Identification and characterization of a juvenile hormone (JH) response region in the JH esterase gene from the spruce budworm, Choristoneura fumiferana.

Using a differential display of mRNA technique we discovered that the juvenile hormone (JH) esterase gene (Cfjhe) from Choristoneura fumiferana is directly induced by juvenile hormone I (JH I), and the JH I induction is suppressed by 20-hydroxyecdysone (20E). To study the mechanism of action of these two hormones in the regulation of expression of this gene, we cloned the 1270-bp promoter region of the Cfjhe gene and identified a 30-bp region that is located between -604 and -574 and is sufficient to support both JH I induction and 20E suppression. This 30-bp region contains two conserved hormone response element half-sites separated by a 4-nucleotide spacer similar to the direct repeat 4 element and is designated as a putative juvenile hormone response element (JHRE). In CF-203 cells, a luciferase reporter placed under the control of JHRE and a minimal promoter was induced by JH I in a dose- and time-dependent manner. Moreover, 20E suppressed this JH I-induced luciferase activity in a dose- and time-dependent manner. Nuclear proteins isolated from JH I-treated CF-203 cells bound to JHRE and the binding was competed by a 100-fold excess of the cold probe but not by 100-fold excess of double-stranded oligonucleotides of unrelated sequence. JH I induced/modified nuclear proteins prior to their binding to JHRE and 20E suppressed this JH I induction/modification. These results suggest that the 30-bp JHRE identified in the Cfjhe gene promoter is sufficient to support JH induction and 20E suppression of the Cfjhe gene.

Although the biological actions of Juvenile hormones (JHs) 1 in insect development and reproduction are well documented, the molecular mechanisms underlying JH action are poorly understood (1)(2)(3)(4)(5). Several possible modes of actions have been proposed for JH. Direct action of JH in regulation of genes such as jhp21 (6), juvenile hormone esterase (7,8), calmodulin (9), vitellogenin (10), and several others have been reported (11,12). Through indirect action, JH was shown to modulate 20hydroxyecdysone (20E) action by affecting the expression of genes in a 20E-induced cascade (13,14). Totally different from the above genomic actions, in ovarian follicular epithelium, JH acts through a membrane receptor to bring about rapid enzyme activation without the need for new transcription (15).
Numerous attempts have been made to identify JH receptors. Palli et al. (16) used human retinoic acid receptor cDNA as a probe and identified a steroid/thyroid superfamily member from Manduca sexta. Further characterization of this cDNA revealed that this is not a JH receptor but, rather, an ecdysoneinduced transcription factor that plays a critical role in ecdysone signal transduction and is related to Drosophila hormone receptor 3 (17), subsequently named Manduca hormone receptor 3 (18). The 29-kDa nuclear protein identified in M. sexta epidermis turned out to be a low affinity JH-binding protein (19,20).
The mammalian retinoid X receptor (RXR) forms a heterodimer with several nuclear receptors including the farnesoid X-activated receptor (FXR). JH III but not JH acid or methoprene can bind/activate the FXR and RXR heterodimer (21). Methoprene and methoprene acid but not JH III can activate RXR (22). These two studies suggested that RXR or its insect homologue ultraspiracle (USP) could play an important role in signal transduction of JH or JH-related compounds. Jones and Sharp (23) showed that both JH III and JHB 3 bind to a USP homodimer from Drosophila melanogaster. Subsequent studies showed that USP from D. melanogaster can bind to the DR12 response element and a reporter gene placed under the control of the DR12 response element fused to the jhe core promoter was induced by JH III (24).
A D. melanogaster mutant tolerant to methoprene (Met) was identified (25). An 85-kDa protein isolated from Met flies showed a 6-fold lower affinity than the wild-type protein for JH III (26). The Met gene was cloned and found to be a member of the basic helix-loop-helix-PER-AHR/ARNT-SIM (PAS) family of transcriptional regulators (27). Met is not a vital gene, as shown by the production of a null mutant allele that is viable (28), but this finding could reflect functional redundancy of Met. The Met gene product was detected in several tissues, including known JH response tissues (29).
To clone JH receptor cDNA, several approaches, including characterization of JH response gene promoters to identify JH response elements to be used for screening expression libraries to isolate JH receptors, are being pursued. Several JH-responsive genes, including calmodulin, vitellogenin, and diapause protein have been identified (9,11,30). Another JH response gene, jhp21, was identified in Locusta migratoria, and a response element has been identified in the promoter region of this gene (6). Characterization of protein that binds to this response element lead to the hypothesis that this protein is a transcription factor activated by JH and that participates in regulation of expression of JH response genes (31). Most of these genes show a relatively slow rate of induction in response to JH application indicating that they may not be directly regulated by a JH-receptor complex. In Manduca sexta fat body cells, a JH-binding protein gene is induced by JH I within 2 h of application and appears to be a primary response gene (32).
CF-203 cells were developed from larval midguts of Choristoneura fumiferana and respond to both 20E and JH (8,(33)(34)(35). We have used differential display of mRNAs technique and identified Cfjhe gene as a primary JH response gene (8). This gene is directly induced by JH I within 1 h after adding the hormone even in the presence of protein synthesis inhibitor, cycloheximide. The expression of Cfjhe gene is completely suppressed by 20E even in the presence of cycloheximide. Both induction by JH I and suppression by 20E are dose-and timedependent (8). Thus, the Cfjhe gene is one of the few examples of genes whose expression is directly regulated by both JH and 20E.
Identification and characterization of the promoter region of Cfjhe gene is essential for studying the mechanism of JH induction and 20E suppression of Cfjhe gene expression. We screened a C. fumiferana genomic library and identified a 1270-bp fragment that is located upstream of the Cfjhe gene transcription start site. Analysis of this 1270-bp genomic fragment using reporter assays in CF-203 cells identified a 30-bp region located between Ϫ604 and Ϫ574 that is sufficient to support both JH induction and 20E suppression of JH induction of Cfjhe gene. Nuclear proteins isolated from CF-203 cells that were exposed to JH specifically bound to this 30-bp JH response region.

EXPERIMENTAL PROCEDURES
CF-203 Cells-CF-203, a continuous cell line was developed from the midgut of C. fumiferana (33). These cells were grown in SF 900 (Invitrogen, Rockville, MD) medium supplemented with 10% fetal bovine serum. The cells grow attached to the substrate and were subcultured by trypsinization for a short time using 0.05% trypsin solution (Invitrogen). New cultures were seeded at 1.5 ϫ 10 5 cells/ml medium in 5-ml flasks and kept at 28°C. They were subcultured every 3-4 days during which time they grew to about 1.5 ϫ 10 6 cells/ml.
Transfection-CF-203 cells were set up at 4 ϫ 10 5 cells/well in 6-well tissue culture plates. The next day, 5 g of DNA was mixed separately and made up to 100 l with neat SF900 medium. In a separate tube 15 l of LipofectAMINE 2000 was mixed with 85 l of neat SF900 medium and was left for 30 min at room temperature. Both the solutions (100 l of DNA mix plus 100 l of lipofectomine 2000 mix equals 200 l of transfection mix) were combined and incubated for 10 min at room temperature. The cells were treated with trypsin for about 10 s and were washed twice with neat SF900 medium. Then, 800 l of neat SF900 was mixed with 200 l of transfection mix, and the mixture was added to the cells in each well. After rocking the cells for 24 h, the transfection mixture was removed and 2 ml of fresh growth medium (SF900 medium with 10% fetal bovine serum) containing hormones was added to each well. Cells were collected at 24 h after adding the hormones. To harvest cells, the medium was removed, and the cells were washed with phosphate-buffered saline, 200 l of reporter lysis buffer was added to each well and the cells were collected and assayed for luciferase reporter activity using the Luciferase TM reporter assay system from Promega (Madison, WI).
Preparation of Nuclear Extracts-The cells were collected and centrifuged for 5 min at 4°C. The pellet was disrupted in homogenization buffer (20 mM Tris, pH 7.0, 50 mM KCl, 300 mM sucrose, 1 mM EDTA, 1 mM DTT, and 1 mM phenylmethylsulfonyl fluoride, homogenized, and centrifuged at 10,000 ϫ g for 60 min at 4°C. The pellet was resuspended in nuclear lysis buffer (10 mM HEPES, pH 7.6, 100 mM KCl, 100 mM EDTA, 10% (w/v) glycerol, 3 mM MgCl 2 , 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, and 0.4 M (NH 4 ) 2 SO 4 ). After incubation for 30 min on ice, the lysate was centrifuged at 14,000 ϫ g for 60 min at 4°C. The nuclear proteins were precipitated by adding 0.3 g/ml (NH4) 2 SO 4 followed by incubation at 4°C for 30 min. After centrifugation at 14,000 ϫ g for 30 min at 4°C, the precipitate was dissolved in nuclear dialysis buffer (25 mM HEPES, pH 7.6, 0.1 mM EDTA, 40 mM KCl, 10% (w/v) glycerol, 1 mM DTT) and dialyzed against 200 ml of the same buffer for 12 h. After a brief centrifugation, the supernatant was stored at Ϫ80°C.
Electrophoretic Mobility Shift Assay-To test the binding of JHRE to nuclear proteins isolated form CF-203 cells, we produced doublestranded DNA probes by synthesizing complimentary oligonucleotides based on 30-bp JHRE (5Ј-CCCTTATAAAAAGATTATTATAGAT-TATTA-3Ј). The oligonucleotides were end-labeled using T4 polynucleotide kinase and [␥-32 P]dATP (6000 Ci/mmol) and purified by passing them through a Sephadex G-50 column. The labeled oligonucleotides were then annealed to produce a double-stranded probe. DNA-binding reactions were carried out in a 20-l volume containing 5-6 g of nuclear extract, 2 l of 10ϫ assay buffer (10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM MgCl 2 , 0.5 mM DTT, 0.5 mM EDTA, 4% glycerol), 1 g of poly(dI-dC), and 20 M single-stranded, nonspecific DNA. After 20min incubation at room temperature, 0.05 pmol of labeled probe was added and the incubation continued for another 20 min at room temperature. For the competition experiments, unlabeled double-stranded probe or double-stranded oligonucleotides of unrelated sequence, but of the same length as the probe and used at a concentration of 100-fold molar excess, were added to the binding reaction at the same time as the labeled probe. Free and protein-bound DNA complexes were separated on 6% non-denaturing polyacrylamide gels in TBE buffer at a constant voltage of 100. The gels were fixed, dried, and exposed to x-ray film using an intensifying screen and kept at Ϫ80°C.
Genomic Library Construction and Screening-A genomic library was constructed using genomic DNA isolated from the spruce budworm larvae and EMBL3 genomic library construction kit form Promega (Madison, WI) following the manufacturer's protocol. A library containing 500,000 clones with an average insert size of 20 kb was prepared. The genomic library was screened using CfJHE cDNA as probe and following the methods described for cDNA library screening (35).

RESULTS
Identification Cfjhe Promoter Region-To identify JH and 20E response regions in the Cfjhe promoter, we screened a C. fumiferana genomic library with Cfjhe gene cDNA as a probe and identified six genomic clones with an average insert size of 20 kb. Restriction digestion of phage DNA followed by Southern blotting and hybridization using the Cfjhe gene cDNA as a probe identified a genomic fragment containing a 1270-bp region that is located upstream to the transcription start site. This genomic fragment was subcloned into pBSKϪ (Stratagene Cloning Systems, La Jolla, CA) and sequenced completely from both directions (Fig. 1). The sequence of this genomic fragment showed a conserved transcription start site at 27 bp upstream from the JHE mRNA translation start site (8) and a typical TATA box at 22 bp upstream to the transcription start site. A search of the 1270-bp sequence (Ϫ1 to Ϫ1270) that is present upstream to the transcription start site using Patch, Match, and AliBaba2 programs available at TRANSFAC public site (transfac.gbf.de/) showed a match with half-sites of several known response elements (REs), including hsp27EcRE, TATAA-binding protein, FXRRE, RXRRE, and E74RE. However, when considered together, both half-sites of none of these REs showed a significant match with the 1270-bp sequence. Some of the putative REs are shown in Fig. 1. This sequence also contained a repeat of 161-bp sequence separated by 93-bp sequence (boxed in Fig. 1). This 1270-bp sequence showed 61% identity with the 1200-bp nucleotide sequence found in the 5Ј flanking region of Trichoplusia ni jhe (36). But, a hormone response element that is similar to DR-1 RE identified in T. ni jhe promoter is not found in the Cfjhe promoter.
Identification of the JH Response Region-We prepared six 5Ј to 3Ј truncations of the 1270-bp Cfjhe promoter region and cloned them into pGL3-basic luciferase reporter vector (Promega Corp.). The nucleotide sequence is numbered relative to the transcription start site of CfJHE mRNA. The 27-nucleotide sequence that is present between transcription and translation start site of CfJHE mRNA was included in all constructs. These six constructs were assayed in CF-203 cells. The constructs that contained the Cfjhe promoter region from Ϫ1270 to ϩ1, Ϫ1013 to ϩ1, Ϫ956 to ϩ1, and Ϫ641 to ϩ1 showed 10-to 15-fold induction of luciferase reporter activity in the presence of JH I when compared with the activity in the presence of Me 2 SO (Fig. 2). On the other hand, the constructs that contained the Cfjhe promoter region from Ϫ424 to ϩ1 showed only 2-fold induction, and the constructs that contained Ϫ74 to ϩ1 did not show any induction of reporter activity in the presence of JH I. These results showed that the JH I response region in the Cfjhe promoter is located between Ϫ641 and Ϫ424 bp upstream from the transcription start site. The steroid hormone 20-hydroxyecdysone (20E) did not induce reporter activity through any of the constructs tested, but it was able to prevent JH I-induced reporter activity when both hormones were administered simultaneously. These results showed that the 217-bp region of the Cfjhe promoter that lies between Ϫ641 and Ϫ424 was able to support both JH induction and 20E suppression as observed for Cfjhe gene (8). Although, the construct containing Ϫ424 to ϩ1 showed a small increase in reporter activity in the presence of JH I, further analysis of this region did not show any JH I-mediated response. As will be shown later, the only recognizable hormone response element (a half-site) in this region, AGGTCA, present at Ϫ208 appears to play no role in JH 1 induction of the reporter gene regulated by the 1270-bp Cfjhe promoter.
The 217-bp fragment that lies between Ϫ641 and Ϫ424 was further analyzed by preparing three truncations containing lengths of 217 bp (Ϫ641 to Ϫ424), 150 bp (Ϫ574-to Ϫ424), and 75 bp (Ϫ499 to Ϫ424). Each of these three fragments was fused with the Cfjhe gene core promoter (which includes a 46-bp sequence containing the TATA box and the transcription start site, Ϫ30 to ϩ15) and cloned into pGL3-basic vector. These three truncation constructs were assayed in CF-203 cells. As shown in Fig. 3, only the construct containing the 217-bp Cfjhe promoter fragment showed induction by JH I suggesting that the JH response region in the Cfjhe promoter is located between Ϫ641 and Ϫ574 bp upstream from the transcription start site.
We prepared three truncations of the 67-bp fragment that lies between Ϫ641 and Ϫ574. Three fragments of the Cfjhe promoter 67 bp (Ϫ641 to Ϫ574), 50 bp (Ϫ624 to Ϫ574), and 30 bp (Ϫ604 to Ϫ574) were fused to the Cfjhe gene core promoter (Ϫ30 to ϩ15) and cloned into pGL3-basic vector and assayed in CF-203 cells. All three constructs showed 10-to15-fold induction of reporter activity in JH I-treated cells when compared with the reporter activity in Me 2 SO-treated cells indicating that the 30-bp region between Ϫ604 and Ϫ574 is sufficient for a JH induced response observed for Cfjhe gene (Fig. 4). This sequence contains direct repeat elements separated by a fournucleotide spacer and shows 100% identity with consensus direct repeat 4 (DR4) element (Fig. 1). This 30-bp region is designated as a putative juvenile hormone response element (JHRE).
JHRE Is Sufficient to Support JH Induction and 20E Suppression-To test if the 30-bp fragment that supported JH I induction is also sufficient for 20E suppression of JH I-induced reporter activity, we transfected pGL3JHRE(1270) (where the luciferase reporter was regulated by the 1270-bp fragment) or pGL3JHRE (30) (where the luciferase reporter gene is regulated by 30-bp JHRE fused to the Cfjhe gene core promoter, Ϫ30 to ϩ15) or pGL3 alone into CF-203 cells. The transfected cells were exposed to Me 2 SO, JH I, 20E, or JH I plus 20E. As shown in Fig. 5A, the 30-bp region from the Cfjhe promoter is sufficient to support JH I induction as well as 20E suppression of JH I-induced reporter activity. The luciferase reporter in pGL3 vector was not induced by either JH I or 20E or both in combination.

Dose response and Time Course of JH I Induction-
The JH I induction of reporter activity through the 30-bp Cfjhe promoter is both JH I dose-and exposure time-dependent. The induction of reporter activity started at as low as 40 nM JH I and increased with the increased concentration of JH I and reached peak levels at 1000 nM concentration of JH I (Fig. 5B). There was a slight decrease in reporter activity at higher concentration of JH I (5000 nM). This may be due to the cytotoxicity of hormone to the cells. The induction of reporter activity increased with an increase in time and reached peak levels by 24 h after adding the hormone (Fig. 5C). There was a decrease in reporter activity by 48 and 72 h after adding hormone.
Nuclear Proteins Isolated from CF-203 Cells Specifically Bind to JHRE-To determine whether JHRE from the Cfjhe promoter binds to nuclear proteins that are present in CF-203 cells, we performed an electrophoretic mobility shift assay (EMSA). Nuclear extracts were prepared from JH I-treated and Me 2 SO-treated CF-203 cells, and the nuclear proteins were incubated with end-labeled double-stranded oligonucleotides synthesized based on 30-bp JHRE sequence (Table I). As shown in Fig. 6A, the nuclear proteins isolated from JH I-treated CF-203 cells bound to the JHRE, and the binding was competed by a 100-fold excess cold probe but not by a 100-fold excess double-stranded oligonucleotides of unrelated sequence indicating that the binding is specific. To determine if nuclear proteins that bind to JHRE need prior exposure to JH, we cultured CF-203 cells in the presence of Me 2 SO or 1 M JH I for 3, 6, 12, and 24 h, and the nuclear proteins were isolated and assayed by EMSA. Nuclear proteins from the cells that were exposed to JH I, but not from the cells that were exposed to Me 2 SO, bound to JHRE suggesting that JH I induces/modifies one or more nuclear proteins that bind to JHRE (Fig. 6B). To test whether 20E suppresses JH I induction/modification of nuclear proteins prior to their binding to JHRE, we cultured CF-203 cells in medium containing Me 2 SO or 1 M JH I or 1 M 20E or 1 M each of JH I and 20E for 24 h, the nuclear proteins were isolated and analyzed for their binding to JHRE by EMSA. As shown in Fig. 6C, nuclear proteins isolated from JH I-treated cells bound to JHRE, but the nuclear proteins isolated from Me 2 SO, 20E, or 20E plus JH I treated cells did not bind to JHRE. These data show that JH I induces/modifies nuclear proteins prior to their binding to JHRE and that 20E can suppress this action of JH I. Identification of Nucleotides Critical for Binding of 30-bp JHRE to Nuclear Proteins-To further characterize JHRE and to identify nucleotides critical for binding to nuclear proteins, we produced mutant versions of 30-bp JHRE (Table I)  Nuclei isolated from JH I-treated and Me 2 SO-treated CF-203 cells were incubated with 32 P-labeled double-stranded 30-bp oligonucleotides corresponding to the Ϫ604 to Ϫ574 JH response sequence of the Cfjhe promoter. The DNA-protein complexes were separated on 6% polyacrylamide gels. A, 32 P-labeled 30-bp probe alone, probe incubated with nuclear proteins isolated from JH I-treated cells, and 100ϫ excess cold probe or 100ϫ excess double-stranded oligonucleotides of unrelated sequence were analyzed. The nuclear proteins isolated from JH Itreated CF-203 cells bound to the 30-bp fragment and 100X excess cold probe but not nonspecific oligonucleotides competed for the binding. (B) The nuclear proteins isolated from CF-203 cells that were grown in the presence of Me 2 SO or JH I for 3 or 6 or 12 or 24 h were analyzed to assess their binding to the 30 bp probe. The nuclear proteins isolated from CF-203 cells that were grown in the presence of Me 2 SO did not bind to JHRE, but the nuclear proteins isolated from CF-203 cells that were exposed to JH I for 3, 6, 12, or 24 h bound to JHRE. C, the nuclear proteins isolated from CF-203 cells, which were grown in the presence of Me 2 SO, JH I, 20E, or JH I plus 20E for 24 h, were analyzed to assess their binding to 30-bp JHRE probe. The nuclear proteins isolated from CF-203 cells that were grown in the presence of Me 2 SO did not bind to JHRE, but the nuclear proteins isolated form CF-203 cells that were exposed to JH I for 24 h bound to JHRE. However, nuclear proteins isolated from cells that were exposed to JH I plus 20E or 20E alone did not bind to JHRE.  TAAAAAGGTCATTATAGGTCATTATA was competed by 25-fold excess cold probe (Fig. 7A). A 25-fold excess of a 30-bp oligonucleotide containing a mutation changing G to A in the left half-site (MI , Table I) or in both the half-sites (M2 , Table I) or in the right half-site (M3 , Table I) showed reduced competition (Fig. 7B). These results showed that G residues present in repeat elements are important for binding of nuclear proteins to this JHRE. To determine the influence of nucleotides upstream to the repeat elements, truncated oligonucleotides where some of the nucleotides at the 5Ј end of JHRE were eliminated were synthesized (M5-M8, Table  I). These truncated oligonucleotides showed reduction in competition as more nucleotides were eliminated (Fig. 7). Mutant 8, in which all nucleotides upstream to the repeat elements were eliminated, showed significantly less competition when compared with the cold probe. The 30-bp JHRE we identified through promoter analysis had two nucleotides (TA) missing at the 3Ј end of the second repeat. We produced a 32-bp oligonucleotide that included these two nucleotides (M4, Table I). The 32-bp JHRE competed well for the binding of 30-bp JHRE to nuclear proteins.
The above results showed that the G residue in the AGATTA repeat is critical for binding of nuclear proteins to JHRE. These results also showed that most of the residues that are present upstream to the AGATTA sequence are not very important in binding of nuclear proteins to JHRE suggesting that the two AGATTA direct repeat elements with a 4-nucleotide spacer is probably the binding site for nuclear proteins. Consequently, we prepared six additional mutants of JHRE (M9 -M14) chang-ing conserved nucleotides in the AGATTA element to nonconserved residues (the consensus sequence for DR4 is RGRN-YANNNNRGRNYA, where R ϭ A or G, N ϭ A, G, T, or C, and Y ϭ A or T). In mutant 14, the AGATTA was changed to AGGTCA, a common half-site found in hormone response elements. Analysis of these mutants by EMSA showed that Mutants 10, 11, and 12 showed significantly less competition when compared with the other mutants. These three mutants had changes in conserved residues of the DR4 element (mutant 10; G in AGATTA was changed to A, mutant 11; the sixth residue, A in AGATTA was changed to T and mutant 12; the third residues in AGATTA changed to T) (Fig. 8, A and B). Mutant 14, where the AGATTA elements were changed to AGGTCA, competed well with the probe for binding of nuclear proteins (Fig. 8, A and B). These data suggest that the DR4 element present in the 30-bp JHRE is responsible for binding to nuclear proteins isolated from CF-203 cells.
To determine whether the G residue in the AGATTA element that is critical for binding of nuclear proteins is also important for JH I induction of a reporter gene placed under the control of JHRE, we prepared three mutants of pGL3JHRE(1270) where the G residues in each AGATTA element (G-588A and G-598A) as well as the G residue in AGGTCA found at Ϫ208 were changed to A (G-208A). Analysis of mutant and wild type (WT) constructs in CF-203 cells showed that both mutants where G residues in AGATTA element was changed to A (G-588A and G-598A) reduced JH I induction of reporter gene by 70% when compared with WT (Fig. 9). The suppression by 20E worked in these mutants, because 20E was able to reduce JH I-induced FIG. 7. Identification of residues critical for binding of nuclear proteins. Nuclear proteins were isolated from JH I-treated CF-203 cells and incubated with 32 P-labeled double-stranded 30-bp oligonucleotide probe corresponding to the JHRE sequence or its mutants (M1-M8, Table I). The DNA-protein complexes were separated on 6% polyacrylamide gels. The nuclear proteins from JH I-treated cells that bound to the 30-bp fragment were competed with 25-fold excess cold probe or its mutant versions. A, an autoradiogram of the EMSA gel. B, the radioactivity in the retarded bands was quantified using a PhosphorImager reporter activity. On the other hand, changes to G residues in AGGTCA at Ϫ208 (G-208A) did not effect JH I induction or 20E suppression. These data suggest that the G residues in the DR4 element present in the Cfjhe promoter are important for JH I induction of this gene. DISCUSSION The major contribution of current study is the identification of a 30-bp JHRE within the Cfjhe promoter that is sufficient for JH induction and 20E suppression of this induction observed for Cfjhe gene. Several lines of evidence support this conclusion. First, when the luciferase gene was placed under the control of this 30-bp JHRE and the Cfjhe core promoter (Ϫ25 to ϩ15), JH I induced reporter activity, and, in turn, 20E suppressed this JH I-induced reporter activity. Second, nuclear proteins isolated from CF-203 cells exposed to JH I specifically bound to this 30-bp JHRE. Third, other regions of the 1270-bp Cfjhe promoter did not support JH I induction. Taken together, these data conclusively show that the identified 30-bp JHRE is responsible for the JH induction and 20E suppression observed for the Cfjhe gene (8).
The 30-bp JHRE contains two direct repeats of AGATTA with a four nucleotide spacer and these elements show 100% similarity with the consensus DR4 element (RGRN-YANNNNRGRNYA, where R ϭ A or G, N ϭ A or G or T or C and Y ϭ A or T). Mutagenesis experiments showed that changing the two residues G and A that are present in the consensus DR4 elements to A and T respectively, significantly reduced its competition with WT probe for binding to nuclear proteins suggesting that these two residues are critical for binding of nuclear proteins to these JHRE. Mutagenesis experiments also showed that the oligonucleotides containing two AGGTCA (common hormone response element half-site) elements that are separated by a 4-nucleotide spacer competed well with WT 30-bp JHRE probe for binding of nuclear proteins isolated from CF-203 cells. These results suggest that the DR4 elements present in the Cfjhe promoter are involved in binding of nuclear proteins isolated from CF-203 cells. In transactivation assays, the mutant versions of the 1270-bp Cfjhe promoter in which the G in each of the AGATTA element in the 30-bp JHRE was mutated to A (G-588A and G-598A) reduced JH I induction by 70%. On the other hand, changing of G to A (G-208A) in AGGTCA single element found at Ϫ208 of Cfjhe promoter did not effect JH I induction of luciferase gene regulated by the Cfjhe promoter. These data suggest that the DR4 elements present in the promoter region of the Cfjhe gene are involved in JH induction. The JHRE (GAGGTTCGAG(A/T)CCT(T/C)) found in the promoter region of another JH-induced gene, jhp21, from L. migratoria (6,31) resemble known hormone response elements especially the IR-1 (inverted repeat elements with one spacer). Ecdysone receptor and ultraspiracle complexes bind to both DR4 and IR-1 elements (37).
The aGATTa motif present in each of the two repeat sequences in JHRE is similar to some of the GATA binding sites identified in the promoter region of the vitellogenin gene from Aedea aegypti (38). To determine if the GATA family of transcription factors can bind to JHRE identified in the Cfjhe promoter, we performed supershift in EMSA using antibodies made against BmGATA␤2 protein (39). These antibodies did not supershift the JHRE-nuclear protein complexes indicating that the nuclear proteins binding to JHRE are not similar to BmGATA␤2 protein (data not shown).
Nuclear proteins isolated from CF-203 cells that were exposed to JH I but not to Me 2 SO bound specifically to the 30-bp JH response region of the Cfjhe promoter indicating that JH I either induces the expression of mRNA or modifies the nuclear proteins prior to their binding to JHRE. Because only short exposure of 3 h to JH I is sufficient to increase the nuclear protein binding to JHRE, we favor the hypothesis that the nuclear proteins that bind to JHRE are modified rather than their mRNA expression being induced by JH I. Nuclear proteins that bind to the JHRE present in the promoter region of jhp21 gene from L. migratoria are phosphorylated through C-type protein kinase that prevents them from binding to JHRE (31). It is likely that the nuclear proteins that bind to JHRE present in the Cfjhe promoter are also phosphorylated, and phosphorylated nuclear proteins may not be able to bind to JHRE. However, exposure to JH I might result in dephosphorylation of nuclear proteins leading to their binding to JHRE. Experiments are in progress to test this hypothesis.
The identified JHRE would be useful for cloning cDNAs for receptors/transcription factors through which JH/20E transduce their signals to bring about opposite effects on the expression of Cfjhe gene. The JHRE identified would also be useful for developing reporter-based assays to screen for new more potent environmentally friendly JH analogs for controlling pests and disease vectors.