse primer
described above (Table I) and a new forward primer
(5'-GCACTGCAGTTTTGTTTTGATGAA-3') containing a PstI site at
position 318. The amplified product was digested with PstI and XhoI and cloned into the pBluescript vector that was
digested with the same restriction enzymes, generating pBS-PI. The
construct was then digested with ClaI; end-filled with
Klenow DNA polymerase; and religated to obtain pBS-PImut, in which the
DREF-binding site is changed from TATCGATT to TATCgcGATT. The
BamHI/XhoI fragments of pBS-PI and pBS-PImut were
cloned into pxp2 that was digested with
BamHI/XhoI to generate the 318/+467 and
318/+467mut constructs, respectively.
The BamHI/XhoI fragments of pBS-PI and pBS-PImut
were also cloned into pxp2 that was digested with
BamHI/XhoI to generate constructs rpII715/+71
and rpII715/+71mut, with the inverse orientation of the DNA
fragments. These constructs are numbered with respect to the
transcriptional start site (+1) of the rpII33 gene.
orc5/sop2 Promoter (P2)--
Deletions of P2 were
generated by digestion of the parental PCR fragment as follows. The
HindIII/XhoI and SalI/XhoI
fragments were cloned into pxp2 that was digested with the same enzymes to generate constructs sop1616/+307 and sop604/+307. (+1 represents the transcriptional start site in the sop2 gene that maps at
a position 326 nucleotides upstream of the translational start codon. This +1 position is shared in the complementary strand by the orc5 gene.) A 627-bp DNA fragment from positions 326 to
+473 was amplified by PCR using two oligonucleotides as primers: the same reverse primer described above (Table I) and a forward primer (5'-CCAGAGCTCTCGTCTGCTTT-3') containing a PvuII site at
position 306. The PvuII/XhoI fragment was
cloned into the pBluescript vector that was digested with
EcoRV/XhoI to obtain construct pBS-PII. The
construct was then digested with ClaI; end-filled with
Klenow DNA polymerase; and religated to obtain construct pBS-PIImut, in
which the region containing the two DREF sites is modified by a small
deletion, changing the sequence
TATCGATataacgaaaTAACGATAgtattgagggccatcgatt, where the ClaI sites are underlined and the DREF sites
are in uppercase letters. The BamHI/XhoI
fragments of pBS-PII and pBS-PIImut were cloned into pxp2 that was
digested with BamHI/XhoI to obtain constructs
sop303/+307 and sop303/+307mut. A 2230-bp DNA fragment from
positions 2080 to +351 (where +1 corresponds to the transcriptional start site of the orc5 gene) was amplified by PCR using the
two oligonucleotide primers 5'-CCAGAGCTCTCGTCTGCTTT-3' (forward)
and 5'-GGCCCTCGAGGGTGGCTAAATGCG-3' (reverse), in which a
PvuII site was introduced at a position corresponding to
+343. The SalI/PvuII fragment was cloned into
pxp2 that was digested with SalI/SmaI to generate
construct orc1405/+346. The SalI/KpnI fragment
was cloned into pxp2 that was digested with
SalI/KpnI to generate construct orc1405/+117.
Finally, the BamHI/EcoRV fragments of pBS-PII and
pBS-PIImut were cloned into pxp2 that was digested with
SmaII/BglII to obtain the orc170/+346 and
orc170/+346mut constructs, respectively.
DREF and Erect Wing Constructs--
For cotransfection
experiments, we cloned the complete Erect Wing (EWG) and DREF cDNAs
in the vector pMK26 (32), which allows strong expression under the
control of the actin 5C promoter. A BamHI/XhoI
fragment containing the complete DREF cDNA (30) was end-filled with
Klenow DNA polymerase and subcloned into the pBluescript vector that
was digested with EcoRV to obtain pBS-DREF. For EWG plasmid
construction, the pET3dEWG6His clone (a kind gift of Dr. Richard
Scarpulla) was first digested with XbaI/BamHI, end-filled with Klenow DNA polymerase, and then inserted into the
pBluescript vector that was digested with EcoRV to obtain pBS-EWG. Finally, the HindIII/PstI fragments
obtained from pBS-DREF and pBS-EWG were cloned into the pMK26 vector
that was digested with HindIII/PstI, generating
constructs pMK-DREF and pMK-EWG, respectively.
Cell Transfection and Reporter Activity
Transfection in Schneider's S2 cells was carried out according
to Soeller et al. (33) with some modifications. Streptomycin (100 µg/ml) and penicillin (100 IU/ml) were added to the medium, and
cells were transfected with 10 µg of the vector pSVgal (Promega) and 5 µg of the corresponding pxp construct. After transfection, cells were incubated for 24 h at 25 °C, washed twice with
phosphate-buffered saline, resuspended in 5 ml of fresh medium, and
incubated for 60-80 h at 25 °C. To prepare extracts, cells were
harvested by centrifugation; washed with phosphate-buffered saline and
then with fresh buffer containing 40 mM Tris-HCl (pH 7.5),
1 mM EDTA, and 150 mM NaCl; resuspended in 100 µl of 0.1 M Tris-HCl (pH 7.5); and subjected to five
cycles of freezing for 60 s at 70 °C and heating for 60 s at 37 °C. Cell debris was removed by centrifugation at 13,000 × g for 5 min. Promoter activities were calculated by normalizing luciferase activity (pxp constructs) to -galactosidase activity (pSVgal). Luciferase activity was determined using the luciferase assay system (Promega) according to the manufacturer's recommendations, and -galactosidase activity was measured as described previously (34).
Electrophoretic Mobility Shift Assays
Electrophoretic mobility shift assays (EMSAs) were carried out
using protein prepared from two sources: the in vitro TNT
transcription/translation system (Promega) for DREF binding assays and
Schneider's cell nuclear extracts for DREF and NRF1 binding assays.
The binding reactions were carried out using 2 µl of in
vitro transcription and translation product or 5 µg of nuclear
protein and 50,000 cpm of the DNA probe in binding buffer containing
20% glycerol, 1 mM dithiothreitol, 20 mM HEPES
(pH 9.0), 5 mM MgCl2, 0.2 mM EDTA,
and 200 mM KCl. After incubation for 30 min at 4 °C,
reaction products were electrophoresed on 4.5% polyacrylamide gels
(for PCR probes) or 6% polyacrylamide gels (for oligonucleotide
probes) containing 0.5× Tris borate/EDTA. DNA probes were
labeled using [-32P]ATP and T4 polynucleotide kinase.
For the P1 probes, two DNA fragments containing the DRE were used as
probes: a double-stranded 30-mer oligonucleotide from positions 225
to 204 and a 173-bp fragment from positions 325 to 153 that was
obtained by PCR amplification (where +1 corresponds to the
transcriptional start site for pol -). For the P2 probe, a 120-bp
DNA fragment from positions 132 to 13 containing the DREs was
amplified by PCR (where +1 corresponds to the transcriptional start
site for sop2). In DREF EMSA experiments, a 100-fold molar
excess of various oligonucleotides was used as competitor, including
unlabeled DNA probes and oligonucleotides from the mtSSB promoter
containing wild-type or mutated DRE sites (30). For the P3 probe, a
double-stranded 35-mer oligonucleotide from positions +42 to +8
containing the NRF1-like site and its mutated version (described above)
was used. DREF antiserum was prepared as described (30). Antiserum
against EWG was kindly provided by Dr. Kalpana White. In supershift
assays, 2 µl of a 1:100 dilution of the relevant antiserum was used.
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RESULTS |
Differential Expression of the Genes Encoding the pol Catalytic
and Accessory Subunits during D. melanogaster Development--
The
genes encoding the two subunits of the mitochondrial DNA polymerase map
within a cluster of genes, the pol cluster, that extends over a
region of 10.5 kilobases in the alcohol dehydrogenase region
(34D-E) of the second chromosome of D. melanogaster (27, 28). The cluster contains five genes that encode proteins involved in
essential cellular processes, including the two pol subunits (pol
- and pol -), one subunit of RNA polymerase II (RpII33), one subunit of the origin recognition complex (ORC5), and one subunit
of the actin-related protein 2/3 complex (SOP2/ARC41). The pol cluster is extremely compact: the genes are tightly packed with very
little intergenic space, with some overlap in their 5'- and 3'-ends
(Fig. 1). The cluster contains three
promoter regions (P1-P3), and two of them direct bidirectional
transcription (28). This tight organization suggests that the structure
of the pol cluster plays an important role in regulating the
expression of its genes. This is a particularly important consideration
given that two of the genes encode the subunits of the key enzyme in the mitochondrial DNA replication process (pol - and pol
-), representing a rare example in eukaryotes of genes encoding
functionally related proteins that are linked in the genome. Moreover,
a third gene of the pol cluster, orc5, encodes an
essential component of the nuclear DNA replication machinery,
suggesting the presence of potential common regulatory mechanisms for
nuclear and mitochondrial DNA replication.
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Fig. 1.
Schematic representation of the
Drosophila pol gene
cluster. The structure and direction of transcription of the five
genes in the pol cluster are shown schematically. Thick
arrows represent the protein coding regions, and thin
arrows indicate the transcriptional start sites. Stippled
boxes represent 5'- and 3'-untranslated regions, and open
boxes represent introns. P1-P3 correspond to the three
promoter regions that direct transcription within the cluster.
Kb, kilobases.
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As a first step to study the factors involved in regulating the
expression of the pol genes, we determined (by Northern analysis)
their patterns of expression during Drosophila development and compared them with the expression profiles of the other genes encoded in the pol cluster. The level of pol - mRNA was
relatively high in eggs and decreased rapidly during embryonic
development (Fig. 2A). This
result is perhaps surprising because mtDNA replication started ~10 h
after egg laying (AEL); and at this developmental stage, the level of
pol - mRNA has declined substantially. During the larval
stages, a period characterized by increases in body weight and cell
ploidy, the steady-state level of pol - mRNA remained very
low, whereas that in adults was comparatively higher. The
spatiotemporal pattern of expression of pol - mRNA, examined by whole-mount RNA in situ hybridization, is in good
agreement with the Northern results. pol - mRNA was
distributed homogeneously in early blastoderm, and the intensity of the
signal decreased through gastrulation and was very faint or
undetectable in older embryos (data not shown).
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Fig. 2.
Developmental pattern of expression of the
five genes encoded in the Drosophila pol
cluster. Total RNAs from 0-18-h embryos,
larvae (first-, second-, and third-instar (L1-L3,
respectively)), pupae (P1-P3), and adults (A) of
D. melanogaster were analyzed by Northern blotting using the
corresponding cDNAs as probes. RNAs were fractionated on
formaldehyde-containing 1.2% agarose gels, transferred to nylon
membrane, and hybridized with the corresponding radiolabeled probes,
and the blots were autoradiographed. Levels of RNA loaded were
evaluated by ethidium bromide (EthBr) staining. The
expression patterns of the pol genes are shown in A, and
those of rpII33, sop2, and orc5 are
shown in B.
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pol - mRNA showed a different developmental profile (Fig.
2A). The level present in eggs was relatively low, but its
steady-state level increased in early embryonic stages, reaching its
maximum between 6 and 12 h AEL. In late embryos, its steady-state
level decreased; and in first-instar larvae, there was a moderate
increase. These results suggest that the expression of the pol -
subunit is regulated precisely during Drosophila
development. Moreover, the steady-state level of pol - reached
its maximum just before the start of mtDNA synthesis that occurred
10-12 h AEL.
To determine whether the differences observed in the pattern of
expression of pol - and pol - mRNAs are due to
different rates of transcription, we performed a series of run-on
transcription experiments using nuclei extracted from 8-10-h AEL
embryos and adults. A higher rate of mRNA synthesis was detected in
the pol - gene in both embryos and adults (Fig.
3). Although we have not evaluated
possible differences in mRNA stability, the different rates of
mRNA synthesis correspond well with the mRNA steady-state levels detected by Northern analysis (Fig. 2A), documenting
a differential regulation of the pol - and pol - genes at
the transcriptional level.
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Fig. 3.
Nuclear run-on transcription analysis of the
pol - and pol
- genes. The relative
rates of transcription of the pol - and pol - genes were
evaluated in run-on experiments as described under "Experimental
Procedures." A, nuclei obtained from adults and 8-10-h
AEL embryos were labeled with [-32P]UTP. The RNA
synthesized de novo was hybridized to 5 µg of 18 S rDNA
(as a control), pol - cDNA, and pol - cDNA
immobilized on a nylon membrane. B, shown is the
densitometric quantitation of the data obtained in four independent
experiments. Values are expressed in arbitrary units.
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Unfortunately, we were unable to detect pol - mRNA by
in situ hybridization despite systematic attempts using a
variety of experimental conditions (data not shown). A lack of
sensitivity of the technique is an unlikely explanation because
Northern analysis showed that the steady-state level of pol -
mRNA was similar or higher than that of pol - mRNA. We
have also used in parallel several control probes that detect other
unrelated mRNAs, obtaining in each case the expected pattern.
Although the reason for the lack of in situ detection of pol
- mRNA is presently unknown, our data suggest the possibility
of some type of sequestration of pol - mRNA that does not
occur with pol - mRNA.
The rpII33 gene is divergently transcribed from the same
promoter as the pol - gene, yet its developmental pattern of
expression is entirely different (Fig. 2B).
rpII33 mRNA was accumulated in eggs, and its level was
maintained fairly constant during early embryogenesis up to 6 h
AEL. From 9 h AEL onward, its steady-state level decreased and was
very low during the larval and pupal periods. Adults also contained a
relatively low amount of the rpII33 transcript. A similar
result was observed in the case of the orc5/sop2
genes, and the pattern of expression was also different for each gene (Fig. 2B). sop2 mRNA was present at
relatively high levels in all developmental stages, whereas the
steady-state level of orc5 mRNA was very high in eggs
and decreased sharply as development proceeded. In larvae and pupae,
its concentration was low and increased again in adults. This pattern
is generally similar to that for pol -. Our results indicate
clearly that the pairs of genes transcribed from the bidirectional
promoters P1 and P2 are differentially regulated during development.
Analysis of the rpII33/pol - Promoter Region (P1)--
The
developmental pattern of expression of the pol - gene suggests
that the accessory subunit of the pol enzyme may play an important
role in regulating mtDNA replication. The pol - gene is
divergently transcribed from the rpII33 gene, and their 5'-ends are separated by only 245 bp (Fig. 1). To identify and characterize transcriptional regulatory elements in the
rpII33/pol - intergenic promoter region (P1), we
cloned defined DNA fragments in the pxp2 vector and determined their
promoter activities in transient transfection experiments in
Schneider's S2 cells, measuring luciferase activity in cell
extracts (see "Experimental Procedures").
A construct containing the 5'-UTR and 1.8 kilobases of the proximal
5'-upstream region of the pol - gene (1806/+467, where +1
corresponds to the pol - transcriptional start site), which includes the complete adjacent rpII33 gene, directed a
substantial level of luciferase activity in Schneider's cells,
>1000-fold as compared with the pxp2 vector (Fig.
4). A similar level of activity was
maintained in shorter constructs, including 318/+467, which
contains the rpII33/pol - intergenic region and the
5'-UTRs of both genes. Computer analysis of the DNA sequence spanning positions 318 to +467 revealed the presence of a DRE motif (TATCGATT) located at position 216 within the rpII33/pol -
intergenic region, close (~30 bp) to the transcriptional initiation
site of the rpII33 gene. The DRE is recognized by DREF, a
transcription factor critical in the regulated expression of several
genes encoding proteins involved in nuclear DNA replication (29). We
have also demonstrated recently that it is essential for the activity
of the D. melanogaster mtSSB promoter (30). Mutation of the
DRE-binding site reduced the activity of construct 318/+467mut by
80% (p < 0.05), suggesting strongly that DREF is
critical to achieve full pol - promoter activity. Furthermore,
construct 212/+417, with the DRE site eliminated, abolished
promoter activity in Schneider's cells.
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Fig. 4.
Functional analysis of the P1 region.
A schematic representation of the divergent P1 region and the
adjacent pol - and rpII33 genes is shown in the
middle. Thick arrows represent the protein coding regions;
stippled boxes represent 5'-untranslated regions; and
open boxes represent introns. Transcriptional initiation
sites (+1) are indicated by thin arrows, and the positions
of translational initiation sites are indicated as ATG with
a corresponding number that indicates the length of the
5'-untranslated region. The DRE sequence is represented as an
ellipse. DNA fragments cloned upstream of the luciferase
reporter gene and used in the analysis of the pol - promoter
activity are shown above the schematic, and those used in the analysis
of the rpII33 promoter activity are shown below. Plasmid
designations indicate the specific DNA sequences present in the
constructs, with numbers relative to the position of the
transcriptional start site. The internal X in the DRE
sequence indicates that the site is mutated. The relative promoter
activities of the constructs, as measured in the luciferase assay, are
indicated to the right. The luciferase activity of the pxp2 vector was
defined as 1.0 and was used as the standard for comparison. Luciferase
activity was normalized to -galactosidase activity for each
construct. Values are means ± S.D. of at least three independent
experiments. Statistical analysis was carried out using the Wilcoxon
signed-rank test. * indicates p < 0.05.
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To determine whether the DRE sequence is also important for the
activity of the rpII33 promoter, we determined the
luciferase activity of the 318/+467 fragment cloned in the pxp2
vector in the opposite orientation (rpII715/+71). An 800-fold
induction in luciferase activity was detected, indicating that the
rpII33/pol - intergenic region divergently directs the
transcription of the two genes. Importantly, when the assay was carried
out with a construct containing the same DNA fragment with the DRE site mutated (rpII715/+71mut), no significant change in luciferase activity was observed. This result demonstrates that the DRE site located in the P1 region is essential only for the promoter activity of
the pol - gene.
Analysis of the orc5/sop2 Promoter Region (P2)--
Two of the
genes in the pol cluster, orc5 and sop2,
overlap in their 5'-ends (Fig. 1). We found that a DNA fragment
encompassing the 5'-upstream region of the two genes contains potent
bidirectional promoter activity. Construct sop1616/+307, which
contains the sop2 5'-UTR and most of the orc5
gene, directed a high level of luciferase activity, >9000-fold as
compared with the pxp2 vector (Fig. 5). A
high level of activity was maintained in the sop303/+307 construct,
which contains the complete 5'-UTRs of the two overlapping genes. The
5'-UTR of the orc5 gene contains two DRE sites. To examine
the functional relevance of the DRE sites in sop2 promoter activity, we mutated both sites in construct sop303/+307mut. We
observed no change in the level of luciferase activity, indicating that
the DRE sites do not play an essential role in the sop2
promoter activity in Schneider's cells.
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Fig. 5.
Functional analysis of the P2 region. A
schematic representation of the divergent P2 region and the adjacent
orc5 and sop2 genes is shown in the
middle. DNA fragments cloned upstream of the luciferase
reporter gene and used in the analysis of the sop2 promoter
activity are shown above the schematic, and those used in the analysis
of the orc5 promoter activity are shown below. The relative
promoter activities of the constructs, as measured in the luciferase
assay, are indicated to the right. The luciferase activity of the pxp2
vector was defined as 1.0 and was used as the standard for comparison.
Luciferase activity was normalized to -galactosidase activity for
each construct. Values are means ± S.D. of at least three
independent experiments. Statistical analysis was carried out using the
Wilcoxon signed-rank test. * indicates p < 0.05.
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To evaluate the orc5 promoter, we assayed the activity of
the orc170/+346 construct and observed a high increase in luciferase activity, >8000-fold as compared with the pxp2 vector. The same region
containing mutated DRE sites (orc170/+346mut) directed significantly
reduced activity, only 30% of that detected in the orc170/+346
construct (p < 0.05). Thus, similar to the results obtained in the bidirectional rpII33/pol -
promoter region, the DRE sites located in the 5'-UTR of the
orc5 gene are essential for the activity of one promoter
(orc5), but have no effect on the activity of the other
(sop2). More importantly, these data establish a common
molecular mechanism involved in the regulated expression of two genes
in the pol complex that are involved in nuclear and mitochondrial
DNA replication, orc5 and pol -, respectively.
DREF Binds to the pol - and orc5 Promoter Regions--
To
characterize further the DRE sequences in P1 and P2, we carried out
electrophoretic mobility shift assays using probes covering the P1 and
P2 regions that contain the DRE sites. We used either DREF produced by
in vitro transcription/translation or nuclear extracts
prepared from cultured Schneider's cells as the protein source. When a
radiolabeled 173-bp fragment containing the single DRE site present in
the P1 region (P1-DRE) was incubated with recombinant DREF protein, a
single retarded protein-DNA complex was detected, which was eliminated
when a 100-fold molar excess of unlabeled probe was included in the
binding reaction (Fig. 6A).
Inclusion of rabbit anti-DREF serum produced a clear supershift, demonstrating that DREF interacts with the DRE site in the pol -
promoter region. The supershift was also eliminated by inclusion of a
100-fold molar excess of unlabeled probe. When a similar experiment was
carried out in which a 120-bp fragment containing the two DRE sites
present in the P2 region (P2-DRE) was used as probe, similar results
were obtained (Fig. 6B). In this case, two retarded
protein-DNA complexes were apparent, and both were supershifted by the
anti-DREF antiserum. As a control, we included preimmune serum in the
binding reaction, and no supershift was produced. The same EMSA
patterns were observed with both promoter fragments when the source of
DREF was a nuclear extract prepared from Schneider's cells (Fig. 6,
A and B).
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Fig. 6.
DREF binds to the DRE sites in the
rpII33/pol
- and
orc5/sop2 promoters. DREF
binding was evaluated by EMSAs using recombinant DREF and nuclear
extract from cultured Schneider's cells. Black arrows
indicate the positions of retarded bands, and white arrows
indicate supershifted bands. The probe for P1 (A) was a
173-bp DNA fragment spanning positions 325 to 153 (relative to the
transcriptional start site at +1), and that for P2 (B) was a
120-bp DNA fragment from positions 132 to 13. The
asterisk in the Ab AntiDREF lane in B
indicates that preimmune serum was used. The competitors used were a
100-fold molar excess of the unlabeled DNAs used as probes.
Ab, antibody.
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Additional EMSA experiments were carried out with Schneider's cell
nuclear extracts and functional DRE sequences that were characterized
previously in the Drosophila mtSSB promoter as competitors (30). Here the probe for P1 was a double-stranded 30-mer
oligonucleotide spanning positions 225 to 204 (Fig.
7A), and that for P2 was as
described above (Fig. 7B). Both probes gave rise to shifted bands, and inclusion of anti-DREF antiserum in the binding reaction produced supershifts not observed upon inclusion of preimmune serum.
When competition of DREF binding to P1 and the corresponding anti-DREF
antiserum-mediated supershift was carried out using a 100-fold molar
excess of unlabeled P1, P2, or ssb (a double-stranded oligonucleotide containing the two functional DRE sites in the mtSSB
promoter (30)) DNA, the retarded bands and supershifts were abolished.
However, if the competition was performed with ssb / DNA (a double-stranded
oligonucleotide containing mutated DRE sites from the ssb promoter),
neither the retarded bands nor the supershifts were eliminated,
providing further support for the binding of DREF to P1. Similar
results were obtained with DREF binding to P2. A 100-fold molar excess
of P2 or ssb DNA eliminated the retarded bands and supershifts, but
competition with ssb/ DNA had no effect.
Notably, however, only partial competition of DREF binding to the P2
region was observed with P1 DNA, likely due to the fact that P1
contains a single DRE site, whereas P2 contains two. Taken together,
the EMSA experiments demonstrate that the DRE sites present in the pol
- and orc5 promoters are bound specifically by
DREF.
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Fig. 7.
Functional DRE sequences compete for binding
of DREF to the rpII33/pol
- and
orc5/sop2 promoters. DREF
binding to the P1 (A) and P2 (B) regions was
examined by EMSAs with DRE-containing competitor DNAs. The probe for P1
was a double-stranded 30-mer oligonucleotide spanning positions 225
to 204, and that for P2 was as described in the legend Fig. 6.
Black arrows indicate the positions of retarded bands, and
white arrows indicate supershifted bands. The
asterisk in the Ab AntiDREF lane in
B indicates that preimmune serum was used. The competitors
used were a 100-fold molar excess of the unlabeled P1 and P2 probes as
indicated, ssb (a double-stranded oligonucleotide containing the two
functional DRE sites in the mtSSB promoter (30)), and
ssb/ (the ssb probe with mutated DRE
sites). Ab, antibody.
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Analysis of the pol - Promoter Region (P3)--
A construct
(1710/+113) containing the complete coding sequence of the
sop2 gene, the ~140-bp sop2/pol -
intergenic region, and 119 bp of the 5'-UTR of the pol - gene
directed 300-fold higher luciferase activity than the pxp2 vector (Fig.
8). Constructs containing shorter DNA
fragments maintained a similar level of activity, including construct
407/+113, which contains only the sop2/pol -
intergenic region and the pol - 5'-UTR. Luciferase activity was
abolished in construct 1197-183, which has the transcriptional
start site eliminated. These data indicate that the proximal
5'-upstream region of the pol - gene has weak but significant
promoter activity in Schneider's cells.
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Fig. 8.
Functional analysis of the P3 region. A
schematic representation of the P3 region, the adjacent sop2
gene, and the 5'-region of the pol - gene is shown. The NRF1-like
sequence element is shown as an ellipse. DNA fragments
cloned upstream of the luciferase reporter gene and used in the
analysis of the pol - promoter activity are shown below. The
relative promoter activities of the constructs, as measured in the
luciferase assay, are indicated to the right. The luciferase activity
of the pxp2 vector was defined as 1.0 and was used as the standard for
comparison. Luciferase activity was normalized to -galactosidase
activity for each construct. Values are means ± S.D. of at least
three independent experiments. Statistical analysis was carried out
using the Wilcoxon signed-rank test. * indicates p < 0.01.
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The proximal 5'-upstream region of the pol - gene does not
contain DNA sequence motifs with significant homology to the DRE site.
However, its 5'-UTR contains a proximal binding motif potentially
recognized by the transcription factor NRF1. To evaluate its functional
relevance, we mutated the NRF1-like site. We observed a moderate but
significant decrease (50%; p < 0.01) in the promoter activity of construct 407/+113mut compared with the native
construct 407/+113, suggesting that the NRF1-like site might be
functional in Schneider's cells.
NRF1 likely plays a key role in nucleo-mitochondrial communications in
mammals (12). Interestingly, its DNA-binding motif is conserved in two
regulatory proteins identified in Drosophila (Erect Wing)
and sea urchin (P3A2). To study the binding of putative regulatory
proteins to the NRF1-like site, we pursued EMSA experiments using, as
probes, a double-stranded 35-mer oligonucleotide spanning positions +42
to +8 from the P3 region that contains the NRF1-like site
(NRF1+) and a similar oligonucleotide containing a mutated
NRF1-like site (NRF1). When Schneider's cell nuclear
extracts were incubated with the NRF1+ probe, several
retarded bands were observed that were eliminated specifically by a
250-fold molar excess of unlabeled NRF1+ oligonucleotide as
competitor (Fig. 9, left
panel). Inclusion of anti-EWG antiserum in the binding reaction
did not produce a supershift, suggesting that the EWG protein is not
present in the retarded complexes. Competition with a 250-fold molar
excess of the NRF1 oligonucleotide eliminated the
retarded fragments, indicating that the protein bound to the probe
likely interacts with sequence elements other than the NRF1-like site.
Furthermore, when the NRF1 oligonucleotide was used as
probe, several retarded bands were clearly visible, including an extra
band not observed in the EMSAs with NRF1+ (Fig. 9,
right panel). Whereas an excess of the NRF1
probe competed completely, only the shared retarded complexes were
eliminated by a 250-fold excess of the NRF1+
oligonucleotide. These results suggest that there is protein binding to
NRF1 that is mediated specifically through the
mutant sequence, possibly explaining the inhibition of promoter
activity detected in transient transfection experiments with the mutant
NRF1-like construct (Fig. 8). This protein is not EWG because no
supershift was observed in the EMSAs in the presence of anti-EWG
antiserum. Overall, the data suggest that neither the EWG protein nor
the native NRF1-like site per se is likely involved in the
control of pol - promoter activity. Consistent with this
interpretation, overexpression of EWG in Schneider's cells had no
effect on the activity of the pol - constructs examined in
transient transfection assays (data not shown).
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Fig. 9.
Regulatory protein binding in the P3
region. Protein binding to the P3 region was examined by EMSAs.
The probes were a double-stranded 35-mer oligonucleotide containing the
NRF1-like site (NRF1+) and a derivative in which the
NRF1-like site was mutated (NRF1).
Arrowheads indicate the positions of shared retarded
bands, and the arrow indicates the position of the retarded
band produced only with the NRF1 probe. The competitors
used were a 250-fold molar excess of the unlabeled NRF1
(1) and NRF1+ (2) probes as
indicated. Ab, antibody.
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DISCUSSION |
Eggs are highly enriched in mitochondria that are deposited by the
mother during oogenesis. As development proceeds, important changes in
the morphological and functional maturation of mitochondria occur (3)
in a process that requires a careful orchestration of
nucleo-mitochondrial interactions. Evidence from studies in different
systems including mammals shows that the mitochondria stored in the egg
are sufficient for the first stages of embryonic development (35).
Later on, mitochondria are distributed throughout the various embryonic
territories, which exhibit specific patterns of organelle
differentiation (36). In Drosophila, the developmental pattern of expression of several genes encoding mitochondrial proteins,
including the nuclearly encoded - and -H+-ATP
synthase subunits and the mitochondrially encoded ATPase-6 and -8 subunits, has been analyzed (37, 38). In each of these cases, gene
expression is activated coordinately 6-12 h AEL, suggesting that
during this period, the maternal contribution becomes limiting, and
there is an activation of mitochondrial biogenesis in the embryo. Accordingly, it has been shown that mtDNA replication and
likely mitochondrial proliferation start around this time of
development (39).
In this work, we have evaluated the expression of the genes encoded in
the pol cluster during Drosophila development and, in
particular, those encoding the two subunits of the pol holoenzyme. Both pol - and pol - mRNAs are stored in the egg,
probably as a result of the high level of mtDNA replication occurring
during oogenesis. The steady-state level of pol - mRNA
declines rapidly during development, reaching a minimum level 10 h
AEL and onward. In contrast, the pol - mRNA level increases
during the first hours of development and reaches its maximum level
6-10 h AEL. Nuclear run-on transcription analyses indicate that the
expression of the genes encoding the two pol subunits is regulated
differentially at the transcriptional level during
Drosophila development. Notably, only the pol -
mRNA level is high in the period of active mtDNA replication. This
might seem surprising because the pol - subunit contains the
catalytic activities of the enzyme. However, the lack of correlation
between the pol - mRNA level and mtDNA replication during
Drosophila development is consistent with several results obtained previously in mammals. First, the pol - mRNA
steady-state levels in different cell types do not correlate with their
mtDNA content (40). Second, cells without mtDNA
( 0) contain similar levels of pol - mRNA and
protein compared with wild-type cells (41), rendering it unlikely that
pol - serves a key role in the control of mtDNA replication.
Although the steady-state level of pol - mRNA has not been
determined in mammals, its developmental profile in
Drosophila suggests that the pol - subunit
participates in the reactivation of mtDNA synthesis during development.
Interestingly, the pol |
TOP
ABSTRACT
INTRODUCTION