Cloning and Characterization of Drosophila melanogaster Juvenile Hormone Epoxide Hydrolases (JHEH) and Their Promoters

Juvenile hormone epoxide hydrolase (JHEH) plays an important role in the metabolism of JH III in insects. To study the control of JHEH in female Drosophila melanogaster, JHEH 1, 2 and 3 cDNAs were cloned and sequenced. Northern blot analyses showed that the three transcripts are expressed in the head thorax, the gut, the ovaries and the fat body of females. Molecular modeling shows that the enzyme is a homodimer that binds juvenile hormone III acid (JH IIIA) at the catalytic groove better than JH III. Analyses of the three JHEH promoters and expressing short promoter sequences behind a reporter gene (lacZ) in D. melanogaster cell culture identified a JHEH 3 promoter sequence (626 bp) that is 10- and 25-fold more active than the most active promoter sequences of JHEH 2 and JHEH 1, respectively. A transcription factor (TF) Sp1 that is involved in the activation of JHEH 3 promoter sequence was identified. Knocking down Sp1 using dsRNA inhibited the transcriptional activity of this promoter in transfected D. melanogaster cells and JH III and 20HE downregulated the JHEH 3 promoter. On the other hand, JH IIIA and farnesoic acid did not affect the promoter, indicating that JH IIIA is JHEH’s preferred substrate. A transgenic D. melanogaster expressing a highly activated JHEH 3 promoter behind a lacZ reporter gene showed promoter transcriptional activity in many D. melanogaster tissues.


Introduction
Juvenile hormone (JH) in insects plays a pivotal role in larval metamorphosis [1], regulation of pheromone biosynthesis and vitellogenesis [2]. JH biosynthesis in insects is regulated by neuropeptides (allatotropins and allatostatins) secreted from the brain [3] and by JH esterase (JHE; EC 3.1.1.1) and JH epoxide hydrolase (JHEH; EC 3.3.2.3) [4][5][6]. JHE is a carboxylesterase and hydrolyzed the methyl ester group of JH, converting the enzyme to JH acid (JHA) that can be converted back to JH by JHA methyl transferase (JHAMT) [7][8][9], whereas the metabolism of JHA into JHA diol (JHAD) is irreversible [10][11][12]. Both JHA and JHAD are inactive, and this degradative pathway was reported for Drosophila melanogaster, Manduca sexta, Culex quinquefasciatus and Aedes aegypti [6,[13][14][15]. The crystal structure of JHEH from Bombyx mori has been described showing a homodimeric conformation of JHEH [16]. The jheh gene was cloned from M. sexta, Bombyx mori and Apis mellifera [17][18][19]. The expressed jheh in the honeybee did not show activity against JH III and probably does not participate in the JH III degradation pathway. A cDNA clone from D. melanogaster third instar larva expressing JHEH showed no activity against JH III and was proposed to be involved in xenobiotic biotransformation but not in JH metabolism [20]. Borovsky et al. [12] showed that female Ae. aegypti that was treated with [12-3 H]-(10R)-JH III preferentially metabolized the radioactively labeled JH III into JH IIIAD and JH IIIA at a ratio of acid diol/acid/diol of 17/4/1, showing that JHEH prefers to metabolize JH IIIA and JH III is metabolized first to JH IIIA by JHE and not to JH IIID. These authors also showed that female mosquitoes treated with  H]JH IIIA metabolized JH IIIA 17-fold faster into JH IIIAD than they metabolized JH III into JH IIID. To find out how jheh is regulated in D. melanogaster, it is necessary to study both the jheh promoter region that controls jheh and its transcription factors (TFs) that regulate the transcription of jheh in response to physiological and external stimuli [21,22]. Most eukaryotic TFs have a DNA-binding domain (DBD) and recent studies showed that TFs promote regulatory elements such as enhancers and promoters form looped structures to facilitate transcription regulation [23].
To study the function of promoters in insects, several techniques have been developed in which potential promoters are linked to reporter genes such as Luciferase (luc) or green fluorescent protein (gfp) to study their activity in insect cell lines such as D. melanogaster (D.Mel2). Using this approach, a novel 121 promoter from anhydrobiotic midge Polypedilum vanderplanki was identified, which was expressed in various insect cell lines [24]. In Spodoptera frugiperda, heat shock proteins promoters have been analyzed using Sf9 cells and various heat shock (SfHsp) genes that were cloned into a plasmid with a luc reporter. Using this technique, a strong heat shock promoter that drove the luciferase gene was found [25], and RNA polymerase-II-dependent promoters were isolated in order to facilitate more efficient protein expression in Sf21 and Hi5 cells [26]. The role of promoters in insect immunity was reported for D. melanogaster, in which the diptericin promoter plays an important role in the insect's immunity [27,28]. To characterize the Drosophila core promoter, Qi et al. [29] used synthetic promoters to facilitate large-scale analysis in S2 cell.
To find out how jheh is controlled in D. melanogaster, we cloned the cDNAs of jheh 1, 2 and 3 and followed their expressions using Northern blot analyses of the ovary, gut and head thorax of female D. melanogaster. Molecular 3D modeling of the three JHEHs and docking of JH IIIA and JH III into the catalytic groove of JH III allowed us to follow their binding specificities to the catalytic groove of JHEH 3. To find out the JHEH active promoter region, jheh 1, 2 and 3 promoters were cloned behind a lacZ reporter gene and analyzed in D. melanogaster D.Mel2 cells. The effects of JH III, JH IIIA, 20Hydroxyecdysone (20HE) and farnesoic acid on the jheh 3 promoter were studied. This report is the first to show how JH III and TF Sp1 regulate the jheh 3 promoter of D. melanogaster.

Insects, Cells, Chemicals and Incubation Conditions
D. melanogaster were reared at 21 • C on a diet of instant Drosophila medium containing blue color (Carolina Biological Supply Company, North Carolina USA). Drosophila melanogaster D.Mel2 cells (2 × 10 5 cells/mL) were grown in serum-free medium (SFM) containing 9% Glutamine (1.5 mL) in 24-well sterile plates (Invitrogen, Waltham, MA, USA) at 27 • C following the manufacturer's guidelines. Cells were stored in liquid N 2 and were split no more than 10-15 times. The cells were checked often for viability with trypan blue, as recommended by the manufacturer. To transfect D. melanogaster cells with different promoter sequences, Cellfectin (10 µL) was mixed with SFM (500 µL) containing Glutamine (9%) (Invitrogen, Waltham, MA USA) and plasmid pCaSpeR-AUG-βgal (2 µg) carrying promoter's sequence to be tested, and the mixture was incubated with D.Mel2 cells (2 × 10 5 cells) for 3 h. After incubation, the medium was removed, and fresh SFM containing 9% Glutamine was added to each well and the cells were incubated for 72 h at 27 • C before testing for β-galactosidase activity. Transfected D.Mel2 cells were lysed in 0.25 M Tris-HCl pH 8.0 buffer (Invitrogen), and the enzymatic activity of β-galactosidase was followed by measuring the absorbance at 420 nm. The absorbance reading was converted into β-galactosidase activity expressed in milliunits (mU), equivalent to 1 nmole of β-D-galactose hydrolyzed per min, using a purified β-galactosidase with known activity (Sigma, St Louis, MO, USA), and a calibration curve was constructed following the manufacturer's guidelines. pCaSpeR-AUG-βgal [30] was obtained from the Drosophila Genomics Resource Center Indiana University (Bloomington, IN, USA) and the D. melanogaster λ-ZAP II cDNA library of 2-week-old males and females was obtained from Stratagene (La Jolla, CA, USA). Farnesoic acid and [12-3 H](10R) JH III were provided by Professor G. Prestwich (University of Utah). JH III acid (JH IIIA) and  H]JH IIIA were synthesized by hydrolyzing the methyl ester of JH III, converting it into JH IIIA by incubating JH III with 0.5 N NaOH in ethanol for 24 h at room temperature. The JH IIIA was purified by reversed phase C 18 HPLC and stored in hexane [9,31]. Next, 20-Hydroxy ecdysone (20HE) was obtained from Sigma and its mimic (RH5992) was provided by Professor G. Smagghe (university of Ghent, Ghent, Belgium). TRIzol for RNA extraction from female D. melanogaster and D.Mel2 cells was obtained from GIBCO BRL (Gaithersburg, MA, USA), and o-nitrophenyl,β-D galactopyranoside (ONPG) was obtained from Sigma (St. Louis, MO, USA). dsRNA was synthesized using a HiScribe RNAi transcription kit (New England BioLabs, Beverly, MA, USA). A dsDNA was cloned into pLitmus 28i and the dsDNA was converted into dsRNA using T7 and PCR. The integrity of the dsRNA was determined by agarose gel electrophoresis, following the manufacturer's guidelines.

Injections into Embryos
D. melanogaster w 1118 /yw (white eye) embryos were injected with pCaSpeR-AUGβgal carrying JHEH 3 promoter sequence (627 bp) by BestGene (Chino Hills, CA) using p-element transformation. The surviving G 0 adults were crossed with w 1118 /yw and the G 1 adults were crossed again with w 1118 /yw, and the resultant G 2 transformants were balanced. Thirty-two transformed D. melanogaster males and females were then assayed for β-galactosidase activity by lightly anesthetizing them with ether. The flies were added to a 96-well microtiter plate containing 50 mM sodium phosphate buffer pH 8.0, 2 mM potassium ferrocyamide, 0.3% X-Gal and 15% Ficoll (100 µL) in each well. D. melanogaster abdomens were poked with a finely drawn glass capillary and the staining solution was allowed to penetrate and absorb inside each fly overnight at room temperature in the dark. After incubation, transformed flies were observed under a Nikon-dissecting microscope for areas that were stained blue. Controls that were not transformed were treated similarly.

RNA Extraction and Purification
Total RNA was extracted from D. melanogaster tissues and adults (150 per group) with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Northern Blot Analysis
An Ambion Northern Max kit (Ambion, Foster City, CA, USA) was used for the Northern blot analysis. Total RNA was extracted in Trizol from female D. melanogaster isolated guts, fat bodies ovaries and intact females (150 per group) 3 days after emergence. RNA (15 µg/lane) samples that were extracted were separated on denaturing 1.0% formaldehyde agarose gel at 100 V for 1.5 h [35], transferred to Hybond-N + nylon membrane and hybridized with [ 32 P]-labeled probes for JHEH 1, 2 and 3 (Tables S1-S3). Hybridized membranes were exposed to X-ray film for 48 h at −80 • C and then developed [35,36] (Figure 3a-c). Radioactively labeled probes were stripped from the membrane in a boiling solution of 0.1% SDS, and the membrane was scanned with a Geiger counter to confirm that the probes were completely removed. The stripped membranes were then hybridized with D. melanogaster actin transcript probe (355 bp) (accession number BT050557.1) to show equal transfer to the membrane in all lanes (Figure 3a-c). The actin probe cDNA was prepared by PCR using D. melanogaster λ-Zap II cDNA library (Stratagene) with primer pair DB657 (forward) 5 CAGGTGATCACCATTGGCAACGGAGCG 3 (t m 62 • C) and DB658 (reverse) 5 CCTGCTTCGAGATCCACATCTGCTG 3 (t m 63 • C). The cDNA actin probe was labeled with [ 32 P] using a Rediprime II DNA labeling system (Amersham, Chicago, IL, USA) [35,36]. The Northern blot analyses were repeated twice, showing similar results.
Biomolecules 2022, 12, 991 5 of 26 Figure 1. jheh 1, 2 and 3 cDNA cloning strategies and primers (arrows) that were used in the cloning and the sequencing. (a). jheh 1 cDNA amino acids and nucleotides sequence, the membrane anchor sequence WWG is single underlined, the catalytic amino acids triad DYD is double underlined, the H that participates in the catalytic activity is double underlined and the polyadenylation signal sequence AATAAA is single underlined. (b). jheh 2 cDNA amino acids and nucleotides sequence, the membrane anchor sequence YWG is single underlined, the catalytic amino acids triad DYE is double underlined, the H that participates in the catalytic activity is double underlined and the poly adenylation signal ATTAAA is single underlined. (c). jheh 3 cDNA amino acids and nucleotides sequence, the membrane anchor sequence YWG is single underlined, the catalytic amino acids triad DYD is double underlined, the H that participates in the catalytic activity is double underlined and the poly adenylation signal is not shown.

Sequencing of JHEH 2
JHEH 2 cDNA was amplified and sequenced using forward and reverse primers (Table S2) generating overlapping amplicons of 432, 404, 515, 315, 292, 492, 413 and 386 nt covering the entire JHEH 2 cDNA (Figure 1b). The 3′ and 5′ ends were amplified using Figure 1. jheh 1, 2 and 3 cDNA cloning strategies and primers (arrows) that were used in the cloning and the sequencing. (a). jheh 1 cDNA amino acids and nucleotides sequence, the membrane anchor sequence WWG is single underlined, the catalytic amino acids triad DYD is double underlined, the H that participates in the catalytic activity is double underlined and the polyadenylation signal sequence AATAAA is single underlined. (b). jheh 2 cDNA amino acids and nucleotides sequence, the membrane anchor sequence YWG is single underlined, the catalytic amino acids triad DYE is double underlined, the H that participates in the catalytic activity is double underlined and the poly adenylation signal ATTAAA is single underlined. (c). jheh 3 cDNA amino acids and nucleotides sequence, the membrane anchor sequence YWG is single underlined, the catalytic amino acids triad DYD is double underlined, the H that participates in the catalytic activity is double underlined and the poly adenylation signal is not shown.

Identification of JHEH 1, 2 and 3 Promoter Regions and Introns
The D. melanogaster genomic scaffold (1420000133386047 Section 8, GenBank accession number AE003798.1) was analyzed using Lasergene Genomic Suite software (DNASTAR) to determine the location of introns, exons and promoter regions of JHEH 1, 2 and 3 ( Figure 2).  An Ambion Northern Max kit (Ambion, Foster City, CA, USA) was used for the Northern blot analysis. Total RNA was extracted in Trizol from female D. melanogaster isolated guts, fat bodies ovaries and intact females (150 per group) 3 days after emergence. RNA (15 μg/lane) samples that were extracted were separated on denaturing 1.0% formaldehyde agarose gel at 100 V for 1.5 h [35], transferred to Hybond-N + nylon membrane and hybridized with [ 32 P]-labeled probes for JHEH 1, 2 and 3 (Tables S1-S3). Hybridized membranes were exposed to X-ray film for 48 h at −80 °C and then developed [35,36] (Figure  3a-c). Radioactively labeled probes were stripped from the membrane in a boiling solution of 0.1% SDS, and the membrane was scanned with a Geiger counter to confirm that the probes were completely removed. The stripped membranes were then hybridized

Sp1Transcription Factor (TF)
Northern blot analysis was performed on D.Mel2 cell (2 × 10 5 cells/mL) that were transfected for 3 h at 27 • C in the presence of Cellfectin with pJHEH#3L3 short promoter (627 bp) cloned in pCaSpeR-AUG-βgal and Sp1 TF dsRNA (363 bp, accession number NM_132351). The Sp1 TF dsRNA was amplified by forward primer DB942 (5 ATGT-TATTGATATTGAAATATAAT 3 ) and reverse primer DB943 (5 TCGTGGTACCAATTG-TAGTGTCGATC 3 ). After transfection, washing and removal of Cellfectin, the cells were grown for 72 h at 27 • C and the RNA extracted with TRIzol and assayed by Northern blot analysis using a [ 32 P] labeled DNA probe of TF Sp1 (363 bp) and [ 32 P] labeled actin probe, as discussed above (Section 2.5.1). Control cells were transfected with pJHEH#3L3 promoter, but were not treated with TF Sp1 dsRNA. The Northern blot analysis was repeated twice, showing similar results.
The geometric and thermodynamic qualities of the three-dimensional models are summarized in Table 1. Although modeling of extended loops gives conformations of poorly reliable geometric and thermodynamic qualities, the calculated QMEAN scores nevertheless gave acceptable values of 0.72, 0.75 and 0.70 for the JHEH 1, JHEH 2 and JHEH 3 homodimers models, respectively ( Table 1). Docking of JH III and JH IIIA to the 3D model of JHEH 3 was performed with the YASARA structure program. Docking experiments were also performed at the SwissDock web server (http://www.swissdock.ch (accessed on 14 June 2022)) [44,45], to confirm our docking results, and molecular cartoons were drawn with Chimera [45] (Figure 4). Table 1. Geometric and thermodynamic quality of the three-dimensional models built for JHEH 1, 2 and 3 homodimers (A + B chain).

Cloning and Characterization of JHEHs Promoters
The promoter regions of JHEH 1, 2 and 3 were identified by blasting the D. melanogaster genome using Lasergene Genomic Suite software (DNASTAR) (see Section 2.4 and Figure 2). Promoter sequences of JHEH 1, 2 and 3 were tested for transcriptional activities by sequentially cutting the promoters into smaller-length sequences using PCR and primers at different 5 end positions of the promoter and the same primer at the 3 end. Each primer carried a restriction site enzyme to allow unidirectional cloning into plasmid pCaSpeR-AUG-βgal at the multiple cloning site ( Figure S1). The recombinant plasmid (2 µg) carrying the short promoters was transfected into D.Mel2 cells using Cellfectin for 3 h at 27 • C.
The medium with the Cellfectin was removed and replaced with new serum-free medium (SFM), the cells were incubated at 27 • C for 72 h and promoter transcriptional activity was tested using a β-galactosidase assay, as described earlier (Section 2.1). JH III, JH IIIA and farnesoic acid in hexane (5 µL) were each added to the medium (1.5 mL) after Cellfectin was removed, and the cells were incubated for 72 h at 27 • C. The same amount of hexane (5 µL) without JH III, JH IIIA or farnesoic acid was added to the control cells. Ecdysone was dissolved in sterile medium and (5 µL) was added to the transfected cells after Cellfectin was removed, and the cells were incubated for an additional 72 h at 27 • C.

Statistical Analysis
Data were analyzed using Student's t-test using GraphPad Prism v5.0. Results were considered statistically significant when p < 0.05 and expressed as means of 3 determinations ± SEM., except where otherwise stated.

cDNA Sequencing of JHEH 1, 2 and 3
Full-length cDNA sequences were generated using PCR and RT-PCR strategies described in the Materials and Methods section (Section 2.4) (Figure 1a (Figure 1a-c) and poly adenylation sequences of AATAAA at 1429, 1478 and ATAAA at 1419 for JHEH 1, 2 and 3, respectively. The three JHEH sequences exhibit a typical catalytic groove of D241, D417 and H444 for JHEH 1, D236, E412 and H439 for JHEH 2 and D236 and D412 and H439 for JHEH 3 (Figure 1a-c), including W166, W163 and W163 for JHEH 1, 2 and 3, respectively, which plays an important role in binding the substrate in the active groove of JHEH. To find the promoter regions, Network Promoter Prediction software

Statistical Analysis
Data were analyzed using Student's t-test using GraphPad Prism v5.0. Results were considered statistically significant when p < 0.05 and expressed as means of 3 determinations ± SEM., except where otherwise stated.

cDNA Sequencing of JHEH 1, 2 and 3
Full-length cDNA sequences were generated using PCR and RT-PCR strategies described in the Materials and Methods section (Section 2.4) (Figure 1a-c). The sequences were deposited in the GenBank (accession numbers AF517545, AF AF517546 and AF517547).  (Figure 1a-c) and poly adenylation sequences of AATAAA at 1429, 1478 and ATAAA at 1419 for JHEH 1, 2 and 3, respectively. The three JHEH sequences exhibit a typical catalytic groove of D241, D417 and H444 for JHEH 1, D236, E412 and H439 for JHEH 2 and D236 and D412 and H439 for JHEH 3 (Figure 1a-c), including W166, W163 and W163 for JHEH 1, 2 and 3, respectively, which plays an important role in binding the substrate in the active groove of JHEH. To find the promoter regions, Network Promoter Prediction software was used [46] (http://www.fruitfly.org/seq_tools/promoter.html (accessed on 15 June 2022)), predicting promoter regions of 0.84 kb, 2.6 kb and 3.0 kb for jheh 1, 2 and 3, respectively. Three introns were found in the jheh 1 and 2 exons and 2 intron in the jheh 3 exon. The jheh sequence is directionally following the D. melanogaster genome (Figure 2)

Northern Blot Analyses
To detect jheh 1, 2 and 3 transcripts in female D. melanogaster, head thoraxes, gut, ovaries, fat bodies and whole females were removed and assayed using Northern blot analyses with jheh 1, 2 and 3-specific [ 32 P] labeled probes (Tables S1-S3). Northern blot analyses show that jheh 1, 2 and 3 transcripts of 1.47, 1.54 and 1.44 kb, respectively, are found in the head thorax, gut, ovary, fat bodies and whole insect RNA extracts. These results show that jheh 1, 2 and 3 transcripts are synthesized in many tissues (Figure 3a-c). Because JH has many physiological targets, and JHEH is the main enzyme that irreversibly inactivates JH III, it is found to be ubiquitously distributed. The densities of the jheh 1 and 3 transcript bands after Northern blot analyses are similar, whereas the jheh 2 transcript bands are 3-4-fold denser, indicating that more jheh 2 transcript is synthesized by female D. melanogaster head thorax, gut, ovary and fat body compared with jheh 1 and 3 transcripts (Figure 3a-c). Northern blot analyses of the act transcript indicate that the transfer was even in all the lanes.

Molecular Modeling of JHEH 1, 2 and 3
The three-dimensional models built for the three JHEH homodimers JHEH 1, JHEH 2 and JHEH 3 of D. melanogaster are very similar (Figure 4a-c). They consist of two dimers associated by non-covalent bonds as a homodimer. Each monomer conformation predominantly contains α-helices associated with a single β-sheet structure. Because of these structural similarities, JHEH 1, JHEH 2 and JHEH 3 are closely superimposed on each other, with a root mean square deviation (RMSD) of 0.87 A between 381 pruned atom pairs corresponding to the structurally conserved α-helices and β-sheets of the three models (Figure 4d). A catalytic groove containing the catalytic triad D236, D412 and H439 occurs at the surface of each monomer. The H161 G162 W163 P164 motif characteristic of the epoxide hydrolases enzymes, and a tyrosine residue (Y382) occurs on the edge of the catalytic groove (Figure 4e,f). In each monomer, the catalytic groove is well exposed and leads to a depression that separates both monomers in the homodimeric structure. Docking experiments performed with JH III and JH IIIA resulted in the anchoring of both hormones to the catalytic groove of JHEH 3 via a network of four and seven hydrogen bonds, respectively (Figure 4e,f). Both hormones are similarly linked to the amino acid residues D236 and D412 of the catalytic triad, and to Y382 located on the edge of the catalytic groove. The linkage of JH III and JH IIIA only differs by the number of hydrogen bonds connecting the epoxide group to D236 (a single H-bond for JH III compared with two H-bonds for JH IIIA) and the acidic group to D412 (two H-bonds for JH III compared with four H-bonds for JH IIIA), showing a different accommodation of both hormones in the catalytic groove of JHEH 3 (Figure 4e,f). Therefore, JH IIIA shows a better affinity for JHEH 3 compared with JH III.

Activities of JHEH 1, 2, 3 Promoters
To follow the activity of each JHEH promoter, shorter promoter sequences were amplified by PCR (Tables S4-S6, Figures S2-S4) and cloned into plasmid pCaSpeR-AUG-βgal at the multiple cloning site behind lacZ ( Figure S1), and the β-galactosidase transcriptional activity driven by each truncated promoter sequence was assayed (Section 2.8)

JHEH 3 Promoter
JHEH 3 promoter sequences 1562 bp and 852 bp (pJHEH#3L1 and pJHEH#3L2, respectively (Figure 7a), were each cloned into plasmid pCaSpeR-AUG-βgal and expressed in D.Mel2 cells, and β-galactosidase activities of 19 mU and 22 mU were determined in cells that were transfected with pJHEH#2L1 and pJHEH#3L2 promoters (Figure 7b). When the promoter sequence was shortened to 627 bp (pJHEH#3L3, Figure 7b), the β-galactosidase activity expressed in D.Mel2 cells increased by 4.8-fold to 106 mU. Shortening the promoter sequence to 452, 332, 212 and 112 bp and expressing the short promoter sequences in D.Mel2 cells reduced the β-galactosidase activity to 23 mU, 18 mU, 5 mU and 1 mU, respectively (pJHEH#3L4, pJHEH#3L5, pJHEH#3L6, pJHEH#3L7, Figure 7b). The increase in β-galactosidase activity expressed by promoter pJHEH#3L3 is significantly higher (p < 0.05) than the longer and shorter segments of the JHEH 3 promoter sequences that were tested (Figure 7a,b). These results prompted us to examine the JHEH 3 promoter sequence between pJHEH#3L2 and pJHEH#3L3 (225 bp, Figure 8a) for TFs or inhibitory sequences. Several TFs were identified (bHLH-CS, H2B-CCAAT, NF-E1-CS2, CAAP-site, TCF-1 and sp1) (Figure 8a). JHEH 3 promoter sequences between pJHEH#3L2 and pJHEH#3L3 ( Figure 8a) were amplified as discussed above, using forward and reverse primers (Table S6) to exclude some of the TF or mutate them by changing the promoter's nucleotides sequence or removing a whole segment of nucleotides (DB876 and DB877, respectively (Figure 8a). The amplified promoter sequences carrying restriction enzyme sequences at the 5 and the 3 ends BamHI and KpnI, respectively (Table S6), were cloned into pCaSpeR-AUG-βgal. The β-galactosidase activity showed a two-fold increase in activity when DB 843, DB875, DB876 and DB877 (Figure 8a-c) were used compared with full length pJHEH#3L2. However, the increase in activity was still 2 to 2.5-fold lower than the activity of pJHEH#3L3. These results indicate that TF Sp1 plays a major role in activating the pJHEH#3L3 promoter sequence (627 bp) ( Figures 7 and 8). In control D.Mel2 cells that were not transfected or transfected with an empty pCaSpeR-AUG-βgal plasmid, no β-galactosidase activity was found, indicating that the observed enzymatic activities were due to JHEH 3 promoter sequences driving lacZ.  Figure  7, by testing different promoter lengths and mutating or deleting TF TCF-1 DNA-binding site using primers DB877 and DB876, respectively (b,c).

Northern Blot Analysis and dsRNA Knockdown of TF Sp1
To find out if TF Sp1 plays a major role in the activation of promoter pJHEH#3L3, Sp1 dsRNA was co-transfected with pCaSpeR-AUG-βgal carrying promoters pJHEH#3L2 or pJHEH#3L3 (materials and methods Section 2.5.2). The D.Mel2 transfected cells were grown for 72 h and analyzed using Northern blot analysis for Sp1transcript and β-galactosidase activity. Northern blot analysis of D.Mel2 cells that were transfected with pJHEH#3L3 and not treated with dsRNA (control) detected a TF Sp1 transcript above 3.0 kb, whereas cells that were transfected with dsRNA showed degraded, shorter transcripts at 2.5 kb and 1.5 kb (Figure 9a, right lane). The similar actin bands in each lane indicate an even transfer of the transcripts by the Northern blot (Figure 9a). For cells that were transfected with pCaSpeR-AUG-βgal carrying promoters pJHEH#3L2 or pJHEH#3L3 and not

Northern Blot Analysis and dsRNA Knockdown of TF Sp1
To find out if TF Sp1 plays a major role in the activation of promoter pJHEH#3L3, Sp1 dsRNA was co-transfected with pCaSpeR-AUG-βgal carrying promoters pJHEH#3L2 or pJHEH#3L3 (materials and methods Section 2.5.2). The D.Mel2 transfected cells were grown for 72 h and analyzed using Northern blot analysis for Sp1transcript and β-galactosidase activity. Northern blot analysis of D.Mel2 cells that were transfected with pJHEH#3L3 and not treated with dsRNA (control) detected a TF Sp1 transcript above 3.0 kb, whereas cells that were transfected with dsRNA showed degraded, shorter transcripts at 2.5 kb and 1.5 kb (Figure 9a, right lane). The similar actin bands in each lane indicate an even transfer of the transcripts by the Northern blot (Figure 9a). For cells that were transfected with pCaSpeR-AUG-βgal carrying promoters pJHEH#3L2 or pJHEH#3L3 and not incubated with dsRNA, their β-galactosidase transcriptional activity was 41 and 78 mU, respectively. On the other hand, for cells that were transfected with pJHEH#3L2 and pJHEH#3L3 in the presence of dsRNA, their β-galactosidase transcriptional activity was low (1 and 2 mU, respectively) (Figure 9b). Drosophila cells that were not transfected or transfected with empty plasmid (Controls) expressed very low β-galactosidase activity (Figure 9b). These results indicate that TF Sp1 plays an important role in upregulating JHEH 3 promoter. incubated with dsRNA, their β-galactosidase transcriptional activity was 41 and 78 mU, respectively. On the other hand, for cells that were transfected with pJHEH#3L2 and pJHEH#3L3 in the presence of dsRNA, their β-galactosidase transcriptional activity was low (1 and 2 mU, respectively) ( Figure 9b). Drosophila cells that were not transfected or transfected with empty plasmid (Controls) expressed very low β-galactosidase activity (Figure 9b). These results indicate that TF Sp1 plays an important role in upregulating JHEH 3 promoter.

Effect of JHEH 3 pJHEH#3L3 Promoter on Transgenic D. Melanogaster
Our Northern blot analyses showed that JHEH transcripts are found in the head thorax, gut, ovary, the fat body and the whole-body extract of female D. melanogaster. Therefore, the most active JHEH promoter pJHEH#3L2 (627 bp, Figure 7a) was cloned into pCaSpeR-AUG-βgal and injected with a helper plasmid into D. melanogaster embryos. Transformed flies that were assayed for β-galactosidase activity and observed under a dissecting microscope exhibited enzymatic activity in many parts of the transgenic flies, in the abdomen, thorax, leg muscle and the junction between the abdomen and thorax (Figure 12), indicating that the JHEH 3 promoter is active in many D. melanogaster tissues, confirming our Northern blot analysis (Figure 3c). Control flies that were transformed

Effect of JHEH 3 pJHEH#3L3 Promoter on Transgenic D. Melanogaster
Our Northern blot analyses showed that JHEH transcripts are found in the head thorax, gut, ovary, the fat body and the whole-body extract of female D. melanogaster. Therefore, the most active JHEH promoter pJHEH#3L2 (627 bp, Figure 7a) was cloned into pCaSpeR-AUG-βgal and injected with a helper plasmid into D. melanogaster embryos. Transformed flies that were assayed for β-galactosidase activity and observed under a dissecting microscope exhibited enzymatic activity in many parts of the transgenic flies, in the abdomen, thorax, leg muscle and the junction between the abdomen and thorax (Figure 12), indicating that the JHEH 3 promoter is active in many D. melanogaster tissues, confirming our Northern blot analysis (Figure 3c). Control flies that were transformed with an empty plasmid did not show transcriptional activity of β-galactosidase (results not shown). with an empty plasmid did not show transcriptional activity of β-galactosidase (results not shown).

Discussion
We identified and sequenced the cDNAs of JHEH 1, 2 and 3 from female D. melanogaster using extracted RNA and a λ Zap cDNA library. The cDNAs code for proteins of 474, 463 and 468 amino acids, respectively (Figure 1a-c). The proteins contain XWG anchor motif (W40, W41 and G42 for JHEH 1; Y41, W42 and G43 for JHEH 2 and Y42, W43 and G44 for JHEH 3) (Figure 1a-c), which is involved in subcellular localization of JHEH in Bombyx mori, Lymantria dispar and Apolygus lucorum, and is highly conserved in higher organisms, but absent in fungi, bacteria and protozoa [38,47,48]. JHEH 1, 2 and 3 amino acid sequences contain a HGWP motif and W166, W163 and W163 as part of the motif in JHEH 1, 2 and 3, respectively (Figure 1a-c). The catalytic amino acids triad of JHEH 1, and 3 is D, D, H and for JHEH 2 D, E, H (Figure 1a-c). Substitution of D with E is found in the late trypsin of Aedes aegypti, E196 replaced D at the specificity pocket of A. aegypti late trypsin without affecting the enzyme's activity [49]. The jheh of D. melanogaster sequence exhibits three related genes, jheh 1, jheh 2 and jheh 3, with introns and promoters of different lengths (0.84, 2.6 and 3.0 kb, respectively) ( Figure 2). The jheh 2 of D. melanogaster larvae was suggested to function as a microsomal Epoxide Hydrolase (mEH) that does not participate in the JH III metabolism but works in concert with other xenobiotic metabolizing enzymes [20]. It is interesting to note that these authors also reported that a whole-body

Discussion
We identified and sequenced the cDNAs of JHEH 1, 2 and 3 from female D. melanogaster using extracted RNA and a λ Zap cDNA library. The cDNAs code for proteins of 474, 463 and 468 amino acids, respectively (Figure 1a-c). The proteins contain XWG anchor motif (W40, W41 and G42 for JHEH 1; Y41, W42 and G43 for JHEH 2 and Y42, W43 and G44 for JHEH 3) (Figure 1a-c), which is involved in subcellular localization of JHEH in Bombyx mori, Lymantria dispar and Apolygus lucorum, and is highly conserved in higher organisms, but absent in fungi, bacteria and protozoa [38,47,48]. JHEH 1, 2 and 3 amino acid sequences contain a HGWP motif and W166, W163 and W163 as part of the motif in JHEH 1, 2 and 3, respectively (Figure 1a-c). The catalytic amino acids triad of JHEH 1, and 3 is D, D, H and for JHEH 2 D, E, H (Figure 1a-c). Substitution of D with E is found in the late trypsin of Aedes aegypti, E196 replaced D at the specificity pocket of A. aegypti late trypsin without affecting the enzyme's activity [49]. The jheh of D. melanogaster sequence exhibits three related genes, jheh 1, jheh 2 and jheh 3, with introns and promoters of different lengths (0.84, 2.6 and 3.0 kb, respectively) ( Figure 2). The jheh 2 of D. melanogaster larvae was suggested to function as a microsomal Epoxide Hydrolase (mEH) that does not participate in the JH III metabolism but works in concert with other xenobiotic metabolizing enzymes [20]. It is interesting to note that these authors also reported that a whole-body extract of D.
melanogaster larva converted JH III into its diol, indicating that D. melanogaster larvae have functional JHEHs that can metabolize JH III. The exact function(s) of JHEH 2 in adult D. melanogaster are yet to be determined. Northern blot analyses show that JHEH 1, 2 and 3 transcripts are found in the head thorax, gut, ovary, fat body and whole extract of females, confirmed by our sequencing data, which show that JHEH 1, 2 and 3 contain membrane anchoring sequences that allow the enzymes to be anchored to membranes of many tissues of female D. melanogaster. It is interesting to note that the intensities of JHEH 2 transcript bands are much higher than the other transcript bands of JHEH 1 and JHEH 3 (Figure 3a-c), indicating that perhaps more JHEH 2 is synthesized by female D. melanogaster, and that the enzyme may have a dual function as a JH III metabolizing enzyme and also as a mEH [20]. Three-dimensional molecular models of the three JHEHs show that the proteins are associated as homodimers by non-covalent bonding, and are made up primarily of α-helices associated with a β-sheet showing high similarity, which allows them to be superimposed on each other (Figure 4a-d). A homodimer structure for JHEH has been determined by X-ray crystallography for the silkworm Bombyx mori [16]. Our 3D models predict that JHEH 1, 2 and 3 can be superimposed, as they have similar spatial conformations. To study the substrate specificity of JHEH 3, we docked JH III and JH IIIA into the catalytic groove of JHEH 3. The binding of the two substrates to JHEH 3 is different; JH IIIA binds stronger than JH III to JHEH 3, using two H-bonds at the epoxide group to bind to D236 compared with one for JH III, and four hydrogen bonds to bind to D412 compared with two H-bonds for JH III (Figure 4g,h). Zhou et al. [16] used X-ray crystallography to show that JH II binds in the active groove of B. mori JHEH using two H-bonds at the epoxide group and one H-bond at D387 in their active site; however, they did not use JH IIIA in their studies. Our results indicate that the binding of JHEH III to JH IIIA is much tighter than the binding to JH III, and therefore, JH IIIA is probably the preferred JHEH substrate and not JH III. To find out if JHEH(s) in female D. melanogaster preferred substrate is JH IIIIA, we treated female D. melanogaster at different times after adult eclosion (3,24,48 and 72 h) with [12-3 H]JH IIIA and separated the radioactive-labeled metabolites 1 h after the treatment using reversed phase C 18 HPLC [6]. The ratio between JH IIIA and JH IIIAD at 3, 24, 48 and 72 h after adult eclosion was 2.3, 1.25, 0.79 and 0.85, respectively ( Table 2), indicating that female D. melanogaster JHEH(s) metabolize JH IIIA into JH IIIAD, confirming our 3D molecular modeling (Figure 4h). In mosquitoes, it was shown that JH IIIA is also the preferred substrate for JHEH. Treating female A. aegypti with [12-3 H](10R)-JH III and analyzing the extracts using C 18 reversed phase HPLC determined the ratio between JH IIIAD, JH IIIA and JH IIID as 17/4/1, and treating female A. aegypti with [12-3 H]JH IIIA converted 50% of the initial JH IIIA in 1 h into JH IIIAD [6]. The JH III titer in D. melanogaster rapidly declines after adult eclosion [50] and JHEH, in concert with JHE play an important role in JH III metabolism. To understand how JHEH 1, 2 and 3 are controlled by their promoters, we reduced the length of the promoter sequences and cloned the short sequences into plasmid pCaSpeR-AUG-βgal ( Figure S1) and transfected D.Mel2 cells. Full-length JHEH 1 promoter (845 bp, pJHHE#1) exhibited the highest transcriptional activity, whereas shorter promoter sequences reduced the transcriptional activity, and the shortest promoter (145 bp, pJHEH#1L5) exhibited the lowest transcriptional activity, which was 5.6-fold lower than pJHEH#1 (Figure 5a,b). Similar approaches were used to study the anhydrobiotic midge, Polypedilum vanderplanki 121 promoter that allows survival under desiccated conditions [24], as well as to identify and functionally analyze heat shock promoters from S. frugiperda. [25]. JHEH 2 promoter pJHEH#2L1 (1325 bp), on the other hand, showed that shorter promoter length increased the promoter's transcriptional activity, reaching a maximum at a promoter length of 245 bp (pJHEH#2L5, Figure 6a,b). These results indicate that shortening the promoter removed DNA sequences that were used as TF(s)-binding sites to downregulate the transcriptional activity of the promoter [23,24]; however, a shorter promoter of 148 bp was not active. The effect of TF(s)-binding sites on the JHEH 3 promoter's transcriptional activity was studied by shortening the promoter and assaying its transcriptional activity. The initial promoter length that we studied (pJHEH#3L1 1562 bp, Figure 7) exhibited transcriptional activity (24 and 10-fold) higher when compared with JHEH 1 and 2 promoters (Figures 5  and 6). A shorter promoter sequence (pJHEH#3L3, 627 bp, Figure 7) exhibited higher transcriptional activity than all the other promoter sequences (Figure 7a,b), indicating that the sequence between pJHEH#3L2 and pJHEH#3L3 (225 bp) may have sequences that bind TF(s) that downregulate or upregulate the promoter. The promoter sequence between pJHEH#3L2 and pJHEH#3L3 contains several TF(s)-binding sites (bHLH-CS, HZB-CCAAT,CAP-site, TCF-1 and Sp1). All the TFs DNA-binding sites were between pJHEH#3L2 and pJHEH#3L3 except for the Sp1 DNA-binding site, which is located at the 5 end of pJHEH#3L3 (Figure 7a). Analysis of the promoter TF(s)-binding sites indicates that only a two-fold increase in promoter transcriptional activity was observed when several of the promoter DNA sequences were shortened and mutated, especially the DNA sequence that binds TF TCF-1 (Figure 8a,b). The highest promoter's transcriptional activity was observed with promoter pJHEH#3L3, indicating that TF Sp1 upregulates that promoter. TF Sp1 is involved in basal transcriptional regulation of various genes that are involved in many cellular processes, including cell differentiation, cell growth, apoptosis, immune responses, response to DNA damage and chromatin remodeling. Post-translational modifications such as phosphorylation, acetylation, glycosylation and proteolytic processing significantly affect the activity of this protein, which can act as an activator or a repressor [51]. Knocking down Sp1 in D.Mel2 cells that were transfected with pJHEH#3L2 and pJHEH#3L3 by dsRNA stopped both promoters' transcriptional activities and degraded the Sp1 transcript (Figure 9a,b). These results show, for the first time, that the role of Sp1 is to activate the JHEH 3 promoter. JH metabolism is an important aspect of insect development [1,2] during in the life of male and female D. melanogaster [52]. The activities of JHEH 3 promoter sequences that were tested in the presence of JH III (1 µM) were significantly inhibited compared with controls (p < 0.05), whereas incubation with JH IIIA at five-fold higher concentration (5 µM) did not significantly inhibit transcriptional activities of the tested promoter sequences (Figure 10a,b). These results indicate that JH IIIA is the preferred substrate for JHEH 3 and not JH III. In insects, larval-larval molting and larvalpupal-adult metamorphosis are elicited by 20 HE [50,53]. During this period, the level of JH III in Drosophila is low, whereas the level of 20HE is high [52]. A relationship between JH III and 20HE exists, in which JH III stays at a low level, and it is not advantageous for the insect to metabolize JH III at this time. We tested this hypothesis by incubating 20HE and 20HE mimic RH5992 with the most active promoter's sequence (pJHEH#3L3, Figure 7a,b) of JHEH 3. We showed that 20HE at low concentrations (1 and 5 µM) and its mimic (5 µM) significantly inhibited the JHEH 3 promoter's activity, suggesting that in D. melanogaster 20HE inhibits the activity of JHEH 3 promoter, and probably the activities of JHEH 1 and 2 promoters. However, the suggested effect of 20HE on JHEH 1 and 2 promoters needs to be tested in future work. Farnesoic acid (1 µM), which is several steps away in the biosynthetic pathway of JH III, did not have an effect on the transcriptional activity of any of the JHEH 3 promoter sequences ( Figure S5). Our Northern blot analyses of JHEH 1, 2 and 3 transcripts show that the transcripts are ubiquitously expressed in many tissues (head thorax, gut, ovary fat body and the whole insect). To find out where JHEH 3 promoter is active, transgenic D. melanogaster that were transformed with JHEH 3 promoter (pJHEH#3L3, 627 bp, Figure 7a) were cloned in pCaSpeR-AUG-βgal, tested for βgalactosidase activity and observed under a dissecting microscope. β-galactosidase activity was found in the abdomen, thorax, leg muscle and the junction between the abdomen and thorax (Figure 12a-d), confirming that the JHEH 3 promoter and its transcript are located in many tissues. These results indicate that JHEH 3, and probably JHEH 1 and 2 promoters, are distributed throughout D. melanogaster, and their function is to control the metabolism of JH III in many of its target tissues. Our results also indicate that 20HE and JH III play an important role in the control of JHEH 3 promoter. When the 20HE titer is high during adult stages and larval stages, it inhibits JHEH III promoter (Figure 13a). During the initial metabolism of JH III, JHEH does not convert JH III into the JH IIID because JH III inhibits the transcriptional activity of the JHEH promoter (Figure 13a).  Figure S5). Our Northern blot analyses of JHEH 1, 2 and 3 transcripts show that the transcripts are ubiquitously expressed in many tissues (head thorax, gut, ovary fat body and the whole insect). To find out where JHEH 3 promoter is active, transgenic D. melanogaster that were transformed with JHEH 3 promoter (pJHEH#3L3, 627 bp, Figure 7a) were cloned in pCaSpeR-AUG-βgal, tested for β−galactosidase activity and observed under a dissecting microscope. β−Galactosidase activity was found in the abdomen, thorax, leg muscle and the junction between the abdomen and thorax (Figure 12a-d), confirming that the JHEH 3 promoter and its transcript are located in many tissues. These results indicate that JHEH 3, and probably JHEH 1 and 2 promoters, are distributed throughout D. melanogaster, and their function is to control the metabolism of JH III in many of its target tissues.
Our results also indicate that 20HE and JH III play an important role in the control of JHEH 3 promoter. When the 20HE titer is high during adult stages and larval stages, it inhibits JHEH III promoter (Figure 13a). During the initial metabolism of JH III, JHEH does not convert JH III into the JH IIID because JH III inhibits the transcriptional activity of the JHEH promoter (Figure 13a).  After JHE converts JH III into JH IIIA, only then does JHEH promoter, in concert with Sp1, transcriptionally activate jheh 3, synthesizing JHEH 3 that converts JH IIIA into JH IIIAD (Figure 13b). This sequence of events is similar to what was shown for female A. aegypti [6]. In larval D. melanogaster cells, it was shown that jhe is stimulated by 1 µM JH III and inhibited by 1 µM 20HE [54], supporting our results that JHEH does not act on JH III because it inhibits the jheh promoter. These results, together with our 3D molecular modeling (Figure 4g,h) suggest that JH IIIA is the preferred substrate of JHEH 3 and jheh promoter is controlled by JH III and is activated by TF Sp1.

Conclusions
Our results show that JHEH 1, 2 and 3 promoter sequences exhibit transcriptional activities in transfected D.Mel2 cells. Testing of the JHEH 3 promoter identified, for the first time, a TF Sp1 DNA-binding sequence that upregulates the promoter and plays an important role in activating the JHEH 3 promoter of D. melanogaster. A highly transcriptional active promoter sequence of JHEH 3 (627 bp) was shown to be active in many tissues of transgenic D. melanogaster, indicating that JHEH 3 is ubiquitously distributed in many tissues.