Abstract
Ecdysteroids are steroid hormones that control many aspects of development and physiology. During larval development, ecdysone is synthesized in an endocrine organ called the prothoracic gland through a series of ecdysteroidogenic enzymes encoded by the Halloween genes. The expression of the Halloween genes is highly restricted and dynamic, indicating that their spatiotemporal regulation is mediated by their tight transcriptional control. In this study, we report that three zinc finger-associated domain (ZAD)-C2H2 zinc finger transcription factors—Séance (Séan), Ouija board (Ouib), and Molting defective (Mld)—cooperatively control ecdysone biosynthesis in the fruit fly Drosophila melanogaster. Séan and Ouib act in cooperation with Mld to positively regulate the transcription of neverland and spookier, respectively, two Halloween genes. Remarkably, loss-of-function mutations in séan, ouib, or mld can be rescued by the expression of neverland, spookier, or both, respectively. These results suggest that the three transcription factors have distinct roles in coordinating the expression of just two genes in Drosophila. Given that neverland and spookier are located in constitutive heterochromatin, Séan, Ouib, and Mld represent the first example of a transcription factor subset that regulates genes located in constitutive heterochromatin.
IN insects, ecdysteroids are the principal steroid hormones that control many aspects of development and physiology, including molting, metamorphosis, longevity, and neuronal functions (Ishimoto and Kitamoto 2011; Yamanaka et al. 2013; Niwa and Niwa 2014; Uryu et al. 2015). Among endogenously identified ecdysteroids, including 20-deoxymakisterone A and 24(28)-dehydromakisterone A (Lavrynenko et al. 2015), the best-characterized biologically active ecdysteroid is 20-hydroxyecdysone (20E), which is derived from the prohormone ecdysone.
Similar to vertebrate steroid hormones, 20E is synthesized in vivo through a series of enzymatic steps from suitable sterol precursors such as cholesterol. Although ecdysteroidogenic genes have been intensively studied over the last 15 years, the ecdysone biosynthetic pathway is still not completely understood (Rewitz et al. 2006; Niwa and Niwa 2014). During larval development, ecdysone is synthesized in an endocrine organ called the prothoracic gland (PG), whereas the conversion of ecdysone to 20E occurs in peripheral tissues via the cytochrome P450 monooxygenase Shade (Shd) (Petryk et al. 2003; Yamanaka et al. 2013). In the first step toward ecdysone synthesis in the PG, cholesterol is converted to 7-dehydrocholesterol (7DC) by the Rieske oxygenase Neverland (Nvd) (Yoshiyama et al. 2006; Yoshiyama-Yanagawa et al. 2011). Although the intermediate steps that convert 7DC to 5β-ketodiol are not entirely understood (Ono et al. 2012; Saito et al. 2016), at least three enzymes are thought to be involved in this conversion, including Shroud (Sro) (Niwa et al. 2010), Spook/Spookier (Spok) (Namiki et al. 2005; Ono et al. 2006), and CYP6T3 (Ou et al. 2011). The conversion from 5β-ketodiol to ecdysone is subsequently catalyzed by three P450 enzymes (Warren et al. 2002, 2004; Niwa et al. 2004, 2005). We define here “Halloween genes” collectively as genes encoding enzymes involved in the conversion of dietary sterols to ecdysteroids. Null mutations in most of the Halloween genes (except nvd, spok, and Cyp6t3) cause characteristic embryonic phenotypes, where a deficiency in ecdysteroids causes the cuticle to remain undifferentiated (Rewitz et al. 2006).
The temporal profiles of the Halloween genes correlate well with the changes in ecdysteroid titer during larval development (Niwa and Niwa 2016a,b). In addition, all known Halloween genes, except for shd, display high tissue specificity, as they are predominantly expressed in the PG (Niwa and Niwa 2014; Christesen et al. 2016; Ou et al. 2016; Nakaoka et al. 2017). Such temporally dynamic and spatially restricted expression profiles of the Halloween genes imply a tight transcriptional control network. To date, several transcription factors (TFs) have been implicated in the PG-specific regulation of the Halloween genes, including βFTZ-F1 (Parvy et al. 2005; Talamillo et al. 2013), Broad (Moeller et al. 2013), the CncC-dKeap1 complex (Deng and Kerppola 2013), DHR4 (Ou et al. 2011), Knirps (Danielsen et al. 2014), Molting defective (Neubueser et al. 2005; Ono et al. 2006; Danielsen et al. 2014), and Ventral veins lacking (Cheng et al. 2014; Danielsen et al. 2014). Although all these TFs are essential for the expression of ecdysteroidogenic genes in the PG, the tissue distribution of these TFs is not restricted to the PG, raising the question as to how the tissue specificity of ecdysone production is ensured.
In the fruit fly, the most recently identified ecdysteroidogenic TF is Ouija board (Ouib), which displays unique characteristics regarding spatial expression and in vivo function (Komura-Kawa et al. 2015). The ouib gene encodes a DNA-binding protein with five C2H2-type zinc finger motifs and an N-terminal protein domain known as zinc finger-associated domain (ZAD) (Chung et al. 2002). In contrast to other ecdysteroidogenic TFs, ouib is specifically expressed in the PG of Drosophila melanogaster. Null mutations of ouib resulted in developmentally arrested larvae and caused sharply reduced expression of a single Halloween gene, spok. Consistent with this finding, the regulatory region of spookier harbors a response element that appears to be specific to Ouib. Strikingly, the developmental arrest phenotype of ouib mutants was rescued by the overexpression of spo, a paralog of spok (spok overexpression had failed for technical reasons). These observations imply that the primary biological function of Ouib is to specifically regulate spok transcription during Drosophila development, which led us to propose that Ouib is the first identified invertebrate TF that is specialized for steroid hormone biosynthesis (Komura-Kawa et al. 2015; Niwa and Niwa 2016b).
The family of the ZAD-C2H2-type zinc finger genes underwent extensive duplication events and expansion during insect evolution (Chung et al. 2002). In the D. melanogaster genome, there are at least 98 ZAD-C2H2-type zinc finger genes (Chung et al. 2007). Besides Ouib, Molting defective (Mld) is another ZAD-C2H2-type zinc finger protein that is required for ecdysone biosynthesis (Neubueser et al. 2005; Ono et al. 2006; Danielsen et al. 2014). These findings raise the question as to whether additional ZAD-C2H2 zinc finger genes are involved in the control of ecdysteroidogenic gene expression in the PG and, if so, how these ZAD-C2H2 zinc finger family members functionally interact with each other.
Here, we describe a third ecdysteroidogenic ZAD-C2H2 zinc finger gene, designated séance (séan), which is crucial for ecdysone biosynthesis in the D. melanogaster PG. Remarkably, PG-specific expression of nvd rescues the lethality associated with a séan mutation. We demonstrate that Séan is of particular importance for the control of nvd expression through a specific element in the nvd promoter region. Moreover, both Séan and Ouib cooperatively act with Mld to positively regulate transcription of nvd and spok, respectively. Our genetic analysis also showed that we could rescue the larval arrest phenotype of mld mutants by the simultaneous overexpression of both nvd and spok. From an evolutionary perspective, Séan, Ouib, and Mld are found only in Drosophilidae species. Our study raises the intriguing possibility that the three ZAD-C2H2 zinc finger proteins Séan, Ouib, and Mld have specifically evolved to regulate the transcriptional activity of just two Halloween genes, nvd and spok, in D. melanogaster.
Materials and Methods
Drosophila strains
Drosophila melanogaster flies were reared on standard agar-cornmeal medium at 25° under a 12:12 hr light/dark cycle. w1118 served as a control strain. y1 v1 nos-phiC31; attP40, v1 and y2 cho2 v1; attP40{nos-Cas9}/CyO (Kondo and Ueda 2013) were obtained from the National Institute of Genetics, Japan. The Cas9-expressing line y[1]M{vas-Cas9}ZH-2Aw[1118]/FM7c (stock number #51323), a deficiency (Df) strain w1118; Df(3R)BSC197/TM6B Tb that lacks a genomic region that includes the séan locus (#9623) (Cook et al. 2012), and upstream activating sequence (UAS)-CG8145(séan)-IRTRiP.GL00720 (#43551) (Perkins et al. 2015) were all obtained from the Bloomington Drosophila Stock Center. We received UAS-CG8145(séan)-IR (#35840 and #100854), UAS-CG11762(ouib)-IR (#108919), UAS-CG8159-IR (#35841), UAS-CG9793(ranshi)-IR (#197393), and UAS-CG9797(M1BP)-IR (#110498) from the Vienna Drosophila RNAi Center (VDRC). The UAS-nvd-IR-1b strain was previously described (Yoshiyama et al. 2006). phm–GAL4#22 (McBrayer et al. 2007), w; UAS-dicer2; phm-GAL4#22/TM6 Ubi-GFP, mld47 (Neubueser et al. 2005), and mld4425 (Ono et al. 2006) were kind gifts from Michael B. O’Connor (University of Minnesota). phm-GAL4 and Feb36-GAL4 (Siegmund and Korge 2001; Andrews et al. 2002) were used as the strains to drive forced gene expression in the PG. UAS-spo-HA and UAS-nvd-Bm[WT]-HA were previously described (Namiki et al. 2005; Yoshiyama-Yanagawa et al. 2011).
Generation of the CG8145/séance alleles
We generated séan alleles via a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system using the pBFv-U6.2 vector (Kondo and Ueda 2013) provided by the National Institute of Genetics, Japan. We selected three independent target sites (Figure 1D). To minimize off-target effects of the CRISPR/Cas9 system, we designed the single-guide RNA (sgRNA) sequence so that no match existed for any stretch of 15 bases on the third chromosome, using the CRISPR Optimal Target Finder at http://tools.flycrispr.molbio.wisc.edu/targetFinder/ (Gratz et al. 2014). Sense and antisense oligonucleotides corresponding to sgRNA target sequences are listed in Supplemental Material, Table S1 in File S2. We inserted annealed 5′-phosphorylated oligonucleotides into BbsI-digested pBFv-U6.2 and pU63-BbsI-chiRNA vectors (Addgene). We injected vectors into the embryos of the y1 v1 nos-phiC31; attP40 or y1 M{vas-Cas9}ZH-2A, w[1118]/FM7c strains, and performed Cas9-based gene targeting as previously described (Kondo and Ueda 2013). Genetic crosses and detection of indel (insertion/deletion) mutations at the séan locus were either conducted with T7 endonuclease (New England Biolabs, Beverly, MA) as previously described (Kondo and Ueda 2013; Komura-Kawa et al. 2015) or by PCR screening of F1 males after they had produced sufficient offspring (for primers see Table S1 in File S2). DNA fragments including the Cas9 target site were amplified by PCR with the extracted genome DNA from each strain, KOD FX Neo (TOYOBO, Osaka, Japan), and the primers (Table S1 in File S2) . Eventually, we isolated one strain for each target site for further analyses. These strains were renamed séan33, séan60, and séan557, all of which caused frameshift mutations within the séan ORFs (Figure 1, E and F and Figure S1 in File S1).
Generation of séance (CG8145) mutant alleles by the CRISPR/Cas9 system. (A) The genomic structure of séance and surrounding genes. The data are derived from the FlyBase GBrowse website (http://flybase.org/cgi-bin/gbrowse2/dmel/?Search=1;name=FBgn0037617). Numbers indicate the nucleotide positions at the 85A9 cytological location of the chromosome 3R scaffold. Boxed arrows represent gene spans and their directions. séan is shown in magenta. Four other ZAD-zinc finger protein genes are shown in cyan. (B) RNA in situ hybridization of Drosophila embryo with a séan-probe. (C) Séan expression profile based on four time points during the L3 stage, data retrieved from our previous study (Ou et al. 2016). (D) A schematic representation of the séan gene showing the sgRNA target sites. Exons are shown as black boxes, the transcription initiation site as an arrow, and sgRNA target sites as green triangles. (E) Sequences of sgRNA target sites and deleted regions of three isolated séan alleles: séan33, séan60, and sean557. The 20-bp target sequence corresponding to each target site is indicated in orange, the neighboring 5′-NGG (or 5′-CCN on the other strand) PAM in green, and the cleavage site of Cas9 is shown as red characters. Deleted regions are indicated by hyphens. (F) Predicted protein structures of séan alleles. Séan33 and Séan60 are composed of 33 and 60 amino acids, respectively. Séan557 is four amino acids longer than the wild-type protein, but lacks the first zinc finger domain entirely and part of the second zinc finger domain with an in-frame inappropriate amino acid stretch (dotted line). Also, see Figure S2 in File S1. CRISPR, clustered regularly interspaced short palindromic repeats; L3, third instar; PAM, protospacer adjacent motif; sgRNA, single-guide RNA; VDRC, Vienna Drosophila RNAi Center; wt, wild-type; ZAD, zinc finger-associated domain.
Analyzing developmental progression of séan mutants
We crossed séan33/TM3 Act-GFP flies, séan60/TM3 Act-GFP flies, and w1118 flies with each other. Eggs were laid on grape plates with yeast pastes at 25° for 8 hr. Thirty-six hours after egg laying (AEL), 100 hatched GFP-negative (séan33/+, séan60/+, and séan33/séan60) first-instar (L1) larvae were transferred to vials with a standard cornmeal diet (25 larvae per vial). Every 24 hr, larval stages were scored by tracheal morphology as previously described (Niwa et al. 2010).
Quantification of 20E
For quantification with mass spectrometry, we collected L1 (36 hr AEL) for each genotype and determined the wet weight of each sample, after which samples were frozen in liquid nitrogen and stored at −80° until measurement. Extraction of steroids from whole larval bodies, HPLC fractionation, and mass-spectrometric analyses were previously described (Igarashi et al. 2011; Hikiba et al. 2013). In this study, the quantification range was 0.49–31.25 ng/ml, with a detection limit of 3.68 pg of 20E/mg (wet weight).
For quantification via enzyme-linked immunosorbent assay (ELISA), we harvested séan557 embryos that were laid on grape juice plates within a 6-hr window and transferred them to food plates (standard cornmeal media) at 25°. Control (#51323; Bloomington) and séan557 second-instar (L2) larvae were collected 12–18 hr after the L1/L2 molt. For PG-specific séan-RNAi (RNA interference), control (phm>w1118) and phm>séan-RNAi (#100854; VDRC) were staged at the L2/L3 (third instar) molt and collected 40–44 hr after the molt. Samples were then processed as previously described (Ou et al. 2011).
RNA in situ hybridization
To generate the séan RNA probe, we constructed a pBluescriptII SK(−) (Promega, Madison, WI) plasmid containing the séan coding sequence (CDS), designated here as séan-pBluescript. Using the ReverTra Ace qPCR RT Kit (TOYOBO), we synthesized cDNAs from total RNA isolated from w1118 larval ring glands. A DNA fragment representing the séan CDS was amplified by PCR from the cDNAs, and ligated to SmaI-digested pBluescriptII SK(−), yielding séan-pBluescript. Digoxigenin (DIG)-labeled antisense RNA probes were synthesized using DIG RNA-labeling mix (Roche Diagnostics, Basel, Switzerland) with T3 and T7 RNA polymerases (Thermo Fisher Scientific, Waltham, MA). Fixation, hybridization, and detection were performed as described previously (Lehmann and Tautz 1994; Niwa et al. 2004).
RNA sequencing (RNA-Seq)
We carefully staged control (phm>w1118) and phm>séan-RNAi (VDRC #100854) larvae at the L2/L3 molt, and we dissected ring glands at 44 ± 0.5 hr after the molt. These samples were then transferred to ice-cold TRIzol reagent (Thermo Fisher Scientific). For each sample, the lysates of 20 ring glands were vortexed at room temperature for 5 sec, briefly spun down, and stored at −80° until use. Total RNA was isolated by NucleoSpin RNA (Macherey-Nagel, Düren Germany), quantified by RiboGreen Quanti Kit (Thermo Fisher Scientific), and RNA integrity was analyzed by Agilent Bioanalyzer Pico chips. We used 100 ng of total RNA per sample as input for cDNA library synthesis. Each genotype was analyzed by two independent biological replicates for analysis. The Encore Complete RNA-Seq library systems (NuGEN Technologies, San Carlos, CA) were used to produce the cDNA libraries for next-generation sequencing, following the manufacturer’s instructions. The cDNA libraries resulting from the Encore RNA-Seq systems were pooled together in equal concentrations for sequencing at McGill University and the Génome Québec Innovation Centre (Montréal, Canada). We normalized raw data with ArrayStar 4.0 (DNASTAR), and data were analyzed by ArrayStar 4.0, Access (Microsoft, Redmond, WA), and the Database for Annotation, Visualization and Integrated Discovery (DAVID) (Huang et al. 2009).
Quantitative real-time PCR (qPCR)
RNA samples of wild-type adult ovaries, séan30/sean60 L1 larvae, and Schneider 2 (S2) cells were isolated using the RNAiso Plus reagent (TaKaRa, Shiga, Japan). Genomic DNA digestion and cDNA synthesis were performed using the ReverTra Ace qPCR RT Kit (TOYOBO). qPCR was performed using the THUNDERBIRD SYBR qPCR Mix (TOYOBO) or Universal SYBR Select Master Mix (Applied Biosystems, Foster City, CA), with a Thermal Cycler Dice TP800 or TP870 system (TaKaRa). We used serial dilutions of a plasmid containing the ORF of each gene as a standard. The expression levels of the target genes were normalized to an endogenous control, ribosomal protein 49 (rp49), (Foley et al. 1993) in the same sample. The primers for quantifying séan and mld are described in Table S1 in File S2. Primers used to amplify nvd, sro, spok, phm, dib, and sad were previously described (McBrayer et al. 2007; Niwa et al. 2010).
For all other samples, total RNA of whole larvae was isolated following a modified TRIzol protocol, where we substituted sodium acetate with lithium chloride for RNA precipitation. First, 10 ring glands or 10 CNS-ring gland complexes were dissected in ice-cold phosphate-buffered saline (PBS), rinsed twice with fresh PBS, transferred into TRIzol, and snap-frozen in liquid nitrogen. RNA of dissected tissues was extracted using RNeasy Mini (QIAGEN, Valencia, CA) or NucleoSpin RNA kits, following the manufacturers’ instructions. RNA samples (0.1–2 μg/reaction) were reverse-transcribed using an ABI High Capacity cDNA Synthesis kit (Thermo Fisher Scientific), and the synthesized cDNA was used for qPCR (QuantStudio 6 Flex Real-Time PCR System; Thermo Fisher Scientific) using KAPA SYBR Green PCR master mix (D-Mark) with 5 ng of cDNA template, with a primer concentration of 200 nM. Samples were calibrated to rp49 based on the ∆∆Ct method. Primer sequences used for these samples are listed in Table S1 in File S2. The primer design ([Tm] = 60 ± 1°) was based on the Roche online assay design center.
Immunostaining
Immunostaining of ring glands was essentially performed as described previously (Imura et al. 2017). Dissected larval tissues were fixed in 4% paraformaldehyde in PBS + 0.3% Triton X-100 for 20 min at room temperature. Samples were then washed with PBS and incubated overnight at 4° with primary antibodies: guinea pig anti-Nvd (1:200) (Ohhara et al. 2015) and rabbit anti-Phantom (1:200; Phm) (Parvy et al. 2005). For this study, we used goat anti-guinea pig Alexa Fluor 488 and goat anti-rabbit Alexa Fluor 555 (Life Technologies, Carlsbad, CA) as fluorescent secondary antibodies. Secondary antibodies were diluted 1:200 and incubated for 1 hr at room temperature. Confocal images were captured using an LSM 700 laser scanning microscope (Zeiss [Carl Zeiss], Thornwood, NY).
Sterol supplementation experiments
Twenty milligrams of dry yeast was mixed with 38 μl H2O and 2 μl ethanol, or supplemented with 2 μl of the following sterols dissolved in 100% ethanol: cholesterol (150 mg/ml; Wako, Osaka, Japan), 7DC (150 mg/ml; Sigma [Sigma Chemical], St. Louis, MO), ecdysone (10 mg/ml; Steraloids, Newport, RI), and 20E (50 mg/ml; Sigma). We crossed séan33/TM3 Ser1 GMR2 Act-GFP flies with séan60/TM3 Ser1 GMR2 Act-GFP flies. Eggs were laid on grape plates with yeast pastes at 25° for 12 hr. We distinguished séan33/séan60 from other progenies by the presence or absence of GFP signal of the balancer chromosome. At 36 hr AEL, 50 hatched séan33/séan60 L1 larvae were transferred to the yeast paste on grape plates and kept at 25°. Every 24 hr, developmental stages were scored by tracheal morphology.
UAS vectors, overexpression of genes, GFP reporter constructs, and the generation of transgenic strains
The GAL4-UAS system (Brand and Perrimon 1993) was used to overexpress cDNAs in D. melanogaster both in vivo and in cultured S2 cells. For all vector constructions in this study, PCR was performed using KOD Plus Neo (TOYOBO).
To generate the pUAST-séan-cDNA construct, a séan cDNA clone (IP14660; Drosophila Genomics Resource Center, Indiana University) was first PCR-amplified, partially digested with EcoRI and XbaI, and cloned into the pUAST vector using the same enzymes. Transgenic flies carrying the pUAST-séan-cDNA construct were generated using conventional P-element transformation.
To generate a UAS vector to overexpress a cDNA encoding N-terminal V5 (GKPIPNPLLGLDST)-tagged Séan protein, we first made a pWALUM10-moe vector (Ni et al. 2011) with a V5-tag sequence. The oligonucleotides pWAL-V5-N-F and pWAL-V5-N-R (Table S1 in File S2) were annealed and then ligated to EcoRI-BglII-digested pWALIUM10-moe, leading to N-V5-pWALIUM10-moe. In parallel, we amplified the séan CDS by PCR with séan-pBluescript and specific primers [CG8145_N-V5_F and CG8145_N-V5_R (Table S1 in File S2)] to add NdeI and NheI sites to the 5′ and 3′ ends of the CDS, respectively. The PCR fragment was digested with NdeI and NheI, and then ligated into a NdeI-NheI-digested N-V5-pWALIUM10-moe.
To generate a UAS vector to overexpress HA-mld, which encodes N-terminal 3xHA-tagged Mld protein, we obtained a Drosophila Gateway vector (pTHW containing 3xHA sequences) from the Drosophila Genomics Resource Center (https://emb.carnegiescience.edu/drosophila-gateway-vector-collection#_References) (#1099). Specific primers, including a Gateway technology recognition sequence (CACC) at the N-terminus, were used for PCR (Table S1 in File S2). The mld-pUAST vector (Neubueser et al. 2005) (a gift from Stephan M. Cohen, University of Copenhagen, Denmark) was used as template DNA. We performed PCR using KOD Plus Neo (TOYOBO) and ligated the amplified mld CDS region into the pENTR TOPO vector (Thermo Fisher Scientific). This ENTRY vector and pTHW were mixed with LR clonase (Thermo Fisher Scientific), leading to 3xHA-mld-pTHW.
To generate a UAS vector to produce N-terminal 3xTy1 (EVHTNQDPLD)-tagged CG8159 protein, we first made a pBluescript II SK(−) plasmid with a 3xTy1-tag sequence. The oligonucleotides Ty1_x3_For and Ty1_x3_Re (Table S1 in File S2) were annealed and then ligated to a SmaI-digested pBluescript II SK(−), leading to 3xTy1-pBluescript. We amplified the 3xTy1 fragment with 5′ and 3′ extensions by PCR, using the 3xTy1-pBluescript and the specific primers Ty1ForprimerpBlueGib and Ty1-Rev-primer (Table S1 in File S2). In parallel, we amplified the CG8159 CDS by PCR with female whole-body-derived cDNA and the primers CG8159-Fwd-Gib and CG8159-Fwd-Gib (Table S1 in File S2). These two PCR fragments were ligated into a SmaI-digested pBluescript II SK (−) by the In Fusion Cloning Kit (TaKaRa). This ligated plasmid was digested by EcoRI and XbaI, and ligated it into EcoRI-XbaI-digested pWALIUM10-moe.
To generate a UAS vector to express a D. melanogaster nvd cDNA, we obtained a clone (#RE52861) from the Berkley Drosophila Genome Project (Rubin et al. 2000). The RE clone library was made from RNA extracted from D. melanogaster 0–22 hr mixed-stage isogenic y; cn bw sp strain embryos. Sequencing of the 1.7 kb nvd cDNA (RE52861) in the pFlc-1 vector revealed two point mutations resulting in amino acid substitutions without changing the reading frame: an AAA to AGA mutation in exon 1 resulting in a K17R substitution, and a CCT to ACT change in exon 2 resulting in a P159T substitution. The QuikChange Site-directed Mutagenesis kit (Stratagene, La Jolla, CA) was used to repair the two changes to the wild-type sequence. Primer sequences used to repair pFlc-1-RE52861 back to the wild-type sequence are described in Table S1 in File S2. The database sequence was confirmed by sequencing the background chromosome on which the mutations were induced as well as the wild-type chromosome. The repaired cDNA was subcloned from the pFlc-1 vector into the EagI and KpnI sites of pUAST.
Genetic rescue experiments with séan and nvd
For rescue experiments of séan mutants by nvd overexpression, w1118; séan60 phm-GAL4#22/TM6 Tb and w1118; séan33 UAS-nvd-Bm[WT]/TM6 Tb were established by chromosomal recombination on the third chromosomes. The w1118; séan60 phm-GAL4#22/TM6 Tb flies were crossed with the w1118; séan33 UAS-nvd-Bm[WT]]/TM6 Tb flies. Eggs were laid on standard agar-cornmeal medium at 25° for 24 hr. Tb+ L3 larvae, corresponding to séan60 phm-GAL4#22/ séan33 UAS-nvd-Bm animals, were collected and then survival larvae, pupae, and adults of the animals were scored.
For the rescue experiments of mld mutants by simultaneous expression of nvd and spo, w1118; phm-GAL4#22 mld47/TM3 and w1118; UAS-nvd(without tag) mld4425/TM3 flies were established by chromosomal recombination on the third chromosome. We also generated w1118; UAS-spo-HA; phm-GAL4 mld47/TM3. The flies of w1118; phm-GAL4#22 mld47/TM3 were crossed with w1118; mld4425/TM3, and the flies of w1118; UAS-spo-HA; phm-GAL4 mld47/TM3 were crossed with w1118; UAS-nvd(without tag) mld4425/TM3. The number of rescued adults of mld47/mld4425 was scored.
For the rescue experiments of séan557 mutants by séan overexpression, séan557, phm22-GAL4/TM6B, Tb, Hu flies were established by chromosomal recombination. UAS-séan-cDNA (1M); séan557/TM6B, Tb, Hu flies were crossed to séan557, phm22-GAL4/TM6B, Tb, Hu. As a control, the flies of séan557/TM6B, Tb, Hu were crossed to séan557, phm22-GAL4/TM6B, Tb, Hu. Tb+, and progenies were scored for each cross after eclosion.
For the rescue experiments of séan557 mutants by nvd overexpression, séan557, phm22-GAL4/TM6B, Tb, Hu and séan557, UAS-nvd-cDNA(Bm) /TM6B, Tb, Hu flies were established by chromosomal recombination. For homozygotes, the flies of séan557, phm22-GAL4/TM6B, Tb, Hu were crossed to séan557, UAS-nvd-cDNA(Bm) /TM6B, Tb, Hu. For trans-heterozygotes, the flies of séan557, phm22-GAL4/TM6B, Tb, Hu were crossed to séan60, UAS-nvd-cDNA(Bm) /TM6B, Tb, Hu, and the flies of séan557, UAS-nvd-cDNA(Bm)/TM6B, Tb, Hu were crossed to séan33, phm22-GAL4/TM6B, Tb, Hu. As controls, the flies of séan557/TM6B, Tb, Hu were crossed to séan33, phm22-GAL4/TM6B, Tb, Hu and séan60, UAS-nvd-cDNA(Bm)/TM6B, Tb, Hu, respectively. Tb+ progenies were scored for each cross after eclosion.
Construction of luciferase reporter plasmids
We amplified a series of nvd upstream regions from w1118 genomic DNA using primers (Table S1 in File S2) to add NotI and BglII sites to the 5′ and 3′ ends, respectively. These amplified nvd upstream regions were digested with NotI and BglII, and ligated into a NotI-BglII-digested pGL3-Basic vector luciferase reporter plasmid (Promega). We constructed reporter plasmids with mutated regions from the pGL3-Basic plasmid containing a wild-type upstream 301-bp region by inverse PCR with specific primers (Table S1 in File S2).
The +111 to +32 bp upstream region of spok was amplified from w1118 genomic DNA by specific primers (Table S1 in File S2) to add SacI and BglII sites to the 5′ and 3′ ends, respectively. DNA fragments of the +91 to +32 bp and the +71 to +32 bp upstream regions were prepared by annealing sense and antisense oligonucleotides (Table S1 in File S2) containing SacI and BglII sites to the 5′ and 3′ ends, respectively. The amplified and annealed upstream regions of spok were digested with SacI and BglII, and then ligated into a SacI-BglII-digested pGL3-Basic plasmid. All other pGL3-Basic plasmids containing a series of spok promoter regions were previously described (Komura-Kawa et al. 2015).
Transfection and luciferase reporter assays
S2 cells were seeded in 1 ml Schneider’s Drosophila Medium (Thermo Fisher Scientific) with 10% heat-inactivated fetal calf serum and penicillin–streptomycin solution (Wako) in a 24-well plate (TrueLine) 1 day before transfection. Transfection of S2 cells was performed with an Actin5C-GAL4 construct (a gift from Yasushi Hiromi), UAS constructs, and a series of pGL3-Basic plasmids using the Effectene Transfection Reagent (QIAGEN), as previously described (Niwa et al. 2004). The Copia Renilla Control plasmid (#38093; Addgene) (Lum et al. 2003) was used as the reference. Construction of V5-sean-pWALIUM10-moe and HA-mld-pUAST is described above. The UAS-FLAG-ouib construct was described previously (Komura-Kawa et al. 2015), as was UAS-GFP.RN3 (Niwa et al. 2002). The cells were incubated for 2 days after transfection. They were then processed using a Dual-Luciferase Reporter Assay System (Promega) following the manufacturer’s instructions, and were analyzed with Fluoroskan Ascent FL (Thermo Fisher Scientific).
Data availability
Strains, DNA plasmids, and primers are available upon request. RNA-Seq data for Table 1, Table S2 in File S2, Table S3 in File S2, and Table S4 in File S2 are available at the Gene Expression Omnibus with the accession code GSE104340, associated with GSM2795618, GSM2795619, GSM2795620, and GSM2795621.
Results
The ZAD-C2H2 zinc finger gene CG8145/séance encodes a TF with essential roles in the PG
Given the role of the ZAD-C2H2 zinc finger protein Ouija board (Ouib) in controlling ecdysone biosynthesis in D. melanogaster (Komura-Kawa et al. 2015), we wondered whether other family members of the ZAD-C2H2 zinc finger family would also have roles in steroidal pathways. Interestingly, the ouib locus (85A9 on chromosomal arm 3R) is part of a cluster that comprises four additional ZAD-C2H2 zinc finger-encoding genes (CG8145, CG8159, ranshi, and M1BP). These genes are all paralogous to ouib and are presumably the result of tandem duplications (Figure 1A). When we conducted RNA in situ hybridization for these genes in Drosophila embryos, we noticed that CG8145 had strong and specific expression in the ring gland (Figure 1B). A similar pattern has also been reported by the Berkeley Drosophila Genome Project Experiment IP14660 (Tomancak et al. 2007). Consistent with this, when we mined microarray data from a recently published study (Ou et al. 2016), we found that CG8145 transcript levels were moderately enriched in the larval ring gland, but the expression level in the whole-body sample indicated that CG8145 is expressed in other tissues as well (Figure 1C). In contrast, CG8145 showed low expression levels in ovaries, a known source of ecdysteroids in adults (Figure S1 in File S1).
We then examined whether we could observe larval lethality when each of these ZAD-zinc finger protein genes was knocked down by PG-specific RNAi, for which we used the phm22-GAL4 driver (hereafter phm>). In addition to phm>ouib-RNAi, we found that PG-specific RNAi against M1BP and CG8145 resulted in larval lethality, whereas CG8159-RNAi and ranshi-RNAi did not. We omitted M1BP from further analysis in this study, as M1BP is known to encode a TF that regulates expression of non-TATA-type genes (Li and Gilmour 2013). On the other hand, CG8145, also known as “numerous disordered muscles” (short: nom) (Dobi et al. 2014), has no known roles in steroidogenesis, but given its transcript enrichment in the ring gland, we further investigated the function of this gene. Further testing with additional RNAi lines validated our initial findings, since three independent lines all gave the same results and two of the lines (#GL00720) did not overlap in their respective target sequences (Figure 1D). Given that we show here that CG8145 is essential for regulating steroid hormone production, we renamed CG8145 to séance (short: séan), in accordance with the tradition that genes encoding enzymes acting in the conversion of dietary sterols to ecdysteroids are collectively known as Halloween genes, and that it appears to work in conjunction with ouija board. A séance refers to a ritual that uses a ouija board to attempt communicating with the dead.
séance is essential for larval development
To further assess the functional role of séan, we generated séan loss-of-function alleles by using a CRISPR/Cas9 approach (Kondo and Ueda 2013). We isolated three independent mutant alleles, séan33, séan60, and séan557, each of which was caused by a small deletion induced by different sgRNAs (Figure 1D). The séan33 and séan60 alleles lead to premature stop codons in the putative CDS of séan (Figure 1E), eliminating all five zinc finger domains in the C-terminal region of Séan (Figure 1F). In contrast, the séan557 allele is caused by a deletion that removes part of exon 2 and its downstream intron (Figure 1E and Figure S2 in File S1). This deletion is predicted to cause an in-frame readthrough of the remaining intron 2 sequence, resulting in a stretch of inappropriate amino acids (Figure 1F, dotted line) that are incorporated into the protein, removing zinc finger 1 entirely and part of the second zinc finger (Figure 1F and Figure S2 in File S1).
séan33/séan60 animals exhibited an early larval (L1) arrest phenotype, with no animals developing into final instar larvae, pupae, or adults (Figure 2, A–D). When we combined the séan33 or séan60 allele with a Df line that uncovers the séan locus (séan33/Df or séan60/Df), we obtained comparable results, suggesting that both séan33 and séan60 are null alleles. Eventually, all séan33/séan60 transheterozygous animals died by 180 hr AEL retaining L1-like morphology (Figure 2, C and D). In contrast, the majority of control séan33/+ or séan60/+ heterozygous animals became pupae by this time (Figure 2, A and B).
Larval lethality and developmental arrest phenotype of séance mutant larvae. (A–C) The survival rate and developmental progression of control (A and B) and séan mutant animals (C), each at N = 50. (D) Comparison of body size and developmental stage between control (right and middle) and séan33/séan60 mutants (left) at 108 hr AEL. Control animals developed into L3 larvae and adults (not shown), whereas séan mutants showed arrested development as L1 larvae. (E) Phenotypic comparison of séan557 mutants, séan-RNAi, and a rescue with séan-cDNA. séan557 mutants arrest as L1 and L2, whereas PG-specific expression of séan-RNAi (phm>séan-RNAi, VDRC #100854) results in L3 lethality. Homozygous séan557 mutants were rescued by PG-specific expression of séan557 cDNA. (F) Whole-larvae ecdysteroid quantification. Control and sean557 homozygous mutants were compared during L1 (6–18 hr after egg hatch) and L2 (12–18 hr after L1/L2 molt). PG-specific séan-RNAi (VDRC #100854) L3 was compared to that of control animals at 40–44 hr L3. At least three samples were tested per genotype in each data set, and each sample was tested in triplicate. Error bars represent SE. Percentages were normalized to control levels of each data set. For L1, N = 300 for each genotype; for L2, N = 90 for each genotype; and for L3, N = 24 for each genotype. AEL, after egg laying; L1, first instar; L2, second instar; L3, third instar; PG, prothoracic gland; RNAi, RNA interference; TRIP, Transgenic RNAi Project; VDRC, Vienna Drosophila RNAi Center.
Larvae carrying two copies of the séan557 allele died as L1 and L2 larvae, as did séan575/Df hemizygotes (Figure 2E). Although séan33/séan60 transheterozygotes tended to produce nonmolting oversized L1 larvae (Figure 2C), séan557 homozygotes, as well as séan575/Df hemizygotes, produced oversized L2 larvae, with the latter to a lesser degree. PG-specific expression of the three phm>séan-RNAi lines caused developmental arrest in the L3 stage, which also produced larvae that were larger than controls (Figure 2E).
Apart from the overgrowth phenotype, none of the allelic combinations showed any apparent developmental or morphological defects during embryonic or larval stages, suggesting that these animals lacked a signal to progress with development. Based on its similarity and vicinity to ouib, we hypothesized that séan had a similarly important role and that the developmental arrest in séan loss-of-function animals was caused by a lack or reduction of systemically acting steroid hormones.
The séance loss-of-function phenotype is caused by ecdysteroid deficiency
We next examined whether the larval arrest and lethality phenotype of séan loss-of-function animals was caused by a failure to produce sufficient ecdysteroids. We used two experimental approaches to determine ecdysteroid concentrations. First, we examined ecdysteroid titers in L1 larvae (36 hr AEL) of controls and séan33/séan60 transheterozygotes by mass-spectrometric analysis. In the control larvae, we detected 9.54 ± 0.96 pg of 20E/mg of wet weight (mean ± SEM, N = 4). In contrast, ecdysteroid titers in séan33/séan60 animals (N = 5) were below the detectable limit, suggesting that loss of séan function severely impaired ecdysone biosynthesis during larval stages. Second, to confirm these findings, we used an ELISA approach to quantify ecdysteroid concentrations in séan557 and phm>séan-RNAi animals. This method also showed significantly lower ecdysteroid levels in either of these genotypes when compared to those of controls (Figure 2F).
Although the PG-specific disruption of ∼1200 genes via RNAi caused larval arrest phenotypes (Danielsen et al. 2016), it appears that only a fraction of these are directly involved in ecdysone production and its regulation. Consequently, only a few of these RNAi lines can be rescued all the way to adulthood by feeding ecdysone or 20E, a strategy that works very well for Halloween gene loss-of-function lines (Ou et al. 2016). When we tested whether séan mutants could be rescued with dietary ecdysone supplementation, we found that both séan33/séan60 and séan557/séan557 animals were partially rescued by 20E feeding. Specifically, séan33/séan60 transheterozygotes and séan557 homozygotes now reached pupal stages, some of the latter even reaching adulthood (Figure 3, A and B and Figure S3 in File S1). Taken together, these results indicated that loss of séan function caused ecdysteroid deficiency.
Expression analysis of Halloween genes and feeding rescue experiment in séan mutant larvae. (A) Rescue studies for séan33/séan60 larvae. Mutant animals fed 20-hydroxyecdysone (20E) and 7-dehydrocholesterol (7DC) developed into third-instar (L3) larvae, whereas animals reared on cholesterol- and ethanol-containing food (vehicle control) remained first-instar (L1) larvae. Bar, 1 mm. (B) The survival rate and developmental progression of séan33/séan60 mutant animals by oral administration of sterols and ecdysteroids (each N = 60). (C) Relative expression levels [quantitative PCR (qPCR)] of Halloween genes compared to those of controls (dotted line = 1) and various backgrounds of séan mutant or séan-RNAi (RNA interference) (phm>séan-RNAi, Vienna Drosophila RNAi Center #100854). BRGC, the brain-ring gland complex. Error bars indicate SEM. * P < 0.05 and ** P < 0.01 with Student’s t-test (black columns) and 95% C.I.s (all other columns). (D) Relative expression levels (qPCR) of Halloween genes in nvd-depleted prothoracic gland (PG). Expression levels were normalized to controls (dotted line = 1). nvd-RNAi was driven by the Feb36-GAL4 driver, and the BRGCs were dissected from late L3 larvae, collected at 44 hr after the second-instar (L2)/L3 molt. Error bars indicate 95% C.I.s. (E) Immunostaining of the PG cells from control and séan mutant L1 larvae at 36 hr after egg laying with antibodies against Phm (magenta) and Nvd (green). Bar, 25 μm. (F) Expression of nvd-cDNA in a sean557 mutant background rescued L1/L2 lethality to adulthood. L2 pupae form in rare cases using this genetic background; these animals attempt pupariation directly from L2 larvae.
phm>séance-RNAi ring glands exhibit reduction of neverland expression, but not other ecdysone biosynthetic genes
To identify downstream targets of Séan, we carried out RNA-Seq on samples from hand-dissected ring glands isolated from phm>séan-RNAi and control L3 larvae. We collected carefully staged larvae (44 ± 0.5 hr after L2/L3 molt) and dissected ring gland RNAs for transcriptome analysis. We collected 20 ring glands per sample to average out inherent differences in developmental timing, thus accounting for individuals that deviated from the population mean. Using a threefold cutoff for differentially expressed genes, we identified 360 up- and 248 downregulated genes. When we subjected these gene cohorts to GO term analysis via DAVID (Huang et al. 2009), the top term in the upregulated group was for genes involved in oxidation–reduction processes that included three genes involved in ecdysone biosynthesis: phantom (phm), shadow (sad), and spookier (spok) (Table S2 in File S2). Consistent with this, other terms including “steroid biosynthesis,” “cholesterol homeostasis,” “ecdysone biosynthesis,” and “glutathione metabolism” were also enriched among the upregulated gene set, all of which harbored genes with established links to ecdysone production (Enya et al. 2014; Danielsen et al. 2016). In contrast, the TF term was found to be enriched in the downregulated gene set and also harbored genes with known links to ecdysteroid regulation, such as broad, knirps, and E75 (Table S3 in File S2). Based on these findings, we conducted a manual term enrichment analysis based on either gene function (e.g., the eight known Halloween genes) or protein family (e.g., P450 and nuclear receptor genes) (Table 1) to complement, refine, and correct errors intrinsic to the DAVID analysis. By comparing the same terms for both up- and downregulated genes, we found that not only genes associated with ecdysone biosynthetic processes were upregulated, but that a known inhibitor of ecdysone production, HR4, was downregulated. Notably, however, a single steroid biosynthetic gene, neverland (nvd), was nearly 10-fold downregulated (fold changes for genes listed in Table 1 are shown in Table S4 in File S2), suggesting that ecdysone production was impaired, and that the upregulation of the ecdysone biosynthetic pathway was an attempt by PG cells to compensate for overall low ecdysone production due to reduced nvd expression.
To validate these findings, we performed qPCR analysis to examine expression levels of six Halloween genes in both séan mutants and PG-specific séan-RNAi larvae. Consistent with the RNA-Seq data, the only gene with reduced expression was nvd, which was, dependent on the sample, 7- to 50-fold downregulated (Figure 3C). In contrast, sro, spok, phm, dib, and sad all showed moderate to substantial upregulation (∼2- to 70-fold) in séan loss-of-function animals. Curiously, nvd-RNAi animals also showed substantial upregulation of the same Halloween genes (Figure 3D). These results suggest that upregulation of sro, spok, phm, dib, and sad transcripts are not directly affected by the loss of séan function, but rather by impairment of cholesterol and/or 7DC metabolism in the PG.
Immunohistological analysis using anti-Nvd antibodies demonstrated that the Nvd protein level was also markedly reduced in séan33/séan60 larvae compared to that in control animals, but not that of Phm, another ecdysone biosynthetic enzyme in the PG (Figure 3E). Therefore, we hypothesized that the resulting larval arrest phenotype was caused by reduced expression of nvd and not linked to the increase in the expression of other Halloween genes.
séance is required for the conversion of cholesterol to 7DC
Nvd plays a crucial role in the first step of the ecdysone biosynthesis pathway, namely the conversion of dietary cholesterol to 7DC (Yoshiyama et al. 2006; Yoshiyama-Yanagawa et al. 2011). We have previously demonstrated that the larval arrest phenotype of loss-of-nvd-function animals is rescued by feeding 7DC but not cholesterol (Yoshiyama et al. 2006). If séan is required for the regulation of nvd during Drosophila development, we would expect that the larval arrest phenotype of séan should also be rescued by feeding of 7DC. Indeed, when séan33/séan60 transheterozygotes were fed yeast paste supplemented with 7DC, they were rescued to pupal and adult stages, whereas cholesterol was unable to do so (Figure 3, A and B). Similarly, when we repeated the experiment with séan557 homozygotes, only 7DC could rescue these animals to adulthood efficiently, whereas cholesterol, ecdysone, and 20E all failed to do so (Figure S3 in File S1). We noted that the higher rescuing activity of 7DC as compared to 20E was also observed in previous studies using loss-of-function animals for nvd and other genes required for cholesterol trafficking and metabolism (Table S5 in File S2) (Huang et al. 2005; Yoshiyama et al. 2006; Enya et al. 2014). 7DC can be utilized as a dietary precursor to generate a normal temporal fluctuation of ecdysteroids by a series of the biosynthesis enzymes downstream of Nvd. These results suggest that loss of séan function specifically impairs the catalytic conversion of cholesterol to 7DC. These results also demonstrate that the moderate increase in the expressions levels of nobo, sro, spok, dib, and sad does not contribute significantly to the séan mutant phenotype.
The séance mutant phenotype is caused by the loss of neverland expression in the PG
We next examined whether the séan mutant phenotype was rescued by forced expression of nvd using the GAL4-UAS gene expression system. We utilized the UAS-nvd-Bm strain to overexpress the silkworm Bombyx mori ortholog of the nvd gene, as we have previously reported that nvd-Bm can rescue the larval arrest phenotype of nvd-RNAi in D. melanogaster flies (Yoshiyama-Yanagawa et al. 2011). We found that expression of nvd-Bm in the PG recovered the larval arrest phenotype of séan33/séan60 transheterozygotes, which caused as much as 82.6% of animals to reach the adult stage (Figure 3F and Table 2). These results strongly suggest that the developmental arrest phenotype of séan mutants is caused solely by the loss of nvd expression in the PG. Therefore, in conjunction with our previous identification of Ouib (Komura-Kawa et al. 2015), our data support the idea that Séan and Ouib are functionally specialized to regulate the expression of two distinct Halloween genes, nvd and spok, respectively, during development.
The neverland promoter is activated by cotransfection of séance and molting defective in S2 cells
We have previously identified the Ouib response element in a ∼170-bp genomic region upstream of the spok CDS. Moreover, in D. melanogaster S2 cells, the presence of Ouib is sufficient to drive a reporter luciferase (luc) gene fused with the 170 bp spok promoter (Komura-Kawa et al. 2015). Analogous to our previous findings, we focused on the identification of the cis-regulatory element(s) responsible for the Séan-mediated control of nvd expression using the luc assay system in S2 cells. We generated DNA constructs carrying the upstream region of nvd fused with a luc gene cassette, and then transfected S2 cells using these DNA constructs with or without a plasmid for overexpressing V5-tagged-séan (V5-séan). However, we failed to detect Séan-induced luc expression when we introduced the V5-séan plasmid alone into S2 cells, even with a 5-kb genomic region upstream of the translation initiation site of nvd (data not shown).
Although several possibilities exist that would explain this unsuccessful reconstruction, we reasoned that there might be a coregulator(s) acting in conjunction with Séan that is required to induce nvd expression in S2 cells. A candidate for such a coregulator is another ZAD-C2H2 zinc finger protein, Molting defective (Mld), since mld mutants exhibit nvd expression (Danielsen et al. 2014). Mld is, like Séan, a ZAD-C2H2 zinc finger protein (Neubueser et al. 2005; Ono et al. 2006; Danielsen et al. 2014). We found that transfection of HA-tagged-mld (HA-mld) plasmid fused with 301 bp upstream of the nvd CDS, including the 113 bp promoter region and 188 bp 5′ untranslated region, in S2 cells resulted in the induction of luc reporter activity (Figure 4, A–C). Moreover, coexpression of both V5-séan and HA-mld caused further drastic induction of the 301-bp nvd promoter-luc expression (Figure 4C). These results suggest that the first 301 bp upstream of the nvd CDS might contain one or more essential cis-regulatory elements that are required for Séan and Mld function.
Transcriptional activity of Séan and Mld for the upstream element of nvd. (A) Schematic representation of the location of the element (−113 to −93) in the D. melanogaster nvd promoter region responsible for Séan-dependent transcriptional activation. Numbers indicate the distance from the transcription start site (+1, underlined) of nvd, which is based on FlyBase data (http://flybase.org/reports/FBgn0259697.html). The box indicates the 15-bp Séan-Mld response element. (B) The 15-bp element marked by the box in A exhibits a striking similarity to the Ouib response element in the spok promoter (15 bp). The bold letters and black lines indicate matching bases in the alignment between the element in the nvd promoter and the Ouib response element. (C) Luciferase reporter assay with plasmids containing the series of upstream elements of nvd. Numbers indicate the distance from the transcription start site of nvd. The white box indicates the Séan-Mld response element. The gray box represents the nvd CDS. Reporter activities of progressive deletion constructs are shown on the right (each at N = 3). The GFP expression plasmid was used as a negative control. (D) Luciferase reporter assay with plasmids containing the 9-bp transversion mutation in the −113 to −93 region of the 300-bp upstream element of nvd (each at N = 3). The GFP expression plasmid was used as a negative control. Error bars indicate SEM. *** P < 0.005 using Student’s t-test with Bonferroni correction. CDS, coding sequence; WT, wild type.
Identification of a Séance-molting defective-response element in the neverland promoter region
To narrow down the element(s) responsible for the Séan-Mld-dependent expression of nvd, we tested several constructs carrying the upstream region of nvd with a range of deletions within the 301-bp region. We first generated the deletion constructs in 20-bp increments from the 5′-terminus of the 301-bp region. Whereas the luc construct fused with the nvd promoter lacking the region from −113 to −94 bp was not activated by Mld alone, luc expression from this construct was still highly induced in the presence of both Mld and Séan (Figure 4C). In contrast, the region from −93 to −74 bp was crucial for the Séan-Mld-dependent luc reporter activity (Figure 4, B and C). Strikingly, this 20-bp region contains a DNA sequence 5′-AGCTTTATTGCTCAG-3′ that is nearly identical to the Ouib response element in the spok promoter region (5′-AGCTTTATTATTTAG-3′; underlines indicate the exact matches between the two sites) (Figure 4B) (Komura-Kawa et al. 2015). To clarify the importance of this 15-bp region for Séan-Mld-dependent control of gene expression, we introduced transversion mutations within the first 9 bp of this putative 15-bp recognition site. This mutated construct exhibited no luc reporter induction in the presence of Séan and Mld upon transfection into S2 cells (Figure 4D). These results suggest that the 15 bp of the nvd promoter region (from −95 to −81 bp) might serve as a Séan-Mld response element, while Mld also acts on the region from −113 to −94 bp.
We also examined the evolutionary conservation of séan as well as the Séan-Mld response elements in putative nvd promoter regions from other Drosophila species. Among 12 Drosophilidae species for which genome annotations have been reported (Clark et al. 2007), clear orthologs of séan are found in many, but not all, Drosophilidae species (Figure S4 in File S1). To examine the Séan-Mld response elements, DNA sequences around the nvd loci of D. melanogaster, D. simulans, D. sechellia, and D. willistoni were available. Using EMBOSS Matcher, an algorithm to identify local similarities between two sequences (McWilliam et al. 2013), we found that the Séan-Mld response element-like motifs were located in proximity (within 300 bp) to the nvd coding region in all of these four species (Figure S5 in File S1). In particular, the putative nvd promoter regions of D. simulans and D. sechellia contain the same 15-bp sequence motif. In conjunction with our previous analysis on the spok promoter (Komura-Kawa et al. 2015), these data suggest that both Ouib and Séan-Mld response elements are also evolutionarily conserved, at least in some Drosophila species.
The spookier promoter is synergistically activated by Ouija board in the presence of molting defective in cultured S2 cells
Unlike Séan, as we have previously reported (Komura-Kawa et al. 2015), Ouib alone had the ability to activate gene expression via the Ouib response element present in the spok promoter region (Figure 5A). On the other hand, the previous study has also reported that mld mutants also displayed decreased expression of not only nvd but also spok (Ono et al. 2006; Danielsen et al. 2014). Given that Séan and Mld cooperatively activate nvd promoter activity, we next examined if Ouib-mediated activation on gene expression was also synergistically enhanced in the presence of Mld. Strikingly, coexpression of both FLAG-ouib and HA-mld drastically increased luc induction under control of the 230- and 181-bp spok promoter regions, both of which contain the Ouib response element (Figure 5A). In contrast, the synergistic transactivation by Mld was not observed with the 131-bp spok promoter fragment lacking the Ouib response element (Figure 5A). Moreover, endogenous spok expression in S2 cells was drastically induced by coexpression of both HA-mld and FLAG-ouib (Figure 5B), whereas coexpression of HA-mld and V5-séan could not induce endogenous nvd expression (Figure S6 in File S1). These results indicate that Mld also works with Ouib to induce spok expression.
Transcriptional activity of Ouib and Mld for the upstream element of spok. (A) Luciferase reporter assay using the expression of ouib and mld along with plasmids containing the series of upstream elements of spok. Numbers indicate the distance from the translation, but not transcription, start site (+1) of spok, as a transcription start site for spok has not been defined. The numbering style of this study is exactly the same as that of Komura-Kawa et al. (2015). The white box indicates the Ouib response element (ORE). The gray box represents the spok coding region. The inset is an enlarged view of the transcriptional activity of Ouib, Mld, and GFP for the upstream element of spok. Reporter activities of progressive deletion constructs are shown on the right (each at N = 3). The GFP expression plasmid was used as a negative control. (B) Quantitative PCR analysis to measure expression levels of endogenous spok expression in S2 cells transfected with various expression constructs, each at N = 3. (C) Luciferase reporter assay (each at N = 3) with the mld expression plasmid and luc plasmids containing the series of upstream elements of spok. (D) Luciferase reporter assay (each at N = 3) with séan, ouib, and/or mld expression plasmids, and luc plasmids containing the upstream elements of spok (the +331 to +32-bp region; 301 bp) and nvd (the +300 to +1-bp region; 300 bp). The GFP expression plasmid was used as a negative control. Error bars indicate SEM. * P < 0.01 and *** P < 0.005 using Student’s t-test with Bonferroni correction. Fluc/Rluc, firefly luciferase/Renilla luciferase.
We also found that transfection of the HA-tagged-mld (HA-mld) plasmid alone in S2 cells induced luc reporter gene expression when we used the 170-bp upstream region of the spok CDS that completely lacked the Ouib response element (Figure 5C). We further generated the deletion constructs in 20-bp increments from the 5′ terminus of the 170-bp spok promoter region. We still observed Mld-mediated activation of luc reporter gene expression with the 110-bp region, but not the 90-bp region, of the spok promoter (Figure 5C). These results suggest that Mld acts on distinct regions from Ouib response elements in the spok promoter regions.
There is no cross-reactivity between Séance and Ouija board
We next examined whether Séan and Ouib are specific to their respective recognition elements, or whether they can cross-react to activate either the spok or nvd promoters. Coexpression of HA-mld and FLAG-ouib did not induce nvd promoter-luc expression in cultured S2 cells (Figure 5D). In addition, coexpression of HA-mld and V5-séan did not induce spok promoter-luc expression in S2 cells (Figure 5D). We also observed that the expression of 3xTy1-tagged CG8159, the paralogue of séan and ouib (Figure 1A), induced neither nvd promoter- nor spok promoter-luc expression with or without HA-mld (Figure S7 in File S1). These results suggest that the action of the paralogous ZAD-zinc finger TFs is highly specific to the nvd and spok promoters, respectively.
The larval arrest phenotype of molting defective is rescued by forced expression of both neverland and spookier
Finally, we wondered whether the only essential targets of Mld are nvd and spok, or whether Mld also regulates other genes required for survival. To address this question, we expressed both UAS-nvd and UAS-spo in the PG cells of mld transheterozygotes using the PG-specific GAL4 driver (Table 3). It has previously been reported that spo overexpression can rescue the larval arrest phenotype of spok RNAi animals, confirming that spo and spok are functionally equivalent in vivo (Komura-Kawa et al. 2015), but that spo overexpression alone does not rescue the mld phenotype (Ono et al. 2006). Indeed, we found that the lethality of mld transheterozygotes was rescued by the overexpression of both nvd and spo. These results suggest that nvd and spok are the two major essential targets of Mld.
Discussion
In this study, we demonstrated that three ZAD-C2H2 zinc finger proteins, Séan, Ouib, and Mld, are required for ecdysone biosynthesis in the larval PG. The following points summarize our current findings in light of our previous studies (Danielsen et al. 2014; Komura-Kawa et al. 2015). First, loss-of-function mutations in séan, ouib, or mld severely reduced the expression of nvd, spok, or both nvd and spok, respectively. Second, we could rescue the larval arrest seen in animals without functional séan, ouib, and mld by supplementing intermediate metabolites of the ecdysone biosynthesis pathway in their diet. For example, the developmental arrest of séan mutants was restored by 7DC. Similarly, the larval arrest of ouib and mld mutants was restored by 5β-ketodiol supplementation. Third, the arrest of séan, ouib, or mld mutants was rescued by the PG-specific overexpression of nvd alone, spo alone (an ortholog of spok), or nvd and spo combined, respectively. Fourth, there are specific Séan and Ouib response elements in the nvd and spok promoter regions, respectively. Finally, the presence of Mld promotes synergistic action with Séan and Ouib to stimulate transcriptional upregulation. Based on these data, we propose that Séan, Ouib, and Mld act primarily to transcriptionally regulate just two Halloween genes, nvd and spok, in the PG (Figure 6).
Model for transcriptional interaction between Séan, Ouib, and Mld. Séan and Ouib activate nvd and spok transcription via Séan and Ouib response elements in the nvd and spok promoters, respectively. To properly activate nvd and spok expression, Mld cooperatively interacts with Séan and Ouib.
Our luc reporter plasmid-based assay confirmed that Séan and Ouib, along with Mld, were sufficient to drive gene expression under the control of the nvd and spok promoters, respectively, in cultured S2 cells. However, we should point out that, in our current assay, the inducible activities of Séan and Ouib are considerably different. Although coexpression of ouib and mld exhibited a ∼400-fold induction of luc expression compared to that of controls, the induction caused by séan and mld was only 10-fold. Moreover, although coexpression of ouib and mld in S2 cells induced endogenous spok expression, coexpression of séan and mld did not induce endogenous nvd expression. Currently, it is unclear what causes this difference between Séan and Ouib. One possible explanation is that the difference might be caused by endogenous séan and ouib expression in S2 cells. We found that S2 cells used in this study expressed considerable amounts of séan and mld, but not ouib (Figure S8 in File S1). This implies that the nvd promoter region might be preloaded by endogenous Séan and Mld, and therefore that the overexpression of Séan and Mld may not achieve high induction of the nvd gene. Alternatively, Séan, but not Ouib, may require one or more indispensable TF(s) to sufficiently drive nvd expression. Along these lines, it would be interesting to test whether and how Séan and Mld cooperate with previously identified TFs that are necessary for nvd expression in the PG, including the CncC-dKeap1 complex (Deng and Kerppola 2013), Knirps (Danielsen et al. 2014), and Ventral veins lacking (Cheng et al. 2014; Danielsen et al. 2014).
It is currently difficult to completely rule out the possibility that Séan, Ouib, and Mld are involved in the direct transcriptional regulation of genes other than nvd and spok. While we found no DNA sequences that exactly matched the Séan-Mld response element (5′-AGCTTTATTGCTCAG-3′) elsewhere in the D. melanogaster genome, our previous study had found that some degenerate Ouib response elements exist in the genome, including the regions upstream of the coding regions of some Halloween genes (Komura-Kawa et al. 2015). Indeed, séan mutants show a significant elevation of other Halloween genes. However, we expect that these effects could be indirect because we also saw substantial upregulation of the same Halloween genes when we knocked down nvd alone via PG-specific RNAi. Thus, upregulation of these Halloween genes possibly reflects an attempt to compensate for low ecdysone production. To further clarify whether Séan and Ouib directly regulate other genes, additional studies will be required, such as chromatin immunoprecipitation sequencing analysis, together with an eventual mutational analysis of any identified targets.
Our RNA-Seq analysis also confirmed the upregulation of additional genes linked to ecdysteroid synthesis, including Cyp18a1, sit, Start1, nobo, and ftz-f1, in Séan-depleted ring gland samples (Table S2 in File S2). Consistent with this, we found genes encoding ecdysteroid-linked TFs that are classified as repressors, Broad and knirps, in the set of downregulated genes (Table S3 in File S2). One known repressor of ecdysone biosynthesis, HR4 (Ou et al. 2011), was markedly downregulated in Séan-depleted ring glands (Table S4 in File S2), raising the possibility that Séan regulates the HR4 gene. Loss of HR4 function in the PG is not lethal, consistent with the finding that séan mutants can be rescued by transgenic nvd expression. HR4 depletion in the PG does not result in substantial upregulation of sad or phm, at least not in the early L3 stage (Ou et al. 2011), suggesting that other TFs are inducing the Halloween genes in séan mutants. The RNA-Seq data also revealed increased expression of genes with roles in cholesterol, heme, and ATP metabolism. One would expect these processes to sustain increased Halloween gene expression, since these enzymes need energy, metabolize cholesterol intermediates, and the P450 subset of ecdysone-producing enzymes has heme moieties as protein cofactors.
In this study, it is still unclear whether the three ZAD-C2H2 zinc finger proteins described here play roles in tissues other than the PG. Unlike ouib, which we can only detect in the PG (Komura-Kawa et al. 2015), we found that séan is highly expressed but not limited to the PG. This observation is partly consistent with the earlier finding that séan is expressed in the muscle founder cells of D. melanogaster embryos (Dobi et al. 2014). Interestingly, the Mld protein is also not limited to the PG but is also found in other tissues, including the imaginal discs, fat body, and the salivary gland during larval development (Neubueser et al. 2005). Further, the muscle morphology in séan mutants is disorganized in D. melanogaster embryos (Dobi et al. 2014), but no detailed functional studies have been reported. Thus, it is possible that séan and mld may have other functions that are not essential for viability but may be important for optimal fitness in the wild. On the other hand, séan and mld expression outside the PG raises the question as to why nvd or spok are not expressed in the non-PG tissues where Séan and Mld are present. One possibility is that these ZAD-C2H2 zinc finger proteins require other cofactors, which may be present in the PG, to induce nvd and/or spok expression. Alternatively, considering that most other identified ecdysteroidogenic TFs are not restricted to the PG either (Niwa and Niwa 2016a,b), one would expect that there are repressive mechanisms, such as chromatin states, that keep ecdysteroidogenic genes turned off in tissues other than the PG.
From an evolutionary point of view, any clear orthologs of séan, as well as mld and ouib (Neubueser et al. 2005; Ono et al. 2006; Komura-Kawa et al. 2015), are not found in any species thus far investigated other than Drosophilidae. Previous studies on spok hypothesized that the presence of mld and ouib might be coupled with the evolutionary appearance of spok in Drosophilidae, where duplication from the ancestral gene spo created the two paralogs (Ono et al. 2006; Komura-Kawa et al. 2015). However, our current study on séan suggests that this hypothesis may be untenable. This is because almost all insect genomes examined so far contain a single, unduplicated ortholog of nvd, placing the Mld/Séan/Ouib-nvd/spo axis outside the Drosophilidae (Yoshiyama et al. 2006; Yoshiyama-Yanagawa et al. 2011; Lang et al. 2012). Surprisingly, a gene synteny analysis suggests that séan orthologs are absent in some of the Drosophilidae species, such as D. ananasse, D. virillis, and D. mojavensis (Figure S4 in File S1). Such an evolutionary pattern raises the question as to which TFs regulate nvd expression in species that lack the séan or mld genes. It is noteworthy that nvd expression is spatiotemporally regulated in several lepidopteran species (Iga et al. 2013; Ogihara et al. 2015, 2017), the honeybee (Yamazaki et al. 2011), and even in a noninsect arthropod (Sumiya et al. 2016). It would be interesting to identify and characterize TFs responsible for nvd expression in these species. Individual insect genomes tend to harbor distinct sets of ZAD-C2H2 zinc finger genes, which expanded in an insect lineage-specific manner (Chung et al. 2007). Thus, it is possible that different ZAD-C2H2 zinc finger genes have evolved to regulate nvd expression in different species.
It should be noted that in D. melanogaster, both nvd and spok are located in the pericentromeric region of the third chromosome (Ono et al. 2006; Yoshiyama et al. 2006), which is thought to form constitutive heterochromatin (Fitzpatrick et al. 2005). Constitutive heterochromatin is a fundamental component of eukaryotic genomes and is believed to ensure a condensed and transcriptionally inert chromatin conformation in all cells of an organism [reviewed by Dimitri et al. (2009), Elgin and Reuter (2013), and Saksouk et al. (2015)]. Despite this, studies in the last four decades have revealed that constitutive heterochromatin contains active genes that are essential for viability in many organisms, including D. melanogaster [reviewed by Dimitri et al. (2009)] and mice (Rudert et al. 1995; Probst et al. 2010). To the best of our knowledge, Séan, Ouib, and Mld represent the first example of a set of TFs that regulate genes located in constitutive heterochromatin in a spatiotemporal-specific manner.
How do these three ZAD-C2H2 zinc finger proteins induce nvd and spok expression from a supposedly “inert” chromosomal region? In general, silenced heterochromatin displays distinctive chromatin characteristics including global hypoacetylation, trimethylation of histone H3 on lysine 9, and recruitment of the heterochromatin protein HP1 [reviewed by Elgin and Reuter (2013) and Timms et al. (2016)]. The ZAD domain is thought to serve as a protein–protein interaction domain (Jauch et al. 2003), suggesting that Séan and Ouib, along with Mld, might recruit cofactors that promote histone acetylation, followed by a reduction of H3K9m3 and reduced HP1 binding. Our analyses failed to detect any protein–protein interactions between Séan and Mld or between Ouib and Mld (T. Kamiyama and R. Niwa, unpublished results), suggesting that there are other interacting proteins, such as chromatin factors. Previous studies have reported that the zinc finger-containing proteins GAGA (Tsukiyama et al. 1994), Prod (Platero et al. 1998), and Su(var)3-7 (Cléard and Spierer 2001) associate with the heterochromatic chromocenter in D. melanogaster. Proteomic analysis to examine whether these or other factors physically interact with Séan, Ouib, and Mld are currently underway. These efforts will allow us to further elucidate the mechanisms by which heterochromatic gene expression is controlled.
Acknowledgments
We thank Stephan M. Cohen, Yasushi Hiromi, Shu Kondo, Michael B. O’Connor, Addgene, Berkley Drosophila Genome Project, the Bloomington Drosophila Stock Center, KYOTO Stock Center, the National Institute of Genetics, the Vienna Drosophila RNAi Center, the Drosophila Genomics Resource Center, and the Developmental Studies Hybridoma Bank for stocks and reagents. We also thank Sora Enya, Reiko Kise, Yuko Shimada-Niwa, Chikana Yamamoto, and Rieko Yamauchi for their technical assistance, and Akiyoshi Fukamizu, Aya Fukuda, Seiji Hira, Masanori Mukai, Akira Nakamura, Masanao Sato, and Don Sinclair for helpful discussion. O.U. was a recipient of a fellowship from the Japan Society for the Promotion of Science. This work was supported by a grant from Precursory Research for Embryonic Science and Technology of the Japan Science and Technology Agency to R.N., the program of the Joint Usage/Research Center for Developmental Medicine, Institute of Molecular Embryology and Genetics, Kumamoto University to R.N., and a grant from the Natural Sciences and Engineering Research Council (NSERC) Canada to B.M.H. This work was also supported by grants from NSERC (RGPIN 341543, 50%) and the Canadian Institutes of Health Research (MOP 93761, 50%) to K.K.-J. The authors declare no competing interests.
Author contributions: Study conception; T.K.-K., K.H., B.M.H., K.K.-J., and R.N. Methodology; M.I. and H.K. Formal analysis; O.U., Q.O., T.K., M.I., K.K.-J., and R.N. Investigation: performed the experiments; O.U., Q.O., T.K.-K., T.K., M.I., M.S., K.K.-J., and R.N.; and data/evidence collection; O.U., Q.O., T.K.-K., T.K., M.I., M.S, K.K.-J., and R.N. Resources; T.K.-K., M.S., K.H., H.K., B.M.H., K.K.-J., and R.N. Data curation; B.M.H., K.K.-J., and R.N. Writing/manuscript preparation: writing the initial draft); K.K.-J. and R.N.; critical review, commentary, or revision: O.U., Q.O., T.K., K.H., and B.M.H.; and visualization/data presentation: O.U., Q.O., T.K., K.K.-J., and R.N. Supervision: K.K.-J. and R.N. Funding acquisition: B.M.H., K.K.-J., and R.N.
Footnotes
Supplemental material is available online at www.genetics.org/lookup/suppl/doi:10.1534/genetics.117.300268/-/DC1.
Communicating editor: P. Geyer
- Received September 11, 2017.
- Accepted November 27, 2017.
- Copyright © 2018 by the Genetics Society of America
Available freely online through the author-supported open access option.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
