The competence factor beta Ftz-F1 potentiates ecdysone receptor activity via recruiting a p160/SRC coactivator

Abstract

Hormones provide generalized signals that are interpreted in a specific spatial and temporal manner by a developing or reproducing multicellular organism. The ability to respond to hormones is determined by the competence of a cell or a tissue. The betaFtz-F1 orphan nuclear receptor acts as a competence factor for the steroid hormone 20-hydroxyecdysone (20E) in Drosophila melanogaster metamorphosis and mosquito reproduction. The molecular nature of the betaFtz-F1 action remains unclear. We report that the protein-protein interaction between betaFtz-F1 and a p160/SRC coactivator of the ecdysone receptor, FISC, is crucial for the stage-specific expression of the 20E effector genes during mosquito reproduction. This interaction dramatically increases recruitment of FISC to the functional ecdysone receptor in a 20E-dependent manner. The presence of betaFtz-F1 facilitates loading of FISC and the ecdysone receptor on the target promoters, leading to enhanced local histone H4 acetylation and robust activation of the target genes. Thus, our results reveal the molecular basis of competence for the stage-specific 20E response.

FIG. 1.

FISC resembles a steroid receptor coactivator of the p160 family. (A) Deduced amino acid sequence of FISC. The bHLH domain is underlined in the deduced amino acid sequence of FISC. The PAS and PAS-associated C-terminal domains are shaded. The LXXLL motifs are shown in bold and underlined. The nucleotide sequence has been deposited in the GenBank database under accession number DQ469817. (B) Schematic alignment of mosquito FISC, Drosophila Taiman, and human AIB1. The degree of amino acid sequence identity within the bHLH and PAS domains is expressed as a percentage.

FIG. 2.

Expression of FISC in the mosquito Aedes aegypti. (A) mRNA profile of FISC in mosquitoes of different developmental stages. Total RNAs were isolated from early-fourth-instar larvae (12 to 24 h post-third molt), early pupae (12 to 24 h postpupation), and the indicated adult mosquitoes. FISC transcripts were measured using real-time reverse transcription-PCR and normalized to β-actin expression. Arbitrary units are plotted against developmental time. Representative data (means ± standard errors of the means) from three independent experiments are shown. (B) FISC mRNA profile in the fat body of adult female mosquitoes.

FIG. 3.

FISC and βFtz-F1 are required for a proper 20E response in the fat body after blood ingestion. dsRNAs were injected into the thoraxes of female mosquitoes as described in Materials and Methods. The transcripts of the indicated genes in female mosquitoes were measured using real-time reverse transcription-PCR at the indicated time after blood feeding and were normalized to β-actin expression. WT, uninjected A. aegypti Rockefeller/UGAL strain; Mal RNAi, injected with double-stranded RNA complementary to bacterial malE; FISC RNAi, injected with FISC dsRNA; Ftz-F1 RNAi, injected with βFtz-F1 dsRNA. Arbitrary units are plotted against developmental time expressed as hours PBM. Representative data (means ± standard errors of the means) from at least three independent experiments are shown.

FIG. 4.

βFtz-F1 potentiates 20E activation of the Vg promoter. (A) Schematic illustration of the βFtz-F1 binding sites in the region of the ecdysone response elements on the Vg promoter. (B) Schematic diagram of βFtz-F1 mutants. DBD, DNA-binding domain; LBD, ligand-binding domain; F, Ftz-F1 box. (C) Effects of βFtz-F1 and FISC on EcR/USP-mediated transactivation of the Vg promoter. L57-3-11 cells were transfected with a pVg1.0-Luc reporter construct and the indicated expression plasmids. After transfection, cells were cultured in control medium (CM) or medium with 1 × 10⁻⁶ M 20E. Data represent ratios of firefly luciferase to Renilla luciferase activity (relative luciferase activity), and the values shown are the means from three independent experiments, with error bars representing the standard deviations of the means. (D) Mutation in the DNA-binding domain or Ftz-F1 box abolishes binding of βFtz-F1 to its cognate DNA sequence. EMSA was performed using ³²P-labeled oligonucleotides containing consensus βFtz-F1 binding sites and in vitro-synthesized βFtz-F1, βFtz-F1^C357A, and βFtz-F1^414A2. The bottom panel shows a Western blot of the in vitro-synthesized βFtz-F1 and its derivatives with polyclonal βFtz-F1 antibodies. (E) Overexpression of βFtz-F1 does not affect the levels of AaFISC, AaEcR-B, and AaUSP-B in the cell transfection assay. A portion of the transfected L57-3-11 cells was analyzed with antibodies against AaβFtz-F1, AaEcR, AaUSP, and AaFISC. The expression vectors used in this experiment are indicated at the top of the panel.

FIG. 5.

FISC physically binds to both EcR/USP and βFtz-F1. (A and B) Protein complexes identified by immunoprecipitation (IP) experiments. L57-3-11 cells were transfected with the indicated expression vectors. Cell extracts were incubated with antibody (Ab) against EcR or βFtz-F1 (A) or USP (B). The precipitated proteins were detected by immunoblotting (IB) with anti-V5 antibody. In the bottom of panel A, 50% of the inputs were loaded and subjected to Western blot analysis with the indicated antibodies. An additional protein band (asterisk) reacted to FISC antibodies, presumably representing a FISC derivative generated by using an alternative start codon. (C) Protein interactions in the fat body detected by gel mobility shift assays. Nuclear proteins were extracted from fat bodies of female mosquitoes at 6 h post-blood meal. Gel shift assays were performed with a ³²P-labeled probe corresponding to the EF fragment of the Vg promoter (Fig. 4A). FBNE, fat body nuclear extracts.

FIG. 6.

EcR/USP, FISC, and βFtz-F1 are translocated into the nucleus of the fat body after blood feeding. The fat body was stained with polyclonal antibodies against EcR (green), FISC (red), and βFtz-F1 (green). Cells were also stained with DAPI to visualize nuclei (blue). Stained fat body trophocyte cells were examined using confocal microscopy. Note the altered cellular distribution of EcR and FISC in fat bodies with downregulated βFtz-F1 expression (βFtz-F1 RNAi). WT, uninjected A. aegypti Rockefeller/UGAL strain.

FIG. 7.

βFtz-F1 enhances the 20E-dependent interactions between EcR/USP and FISC. (A) Schematic diagram of EcRb, USPb, βFtz-F1, and FISC. (B to D) Protein interactions were tested in a mammalian two-hybrid system. CV-1 cells were transfected with 5× UAS-TATA-Luc reporter construct, along with expression vectors for GAL4 and VP16 fusions, and full-length USPb and βFtz-F1 proteins, as indicated. After transfection, cells were cultured in medium with or without 1 × 10⁻⁶ M 20E. Data represent ratios of firefly luciferase to Renilla luciferase activity (relative luciferase activity), and the values shown are the means from three independent experiments, with error bars representing the standard deviations of the means. For EcRΔAF2, the AF2 core was removed from GAL4-EcR^348-675. DBD, DNA-binding domain; LBD, ligand-binding domain; CM, control medium.

FIG. 8.

βFtz-F1 is crucial for the recruitment of EcR/USP/FISC to the 20E-responsive promoters. (A) βFtz-F1, FISC, and EcR/USP are associated with the Vg promoter in the fat body after blood feeding. Fat bodies of female mosquitoes were collected at 6 h PE, 96 h PE, and 6 h PBM. ChIP experiments were performed using antibodies against acetylated H4 (Ac-H4), EcR, βFtz-F1, and FISC, followed by PCR analysis of the indicated regions of the Vg promoter. The amounts of input promoter DNA were also examined using PCR. (B) Binding of EcR/USP, βFtz-F1, and FISC is restricted to the proximal region harboring EcRE on the Vg promoter. ChIP was carried out as described for panel A, except that a region about 0.9 kb upstream from the EcRE (positions −1670 to −1282) on the Vg promoter was analyzed. (C) Injection of Mal dsRNA does not affect the recruitment of FISC to the Vg promoter. (D) Protein levels of EcR, βFtz-F1, and FISC in the fat bodies used for ChIP assays. Aliquots of fat body cell lysates were subjected to immunoblot analyses with antibodies as indicated. (E) Cooccupancy of EcR/USP, βFtz-F1, and FISC on the Vg promoter. Chromatin fragments from fat bodies of female adults at 6 h PBM were first incubated with USP antibody. An aliquot of total soluble chromatin was set aside without immunoprecipitation and used as the input fraction. USP-bound DNA complexes were precipitated, eluted, and reimmunoprecipitated (Re-ChIP) with antibody against EcR, βFtz-F1, or FISC. After reverse cross-linking, purified DNA was amplified with primers specific for the Vg promoter. A rabbit immunoglobulin G (IgG) was used to detect any nonspecific immunoprecipitated DNA. The gel shown of the PCR products is representative of two separate experiments. WT, uninjected A. aegypti Rockefeller/UGAL strain.