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

Expression of the Drosophila melanogaster ATP synthase a subunit gene is regulated by a transcriptional element containing GAF and Adf-1 binding sites Ana Talamillo1,*, Miguel Angel Fern

Expression of the Drosophila melanogaster ATP synthase a subunit gene is regulated by a transcriptional element containing GAF

and Adf-1 binding sites

Ana Talamillo1,*, Miguel Angel Ferna´ndez-Moreno1, Francisco Martı´nez-Azorı´n1, Bele´n Bornstein1,2, Pilar Ochoa1and 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

nuc-lear-encoded mitochondrial genes, we have performed a

detailed analysis of the promoter region of the a subunit of

the Drosophila melanogaster F1F0ATP 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

dehy-drogenase distal factor 1 In vitro mutagenesis revealed that

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 addi-tion, 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

The bulk of cellular ATP is synthesized through oxidative

phosphorylation (OXPHOS) that takes place in the

mito-chondria The OXPHOS system is composed of five

multisubunit complexes embedded in the inner

mitochond-rial 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

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 func-tion 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 F1soluble 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-transcrip-tional regulation of gene expression [5,6], changes in Ca2+

Correspondence to R Garesse, Departamento de Bioquı´mica,

Insti-tuto de Investigaciones Biome´dicas ‘Alberto Sols’ CSIC-UAM,

Fac-ultad de Medicina, Universidad Auto´noma de Madrid, C/Arzobispo

Morcillo 4, 28029 Madrid, Spain Fax: +34 91 5854001,

Tel.: +34 91 4975453, E-mail: rafael.garesse@uam.es

Abbreviations: Adf-1, alcohol dehydrogenase distal factor; GAF,

GAGA factor; OXPHOS, oxidative phosphorylation; n-DNA,

nuc-lear DNA; mtDNA, mitochondrial DNA; NRF, nucnuc-lear 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)

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

mito-chondrial 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

regula-tory 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

sys-tems 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

single-stranded 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],

establish-ing 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 68C in ZAP buffer [7% (w/v) SDS, 0.25M phosphate buffer, pH 7.2], washed in 0.5% (w/v) SDS, 2· NaCl/Cit at 55 C (NaCl/Cit: 0.15M NaCl/0.015Msodium citrate) and visualized by autoradio-graphy 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 (http://Flybase.bio.indiana.edu) 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 manu-facturer’s instructions Both DNA strands were sequenced

in their entirety and the sequences were analysed using the GCGprogramme (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 after egg laying (AEL), and 8 lCi

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/7M 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 start codon in the genomic sequence (GenBank

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 probe (3.2 lCi)

3 was hybridized with 75 lg of total RNA extracted from adult Drosophila, for 15 h in 80% (v/v) formamide, 40 m Pipes pH 6.4, 1 m EDTA, 0.4

NaCl After adding four volumes of S1 nuclease buffer

(40 mMsodium acetate, 250 mMNaCl, 4 mMZnSO4), the

sample was incubated with 150 units of S1 nuclease

(Pharmacia) for 60 min The reaction was stopped with

4Mammonium acetate and 0.1MEDTA, 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 90C 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/

7Murea 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

oligo-nucleotide for the a-F1-ATPase cDNA (see Primer

exten-sion 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

TA-3¢), at positions)184/)165

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

recogni-tion 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 and the amplified fragment was cloned into the pXp2

This strategy was also used to mutate the alcohol dehy-drogenase 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 (Pro-mega) according to manufacturer’s recommendations, and b-galactosidase activity was measured as described previ-ously [30]

Results

Transcriptional initiation sites of theD melanogaster a-F1-ATPase gene

We have previously characterized a cDNA encoding the

D melanogastera-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 Chromo-somal 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.

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

32P-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

failure to precisely identify the initiation site typical in housekeeping and TATA-less promoters Finally, we per-formed 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-ATP-asegene 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 canon-ical 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-F1-ATPasegenes, 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), indica-ting 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.

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

substan-tial 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 transcrip-tional 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 site-directed 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.

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-F1-ATPase 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

pseu-doobscura) 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 theD melanogaster a-F1-ATPase transcript

The expression of the a-F1-ATPase gene during develop-ment is coordinated with the expression of the nuclear-encoded b-F1-ATPase gene and the mitochondrial-nuclear-encoded ATPase 6and 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

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.

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

Fig 6 Alternative 3¢ end processing of the a-F1-ATPase mRNA (A) Northern blots of mRNA from different stages of D melanogaster embry-ogenesis 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.

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.

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

accu-mulated in recent years, highlighting the importance of two

critical regulatory processes: changes in chromatin

struc-ture; 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

mito-chondria 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-F1-ATPasegene 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]

Interest-ingly, 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

Further-more, the second transcription initiation site located at)120

also lies within a short element conserved in the

transcrip-tionl 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

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-F1-ATPasegene 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 tran-scribed 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-inde-pendent manner Transfection assays unequivocally dem-onstrate 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 promot-ers, 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-F1-ATPase gene expression is particularly interesting given the significant number of genes regulated by SIN3 (a histone deacetylase complex) that are involved in mito-chondrial energy-generating pathways and encoding components of the mitochondrial translation machinery [55] Adf-1 is a transcription factor containing a myb-related 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

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

demon-strated 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

developmen-tal 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

polyadeny-lation 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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