Agonist-mediated assembly of the crustacean methyl farnesoate receptor

The methyl farnesoate receptor (MfR) orchestrates aspects of reproduction and development such as male sex determination in branchiopod crustaceans. Phenotypic endpoints regulated by the receptor have been well-documented, but molecular interactions involved in receptor activation remain elusive. We hypothesized that the MfR subunits, methoprene-tolerant transcription factor (Met) and steroid receptor coactivator (SRC), would be expressed coincident with the timing of sex programming of developing oocytes by methyl farnesoate in daphnids. We also hypothesized that methyl farnesoate activates MfR assembly. Met mRNA was expressed rhythmically during the reproductive cycle, with peak mRNA accumulation just prior period of oocytes programming of sex. Further, we revealed evidence that Met proteins self-associate in the absence of methyl farnesoate, and that the presence of methyl farnesoate stimulates dissociation of Met multimers with subsequent association with SRC. Results demonstrated that the Met subunit is highly dynamic in controlling the action of methyl farnesoate through temporal variation in its expression and availability for receptor assembly.


Results
SRC cloning. We initially demonstrated that daphnid Met associated with SRC to form the functional MfR using SRC derived from A. aegypti. Here, we cloned SRC from D. pulex for use in our assessment. The derived daphnid SRC nucleotide sequence (Fig. S1) was 97.8% similar to the D. pulex SRC sequence reported by Miyakawa et al. 16 . At 2,357 amino acids the D. pulex SRC is much longer than A. aegypti SRC and contains an extended C-terminal end. Not surprisingly, the gene is more comparable in length and sequence similarity, 79.7%, to D. magna SRC. The deduced amino acid sequence of the D. pulex SRC had 29.0% sequence similarity to the A. aegypti homolog used to locate SRC in the daphnid genome (Fig. S2). The daphnid SRC contains ten "LXXLL" transcription factor binding domains in the C-terminal. The basic helix-loop-helix (bHLH) domain, has 100% sequence similarity to the sequence deduced in D. pulex and in D. magna and 52% to A. aegypti. The Per-Arnt-Sim (PAS) domain, has 100% sequence similarity to that previously deduced in D. pulex and in D. magna and 37.5% to A. aegypti.
MfR subunit expression. We hypothesized that daphnid MfR subunits (Met and/or SRC) would be expressed in a temporal fashion such that mRNAs would be present and available for protein production just prior to or during the period of susceptibility to male sex determination. Analysis of Met mRNA levels revealed that levels of this MfR subunit transcript oscillates over the course of each molt/reproductive cycle, with base level expression at the beginning and end of each cycle, and peak expression at 36 hours post molt (Fig. 1a). SRC mRNA levels did not significantly vary over the course of two reproductive cycles, though expression levels were highest at 36 hours post molt (Fig. 1b). This mRNA accumulation apex occurs approximately 24 hours before the onset of susceptibility to methyl farnesoate. Met self-association and dissociation. Some bHLH-Pas proteins have been shown to exist in cells as homo-multimers 17 . BRET experiments were performed to evaluate whether Met self-associated in the absence or presence of its ligand, methyl farnesoate (Fig. 2a). Co-expression of Rluc2-Met along with mAmetrine-Met (mAme-Met) generated a BRET ratio that was significantly elevated as compared to assays performed with various combinations of the Met fusion proteins along with free Rluc2 or mAmetrine (Fig. 2b). Inclusion of methyl farnesoate in the assay significantly reduced the BRET ratio in assays containing both Met fusion constructs, Rluc2-Met (photon donor) and mAme-Met (fluorophore) (Fig. 2b), but had no significant effect on the BRET ratio in assays containing free photon donor or fluorophore.
Concentration-response analysis revealed that the BRET ratio generated from cells with both Met fusion constructs (photon donor and fluorophore) decreased in response to increasing concentrations of methyl farnesoate, with a maximum decrease at 3 μ M methyl farnesoate (Fig. 3a). The decrease in BRET ratio, in response to methyl farnesoate, represented an approximately 50% dissociation of the Met proteins. Thus, Met proteins exist in cells as homo-multimeric complexes and the Met agonist methyl farnesoate stimulates the partial dissociation of these complexes.
Met and SRC association. BRET assays performed with cells co-expressing various combinations of Rluc2-SRC, mAmetrine-Met, and free Rluc2 or mAmetrine all produced measurable BRET ratios. However, methyl farnesoate significantly increased the BRET ratio only in assays containing Rluc2-SRC along with mAme-Met (Fig. 2c). These results supported the hypothesis that methyl farnesoate stimulated the association of Met and SRC to form an active transcription factor.
Concentration-response analysis with the mAme-Met and Rluc2-SRC constructs confirmed that methyl farnesoate stimulates Met and SRC association, with significant association beginning at 10 μ M (Fig. 3b). Therefore, methyl farnesoate stimulates both the dissociation of Met homo-multimers and the formation of Met:SRC dimers.
Met:SRC ligand-mediated transcriptional activation. Lastly, we evaluated the ability of the cloned Met and SRC proteins to initiate gene transcription in response to methyl farnesoate. These experiments were performed as we previously described 15 but with the daphnid SRC in place of the Aedes SRC. The reporter gene activation increased with increasing concentrations of methyl farnesoate, with significant transcriptional activation at concentrations ≥ 10 μ M (Fig. 3c).

Discussion
Environmental cues, such as photoperiod, temperature, population density and food availability alter reproductive patterns in some crustacean species 5,8,9,11 . The hormone, methyl farnesoate, is recognized as mediating many of these actions 18 . The recent identification of the MfR 15,16 enabled further elucidation of how methyl farnesoate controls reproduction and development.
We previously identified the MfR using the SRC ortholog derived from A. aegypti, and determined that methyl farnesoate activated Met-mediated gene transcription in the presence of the insect SRC 17 . However, cloning SRC from Daphnia sp was essential to more fully understand the interaction between methyl farnesoate and the receptor complex. For example, with only 37.5% sequence similarity in their PAS domains, the daphnid and mosquito SRC may have interacted differently with the daphnid Met subunit. PAS domains play an integral role in heterodimerization of PAS proteins 19 , and upon mutation of these domains those protein-protein interactions are diminished 20 .
SRC family members can function as DNA-binding receptor partners or receptor co-activators. The SRC ortholog in A. aegypti was shown to function as a DNA-binding partner of A. aegypti Met to produce the juvenile hormone-responsive transcription factor 21 . As the A. aegypti SRC ortholog activated reporter gene transcription in combination with the daphnid Met 15 and this activity mimicked the activity of daphnid SRC with daphnid Met (this study), we surmise that SRC also functions as a DNA-binding partner to Met in daphnids and likely other crustaceans.
Members of the SRC family typically house 4-6 "LxxLL" binding motifs (where "L" is leucine and x represents any amino acid), that are responsible for binding of the SRC to ligand-bound partner receptors 22 and transcription coactivator recruitment 23 . Seven LxxLL motifs exist in the daphnid SRC, while the A. aegypti FISC contains only one. The presence of these binding domains may be responsible for the dramatic increase in methyl farnesoate-mediated transcriptional activation (18-fold), compared to that measured in previous assays using the shorter A. aegypti FISC (9-fold) 15 .
The relative accumulation of SRC mRNA did not significantly change over the course of the daphnid molt cycle, conceivably due to its probable involvement in other reproductive 24 and metabolic 25 functions. However, daphnid Met mRNA oscillated over the course of each molt cycle, peaking in expression at 36 hours post molt, just prior to the window of oocyte susceptibility to methyl farnesoate 2 . We postulate, that the increased level of Met mRNA results in the accumulation of Met protein during the developmental window of susceptibility to methyl farnesaote resulting in the programming of oocytes to develop as males 2 .
Some of the protein-protein interactions between subunits of the orthologous juvenile hormone receptor, in insects, have been characterized. For example, Drosophila melanogaster Met proteins form spontaneous homomultimers 26,27 that dissociate in the presence of juvenile hormone 27 and juvenile hormone analogs 26 . Juvenile hormone binds with high affinity to the PAS ligand-binding domain of Met in some species 26 , and activates the functional juvenile hormone receptor to initiate gene transcription of some developmental genes 28,29 . Results from the present study suggest that daphnid Met also accumulates in cells as homo-multimers, although we cannot exclude the possibility that the observed Met complexes were a consequence of overexpression of the protein in our experimental system. BRET analyses revealed that daphnid Met forms homo-multimers that partially dissociate in the presence of methyl farnesaote. Although the level of dissociation never exceeded 50% even with the addition of increasing concentration of the hormone. This partial dissociation of Met multimers may reflect the dissolution of inactive Met complexes (e.g., quadrimers) to hormone-activated complexes (e.g., dimers). Many bHLH-PAS proteins operate as heterodimeric protein complexes 30 , although transcriptionally active homodimeric bHLH-PAS protein complexes have also been reported 31 .
BRET assays also revealed that methyl farnesoate stimulated the association of Met with SRC. The ligand-stimulated dimerization of daphnid Met and SRC is consistent with the reported dimerization of mosquito Met and FISC (SRC ortholog) in the presence of juvenile hormone 28 . We and others have shown that SRC is necessary for the activation of some receptor proteins 16,15,22,32 .
We hypothesized that daphnid Met actively contributes to the assembly of the MfR. Results support this hypothesis. Firstly, Met mRNA accumulates in cells, presumably to provide ample protein, just prior to the period of sensitivity to the Met ligand, methyl farnesoate. The resulting Met protein accumulates in cells as multimers (Fig. 4a), that dissociate in response to methyl farnesoate (Fig. 4b). Upon dissociation, hormone-bound Met binds with SRC (Fig. 4c), and this complex functions as the active MfR transcription factor (Fig. 4d). All measured Met responses to methyl farnesoate (Met dissociation, association with SRC, reporter gene activation) occurred in the range of 3 to 10 μ M methyl farnesoate. Methyl farnesoate levels in various crustacean species have been measured to range from 4 nM to 4.0 μ M 33-36 with the range likely reflecting the nadir and apex of methyl farnesoate production. Thus, the concentration of methyl farnesoate required to activate this signaling event in our experimental system seems biologically relevant.
The identification of the early responses of Met to methyl farnesoate enhances our basic understanding of hormone-receptor interactions in crustaceans, an economically and ecologically important genera. As methyl farnesoate is a critical regulator of crustacean reproduction and development, an understanding of the molecular actions of the hormone may lead to strategies for the enhancement or sustainable maintenance of crustacean populations.

Materials and Methods
Methyl farnesoate (Echelon Biosciences Inc., Salt Lake City, Utah), was dissolved in DMSO for delivery to the assay solutions. Final DMSO concentration in all assay solutions including controls was 0.001% v/v in BRET assays and 0.0005% v/v in luciferase reporter gene assays.  Scientific RepoRts | 7:45071 | DOI: 10.1038/srep45071 food suspension prepared as described previously 37 . Under these conditions, cultured organisms were exclusively female and reproduced parthenogenetically.

Cloning of SRC. SRC was cloned from tissues of
The A. aegypti FISC nucleotide sequence (ABE99837) was used to search for the daphnid SRC in the wFlea-Base: the Daphnia Genome Database (http://wfleabase.org). Total RNA from adult female D. pulex was isolated using the SV Total RNA Isolation System (Promega). RNA integrity was verified by agarose gel electrophoresis (2.0%), and purity by the 260/280 nm ratio. Forward (5′ -GGGATTCTAAAACAAAATTGGTACC-3′ ) and reverse (5′ -GAGTCAAGGTCTTGGTTGGATTC-3′ ) oligonucleotide primers were designed to amplify the entire daphnid SRC open reading frame. Amplification of the daphnid SRC was performed with an iCycler Thermal Cycler MfR subunit expression. Three hundred adult female D. magna were reared as described by Hannas et al. 37 for use in MfR subunit gene expression analysis. Daphnids were kept individually in 40 mL daphnid media and sampled in triplicate where each replicate contained 5-10 daphnids. Beginning at 0 hrs post-molt, daphnids were sampled at defined times over two molt cycles. Replicates were kept in RNAlater ® at 4 °C for 24 hrs, then stored at − 80 °C. Whole animal tissue was homogenized using a Next Advance Bullet Blender ® , and RNA isolation and reverse transcription was completed as previously described 37 .
Met and SRC mRNA levels were quantified using 7300 Real Time PCR System (Applied Biosystems, Foster City, CA) and amplification mixtures consisting of 12.5 μL 2x SYBER green (ThermoFischer Scientific), 300 nM primers, 500 ng DNA in a total volume of 25 μL. Reaction mixtures were heated to 95 °C for 5 min, followed by 40 cycles of 95 °C for 5 sec then 60 °C for 1 min. Mixtures were then heated to 90 °C for 15 sec, cooled to 60 °C for 1 min, reheated to 90 °C for 15 sec and re-cooled to 60 °C for 15 sec. A single melting peak was detected for each sample, indicating amplification occurred only for the target DNA sequence. The comparative C T method (2 −ΔΔCT ) was used to assess relative levels of Met and SRC mRNA (normalized to levels of actin and gapdh within the same cDNA sample). Met and SRC mRNA levels were normalized to the mRNA levels measured in organisms at 0 hr. Fusion protein construction. The association of Met and SRC was assessed using bioluminescence resonance energy transfer (BRET) methodology. Daphnid SRC was fused to the Renilla luciferase 2 protein (Rluc2), which served as the photon donor during BRET (substrate: coelenterazine 400 A, emission: 410 nm). The daphnid SRC gene was amplified (with a stop codon) from the TOPO cloning vector using primers harboring AgeI (forward) and BstBI (reverse) restriction enzyme sites, and sub-cloned into the pMT-B vector (ThermoFischer Scientific). Rluc2 (a gift from Dr. Sanjiv Gambhir, Stanford University, School of Medicine, Stanford, CA) was amplified from its original storage plasmid (pcDNA) using primers harboring XhoI (forward) and BstBI (reverse). The reverse BstBI primer also contained a short 24 nucleotide "linker" sequence (AGCGGAAGTGGTAGCGGAAGTGGC) to lengthen the distance between the two proteins and decrease probability of incorrect folding. The Rluc2-linker sequence was sub-cloned at the 5′ -terminus of the pMT-SRC plasmid, to create pMT-Rluc2-linker-SRC (referred to as Rluc2-SRC).
The previously cloned Met 15 was fused to yellow fluorescent protein mAmetrine (mAme) to serve as the fluorophore during BRET (excitation: 410 nm, emission: 535 nm). The Met gene was amplified (with a stop codon) using primers harboring NotHFI (forward containing linker sequence, (ATAGCGGAAGTGGTAGCGGAAGTGGT) and BStBI (reverse) restriction enzyme sites, and sub-cloned into the pMT-B vector. mAme was amplified from pBad cloning vector using primers harboring KpnI (forward) and ApaI (reverse) restriction enzyme sites, and sub-cloned at the 5′ -terminus of the pMT-Met, to create pMT-mAme-linker-Met (referred to as mAme-Met).
Met was also fused to Rluc2, for use with mAme-Met, to assess spontaneous association and dissociation of Met multimers. The Met gene was amplified (with stop codon) using primers harboring NotHFI (forward containing linker sequence, AGCGGAAGTGGTACCGGAAGTGG) and BstBI (reverse) restriction enzyme sites, and sub-cloned into the pMT-B vector. Rluc2 was amplified from pcDNA storage vector with primers harboring KpnI (forward) and EcoRI (reverse) restriction enzyme sites and sub-cloned at the 5′ -terminus of the pMT-Met, to create pMT-Rluc2-linker-Met (referred to as Rluc2-Met).
Transfections were performed by calcium phosphate DNA precipitation with the relevant plasmids. Total DNA transfected was constant across treatments, while the donor: acceptor ratio was held at an optimized ratio (producing highest energy transfer), 1: 6 (Rluc2-SRC/mAme-Met). Transcription of the transfected genes was induced with CuSO 4 (500 μ M). Twenty-four hours later, cells were treated with methyl farnesoate for 1 hour in phosphate-buffered saline. Coelenterazine 400 A (5 μ M) was then added and light emission was measured immediately at 410 ± 40 nm and 535 ± 15 nm using a FluoroStar fluorimeter (BMG Labtech). The ratio of light emitted at 535 nm/410 nm (corrected for basal level donor emission of Rluc2 38,39 ) was termed the BRET ratio. The BRET ratio was indicative of the level of dimerization between photon emitter-fusion protein and the fluorophore-fusion protein.
Luciferase reporter gene assays. Luciferase-based reporter gene transcription assays were conducted to assess the ability of the activated Met:SRC to initiate gene transcription. S2 cells were transfected with plasmids containing Met fused to the Gal4 DNA binding domain 15 , SRC, Renilla luciferase (pRL-CMV, internal transfection control, Promega) and the reporter gene vector (pGL5-Luc, Promega). Following transfection, transcription was induced with CuSO 4 (500 μ M for 24 hours). Cells then were treated with methyl farnesoate in Ex-cellTM 420 insect serum-free medium with L-glutamine (SAFC Biosciences, Sigma, St. Louis, MO). Cells were harvested after 24 hours of incubation with methyl farnesoate. Firefly and Renilla luciferase activity were assessed using the Dual-Glo ® luciferase system (Promega) and manufacturer's protocol. Firefly luciferase activity was normalized to Renilla luciferase activity, and each methyl farnesoate treatment group was normalized to DMSO control treated cells.
Statistical analysis. Significant differences between data points were evaluated using Student's t test (p < 0.05) or a one-way analysis of variance (ANOVA) followed by a Tukey's multiple comparison procedure (p ≤ 0.05), as indicated. Statistical analyses were performed using Origin software (OriginLab Corp., Northhampton, MA).