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. 2012 Oct 9;109(41):16576-81.
doi: 10.1073/pnas.1214209109. Epub 2012 Sep 24.

bHLH-PAS heterodimer of methoprene-tolerant and Cycle mediates circadian expression of juvenile hormone-induced mosquito genes

Sang Woon Shin  1 , Zhen Zou, Tusar T Saha, Alexander S Raikhel
Affiliations

bHLH-PAS heterodimer of methoprene-tolerant and Cycle mediates circadian expression of juvenile hormone-induced mosquito genes

Sang Woon Shin et al. Proc Natl Acad Sci U S A. .

Abstract

Juvenile hormone (JH) governs a great diversity of processes in insect development and reproduction. It plays a critical role in controlling the gonadotrophic cycles of female mosquitoes by preparing tissues for blood digestion and egg development. Here, we show that in female Aedes aegypti mosquitoes JH III control of gene expression is mediated by a heterodimer of two bHLH-PAS proteins-the JH receptor methoprene-tolerant (MET) and Cycle (CYC, AAEL002049). We identified Aedes CYC as a MET-interacting protein using yeast two-hybrid screening. Binding of CYC and MET required the presence of JH III. In newly eclosed female mosquitoes, the expression of two JH-responsive genes, Kr-h1 and Hairy, was dependent on both the ratio of light to dark periods and JH III. Their expression was compromised by in vivo RNA interference (RNAi) depletions of CYC, MET, and the steroid receptor coactivator SRC/FISC. Moreover, JH III was not effective in induction of Kr-h1 and Hairy gene expression in vitro in fat bodies of female mosquitoes with RNAi-depleted CYC, MET or SRC/FISC. A sequence containing an E-box-like motif from the Aedes Kr-h1 gene promoter specifically interacted with a protein complex, which included MET and CYC from the female mosquito fat body nuclear extract. These results indicate that a MET/CYC heterodimer mediates JH III activation of Kr-h1 and Hairy genes in the context of light-dependent circadian regulation in female mosquitoes during posteclosion development. This study provides an important insight into the understanding of the molecular basis of JH action.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
CYC binds to MET in a JH III-dependent manner. (A) Schematic diagram of MET used to construct yeast bait. MET122–977 contained bHLH, PAS-A and PAS-B domains, and the full C-terminal region. (B) A MET-binding Y2H clone (Y24) encodes A. aegypti CYC. JH III (10 μg/mL) was necessary for the growth of the yeast clone. (C) Full-length CYC specifically bound to MET in the presence of 10 μg/mL JH III. No significant binding was observed between MET and MET in the presence of JH III (MET/MET). Aedes TGO bound to MET at the background level in the presence or absence of JH III (MET/TGO). In both B and C, the yeast colony transformed with pGBKT7-53 and pGADT7-T plasmids was used as a positive control. The two positive control plasmids were a fusion protein of GAL4 DNA-BD with murine p53 and a fusion of GAL4-AD with large T-antigen, respectively. Murine p53 and large T-antigen specifically bound to each other, irrespective of the presence of JH III (P53/Large T). For the negative control, pGBKT7-Lam expressing human lamin C was used in place of the pGBKT7-53 plasmid and to test its interaction with murine p53 as a control for fortuitous interactions. This control was negative under all conditions (Lam/Large T). (D) JH III, but not methoprene, mediated binding between MET and CYC in a dose-responsive manner. Measurements were performed by means of the quantitative yeast β-galactosidase assay.
Fig. 2.
Fig. 2.
Circadian- and JH-dependent expression of Kr-h1 and Hairy genes. Kr-h1 (A) and Hairy (B) genes showed the circadian rhythmic pattern of gene expression when mosquitoes were kept under a 12 h dark–12 h light (12D:12L) cycle, with a higher expression level at circadian time 8 (CT8) than CT0 on each day. Overall, expression levels of both genes reached maximum at 4D PE. This circadian expression was not observed in the mosquitoes kept in constant darkness (24D:0L) and transcripts remains low at 4D PE. (C and D) Female mosquitoes 1-d PE were collected at CT0, the transition time from dark to light in a 12D:12L cycle, and each mosquito was treated with 0.2 μL of either 1 μg/mL JH III in acetone solution or acetone only. RNA samples were isolated at 4-h intervals until CT24 (same time point as CT0 2D PE) and applied to qPCR for quantifying the gene expression of Kr-h1 (C) and Hairy (D). Statistical significance between samples was evaluated using the Student t test (GraphPad 5.0).
Fig. 3.
Fig. 3.
MET, CYC, and FISC are required for the circadian activation of Kr-h1 and Hairy genes. RNAi depletion of CYC (iCYC), MET (iMET), or FISC (iFISC) compromised the circadian activation of Kr-h1 and Hairy genes in fat bodies of Aedes female mosquitoes. After injection of iMET, iCYC, iFISC, and control dsRNA (iLuc) into the newly emerged female mosquitoes (1-d PE), they were subjected to a 12D:12L cycle for 4 d. Total RNA from these 5D PE mosquitoes was extracted at CT0 and CT12 and subjected to qPCR analysis using either Kr-h1 or Hairy gene-specific primers. (C and D) Four days after dsRNA injection of iMET, iCYC, iFISC, or control iLuc into 1 PE-old Aedes female mosquitoes, fat bodies were dissected and incubated in in vitro culture medium either in the presence of 1 μg/mL JH III or solvent (acetone). RNA samples were isolated 6 h after incubation and the levels transcripts were quantified by means of qPCR for Kr-h1 (C) and Hairy (D). Each result represents an average of three biologically independent repeats. Results were normalized against the β-actin transcript. In all experiments, each time point represents an average of three biologically independent repeats. Statistical significance between samples was evaluated using the Student t test (GraphPad 5.0).
Fig. 4.
Fig. 4.
Binding of CYC and MET to the E-box-like motif from the Kr-h1 gene promoter. (A) A map of the 2-kb of 5′ regulatory region of the Kr-h1 gene. It was cloned by means of 5′-RACE PCR based on the nucleotide sequence of AAEL002390 (Kr-h1). It harbored three imperfect E-box-like (CACGCG) motifs—K1, K2, and K4—and a single K3 CACGTG motif. E-box–like motifs (indicated in bold) and their flanking sequences, used for gel mobility shift assay (EMSA) experiments (B), are indicated. (B) The EMSA revealed the presence of a DNA–protein complex between the Kr-h1 K1 sequence and the nuclear extract from fat bodies of female mosquitoes (left panel). The presence of MET and CYC, but not FISC, in the DNA–protein complex was confirmed by adding polyclonal antibodies against each of these proteins to the binding reactions (center panel). NS – nonspecific serum control. For competition tests (Right), a 50-fold molar excess of unlabeled probe or a nonspecific double-stranded oligonucleotide (AP2) was added to the binding mixtures. (C and D) Gel mobility shift assay using nuclear extracts from Schneider Drosophila S2 cells. (C) The assay revealed the presence of a DNA–protein complex between the Kr-h1 K1 sequence and the nuclear extract from Schneider Drosophila S2 cells (Left). This K1 complex shows a different mobility from that of the complex, formed by binding between the ETv motif and the nuclear extract (Right). (D) The presence of CYC in the Kr-h1 K1 complex, but not in the ETv complex, was confirmed by adding polyclonal antibody against Drosophila CYC to the binding reactions (Left). For competition tests (Right), an ∼50-fold molar excess of unlabeled probe or a nonspecific double-stranded oligonucleotide (AP2) was added to the binding mixtures.
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