SEARCH

SEARCH BY CITATION

Keywords:

  • AP-4;
  • E-box;
  • diapause hormone and pheromone biosynthesis-activating neuropeptide;
  • Helicoverpa armigera

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Activated protein 4 (AP-4), an E-box DNA-binding protein, was cloned from the cotton bollworm, Helicoverpa armigera (Har). The expression of Har-AP-4 mRNA and the protein that it encodes are significantly higher in nondiapause pupae than in diapause pupae. In vitro-translated Har-AP-4 can bind specifically to the E-box motif on the promoter of the diapause hormone and pheromone biosynthesis-activating neuropeptide (DH-PBAN). Har-AP-4, fused with the green fluorescent protein (GFP), is localized to the nucleus, and overexpression of Har-AP-4 can significantly activate the promoter of the DH-PBAN gene that is involved in nondiapause pupal development in H. armigera. These results suggest that Har-AP-4, which binds to the promoter of DH-PBAN, may play a role in regulating pupal development in H. armigera.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Insects have evolved a period of developmental arrest (diapause) that enables them to adapt to environments that are unfavourable for survival (Denlinger, 2002). A model species, Bombyx mori, has often been studied in relation to diapause, and, using this model, researchers have found that the neuroendocrine system is deeply involved in the control of diapause, through the transduction of environmental signals into humoral factors (Denlinger et al., 2005). These include the diapause hormone (DH) that is secreted from the suboesophageal ganglion (SG) and can induce the embryonic diapause of B. mori (Yamashita, 1996). The DH cDNA (or gene) encodes a polyprotein precursor. DH, the pheromone biosynthesis-activating neuropeptide (PBAN) and three other neuropeptides are cleaved from this precursor (Sato et al., 1993; Xu et al., 1995). This cDNA (or gene) is therefore referred to as the DH-PBAN cDNA or gene. DH-PBAN genes have since been found in a number of lepidopteran species, but the functions of DH remain unknown, except in B. mori. Interestingly, the DH from the noctuid species Helicoverpa armigera (Har), which is a pupal diapause species, has been shown to stimulate pupal development and break pupal diapause through regulating the synthesis and release of ecdysone (Zhang et al., 2004b, c).

Previous studies have demonstrated that POU-M2, a Pit–Oct–Unc (POU) family transcription factor, can bind to the promoter of the DH-PBAN gene, regulating its expression in H. armigera. However, the transcriptional activity is weaker than in B. mori (Zhang et al., 2005; Zhang & Xu, 2009). Therefore, there must be transcription factors other than POU-M2 that play roles in the regulation of the Har-DH-PBAN promoter regions. Using the electrophoretic mobility shift assay technique, Hong et al. (2006) discovered a transcription factor, Har-DH-modulator binding protein 3 (Har-DHMBP3), that can bind specifically to the classical E-box of the DH-PBAN gene promoter. Transient transfection assays showed that the deletion of the E-box regulator domain halved the activity of the Har-DH-PBAN promoter. Thus, we hypothesized that DHMBP3 might be a candidate for the transcription factor, AP-4, which can activate cellular genes by binding to a symmetrical DNA sequence, the E-box (Hu et al., 1990).

Here, we describe the primary structure and expression of the mRNA that encodes the E-box DNA-binding protein, AP-4. The AP-4 protein can bind specifically to the E-box motif of the Har-DH-PBAN promoter and significantly activates the DH-PBAN promoter in H. armigera. However, AP-4 is smaller than DHMBP3. Thus, we suggest that Har-AP-4 is a component of the DHMBP3 protein complex and involved in the regulation of DH-PBAN gene expression during pupal development in H. armigera.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Cloning and sequencing of H. armigera AP-4 cDNA

When we began to isolate Har-AP-4 cDNA, the AP-4 mRNA from Drosophila melanogaster was the only one to have been reported in insects (King-Jones et al., 1999). A comparison of the nucleotide sequences of D. melanogaster (GenBank accession no. AAD48781), Mus musculus (GenBank accession no. AAH47270) and Homo sapiens (GenBank accession no. AAH10576) AP-4 mRNAs shows that only a short fragment (63 bp) is conserved amongst them. Thus, we designed two primers, AP4-51 and AP4-52, to generate the 5′-AP-4 cDNA, based on the short conserved sequence. We performed 5′-rapid amplification of cDNA ends (5′-RACE) using the following sets of primers: nested universal primer (NUP; Clontech) and the outer primer, AP4-51 (first PCR), and NUP and the inner primer, AP4-52 (second PCR). We amplified a ∼520 bp product at the 5′-end, subcloned the fragment into a vector, and sequenced it. The 5′-end sequences had 77% identity at the amino acid level with the corresponding regions of the AP-4 from D. melanogaster. To obtain the full-length AP-4 cDNA, we performed 3′-RACE using the specific primer universal primer mix (UPM, Clontech) and either AP4-31 (outer primer) or AP4-32 (inner primer), which were based on the sequences of the 520 bp cDNA fragment that we sequenced as described above. A ∼1 kb band at the 3′-end was amplified using PCR. The entire cDNA is shown in Fig. 1.

image

Figure 1. The nucleotide and deduced amino acid sequences of the cDNA that encodes Helicoverpa armigera activated protein 4 (Har-AP-4). The suggested start ATG and stop codon TAG are shown in bold. The conserved sites, R48-R49-E56-R57-R58-R59-K81-K84, E56 and I63-N64-L70-L88-Y94-I95 are shown in bold italics. The arrows below the nucleotide sequences represent the position of the different synthetic primers used in the PCR reaction. This sequence has been deposited in the GenBank database (accession number: DQ224406).

Download figure to PowerPoint

The full-length cDNA is 1435 bp, including a 320 bp 5′-untranslated region, a 993 bp open reading frame, and a 122 bp 3′-untranslated region (Fig. 1). The Har-AP-4 cDNA that we prepared encodes 331 amino acids that contain a conserved basic helix-loop-helix (bHLH) domain located between residues 45 and 103. This sequence has been submitted to GenBank (accession no. DQ224406).

Using homology analysis of the AP-4 protein sequences from four insects and two non-insect animals, we found that Har-AP-4 has the closest identity to that of B. mori (76%, GenBank accession no. ACB32276), followed by Apis mellifera (58%, GenBank accession no. XP 001122450.1), Tribolium castaneum (53%, GenBank accession no. XP 967737.2), D. melanogaster (45%; King-Jones et al., 1999), M. musculus (42%; Tsujimoto et al., 2005) and H. sapiens (43%; Hu et al., 1990) (Fig. 2).

image

Figure 2. The alignment of Helicoverpa armigera activated protein 4 (Har-AP-4) with other insect or animal AP-4s. The basic helix-loop-helix (bHLH) ZIP and ZIP II motif domains are underlined. The numbers on the right indicate the amino acids of each protein. Highly conserved residues are shaded in black. Har., H. armigera, GenBank accession no. DQ224406; Bom., Bombyx mori, GenBank accession no. ACB32276; Tri., Tribolium castaneum, GenBank accession no. XP 967737; Apis., Apis mellifera, GenBank accession no. XP 001122450; Dro., Drosophila melanogaster, GenBank accession no. AAD48781; Mus., Mus musculus, GenBank accession no. AAH47270; Homo. Homo sapiens, GenBank accession no. AAH10576.

Download figure to PowerPoint

The expression of AP-4 in nondiapause and diapause individuals

Diapause-destined pupae completely enter diapause 10 days after pupation. Therefore, we investigated the temporal expression of the AP-4 gene in the brain and the SG or the brain-SG complexes from nondiapause and diapause-destined individuals during early pupal development (day 0–8) using reverse transcription-polymerase chain reaction (RT-PCR) (Fig. 3). The expression level of AP-4 mRNA on day 0 was similar in both nondiapause and diapause-destined individuals, but was significantly higher in the nondiapause pupae than in the diapause pupae from day 1 to day 8. Apparently, the expression of AP-4 is closely correlated with pupal development.

image

Figure 3. Developmental expression of Helicoverpa armigera activated protein 4 (Har-AP-4) mRNA detected by reverse transcription-polymerase chain reaction (RT-PCR). RNA was extracted from the brain and suboesophageal ganglion (SG), or the brain-SG complexes, of nondiapause and diapause-destined pupae. (A) The first strand of cDNA was synthesized: the Har-AP-4 cDNA fragment was amplified using the primers, AP4-AF and AP4-AR, for 27 cycles, and actin cDNA was used as an internal standard. The Arabic numbers represent the number of days after pupation. (B) The DNA bands were quantified using a gel logic 200 imaging system (Kodak, Eastman Kodak Company, Rochester, NY, USA), and normalized to the level of actin mRNA, which was used as an internal standard. The amounts of the Har-AP-4 mRNA were normalized to the highest value (100%). Each point represents the mean ± SE of the independent measured values from three experiments. Asterisks indicate significant differences (* indicates that P < 0.05 and ** indicates that P < 0.01).

Download figure to PowerPoint

Developmental changes of the AP-4 protein

Using antibodies that recognize AP-4 specifically, we investigated whether the AP-4 protein is present in the brain-SG complexes of nondiapause and diapause individuals using Western blots (Fig. 4). The developmental changes of the AP-4 protein in individuals that were in diapause remained at a consistently low level, but they were clearly present in nondiapausal individuals and they increased gradually as development towards the adult form progressed. The expression pattern at the protein level corresponded to that seen at the mRNA level.

image

Figure 4. The expression of the Helicoverpa armigera activated protein 4 (Har-AP-4) protein in nondiapause and diapause-destined individuals. Proteins for the Western blot were extracted from the brain-suboesophageal ganglion complexes of pupae. The numbers represent the number of days after pupation. Actin was used as an internal standard.

Download figure to PowerPoint

The nuclear localization of AP-4

AP-4 is presumed to be a transcription factor that regulates gene expression. Therefore, we investigated the histological location of Har-AP-4 to confirm whether it localizes to the nucleus efficiently. A Har-AP-4-green fluorescent protein (Har-AP-4-GFP) fusion construct was transfected into Helicoverpa Zea cell lines (Hz-am1) cells, and we found that the Har-AP-4-GFP was localized exclusively to nuclei that were marked by 4′,6-diamidino-2-phenylindole (DAPI) (Fig. 5).

image

Figure 5. The nuclear localization of Helicoverpa armigera activated protein 4 (Har-AP-4) (as imaged using an inverted microscope, ×200). The Har-AP-4 green fluorescent protein (GFP)-fusion was transfected into Hz-am1 cells. (A) The expression of GFP-Har-AP-4; (B) 4′,6-diamidino-2-phenylindole (DAPI) staining to show the nuclei of the cells; (C) overlapping of (A) and (B) to show that Har-AP-4-GFP is located exclusively in the nuclei.

Download figure to PowerPoint

AP-4 binds to an E- box element in the Har-DH-PBAN promoter

A previous study has identified a CAGCTG box (ie the E-box element) at positions −360 to −355 bp of the Har-DH-PBAN promoter that can specifically bind the nuclear factor DHMBP3 and modulate the activity of the activator domain (Hong et al., 2006). We synthesized three probes for use in an electrophoretic mobility shift assay (EMSA): the DH-specific probe (HS) is a wild-type sequence that contains an E-box sequence; the E-box mutant (EM) contains a mutation in the E-box sequence; and NS is a nonspecific competitor (Fig. 6A). We detected DHMBP3 using the HS probe, but not using the E-box mutant EM (Fig. 6B, lanes 2 and 3). In vitro-translated Har-AP-4 bound to HS efficiently, and 100-fold unlabelled NS did not bind competitively (Fig. 6B, lanes 6 and 8). Labelled EM did not interact with AP-4 (Fig. 6B, lane 9), and unlabelled EM was not able to compete with AP-4 (Fig. 6B, lane 13). The AP-4 protein-DNA binding site is the same as that for DHMBP3. However, AP-4 is smaller than DHMBP3.

image

Figure 6. Detection of Helicoverpa armigera activated protein 4 (Har-AP-4) binding activity using the electrophoresis mobility shift assay. (A) The E-box is included in the HS probe, and it is mutated in the EM probe. NS is a nonspecific competitor. (B) The HS probe was incubated without competitor (lanes 1, 2 and 6) or with 100-fold unlabelled HS (lanes 4 and 7), 100-fold unlabelled NS (lanes 5 and 8) or 100-fold unlabelled mutants EM (lanes 11 and 13). Abbreviations: AP-4, Har-AP-4 protein expressed in vitro; com., competitor; DHMBP3, diapause hormone modulator binding protein 3; EM, E-box mutant probe; HS, Har-diapause hormone and pheromone biosynthesis-activating neuropeptide specific probe; NS, nonspecific probe; SG, suboesophageal ganglion.

Download figure to PowerPoint

Over-expression of AP-4 activates the Har-DH-PBAN promoter

Different parts of the Har-DH-PBAN promoter were deleted to verify whether the upstream cis-elements (the E-box site) are involved in the activation of the promoter (Fig. 7). The Har-DH-PBAN promoters (HPs), HP1 (without the E-box), HP2 and HP4 were co-transfected with the AP-4 expression plasmid, GFP-AP-4. As shown, the forced expression of AP-4 in the Bm-N cells activated the Har-DH-PBAN promoters, HP2 and HP4, significantly. In contrast, AP-4 did not activate HP1 or Luciferase Reporter Vector (pGL3)-basic.

image

Figure 7. The forced expression of activated protein 4 (AP-4) in the Bombyx mori cell line (Bm-N) activates the Helicoverpa armigera diapause hormone and pheromone biosynthesis-activating neuropeptide (Har-DH-PBAN) promoter. (A) Diagram of the Har-DH-PBAN promoters fused to the luciferase reporter gene. The bent arrows show the transcription initiation site. (B) The activities of Har-DH-PBAN promoters (HPs) HP1, HP2 and HP4. We co-transfected 0.8 µg of the Har-DH-PBAN promoters and Luciferase Reporter Vector (pGL3)-basic without (open bars) or with (black bars) 1 µg of green fluorescent protein-Har-AP-4 added to the Bm-N cell line. The amount of cellfectin was adjusted based on the amount of plasmid DNA used. The cells were harvested for the luciferase assays 48 h after transfection. The results were normalized relative to the luciferase activity of pGL3-basic in the Bm-N cell line, to which a value of 1.0 was assigned. All data are presented as the means ± SEM. The asterisks (**) indicate a highly significant difference (P < 0.01) between the test and the control group as analysed using one-way anova.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

In this study, we cloned AP-4 cDNA from H. armigera using the RACE method. Although identity between H. armigera and D. melanogaster AP-4 is much lower at the amino acid level (45%), the Har-AP-4 cDNA contains the conserved bHLH domains ZIP and ZIP II. The bHLH motif allows for homodimerization and heterodimerization with other HLH proteins. Dimerization is a prerequisite for the DNA binding of a conserved basic region that is located immediately N-terminal to the HLH motif. Members of the bHLH subgroup of bHLH proteins, such as AP-4, possess a leucine zipper (ZIP II) as a second dimerization motif that is immediately C-terminal to the HLH motif (Murre et al., 1994).

Har-AP-4 is widely expressed and occurs in different tissues (data not shown) and developmental stages (Fig. 3). Ubiquitously expressed AP-4 should therefore be expected to have pleiotropic functions. This has been confirmed in previous studies on AP-4, which have proven its involvement in processes that are as diverse as cell proliferation (Jung et al., 2008; Cao et al., 2009) and differentiation (Hu et al., 1990; Badinga et al., 1998), the immune response (Aranburu et al., 2001) and apoptosis (Tsujimoto et al., 2005). Amongst insects, AP-4 has only been found in Drosophila. It has been suggested that dAP-4 contributes to the transcriptional control of the salivary gland secretion protein (Sgs-4) gene, which regulates salivary gland development (King-Jones et al., 1999). In the present paper, AP-4 from H. armigera has been shown to localize exclusively at the nuclei, indicating that Har-AP-4 might function as a transcription factor to regulate pupal development through the control of DH-PBAN gene expression.

The Har-DH-PBAN gene promoter has a potential binding site for AP-4 at the E-box motif. This motif can bind the nuclear factor DHMBP3, as Hong et al. (2006) have reported. Thus, we conclude DHMBP3 is a candidate of the transcription factor AP-4. In the present paper, we have demonstrated that Har-AP-4 binds specifically to the E-box site of the Har-DH-PBAN promoter and that the Har-AP-4 that is expressed as a result may activate the transcription of the Har-DH-PBAN gene significantly. However, Har-AP-4 is smaller than DHMBP3, as demonstrated by the comparative DNA-protein interaction experiment (Fig. 6B). It has been reported that different classes of cellular bHLH proteins (AP-4 and E47) may be involved in regulating the function of the HIV-1 TATA element by either inhibiting or promoting the assembly of different preinitiation transcriptional complexes (Ou et al., 1994). Work on D. melanogaster found that AP-4 and the secretion enhancer binding protein (SEBP3) did not have different binding specificities but there was a difference in the electrophoretic mobilities of the two proteins (King-Jones et al., 1999). King-Jones et al. (1999) demonstrated that AP-4 is a component of SEBP3, and that SEBP3 is a heterodimerized complex that is comprised of AP-4 and another HLH transcription factor, Daughterless. Similarly, Har-AP-4 may also heterodimerize during the regulation of the expression of Har-DH-PBAN, suggesting that DHMBP3 may be a heterocomplexed protein and that Har-AP-4 is a DNA-binding component of DHMBP3.

Previous studies have demonstrated that expression of the Har-DH-PBAN gene is higher in nondiapause pupae than in diapause-destined ones, and that the gene's function is to regulate pupal development, and not to induce diapause (Zhang et al., 2004c). The patterns of expression of both the Har-AP-4 mRNA and its encoded protein in nondiapause and diapause-type individuals are consistent with those of Har-DH-PBAN. The close relationship between Har-AP-4 and Har-DH-PBAN implies that Har-AP-4 may play a role in the regulation of Har-DH-PBAN gene expression. In fact, AP-4 can bind efficiently to the E-box, significantly up-regulating the promoter activity of Har-DH-PBAN. Thus, Har-AP-4 may play a key role in regulating development through changing the Har-DH-PBAN transcript. However, determination of the biological significance of the presence of the AP-4 site in the Har-DH-PBAN gene promoter and the identification of the partner of Har-AP-4 in determining pupal development await further in vivo investigations.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Animals

Helicoverpa armigera were reared on an artificial diet at 20 °C, using a L14:D10 photoperiod for the nondiapause individuals and a L10:D14 photoperiod for the diapause individuals. The developmental stages were synchronized at each moult by collecting new larvae or pupae. All tissues were dissected in insect saline containing 0.75% NaCl and stored at −80 °C.

RACE

We constructed 5′- and 3′-ready-cDNA using the SMART RACE cDNA amplification kit, according to the manufacturer's protocols (Clontech, Mountain View, CA, USA). Two primers, AP4-51 (5′-GC ATT GAT GCT CTG CAT GCG TCT-3′) and AP4-52 (5′-CG CTC GTT GCT GTT GGC GAT TTC-3′), were synthesized for the 5′-RACE reaction based on the short fragment (63 bp) AP-4 mRNA which is conserved amongst D. melanogaster (GenBank accession no. AAD48781), Mus musculus (GenBank accession no. AAH47270) and Homo sapiens (GenBank accession no. AAH10576). The PCR for 5′-RACE was performed using NUP (Clontech) and AP4-51, and using LA Taq with GC buffer (TaKaRa, Kyoto, Japan). The amplification conditions were as follows: after 5 min at 94 °C, we used 30 cycles of 1 min at 94 °C, 1 min at 55 °C and 1 min at 72 °C. Finally, the reactions were left at 72 °C for 10 min. The second PCR was performed using the primers NUP and the inner primer, AP4-52. The amplification conditions were identical to those described above. The PCR product was gel purified, ligated into the pMD18-T vector (TaKaRa) and the recombinant plasmid DNA transformed into XL-1 blue competent cells. The DNA from recombinant clones was isolated and sequenced (Invitrogen, Guangzhou, China).

For the 3′-RACE reactions, we used the specific primers, AP4-31 (5′-GAG GCA TCT GAT CAC GCT TTA GCC-3′) and AP4-32 (5′-G ATG GAG GCA GAG AAA AGG ACT CG-3′) (Fig. 1). These were synthesized based on the 5′-cDNA sequences. The PCRs were performed using the outer primer (AP4-31) and UPM (Clontech), and the inner primer (AP4-32) and UPM for 3′-cDNA. The PCR conditions used were the same as for the 5′-RACE. A positive clone was selected and sequenced as for the 5′-RACE reactions.

Construction of the expression system

The total RNA from H. armigera pupa brain was obtained using the acid guanidinium thiocyanate-phenol-chloroform method (Chomczynski & Sacchi, 2006). One microgram of total RNA was reverse transcribed at 42 °C for 1 h in 25 µl of reaction buffer that contained 1 mM deoxynucleotide triphosphates, 50 pmol oligo-dT18 primer and 10 units of reverse transcriptase Avian Myeloblastosis Virus (AMV) (TaKaRa). The open reading frame of Har-AP-4 was amplified using the primers, AP4-1 (5′-ATG TCA TTA CAC GGT AAT-3′) and AP4-2 (5′-TCA CGA ATG CTT AAC TAG-3′). The full-length sequence contains BamHI and XhoI restriction sites. The PCR products were purified, digested using BamHI and XhoI, and cloned directly into the digested blank plasmids, pBluescript KS (+) or pIZ/V5-His (Invitrogen). The recombinant plasmids were named T7-Har-AP-4 and GFP-Har-AP-4.

Developmental expression of Har-AP-4 mRNA

The developmental expression of AP-4 mRNA was investigated in the brain and suboesophageal ganglion (brain-SG) complexes of H. armigera using PCR, carried out according to previously described procedures (Zhao et al., 2004). The total RNA was extracted from the brain and SG, or from the brain-SG complexes. First-strand cDNA was synthesized using 1 µg total RNA at 42°C for 1 h, prepared with an AMV reverse transcription system kit (TaKaRa). The Har-AP4 cDNA fragment was amplified using the primers AP4-AF and AP4-AR for 27 cycles, and actin was used as an internal standard.

The expression and purification of recombinant Har-AP-4

The cDNA containing the H. armigera AP-4 HLH domain was amplified using PCR and the two primers AP4-PF (5′-AGA GAA ATT GCT AAC AGC-3′) and AP4-PR (5′-TCA CTT CAG AGG TAT TTC ACC-3′), which contain the two restriction sites NdeI and EcoRI, respectively. The product was then directly subcloned into the digested pET28a vector (pET-Har-AP-4). The expression and purification of recombinant pET-Har-AP-4 was carried out as described previously in Cui & Xu (2006). Purified rAP-4 was quantified using the Bradford method (Bradford, 1976).

The generation of the Har-AP-4 polyclonal antibody and the Western blot analysis

The recombinant Har-AP-4 protein, which consists of 101 amino acids (the HLH domain) was used to generate polyclonal antibodies in rabbits as described previously (Cui & Xu, 2006).

Proteins for Western blotting were extracted from 15 brain-SG complexes from pupae by homogenization in phosphate-buffered saline, followed by centrifugation at 12 000 g for 20 min at 4 °C. The supernatants were lyophilized and stored at −80 °C until use. The protein extracts (30 µl, equivalent to 1.5 brain-SG complexes of H. armigera) were separated using 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) membrane (Hybond-P, Amersham, Piscataway, NJ, USA) using the standard protocol. The immunoreactivity was tested using the anti-Har-AP-4 at a 1:1000 dilution. Nonspecific binding was blocked using a 5% fat-free milk solution, and the Har-AP-4 protein was detected using the DAB Stock Stain Kit (Boster Co., Wuhan, China) (Cui & Xu, 2006).

In vitro translation and EMSA

T7-AP-4 plasmid DNA (1 µg) was used as a template for in vitro translation using the TNT Quick Coupled Transcription/Translation System (Promega, Madison, WI, USA) containing 40 µl TNT T7 Quick Master Mix. The translation product (2 µl) was then used for the EMSA assay using procedures that have been described previously (Hong et al., 2006).

Nuclear protein extracts and the reporter plasmid

Nuclear protein extracts were prepared from the SG of day 1 pupae according to the procedures described by Zhang et al. (2004a). The DH-PBAN promoters, HP1, HP2 and the HP4 luciferase reporter plasmid (which starts at position +29 and extends to −291, −371 or −534 bp) were described by Hong et al. (2006).

Cell culture and transfection

The B. mori cell line (Bm-N) was cultured in TC-100 Insect Medium (Sigma, St Louis, MO, USA) with 10% foetal calf serum at 27 °C. For transient transfections, the cells were diluted 1:3. Then, 100 µl cells were plated per well in a 96-well format and cultured for 24 h.

Twenty microlitres of transfection solution [containing 1 µl cellfectin, 0.8 µg reporter plasmid DNA and 0.2 µg internal control plasmid (the pRL-TK Control Vector, Promega)] were transfected in 100 µl serum-free medium for 6 h (Liu et al., 2007). For co-transfection, 2 µl cellfectin, 0.8 µg reporter plasmid DNA, 0.8 µg GFP-Har-AP-4 plasmid and 0.2 µg of an internal control plasmid were used. The serum-free medium was then replaced with the medium containing 10% foetal serum. The cells were incubated for further 48 h and harvested. Each treatment was repeated three times.

The cell nuclei were counter-stained with DAPI and visualized using an inverse fluorescence microscope (Olympus IX70, Guangzhou, China).

Measurement of the luciferase activity

To check whether Har-AP-4 could activate the DH-PBAN promoter, we assayed the activity of luciferase using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. The activity of luciferase was determined in triplicate using three separate experiments and a liquid-scintillation spectrometer (Beckman LS6000 series, Ramsey, MN, USA). The firefly luciferase activities were divided by the Renilla luciferase activities to control the transfection efficiency.

Statistical analyses

One-way anova and Tukey's tests were performed using Origin 8.0 (OriginLab, Northampton, MA, USA). P-values lower than 0.05 were considered significant and P-values of 0.01 or lower were considered highly significant. The error bars represent the SEMs.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

This study was supported by a Grant-in-Aid from the Natural Scientific Foundation (30730014) of the National Natural Science Foundation of China and the Major State Basic Research Developmental Program (2006CB102001) of the Ministry of Science and Technology of China.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  • Aranburu, A., Carlsson, R., Persson, C. and Leanderson, T. (2001) Transcription factor AP-4 is a ligand for immunoglobulin-kappa promoter E-box elements. Biochem J 354: 431438.
  • Badinga, L., Song, S., Simmen, R.C. and Simmen, F.A. (1998) A distal regulatory region of the insulin-like growth factor binding protein-2 (IGFBP-2) gene interacts with the basic helix-loop-helix transcription factor, AP-4. Endocrine 8: 281289.
  • Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248254.
  • Cao, J., Tang, M., Li, W.L., Xie, J., Du, H., Tang, W.B. et al. (2009) Upregulation of activator protein-4 in human colorectal cancer with metastasis. Int J Surg Pathol 17: 1621.
  • Chomczynski, P. and Sacchi, N. (2006) The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nat Protoc 1: 581585.
  • Cui, S.Y. and Xu, W.H. (2006) Molecular characterization and functional distribution of N-ethylmaleimide-sensitive factor in Helicoverpa armigera. Peptides 27: 12261234.
  • Denlinger, D.L. (2002) Regulation of diapause. Annu Rev Entomol 47: 93122.
  • Denlinger, D.L., Yocum, D.G. and Rinehart, J.L. (2005) Hormonal control of diapause. In Comprehensive Molecular Insect Science, Volume 3 (Gilbert, L.I., Iatrou, K. and Gill, S.S., eds), pp. 615650. Elsevier Press, Amsterdam.
  • Hong, B., Zhang, Z.F., Tang, S.M., Yi, Y.Z., Zhang, T.Y. and Xu, W.H. (2006) Protein-DNA interactions in the promoter region of the gene encoding diapause hormone and pheromone biosynthesis activating neuropeptide of the cotton bollworm, Helicoverpa armigera. Biochim Biophy Acta Gene Struct Expr 1759: 177185.
  • Hu, Y.F., Luscher, B., Admon, A., Mermod, N. and Tjian, R. (1990) Transcription factor AP-4 contains multiple dimerization domains that regulate dimer specificity. Genes Dev 4: 17411752.
  • Jung, P., Menssen, A., Mayr, D. and Hermeking, H. (2008) AP4 encodes a c-MYC-inducible repressor of p21. Proc Natl Acad Sci USA 105: 1504615051.
  • King-Jones, K., Korge, G. and Lehmann, R. (1999) The helix-loop-helix proteins dAP-4 and Daughterless bind both in vitro and in vivo to SEBP3 sites required for transcriptional activation of the Drosophila gene Sgs-4. J Mol Biol 291: 7182.
  • Liu, W., Chen, W., Zhang, P., Yu, C., Kong, F., Deng, J. et al. (2007) Molecular cloning and analysis of the human PCAN1 (GDEP) promoter. Cell Mol Biol Lett 12: 482492.
  • Murre, C., Bain, G., Van Dijk, M.A., Engel, I., Furnari, B.A., Massari, M.E. et al. (1994) Structure and function of helix-loop-helix proteins. Biochim Biophys Acta 1218: 129135.
  • Ou, S.H., Garcia-Martinez, L.F., Paulssen, E.J. and Gaynor, R.B. (1994) Role of flanking E box motifs in human immunodeficiency virus type 1 TATA element function. J Virol 68: 71887199.
  • Sato, Y., Oguchi, M., Menjo, N., Imai, K., Saito, H., Ikeda, M. et al. (1993) Precursor polyprotein for multiple neuropeptides secreted from the suboesophageal ganglion of the silkworm Bombyx mori: characterization of the cDNA encoding the diapause hormone precursor and identification of additional peptides. Proc Natl Acad Sci USA 90: 32513255.
  • Tsujimoto, K., Ono, T., Sato, M., Nishida, T., Oguma, T. and Tadakuma, T. (2005) Regulation of the expression of caspase-9 by the transcription factor activator protein-4 in glucocorticoid-induced apoptosis. J Biol Chem 280: 2763827644.
  • Xu, W.H., Sato, Y., Ikeda, M. and Yamashita, O. (1995) Molecular characterization of the gene encoding the precursor protein of diapause hormone and pheromone biosynthesis activating neuropeptide (DH-PBAN) of the silkworm, Bombyx mori and its distribution in some insects. Biochim Biophys Acta 1261: 8389.
  • Yamashita, O. (1996) Diapause hormone of the silkworm, Bombyx mori: structure, gene expression and function. J Insect Physiol 42: 669679.
  • Zhang, T.Y. and Xu, W.H. (2009) Identification and characterization of a POU transcription factor in the cotton bollworm, Helicoverpa armigera. BMC Mol Biol 10: 25.
  • Zhang, T.Y., Kang, L., Zhang, Z.F. and Xu, W.H. (2004a) Identification of a POU factor involved in regulating the neuron-specific expression of the gene encoding diapause hormone and pheromone biosynthesis-activating neuropeptide in Bombyx mori. Biochem J 380: 255263.
  • Zhang, T.Y., Sun, J.S., Zhang, L.B., Shen, J.L. and Xu, W.H. (2004b) Cloning and expression of the cDNA encoding the FXPRL family of peptides and a functional analysis of their effect on breaking pupal diapause in Helicoverpa armigera. J Insect Physiol 50: 2533.
  • Zhang, T.Y., Sun, J.S., Zhang, Q.R., Xu, J., Jiang, R.J. and Xu, W.H. (2004c) The diapause hormone-pheromone biosynthesis activating neuropeptide gene of Helicoverpa armigera encodes multiple peptides that break, rather than induce, diapause. J Insect Physiol 50: 547554.
  • Zhang, T.Y., Sun, J.S., Liu, W.Y., Kang, L., Shen, J.L. and Xu, W.H. (2005) Structural characterization and transcriptional regulation of the gene encoding diapause hormone and pheromone biosynthesis activating neuropeptide in the cotton bollworm, Helicoverpa armigera. Biochim Biophys Acta Gene Struct Expr 1728: 4452.
  • Zhao, J.Y., Xu, W.H. and Kang, L. (2004) Functional analysis of the SGNP I in the pupal diapause of the oriental tobacco budworm, Helicoverpa assulta (Lepidoptera: Noctuidae). Regul Pept 118: 2531.