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 ´ ´ ´ ´ Ana Talamillo1,*, Miguel Angel Fernandez-Moreno1, Francisco Martınez-Azorın1, Belen Bornstein1,2, Pilar Ochoa1 and Rafael Garesse1 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 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: rafael.garesse@uam.es 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 June 2004, revised 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 (or a) and ATPase (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+ Ó FEBS 2004 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) and [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 (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 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 after egg laying (AEL), and 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 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 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, mM EDTA, 0.4 M Ó FEBS 2004 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, mM ZnSO4), the sample was incubated with 150 units of S1 nuclease (Pharmacia) for 60 The reaction was stopped with 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 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/ 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 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 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 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 It contains four exons separated by three 624, 92 and 113 bp introns The first Fig 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 Ó FEBS 2004 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 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 Ó FEBS 2004 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 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) Ó FEBS 2004 Fig 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 and 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 Ó FEBS 2004 Drosophila a-F1-ATPase gene expression (Eur J Biochem 271) 4009 Fig 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 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 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Nucleic Acids Res 25, 2547–2561  ... 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. .. 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... Fig 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,