Báo cáo khoa học: Expression of the Drosophila melanogaster ATP synthase a subunit gene is regulated by a transcriptional element containing GAF and Adf-1 binding sites pptx

Eur. J. Biochem. 271, 4003–4013 (2004) Ó FEBS 2004

doi:10.1111/j.1432-1033.2004.04336.x

Expression of the Drosophila melanogaster ATP synthase a subunit
gene is regulated by a transcriptional element containing GAF
and Adf-1 binding sites
´
´
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Ana Talamillo1,*, Miguel Angel Fernandez-Moreno1, Francisco Martınez-Azorın1, Belen Bornstein1,2,
Pilar Ochoa1 and Rafael Garesse1
1

Departamento de Bioquı´mica, Instituto de Investigaciones Biome´dicas ‘Alberto Sols’, CSIC-UAM, Facultad de Medicina,
Universidad Auto´noma de Madrid, Spain; 2Servicio de Bioquı´mica, Hospital Severo Ochoa, Legane´s, Madrid, Spain

Mitochondrial biogenesis is a complex and highly regulated
process that requires the controlled expression of hundreds
of genes encoded in two separated genomes, namely the
nuclear and mitochondrial genomes. To identify regulatory
proteins involved in the transcriptional control of key nuclear-encoded mitochondrial genes, we have performed a
detailed analysis of the promoter region of the a subunit of
the Drosophila melanogaster F1F0 ATP synthase complex.
Using transient transfection assays, we have identified a
56 bp cis-acting proximal regulatory region that contains
binding sites for the GAGA factor and the alcohol dehydrogenase distal factor 1. In vitro mutagenesis revealed that

The bulk of cellular ATP is synthesized through oxidative
phosphorylation (OXPHOS) that takes place in the mitochondria. The OXPHOS system is composed of five

multisubunit complexes embedded in the inner mitochondrial membrane and two small electron carriers, ubiquinone
and cytochrome c [1]. The OXPHOS system is generated in a
unique manner. The majority of the more than 80 OXPHOS
subunits are encoded by genes in the nuclear DNA (n-DNA),
while 13 essential subunits are encoded in the mitochondrial
DNA (mtDNA), contributing to four out of the five
OXPHOS complexes. The mtDNA consists of a small,
double-stranded, circular DNA molecule that is transcribed
and translated within this organelle. However, all of the

Correspondence to R. Garesse, Departamento de Bioquı´ mica, Insti´
tuto de Investigaciones Biomedicas ‘Alberto Sols’ CSIC-UAM, Fac´
ultad de Medicina, Universidad Autonoma de Madrid, C/Arzobispo
Morcillo 4, 28029 Madrid, Spain. Fax: +34 91 5854001,
Tel.: +34 91 4975453, E-mail:
Abbreviations: Adf-1, alcohol dehydrogenase distal factor; GAF,
GAGA factor; OXPHOS, oxidative phosphorylation; n-DNA, nuclear DNA; mtDNA, mitochondrial DNA; NRF, nuclear respiratory
factor; RACE, rapid amplification of cDNA ends; AEL, after egg
laying; UTR, untranslated region; DPE, downstream promoter
element.
*Present address: Departamento de Anatomı´ a y Biologı´ a Celular,
Facultad de Medicina, Universidad de Cantabria, Santander, Spain.
(Received 2 June 2004, revised 6 August 2004,
accepted 18 August 2004)

both sites are functional, and phylogenetic footprinting
showed that they are conserved in other Drosophila species
and in Anopheles gambiae. The 56 bp region has regulatory
enhancer properties and strongly activates heterologous
promoters in an orientation-independent manner. In addition, Northern blot and RT-PCR analysis identified two

a-F1-ATPase mRNAs that differ in the length of the 3¢
untranslated region due to the selection of alternative
polyadenylation sites.
Keywords: mitochondria; a-F1-ATPase; GAGA; Adf-1;
transcription regulation.

components involved in the replication, maintenance and
expression of the mtDNA, as well as the factors that
participate in the assembly of the respiratory complexes, are
encoded in the nucleus. Therefore, correct OXPHOS function relies on the coordinated expression of numerous genes
encoded in two physically separated genetic systems [2,3].
The multisubunit enzyme ATP synthase (complex V of the
OXPHOS system) is present in the membranes of eubacteria,
mitochondria and chloroplasts. It synthesizes ATP by means
of a rotary mechanism coupled to the electrochemical
gradient generated by the electron transport chain [4]. The
mitochondrial ATP synthase of animals contains 16 subunits
and is responsible for the synthesis of the majority of cellular
ATP, thereby playing a crucial role in energy metabolism. It
is formed by an F1 soluble complex containing five subunits
with a stoichiometry of a3b3dce, and a hydrophobic F0
complex composed of 11 subunits that forms an H+ channel
embedded in the inner mitochondrial membrane [1]. The F1
subcomplex contains the three catalytic sites of the enzyme
located at the interfaces of the a and b subunits where
nucleotide turnover takes place [4]. Two of the subunits are
encoded in the mtDNA; ATPase 6 (or a) and ATPase 8 (or
A6L). In contrast, the remainder are nuclear-encoded and
are translated by cytoplasmic ribosomes before being
imported to the mitochondria.

The molecular basis underlying nucleo–mitochondrial
crosstalk is still poorly understood [3]. During the last few
years a number of processes have been shown to participate
in this process, including transcriptional and post-transcriptional regulation of gene expression [5,6], changes in Ca2+


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4004 A. Talamillo et al. (Eur. J. Biochem. 271)

concentration [7], control of the mitochondrial dNTP pool
[8,9], mitochondrial localization to specific cellular domains
[10], or changes in local ATP concentrations [11]. A
particularly fruitful experimental strategy to identify key
regulatory factors in mitochondrial biogenesis was pioneered
by Scarpulla’s group [5]. This involves characterization of the
promoter regions of mammalian nuclear-encoded mitochondrial genes, and has led to the identification of several
transcription factors and coactivators that regulate the
expression of genes playing key roles in the biogenesis of
the OXPHOS system. In general, the transcription of nuclear
genes encoding proteins involved in OXPHOS biogenesis is
controlled by a combination of transcription factors that
are specific for each promoter [3,12]. However, one DNA
regulatory element that is more common in the 5¢ upstream
regulatory region of respiratory genes is that recognized by
the constitutively expressed Sp1 factor [13]. Additionally,
two other transcription factors have been shown to play a
significant role in mitochondrial biogenesis, the nuclear
respiratory factors (NRFs) 1 and 2 [5,14]. These factors are
likely to be involved in the integration of mitochondrial

biogenesis with other cellular processes related to cell growth
[15,16]. NRF-1 belongs to a novel class of regulatory
proteins, and it contains a DNA binding domain conserved
in two invertebrate developmental regulators, Erect Wing
and P3A2 [17]. Erect Wing is essential for the Drosophila
myogenesis and neurogenesis [18] while P3A2 regulates the
expression of several genes during sea urchin development
[19]. The transcription factor NRF-2 belongs to the ets family
and is the human homologue of the previously described
mouse transcription factor GABP [20]. Other DNA regulatory elements have been identified in the promoter of several
genes involved in mitochondrial biogenesis, such as
OXBOX/REBOX [21], Mts [22] or GRBOX [23]. However
the putative transcription factors that recognize these DNA
motifs remain to be identified.
In contrast, less is known about the mechanisms
controlling mitochondrial biogenesis in other animal systems or in invertebrates. We previously described how the
transcription of several Drosophila melanogaster genes
encoding components of the mtDNA replication machinery
was regulated. These included the mitochondrial singlestranded binding protein (mtSSB), and the catalytic (a) and
accessory (b) subunits of the DNA polymerase c (Pol c)
[24,25]. Interestingly, the expression of the genes encoding
mtSSB and Pol c-b is transcriptionally regulated by the
DNA replication-related-element binding factor (DREF).
Indeed, in Drosophila this transcription factor regulates the
expression of genes that are essential for the cell-cycle and
for the nuclear DNA replication machinery [26], establishing a link between mitochondrial and nuclear DNA
replication [24,25]. Here, we have identified essential
elements that participate in the transcriptional regulation
of the gene encoding the a subunit of the H+ ATP synthase
(a-F1-ATPase) in D. melanogaster.

Materials and methods
Library screenings
We screened a D. melanogaster genomic library prepared in
the vector k-DASH using the previously described a-F1-

ATPase cDNA labelled with [32P]dCTP[aP] as a probe [27].
Two genomic equivalents were transferred to Zeta-probe
filters (Bio-Rad), hybridized at 68 °C in ZAP buffer [7%
(w/v) SDS, 0.25 M phosphate buffer, pH 7.2], washed in
0.5% (w/v) SDS, 2· NaCl/Cit at 55 °C (NaCl/Cit: 0.15 M
NaCl/0.015 M sodium citrate) and visualized by autoradiography with intensifying screens at )70 °C. Positive clones
were purified by two additional rounds of screening, and
positive phages were amplified using standard protocols,
and the inserts analysed by Southern blotting. The complete
sequence of the gene as well as 5¢ upstream and 3¢
downstream regions () were
included in two overlapping phages. Selected fragments
strongly hybridizing with the probe were subcloned into
pBluescript (Stratagene) and further characterized by
sequencing.
DNA sequencing
The nucleotide sequence of the genomic clones was
determined using the dideoxy chain-termination method
with Taq DNA polymerase and automatic sequencing (3T3
DNA sequencer, Applied Biosystems) following the manufacturer’s instructions. Both DNA strands were sequenced
in their entirety and the sequences were analysed using the
GCG programme (University of Wisconsin) [28].
Mapping of transcriptional initiation sites
Identification of the a-F1-ATPase transcription start site

was achieved by three different methods: primer extension,
high-resolution S1 mapping and rapid amplification of
cDNA ends (RACE).
Primer extension analysis. Two different oligonucleotides
were used: a-PE1 (5¢-ACGGCCGGTCTCCTCCAGA
TC-3¢) from bp 216–195 and a-PE2 (5¢-GGACGC
CAGGCGGGCGGAAAAAATCG-3¢) from bp 30–4
from the ATG start codon in the a-F1-ATPase cDNA
sequence, respectively [27] (accession number Y07894). In
the assay, 50 pmol of the oligonucleotides were labelled with
50 lCi of [32P]ATP[cP] and polynucleotide kinase. Total
RNA (30 lg) from adults or from embryos obtained 0–18 h
1 after egg laying (AEL), and 8 lCi of 32P-labelled primer
were used in each experiment. Annealing and reverse
transcription were carried out as described previously [29],
and the extended products were analysed in 8% (w/v)
polyacrylamide/7 M urea gels. Sequencing reactions using
the same oligonucleotides were run in parallel.
S1 analysis. We PCR amplified a 506 bp fragment using
the forward primer 5¢-AGATGACCTGATTCCCTT
GG-3¢ corresponding to bp )476 to )459 from the ATG
2 start codon in the genomic sequence (GenBank accession
number NT_033778) and the reverse primer 5¢-GGACGC
CAGGCGGGCGGAAAAAATCG-3¢ corresponding to
bp 30–4 in the same sequence. The reverse oligonucleotide
was labelled at its 5¢ end with 100 lCi of [32P]ATP[cP] using
T4 polynucleotode kinase under standard conditions. The
3 probe (3.2 lCi) was hybridized with 75 lg of total RNA
extracted from adult Drosophila, for 15 h in 80% (v/v)
formamide, 40 mM Pipes pH 6.4, 1 mM EDTA, 0.4 M

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Drosophila a-F1-ATPase gene expression (Eur. J. Biochem. 271) 4005

NaCl. After adding four volumes of S1 nuclease buffer
(40 mM sodium acetate, 250 mM NaCl, 4 mM ZnSO4), the
sample was incubated with 150 units of S1 nuclease
(Pharmacia) for 60 min. The reaction was stopped with
4 M ammonium acetate and 0.1 M EDTA, and the nucleic
acids extracted with phenol and precipitated with ethanol.
The pellet was resuspended in 75 mM NaOH, and after
incubating for 15 min at 90 °C it was precipitated with
ethanol, resuspended in 98% (v/v) formamide, 25 mM
EDTA, 0.02% (w/v) bromophenol blue, 0.02% (w/v)
xylene cyanol, and analysed in 8% (w/v) polyacrylamide/
7 M urea gels. Sequencing reactions were run in parallel.
5¢ RACE experiments. We used the RLM-RACE kit from
Ambion Inc. (cat. # 1700), following the manufacturer’s
instructions. We used a-PE1 as the outer primer oligonucleotide for the a-F1-ATPase cDNA (see Primer extension analysis) and the inner primer was 5¢-TCTCCTCCA
GATCAGCCTTGGGGG-3¢.
RT-PCR analysis of a-F1-ATPase mRNA-3¢ ends
Total RNA from D. melanogaster embryos 0–18 h AEL or
adult flies was extracted using Trizol (Gibco-BRL) and
treated for 30 min with RNAse-free DNAse I (1 unit per lg
of RNA). Reverse transcription was carried out following a
protocol described previously [24]. Amplification of the 3¢
ends was performed with an oligo(dT) primer and one of
the two primers a-RT1 (5¢-TGCGCGGTCATCTGG

ACAA-3¢) and a-RT2 (5¢-ATCGCCAAGGACGGTGC
4 TA-3¢), at positions )184/)165 and )83/)64 with reference
to the translation stop codon, respectively.
Promoter constructs
A 909 bp DNA fragment from the 5¢ region upstream of the
D. melanogaster a-F1-ATPase gene (from )914 to )5
considering +1 the first nucleotide of the translation start
codon) was amplified by PCR from total DNA using the
primers pADm1 (forward; 5¢-AGCAGTCGACGA
AGCGACGAAGTGAAGCTGCGTGA-3¢) and pADm3
(reverse; 5¢-ATCCGTCGACATGCTTTTTAACTGTT
CG-3¢). After digestion with SalI (which recognizes the
sequence underlined in the oligonucleotides), the DNA
fragment was cloned into the pXp2 vector that contains the
luciferase reporter gene. The construct with the suitably
orientated insert was used as a parental DNA fragment for
the generation of a series of deletion constructs. These were
generated either by ExoIII digestion and blunt-end cloning,
by restriction endonuclease-based cloning, or by PCR
amplification and cloning of selected DNA fragments.
Finally, we obtained the constructs shown below, where +1
represents the transcription start point according to the data
presented here.
Mutagenesis of the GAGA element was achieved by
PCR and subcloning of the amplified fragment. The
oligonucleotides used for PCR were the luciferase gene
internal primer 5¢-GGCGTCTTCCATTTTACC-3¢ and
the oligonucleotide 5¢-CCGTCGACATTAATTTAATTT
ccccAATTATATTGCGTCG-3¢ in which the SalI recognition site is in bold and the GAGA element is replaced by the
sequence underlined (lowercase letters show the nucleotide

changes). The )146/+79 construct was used as a template
5 and the amplified fragment was cloned into the pXp2 vector.
This strategy was also used to mutate the alcohol dehydrogenase distal factor (Adf-1) element using the specific
primer 5¢-CCGTCGACATTAATTTGAGAAATTATAT
TGCGTCGCccgccggcCgcCacgGAGGGTGAC-3¢ (again
the SalI recognition site is in bold, the location of the
Adf-1 element is underlined, and nucleotides in lowercase
have been changed). A similar strategy was carried out to
construct the GAGA or/and Adf-1 mutants in the hybrid
promoters (see Results).
Cell transfection assays
The pXp constructs (5 lg) were transiently transfected into
Schneider S2 cells (as described previously [25]) to assay
their promoter activity. To correct for variations in the
efficiency of transfection, we cotransfected the cells with the
plasmid pSV-bGAL and the quantification of luciferase was
normalized to b-galactosidase activity. Luciferase activity
was determined using the Luciferase Assay System (Promega) according to manufacturer’s recommendations, and
b-galactosidase activity was measured as described previously [30].

Results
Transcriptional initiation sites of the D. melanogaster
a-F1-ATPase gene
We have previously characterized a cDNA encoding the
D. melanogaster a-F1-ATPase subunit [27]. To isolate the
corresponding D. melanogaster a-F1-ATPase gene and
flanking regions, we screened a genomic library using this
a-F1-ATPase cDNA as a probe. Two overlapping clones
containing the entire gene as well as 5¢ upstream and 3¢

downstream sequences were selected for further analysis.
The a-F1-ATPase gene maps to the 2R arm of the
D. melanogaster polytene chromosomes and its structure
is shown schematically in Fig. 1. It contains four exons
separated by three 624, 92 and 113 bp introns. The first

Fig. 1. Structure of the D. melanogaster a-F1-ATPase gene. Chromosomal location and structure of a-F1-ATPase. The gene maps to the
2R arm in D. melanogaster. In the schematic diagram of the gene
structure, white boxes represent introns, black boxes represent exons,
and the grey boxes represent UTRs. The line underneath the gene
shows the position of several restriction endonucleases. E: EcoRI; K:
KpnI; C: ClaI; Ev: EcoRV; S: SalI; P: PstI; B: BamHI.


4006 A. Talamillo et al. (Eur. J. Biochem. 271)

exon encodes the 5¢ untranslated region (5¢-UTR) as well as
the first 22 amino acids of the 23 residues which form the
targeting sequence. The last amino acid of the presequence,
the complete mature protein and the 3¢-UTR region are
encoded in exons 2–4. To determine the transcriptional
initiation sites of the a-F1-ATPase gene we first carried out
primer extension analysis using total RNA extracted from
adults or embryos 0–18 h AEL, and two different
32
P-labelled oligonucleotide primers (a-PE1 and a-PE2).
Both primers produced identical results in embryos and
adults, three transcriptional initiation sites being detected at
positions )86, )91 (the majority) and )120, considering
position +1 as the first nucleotide of the translation

initiation codon ATG (Fig. 2A,C).
This result was confirmed by high resolution S1 mapping
using a 506 bp probe that extended from the coding region
(position +30) to 477 bp upstream of the ATG. In this
analysis, several DNA fragments were protected (Fig. 2B),
with the strongest signal corresponding to position )91, the
prominent position detected by primer extension. The
position )91 is 22 nucleotides upstream of the transcription
startpoint previously described for this gene [27] (GenBank
accession number Y07894). Additionally, more weakly
protected smaller fragments were detected, reflecting the

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failure to precisely identify the initiation site typical in
housekeeping and TATA-less promoters. Finally, we performed a RACE study on the 5¢ end of the a-F1-ATPase
mRNA. All of the clones analysed end in the region )83 to
)115, most of them ending between )83 to )90 (Fig. 2C).
Interestingly, three clones identified in RACE experiments
detected the same nucleotides as the three weaker bands
shown by S1 mapping as the transcription start point.
Hence, we concluded that the D. melanogaster a-F1-ATPase gene contains a heterogeneous region responsible for the
initiation of transcription although a common initiation site
was located at position )91.
As frequently observed in other housekeeping genes,
neither TATA nor CCAAT boxes were found in canonical positions in the Drosophila a-F1-ATPase gene [31].
Moreover, in the 5¢ upstream region of the a-F1-ATPase
gene we did not find the TCAG/TTPy arthropod initiator
element [32]. Nevertheless, in the bovine and human a-F1ATPase genes, the transcriptional initiation sites located at
positions )91 and )120 lie within short conserved

sequences. Indeed, the sequence at the )91 transcriptional
start site, CCATCT, corresponds to a conserved
vertebrate initiator element (Inr; PyPyAT/APyPy), indicating that it may be involved in tethering the basal

Fig. 2. Identification of the transcription start site of the D. melanogaster a-F1-ATPase gene. (A) Primer extension analysis. Transcripts from
a-F1-ATPase mRNA primers were obtained with both a-PE1 and a-PE2 primers, although because the results were identical only those with the
primer a-PE2 are shown. Sequencing was performed with the same primers using a genomic clone as the template. (B) High resolution S1 mapping.
Total RNA from D. melanogaster was hybridized with a 506 bp probe from )476 to +30, the ATG translation start codon being referred to as +1.
Bands that were protected from S1 nuclease were visualized in 8% (w/v) acrylamide/urea gels, close to the sequencing reactions. (C) The nucleotide
sequence of a-F1-ATPase 5¢ upstream region. Black arrows show the transcription start points identified in the primer extension assays. White
arrows represent transcription start points from S1 mapping. The thickness of the black and white arrows is related to the intensity of the band.
Asterisks represent transcription start points from the 5¢ end amplification experiments (see Materials and methods). PCR products were cloned and
six of them were sequenced, identifying the nucleotide shown by asterisk as the 5¢ end of a-F1-ATPase cDNA.


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Drosophila a-F1-ATPase gene expression (Eur. J. Biochem. 271) 4007

transcriptional apparatus to the a-F1-ATPase promoter.
Furthermore, several short sequences commonly found
downstream of transcriptional initiation sites in Drosophila
promoters include ACGT, ACAA, ACAG, and AACA
[32], and these were detected at )17, )18, )36 and )103
positions of the a-F1-ATPase gene (position relative to
ATG). However, the region did not contain a canonical
downstream promoter element (DPE), which is recognized
by the TAFII60 factor [33]. Indeed, the short elements
described above probably substitute for the DPE motif in
the Drosophila a-F1-ATPase promoter.

Functional analysis of the a-F1-ATPase promoter region
The function of the Drosophila a-F1-ATPase promoter
region was characterized by transient transfection into
Schneider SL2 cells. A series of deletions of the 5¢ upstream
region of the gene were cloned in the pXp2 vector that
contains the luciferase reporter gene. A construct containing
the )823/+86 region (position +1 corresponds to the main
transcriptional initiation site located 91 nucleotides
upstream of the ATG initiation codon) promoted substantial luciferase activity in Schneider cells, 2,300-fold higher
than the native pXP2 vector (Fig. 3). This activity was
orientation-dependent and indicates that the a-F1-ATPase
5¢ proximal upstream region contains a strong promoter,
with similar activity in Schneider cells to the promoter of
the b-F1-ATPase gene [34] and 10-fold stronger than
the promoter of the gene encoding the catalytic subunit of
the mitochondrial DNA polymerase [25]. Similar luciferase
activity was maintained even when the upstream region was
reduced and contained only the )146/+86 region. In
contrast, a construct containing the )93/+86 region had
significantly lower promoter activity, reaching only 13% of
the maximal activity, while the )61/+86 construct directed
similar levels of luciferase activity as the pXp2 vector
(Fig. 3).
These results indicated that although the 53 bp DNA
region located between nucleotides )146/)93 does not

itself have promoter activity, it contains DNA elements
critical for the activation of the a-F1-ATPase promoter.
Computer analysis revealed the presence of two DNA
sequence motifs in this region potentially recognized by

the GAGA factor (GAF) and the alcohol dehydrogenase
distal factor (Adf-1), respectively (Fig. 4A). GAF is a
Drosophila regulatory protein that overcomes transcriptional repression produced by histones at the chromatin
level [35]. Adf-1 was initially identified as an activator of
the alcohol dehydrogenase (Adh) promoter and was
subsequently shown to control the expression of several
Drosophila genes [36,37]. Interestingly, it has been shown
that GAF and Adf-1 act together to remodel nucleosome
structure and activate transcription both in vitro and
in vivo [38].
To analyse the involvement of the potential GAF and
Adf-1 binding sites in activating the a-F1-ATPase
promoter, we eliminated the target sequences by sitedirected mutagenesis and examined the activity of the
mutated constructs in cell transfection assays. Mutating
the GAGA or Adf-1 elements individually significantly
reduced promoter activity by up to 40–60%, whereas
when both sites were abolished, the activity of the
promoter was reduced by 75% (Fig. 4B). In addition, we
carried out cotransfection studies in Schneider cells using
different a-F1-ATPase promoter constructs and a plasmid
that express GAF under the control of the actin 5C
promoter. The GAGA factor stimulated at least threefold
the activity of the promoter in constructs )397/+86 and
)146/+86, but had no effect on the activity of the
construct )93/+86, which does not contain the potential
GAF binding site (Fig. 4C).
The combination of GAF/Adf-1 has been shown to
activate transcription in a variety of promoter contexts.
Hence, we generated a construct containing a 56 bp
DNA fragment ()144/)89) that included the GAGA and

Adf-1 elements linked to the basal promoters of the
d-aminolevulinate synthase (ALAS) and b-F1-ATPase
genes [29,34]. In both constructs there was a substantial

Fig. 3. Functional analysis of the D. melanogaster a-F1-ATPase promoter. Scheme of the a-F1-ATPase promoter constructs used for transient
transfection assays in Schneider cells (see Materials and methods). The promoter regions are represented by solid lines and the luciferase reporter
gene is shown as a solid arrow. The numbers to the left of each construct indicate the limit of the promoter fragment with reference to the
transcription start point as established in this study. The relative promoter activities of the constructs measured in the luciferase assay are indicated
on the right by black boxes. The luciferase activity of the vector with no insert was defined as being equal to one. Luciferase activity was normalized
to the b-galactosidase activity of cotransfected control plasmid. Values are the means ± SD of at least five independent experiments.


4008 A. Talamillo et al. (Eur. J. Biochem. 271)

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Fig. 4. The transcription of a-F1-ATPase is controlled by a cassette containing the GAGA and Adf-1 elements. (A) Sequence of the )146/+94
(relative to the main transcription start point) a-F1-ATPase promoter region. The 56 bp region essential for promoter activity is boxed, and the
GAGA and Adf-1 elements in the DNA are underlined. The translation start codon and the main transcription start point are larger and in bold.
(B) Mutations either in the GAGA or Adf-1 binding sites significantly reduced a-F1-ATPase promoter activity. Each DNA element in the )146/
+86 promoter construct (whose luciferase activity was considered as 100%) was mutated and the activity of the mutant constructs was assessed.
Wild type and mutated GAGA and Adf-1 binding sites are shown by open and filled symbols, respectively. (C) Activation of the a-F1-ATPase
promoter was induced in presence of GAF. Different a-F1-ATPase promoter constructs were cotransfected with a plasmid expressing GAF and the
activity of constructs including the GAGA element increased significantly. The data show the luciferase activity of the a-F1-ATPase promoter
constructs cotransfected with GAF relative to that cotransfected with the non-GAF containing vector. Values are the means ± SD of at least three
independent experiments. Wild type and mutated GAGA and Adf-1 binding sites are shown by open and filled symbols, respectively.

increase of promoter activity independent of the
orientation. This increase was sevenfold in the case of
the ALAS promoter and reached 10-fold in the b-F1ATPase promoter (Fig. 5), indicating that this 56 bp

region has enhancer properties. Mutation of the GAF or
Adf-1 binding sites reduced the capacity of this fragment
to stimulate transcription by between 60% and 20%
depending on the promoter context (Fig. 5). In contrast,
cotransfection of the GAGA factor stimulated the
activity of the constructs containing the 56 bp activator
region linked to both heterologous promoters (data not
shown).
We performed phylogenetic footprinting of the 5¢
upstream region of the a-F1-ATPase gene and found that
the combination of GAF and Adf-1 binding sites is present
both in other Drosophila species (D. yakuba and D. pseudoobscura) and in Anopheles gambiae (data not shown). The
conservation of sequence elements during evolution is
indicative of their functional conservation, as has been
repeatedly demonstrated in several genes in the recent years
[39–41].

3¢ RNA processing of the D. melanogaster a-F1-ATPase
transcript
The expression of the a-F1-ATPase gene during development is coordinated with the expression of the nuclearencoded b-F1-ATPase gene and the mitochondrial-encoded
ATPase 6 and 8 genes [27]. Interestingly, two transcripts of
different sizes (roughly 2.2 and 1.8 kb) can be detected at all
developmental stages, as well as in adults [27] (Fig. 6A).
Furthermore, we previously isolated two cDNAs that differ
in their 3¢ region, suggesting that the difference in size may
be due to the alternative selection of polyadenylation sites
[27]. In order to precisely determine the origin of the two
transcripts, we carried out RT-PCR experiments on total
RNA extracted from embryos 0–18 h AEL or adults. In
these reactions, we used two different oligonucleotide

primers to map the 3¢ region of the gene (a-RT1 and
a-RT2; see Materials and methods) and an oligo(dT)
primer. With the combination of oligo(dT)/a-RT1 primers
two 700 and 320 bp fragments were amplified both from
embryos and adults, while the fragments amplified by the


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Drosophila a-F1-ATPase gene expression (Eur. J. Biochem. 271) 4009

Fig. 5. Functional analysis of the GAGA/Adf-1 cassette in heterologous promoters. Hybrid promoters indicate the a-F1-ATPase GAF/Adf-1 binding
cassette has enhancer properties. (A) The basal promoter activity of b-F1-ATPase is greatly increased when the a-F1-ATPase GAF/Adf-1 binding
cassette is cloned upstream, irrespective of its orientation. When either the GAF or Adf-1 binding sites are mutated, promoter activation is
impaired. (B) Similar results were obtained when the d-amino levulinate basal promoter was used. Asterisks represent mutations in the GAGA or
Adf-1 elements. Values are the means ± SD of at least four independent experiments.

Fig. 6. Alternative 3¢ end processing of the a-F1-ATPase mRNA. (A) Northern blots of mRNA from different stages of D. melanogaster embryogenesis and in adults using the a-F1-ATPase cDNA as a probe. Two different types of mRNAs are seen albeit in the same relative proportion, the
larger 2.2 kb mRNA predominating. (B) RT-PCR products from adult and embryo total RNA using a-RT1 (A) or a-RT2 (B) and oligo(dT)
primers. Results are consistent with the mRNA populations identified by Northern blotting. (C) Nucleotide sequence of the a-F1-ATPase gene 3¢
end. The last 12 amino acids and the stop codon are shown under the corresponding nucleotide sequence. The putative polyadenylation signals are
underlined and shown in bold.

oligo(dT)/RT-2 primers were approximately 600 and
230 bp in size (Fig. 6B). We sequenced the four DNA
fragments and confirmed that they corresponded to cDNAs

originated by the alternative selection of polyadenylation
signals located at position 101 and 462 downstream of the
TAA translation stop codon (Fig. 6C). Northern analysis

4010 A. Talamillo et al. (Eur. J. Biochem. 271)

revealed that the proportion of both transcripts remained
similar throughout development, as well as in different
tissues, such as the head, thorax and abdomen (data not
shown).

Discussion
The precise qualitative and quantitative regulation of
eukaryotic gene transcription is an extremely complex
process. A large body of experimental evidence has accumulated in recent years, highlighting the importance of two
critical regulatory processes: changes in chromatin structure; and in the binding of regulatory proteins to DNA
sequence-specific motifs located in the promoter and/or
enhancer regions. Although a plethora of transcription
factors and chromatin-remodelling proteins have been
identified, we still know very little about the detailed
mechanisms involved in regulating the activity of individual
gene promoters [42].
We are interested in understanding the transcriptional
regulation of genes encoding essential components of the
mitochondrial respiratory chain in Drosophila melanogaster.
The energetic demands of the different tissues in an
organism vary depending on their physiology, and can
respond to both environmental and developmental signals
[2,43]. Tissues with large energy demands contain mitochondria that are well differentiated, both structurally and
functionally, with highly developed cristae full of ATP
synthase complexes. For this reason, ATP synthase and in
particular the a-F1-ATPase and b-F1-ATPase catalytic

subunits have been often used as markers for mitochondrial
biogenesis [6,31,44,45]. The Drosophila a-F1-ATPase gene is
organized into four exons that are separated by three
introns, and that map in the 59AB region of the 2R polytene
chromosome. Two mutations have been mapped to the
aF1-ATPase gene, bellwether and colibri, which were
isolated in screens to identify lethal mutations in the 59AB
region [46] and larval growth defects [47], respectively.
We have characterized some of the elements involved in
regulating the transcription of the Drosophila aF1-ATPase
gene. Initially, we mapped the transcription initiation sites
by primer extension, S1 mapping analysis and mRNA 5¢end amplification. In this way, we localized a small region
containing several sites where RNA synthesis commences,
the strongest located 91 nucleotides upstream of the
translation initiation codon. The structural organization
of the transcription initiation region of the Drosophila a-F1ATPase gene constitutes a clear example of the flexibility in
the basal promoter region of animal genes. The promoter is
TATA-less, a characteristic of many housekeeping genes,
but nor does it contain an arthropod Inr element or DPE
element, two DNA motifs that functionally substitute for
the TATA box in several Drosophila genes [48,49]. Interestingly, the main transcriptional initiation site ()91) is located
within a canonical vertebrate Inr element that is conserved
in the promoter of the human a-F1-ATPase gene. Furthermore, the second transcription initiation site located at )120
also lies within a short element conserved in the transcriptionl initiation site of the bovine a-F1-ATPase gene. The
region located between the transcription initiation sites and
the translation start codon (5¢-UTR) contains several short
sequence elements highly represented in Drosophila

Ó FEBS 2004

promoters and that are likely to play an important role in
tethering the basal transcriptional machinery [48].
We previously described how the expression of the a-F1ATPase gene is regulated during Drosophila development
[27]. The a-F1-ATPase mRNA is stored in eggs, and during
the first stages of embryogenesis its steady-state level
decreases, before increasing as development proceeds.
Moreover, the a-F1-ATPase gene is more heavily transcribed in the head and thorax of adult flies in comparison
to the abdomen (data not shown), indicating that its
expression is differentially regulated at the tissue level. Here,
we analysed the function of the a-F1-ATPase 5¢ upstream
region by transiently transfecting Schneider cells with
constructs harbouring specific promoter regions. This
strategy allowed us to define the basal promoter of the
gene to a 93 bp proximal region and identify a region critical
for the transcriptional regulation of the gene. Full promoter
activity is strictly dependent on a 56 bp region located
between position )89 and )144 (relative to the main
transcription start point), that contains binding sites for the
GAGA factor and Adf-1. This small activator region is also
active on heterologous promoters in an orientation-independent manner. Transfection assays unequivocally demonstrate that both sites are functional and suggest that the
activity of this fragment is dependent of the GAF and Adf-1
regulatory proteins.
Both GAF and Adf-1 are ubiquitous transcription factors
critical for Drosophila viability. GAF is encoded by the
trithorax-like locus [50] which is required for the correct
expression of several homeotic genes. It was first identified
as an activator of the engrailed and Ultrabithorax promoters, and was later shown to regulate the activity of several
constitutive, inducible and developmentally regulated genes
(reviewed in [51]). GAF binds to GA-rich sequences and
specifically interacts with the trinucleotide GGA via a single

zinc-finger DNA binding domain [52]. GAF does not
directly regulate the RNA polymerase II basal machinery,
but it does activate transcription by relieving the repression
of histone in chromatin structure, permitting the access of
transcription factors to promoter or enhancer regulatory
regions [35]. Although direct stimulation by GAF has only
been demonstrated in roughly a dozen promoters, the
number of genes transcriptionally regulated by GAF is
likely to be much larger as GAF binds to hundreds of
euchromatic regions in salivary polytene chromosomes [53].
Moreover, it plays a broader role in chromosome structure
and function, including chromosome condensation and
segregation [54], and is therefore essential for nuclear
division.
The involvement of chromatin structure in a-F1ATPase gene expression is particularly interesting given
the significant number of genes regulated by SIN3 (a
histone deacetylase complex) that are involved in mitochondrial energy-generating pathways and encoding
components of the mitochondrial translation machinery
[55]. Adf-1 is a transcription factor containing a mybrelated DNA binding motif that interacts directly with
TAF subunits of the TFIID complex [37]. It is widely
expressed, although it is more concentrated in the
nervous system. Initially identified as an activator of
the Adh distal promoter, it also participates in the
transcriptional regulation of several genes including Dopa


Ó FEBS 2004

Drosophila a-F1-ATPase gene expression (Eur. J. Biochem. 271) 4011

decarboxylase [36] and fushi tarazu [37]. Loss of function
mutations of the Adf-1 gene are lethal, but interestingly
the hypomorphic mutation nalyot produces a mild
phenotype that affects the maturation of neuromuscular
junctions and disrupts olfactory memory [56].
A combination of GAF and Adf-1 factors is critical for
the transcriptional regulation of the alcohol dehydrogenase
gene [57]. Recently, in an elegant study of transgenic flies
harbouring different chimeric combinations of the GAGA
binding site from the hsp26 gene regulatory region and the
Adf-1 binding site from the Adh distal promoter, the
cooperative effect of GAF and Adh-1 was directly demonstrated in vivo [38]. Interestingly, GAF or Adh-1 binding
alone activates transcription but the establishment of
DNaseI hypersensitivity is dependent on the binding of
both factors [38].
Our results suggest that GAF and Adf-1 also act together
to regulate the expression of the Drosophila a-F1-ATPase
gene. Indeed, this is highlighted by the presence of similar
combinations of the two transcription factors in the
promoter proximal region of the orthologous genes of
different insects that diverged several million years ago,
including three Drosophila species and Anopheles gambiae.
We also have identified two a-F1-ATPase mRNAs,
which differ in the length of their 3¢ UTRs, by selection of
alternative polyadenylation sites. The ratios of the two
a-F1-ATPase mRNAs are similar at different developmental stages, as well as in different parts of the body of the
adult flies. Although this may reflect the random selection of
two poly(A) sites with different inherent properties, the
possibility that the tissue-specific regulation of polyadenylation plays an important physiological role can not be ruled
out. Polyadenylation of mRNA is essential for its transport

from the nucleus to the cytoplasm, for the stability of the
transcript, and for the efficiency of translation [58], and thus
poly(A) site selection is important for the control of gene
expression. In the last few years, a variety of transcription
units containing two or more poly(A) sites in their 3¢
terminal exons have been described (reviewed in [59]).
Furthermore, in some cases it has been shown that the ratio
of the transcripts containing different 3¢-UTRs determines
the amount of protein in specific tissues [59]. Experiments
are currently in progress to determine the potential
functional implication of the alternative selection of the
polyadenylation site in the Drosophila a-F1-ATPase
mRNA.

Acknowledgements
This work has been supported by grants from the Spanish ministry of
science and technology (BMC2001-1525) and from the FIS of the
Instituto de Salud Carlos III, Red de Centros RCMN (C03/08),
Madrid, Spain.

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