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. 2022 Sep 23;222(3):iyac137. doi: 10.1093/genetics/iyac137

Su(var)2-10- and Su(var)205-dependent upregulation of the heterochromatic gene neverland is required for developmental transition in Drosophila

Yuya Ohhara 1,2,✉, Yuki Kato 3, Takumi Kamiyama 4,5, Kimiko Yamakawa-Kobayashi 6,7
Editor: R Duronio
PMCID: PMC9630985  PMID: 36149288

Abstract

Animals develop from juveniles to sexually mature adults through the action of steroid hormones. In insect metamorphosis, a surge of the steroid hormone ecdysone prompts the transition from the larval to the adult stage. Ecdysone is synthesized by a series of biosynthetic enzymes that are specifically expressed in an endocrine organ, the prothoracic gland. At the late larval stage, the expression levels of ecdysone biosynthetic enzymes are upregulated through the action of numerous transcription factors, thus initiating metamorphosis. In contrast, the mechanism by which chromatin regulators support the expression of ecdysone biosynthetic genes is largely unknown. Here, we demonstrate that Su(var)2-10 and Su(var)205, suppressor of variegation [Su(var)] genes encoding a chromatin regulator Su(var)2-10 and nonhistone heterochromatic protein 1a, respectively, regulate the transcription of one of the heterochromatic ecdysone biosynthetic genes, neverland, in Drosophila melanogaster. Knockdown of Su(var)2-10 and Su(var)205 in the prothoracic gland caused a decrease in neverland expression, resulting in a defect in larval-to-prepupal transition. Furthermore, overexpression of neverland and administration of 7-dehydrocholesterol, a biosynthetic precursor of ecdysone produced by Neverland, rescued developmental defects in Su(var)2-10 and Su(var)205 knockdown animals. These results indicate that Su(var)2-10- and Su(var)205-mediated proper expression of neverland is required for the initiation of metamorphosis. Given that Su(var)2-10-positive puncta are juxtaposed with the pericentromeric heterochromatic region, we propose that Su(var)2-10- and Su(var)205-dependent regulation of inherent heterochromatin structure at the neverland gene locus is essential for its transcriptional activation.

Keywords: Drosophila, heterochromatin, Su(var)2-10, HP1a, ecdysone, neverland, prothoracic gland

Introduction

Animals develop from juveniles to sexually mature adults during postembryonic development. Similar to puberty in mammals, the developmental transition in arthropods from juveniles to adults is triggered by steroid hormones. In holometabolous insect larvae, metamorphosis is triggered by the action of steroid hormones called ecdysteroids. The best-characterized bioactive ecdysteroid is 20-hydroxyecdysone (20E). 20E activates its specific receptor, a heterodimer of ecdysone receptor (EcR) and ultraspiracle (USP), to induce downstream gene expression cascades and subsequent metamorphic events, such as tissue remodeling and cell death in larval tissues.

A precursor of 20E, ecdysone, is produced from the prothoracic gland (PG), a polyploid endocrine organ. The initial step of ecdysone biosynthesis, the conversion of dietary cholesterol to 7-dehydrocholesterol (7-DHC), is catalyzed by Neverland (Nvd) (Fig. 1a) (Yoshiyama et al. 2006; Yoshiyama-Yanagawa et al. 2011). 7-DHC is then metabolized to 5β-ketodiol by at least 3 enzymes, including Spookier/Spook (Spok/Spo) (Namiki et al. 2005; Ono et al. 2006), Shroud (Sro) (Niwa et al. 2010), and CYP6T3 (Ou et al. 2011) (Fig. 1a). 5β-ketodiol is further converted into ecdysone through sequential enzymatic reactions mediated by Phantom (Phm) (Niwa et al. 2004; Warren et al. 2004), Disembodied (Dib) (Warren et al. 2002; Niwa et al. 2005), and Shadow (Sad) (Warren et al. 2002) (Fig. 1a). Ecdysone is secreted from the PG through a vesicular trafficking mechanism (Yamanaka et al. 2015) and then converted into 20E by Shade (Shd) in peripheral organs (Petryk et al. 2003) (Fig. 1a).

Fig. 1.

Fig. 1.

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PG-selective RNAi screen to identify novel transcriptional regulators of ecdysone biosynthetic genes. a) Schematic diagram of the ecdysone biosynthetic pathway in the PG. b) Scheme of PG-selective RNAi screens. Candidate genes and results of the first and second screens are summarized in Supplementary Tables 3 and 5, respectively. c) A heat map representing the relative expression levels of ecdysone biosynthetic genes in control (phm>+) and knockdown animals (phm>gene-of-interest-RNAi). The gene expression levels were measured using qPCR at 96 h after hatching.

Transcription factors and nuclear receptors that regulate the transcription of ecdysone biosynthetic enzymes have been identified in Drosophila (Niwa and Niwa 2016; Kamiyama and Niwa 2022). Examples of these are EcR, βFtz-F1, Broad, ventral veins lacking, knirps, snail, forkhead box protein O−USP heterodimer, and cap-n-collar and Keap1 complex, which are involved in the transcription of multiple ecdysone biosynthetic enzymes (Parvy et al. 2005, 2014; Xiang et al. 2010; Deng and Kerppola 2013; Moeller et al. 2013; Cheng et al. 2014; Danielsen et al. 2014; Koyama et al. 2014; Zeng et al. 2020). Furthermore, recent studies have revealed that the genes nvd and spok, located in the pericentromeric heterochromatic region (Ono et al. 2006; Yoshiyama et al. 2006; Uryu et al. 2018), are specifically and directly activated by transcription factors Séance and Ouija Board (Ouib), respectively, in cooperation with Molting defective (Mld) (Komura-Kawa et al. 2015; Uryu et al. 2018). In addition, a poly(A) deadenylation complex named CCR4-NOT selectively regulates the expression of spok (Zeng et al. 2018), and a poly(A) binding protein, Pabp, regulates spok expression through the regulation of nuclear translocation of Mld (Kamiyama et al. 2020).

In addition to transcription regulators and poly(A)-binding proteins, chromatin regulators are also involved in the regulation of ecdysone biosynthetic gene expression. Ada2a-containing complex histone acetyltransferase (Pankotai et al. 2010; Borsos et al. 2015), the polycomb repressive complex-2 histone methyltransferase (Yang et al. 2021), and the insulator-associated protein CCCTC-binding factor (Fresán et al. 2015) have been identified as essential for the upregulation of multiple ecdysone biosynthetic genes. Furthermore, given that certain heterochromatin-residing genes require a heterochromatic environment for normal expression (Wakimoto and Hearn 1990; Eberl et al. 1993; Lu et al. 1996, 2000; Yasuhara and Wakimoto 2006), it is possible that the transcription of the heterochromatic ecdysone biosynthetic genes nvd and spok is under the control of inherent epigenetic factors, such as heterochromatin-associated chromatin regulators. However, chromatin regulators that control the transcription of a specific ecdysone biosynthetic enzyme, including heterochromatic nvd and spok, have not been identified.

In the present study, we performed a PG-selective RNAi screen to identify novel regulators of ecdysone biosynthetic genes. We identified Su(var)2-10, which belongs to the suppressor of variegation [Su(var)] genes (Hari et al. 2001; Elgin and Reuter 2013), as a novel regulator of nvd gene expression. Su(var) genes including Su(var)2-10 have been originally isolated as essential genes for heterochromatin formation in the white (w) locus adjacent to pericentric heterochromatin due to chromosomal inversion [In(1)wm4]: In(1)wm4 animals show variegated eyes (red and white patches) because of partial silencing of the inverted white locus, while loss of Su(var) genes in the In(1)wm4 background leads to a red-eye phenotype due to less heterochromatin formation in the inverted white locus (Elgin and Reuter 2013). Su(var)2-10 encodes a nuclear protein called Su(var)2-10 [a.k.a. protein inhibitor of activated STAT (PIAS)] possessing small ubiquitin-related modifier (SUMO) E3 ligase activity (Hari et al. 2001; Rytinki et al. 2009; Rabellino et al. 2017). Recent studies have been elucidated that Su(var)2-10 deposits SUMO on chromosome to recruit the histone methyltransferase, leading to suppression of spurious transcription and ensuring normal gene expression in the heterochromatin (Ninova, Chen, et al., 2020; Ninova, Godneeva, et al., 2020). Our genetic evidence shows that knockdown of Su(var)2-10 in the PG resulted in the third instar arrest phenotype due to downregulation of nvd expression. Furthermore, we found that Su(var)205, encoding heterochromatic protein 1a (HP1a), a major component of heterochromatic chromosomes (Eissenberg et al. 1992; Eissenberg and Elgin 2014; Schoelz and Riddle 2022), also controls pupariation via nvd upregulation. Considering that Su(var) genes are essential for heterochromatin formation and normal expression of heterochromatin-residing genes (Elgin and Reuter 2013), we propose that Su(var)2-10- and Su(var)205-dependent regulation of inherent heterochromatin structure at the nvd gene locus is essential for its transcriptional activation.

Materials and methods

Drosophila stocks and medium

Fly stocks and their genotypes used in this study are listed in Supplementary Tables 1 and 2. Stocks used for the RNAi screen are summarized in Supplementary Table 3. w1118 served as a wild-type strain. Fly stocks were reared on a glucose/cornmeal/yeast medium (1 g glucose, 0.7 g cornmeal, 0.4 g yeast extract, 50 mg agar in 10 ml water) supplemented with 30 µl propionic acid and 35 µl butylparaben (167 mg/ml in 70% ethanol). Fly stocks placed in vials (MKC-20, Hi-tech) with the culture medium were kept in the 25°C culture room at relative humidity levels between 40% and 60% under a 12-h light/dark cycle (Fluorescent light was turn on from 8:00 AM to 8:00 PM JST). The glucose/cornmeal/yeast medium was used for the first RNAi screen (see PG-Selective RNAi Screening).

German food, a nutrient-rich semi-defined medium, was used for all experiments except for the first RNAi screen. A total of 22.5 g German food powder (Genesee Scientific, San Diego, CA, USA, 66–115) was added to 100 ml water, and the mixture was boiled using a microwave. After stirring for 30 min at room temperature (18–25°C), 600 µl propionic acid was added to the mixture to obtain a German food medium.

Staging and analysis of developmental progression

Parent flies were maintained in plastic bottles (Genesee Scientific, San Diego, CA, USA, 32–310) and allowed to lay eggs for 24 h on grape juice agar plates (2 g agar in 100 ml grape juice, poured into a 4.5 cm × 1.6 cm plastic dish) supplemented with dry yeast powder (Oriental Yeast, Japan). Newly hatched 10–35 larvae were transferred to vials (MKC-30, Hi-tech) containing 1–3.5 g of the German food medium. Larvae were cultured in the 25°C incubator (LTI-400E, EYELA) at relative humidity levels between 40% and 60% under a 12-h light/dark cycle (Fluorescent light was turn on from 8:00 AM to 8:00 PM JST), and developmental stages and lethality were scored every 24 h. TM6B/TM6B Gal80 balancer-possessing animals were excluded at 48 h after hatching, and CyO-GFP balancer-possessing animals were excluded at 0 h after hatching using a fluorescent stereomicroscope.

PG-selective RNAi screening

PG-selective RNAi screening was performed using the Gal4/UAS system to identify novel regulators of ecdysone biosynthetic genes. In the first screen, candidate genes were knocked down in the PG and the membrane localization of EGFP-fused Synaptotagmin (Syt) (Syt::EGFP) (Zhang et al. 2002) was observed to exclude genes involved in the regulation of Syt-mediated vesicle trafficking. Ten virgin females carrying PG-selective phantom-Gal4 (phm-Gal4) (Rewitz et al. 2009) and UAS-Syt::EGFP were crossed with 5 UAS-RNAi males to obtain the offspring in which gene of interest was knocked down in the PG (phm>Syt::EGFP gene-of-interest-RNAi). To obtain control animals (phm>Syt::EGFP), females carrying phm-Gal4 and UAS-Syt::EGFP were crossed with w1118 males. Parent flies were cultured on the glucose/cornmeal/yeast medium in a plastic vial for 2 days. At day 6 after crossing, in which most of control larvae were in the wandering stage, Syt::EGFP expression in control and knockdown animals were observed as described in Immunohistochemistry.

In the second screen, newly hatched control (phm>+) and knockdown larvae (phm>gene-of-interest-RNAi) were collected and reared on the German food as described in Staging and Analysis of Developmental Progression. At 96 h after hatching, control and knockdown larvae were sampled, and the expression levels of ecdysone biosynthetic genes were analyzed using qPCR as described in Quantitative RT-PCR.

Immunohistochemistry

Immunohistochemistry was performed to investigate the expression levels/patterns of Syt::EGFP, Nvd, Spok, Sro, and Dib proteins. Larvae were dissected in PBS and fixed for 25 min with 4% paraformaldehyde in 0.01% PBT (0.01% Triton X-100 in PBS). Tissues were washed with 0.1% PBT 3 times for 10 min each and washed with 1% PBT for 5 min to increase antibody permeability. Tissues were then blocked with 1% goat serum (Sigma, G9023) in 0.1% PBT for 30 min, and incubated at 4°C overnight with a primary antibody against GFP (mouse IgG, monoclonal, 3E6) (Thermo Fisher Scientific, A-11120) at a 1:1,000 dilution, Nvd (guinea pig IgG, polyclonal) (Ohhara et al. 2015) at a 1:200 dilution, Spok (guinea pig IgG, polyclonal) (Gibbens et al. 2011) at a 1:500 dilution, Sro (guinea pig IgG, polyclonal) (Shimada-Niwa and Niwa 2014) at a 1:1,000 dilution, or Dib (rabbit pig IgG, polyclonal) (Parvy et al. 2005) at a 1:500 dilution in a blocking solution. Note that anti-Nvd antibody solution was incubated with fixed w1118 larval tissues at 4°C overnight before the experiment to reduce the background signal. Tissues were washed with 0.1% PBT 3 times for 10 min each and incubated at 4°C overnight with Alexa 488-conjugated goat IgG against mouse/rabbit/guinea pig (Thermo Fisher Scientific, A-11001/A-11008/A-11073) at 1:1,000 dilution and Hoechst 33342 (Thermo Fisher Scientific, 62249) at 1:1,500 dilution in 0.1% PBT. After washing with 0.1% PBT for 10 min each, brain-ring gland complexes were dissected and mounted in a mounting medium (2.4 g Mowiol 4-88 [Sigma-Aldrich, 81381], 6 g glycerol, 6 ml distilled water, and 12 ml 0.2 M Tris–HCl [pH 8.5]). Images were taken with a Zeiss LSM700 and a Zeiss LSM800, and image analyses were performed using Image J/Fiji software (Schindelin et al. 2012).

For visualization of GFP-fused Su(var)2-10 proteins, tissues were incubated at 4°C overnight with primary antibodies against GFP (rabbit IgG, polyclonal) (Medical and Biological Laboratories, #598; see the related information including specificity of this antibody at https://ruo.mbl.co.jp/bio/dtl/A/?pcd=598) at a 1:1,000 dilution. We also used anti-HP1a antibody (mouse IgG, monoclonal) (Developmental Studies Hybridoma Bank, C1A9), which can specifically recognize Drosophila HP1a protein (James and Elgin 1986) at a 1:50 dilution in a blocking solution. Alexa 488-conjugated goat IgG against rabbit IgG (Thermo Fisher Scientific, A-11008) and Alexa 647-conjugated goat IgG against mouse IgG (Thermo Fisher Scientific, A-21235) were used as secondary antibodies at 1:1,000 dilution, with Hoechst 33342 (Thermo Fisher Scientific, 62249) at 1:1,500 dilution. Washing, blocking, mounting, and image acquisition were performed in the same procedure as described above.

Quantitative RT-PCR

Quantitative RT-PCR (qPCR) was performed to measure the expression levels of ecdysone biosynthetic genes. Total RNA was extracted from larvae using TRIzol (Thermo Fisher Scientific, 15596026). A total of 0.1–0.5 μg of RNA samples were reverse-transcribed using the SuperScript III kit (Thermo Fisher Scientific, 18080051). The obtained cDNA samples were used as a template for qPCR using the Quantifast SYBR Green PCR kit (QIAGEN, 204056) and Rotor-Gene Q (QIAGEN) with a primer concentration of 1 μM. All reactions were performed at 95°C for 10 min, followed by 50 cycles of 95°C for 10 s and 60°C for 30 s. Dissociation curve analysis was applied to all reactions to ensure the presence of a single PCR product. The expression levels of the target genes were calculated using the relative standard curve method. Stock cDNA used for the relative standard curves was synthesized from pooled RNA derived from larvae raised under the same conditions and diluted serially. The expression levels of the target genes were normalized to an endogenous reference gene, ribosomal protein 49 (rp49) (also known as ribosomal protein L32). The mean expression level of the control was set to 1. The primer sets used for qPCR are listed in Supplementary Table 4.

7-DHC and 20E supplementation experiments

About 50 μl of sterol solution (10 mg/ml 7-DHC [Sigma-Aldrich, 30800] or 20E [Tokyo chemical industry, H1480] in ethanol) was added to 2 g of the German food to obtain a 7-DHC or 20E-containing medium (final sterol concentration: 0.5 mg/g). Two grams of the German food with 50 μl of ethanol served as a control medium. A total of 15–30 larvae reared on the German food were transferred to the control or sterol-containing media at 48 h after hatching. Developmental stages were scored every 24 h.

Image analysis

To quantify DNA signal intensity in the PG, a series of images obtained by immunostaining/histochemistry were processed using Image J/Fiji as follows. First, the PG area was segmented: In transgenic animals expressing mCherry.nls in the PG, the mCherry.nls signal was binarized using a function named “Make binary”; In case of the PGs of wild type and mutant animals stained with anti-Dib antibody, the Dib signal was binarized and then the nuclei of PG cells were filled using “Make binary” and “Fill holes.” Next, DNA signal overlapped with the PG area was calculated using “Image calculator.” The processed images were z-stacked, and PG area in a z-stacked image was selected using “Freehand selection” tool. Integrated density within the selected region was measured and then adjusted using average DNA staining intensity obtained from a z-stacked image of the brain lobe. Adjusted DNA staining intensity was then divided by the PG cell number to calculate mean DNA intensity per PG cell. In accordance with the result of ploidy measurement in the previous study, the mean chromatin values (C values) in the PG of wild type (+/+) and transgenic control animals (phm>mCherry.nls) at 96 h after hatching were set to 58 and 53, respectively (Ohhara et al. 2019).

For quantification of Nvd, Spok, and Sro immunostaining signal in the PG, a series of images were also processed using Image J/Fiji as follows. PG area in a z-stacked image was selected using “Freehand selection,” and average immunostaining signal intensity was measured within the region. The immunostaining signal intensity was adjusted using average immunostaining intensity obtained from a z-stacked image of the brain lobe. The mean value of adjusted signal intensity in control animals was set to 1.

For GFP-fused Su(var)2-10 observations, Su(var)2-10 punctum was defined as a GFP-positive nuclear punctum showing GFP signal intensity 1.5 − 2 times higher than surrounding GFP-positive nuclear region. The number of Su(var)2-10 puncta-positive PG cells was counted manually using the cell counter plugin in Image J/Fiji software (https://imagej.nih.gov/ij/plugins/cell-counter.html).

Statistical analysis

Statistical analyses were performed using R software (version 4.1.0) (Ihaka and Gentleman 1996). Data were analyzed using Fisher’s exact test, Student’s t-test, Mann–Whitney U-test, Dunnett’s test, Tukey’s test, or Steel’s multiple comparison test.

Results

A PG-selective RNAi screen to identify novel transcriptional regulators of ecdysone biosynthetic genes

Previously, we performed a PG-selective RNAi screen to identify novel regulators of ecdysone biosynthesis and identified 449 genes that regulate larval-to-prepupal transition (Ohhara et al. 2019) (Fig. 1b and Supplementary Fig. 1 and Supplementary Table 3). We further analyzed the PG cells of knockdown animals to screen the genes that are involved in the regulation of the cell cycle in the PG (Ohhara et al. 2019), given that proper progression of the endocycle, a variant of the cell cycle that is characterized by the absence of mitosis (Lilly and Duronio, 2005), is essential for ecdysone biosynthesis in the PG (Ohhara et al. 2017, 2019; Zeng et al. 2020). Once the C value in PG cells reaches 32 (which corresponds to 3 rounds of endocycling), the expression levels of ecdysone biosynthetic enzymes are significantly upregulated in the late third instar, between 72 and 96 h after hatching (Ohhara et al. 2017, 2019). Of the 449 genes, 74 were potentially dispensable for endocycle progression in PG cells, given that knockdown of these genes did not cause a decrease in DNA content in PG cells compared to control animals at 72 h after hatching, at which point the C value in PG cells reached 32 (Fig. 1b and Supplementary Fig. 1 and Supplementary Table 3). In addition, knockdown of these genes did not cause morphological defects in the PG (Ohhara et al. 2019). Here, we focused on these genes as candidates for novel transcriptional regulators of ecdysone biosynthetic enzymes and performed the following screening.

Using immunohistochemistry, we determined Syt::EGFP localization, thus excluding genes that are involved in the trafficking of vesicle-mediated ecdysone secretory granules (Yamanaka et al. 2015). A vesicle-localizing protein, Syt::EGFP (Zhang et al. 2002), was expressed in the PG using the PG-selective phm-Gal4 (Rewitz et al. 2009) in control (phm>Syt::EGFP) and knockdown animals (phm>Syt::EGFP gene-of-interest-RNAi). Syt::EGFP was localized to the plasma membrane in the PG of control animals, whereas knockdown of membrane trafficking regulators, such as Sec8 and Sec15, resulted in loss of Syt::EGFP membrane localization, as previously reported (Yamanaka et al. 2015) (Figure S1 in File S2). Furthermore, knockdown of amx, CG10333, CG32069, dwg, Him, not, Nup214, ps, SF2, Trs23, and Vha44 caused a decrease in Syt::EGFP membrane localization (Supplementary Fig. 1), suggesting that these genes are required for membrane localization of Syt::EGFP in the PG. In addition, knockdown of CG32104, MED17, Nurf-38, Rtf1, ubl, and Wdr3 caused a reduction in Syt::EGFP protein levels in the PG (Supplementary Fig. 1). Thus, these genes may be involved in the post-transcriptional regulation of Syt. Therefore, these 19 genes were excluded from the candidate list. In addition, we excluded 10 genes whose knockdown did not cause developmental arrest or delayed pupariation (Supplementary Table 3). The remaining 45 genes were then tested in the second screen (Fig. 1b).

Next, we examined the expression levels of ecdysone biosynthetic genes, nvd, spok, sro, phm, dib, and sad, in control (phm>+) and knockdown animals (phm>gene-of-interest-RNAi) using qPCR at 96 h after hatching. Compared to the control, many knockdown animals showed a decrease in expression of multiple ecdysone biosynthetic genes (Fig. 1c and Supplementary Table 5). The expression levels of ecdysone biosynthetic genes were not significantly reduced in CG4806, CG13692, dare, Dh31-R, and tamo knockdown animals (Fig. 1c and Supplementary Table 5), which suggests that these genes are not involved in the transcriptional regulation of ecdysone biosynthetic enzymes, but rather involved in the post-transcriptional regulation of ecdysone biosynthetic enzymes or steroidogenesis-related biochemical reactions such as the mitochondrial electron transfer system. Actually, dare, which encodes a flavoenzyme that carries electrons for the steroidogenesis in mitochondria, has been identified as essential for ecdysone production in the PG (Freeman et al. 1999). In contrast, the expression level of nvd, but not other ecdysteroidogenic genes, was significantly reduced in Su(var)2-10 and Ugt37A3 knockdown animals (Fig. 1c and Supplementary Table 5). This suggests that Su(var)2-10 and Ugt37A3 selectively regulate nvd transcription. Because Su(var)2-10 serves as a transcriptional coregulator and a chromatin regulator (Rytinki et al. 2009; Rabellino et al. 2017; Ninova, Chen, et al. 2020; Ninova, Godneeva, et al., 2020), we focused on Su(var)2-10 hereafter and performed further genetic analyses to reveal its function.

Su(var)2-10 and Su(var)205 knockdown animals exhibit larval stage arrest phenotype due to a reduction of nvd expression

To elucidate the importance of Su(var)2-10 in ecdysone biosynthesis, we investigated the developmental phenotype and expression levels of ecdysone biosynthetic genes in 3 control animals, possessing only phm-Gal4 (phm>+) or UAS-Su(var)2-10 RNAi-1/2 construct [+>Su(var)2-10-RNAi-1/2], and 2 independent Su(var)2-10 RNAi animals [phm>Su(var)2-10-RNAi-1/2]. We confirmed that knockdown of Su(var)2-10 led to third instar arrest phenotype (Fig. 2, a and b) and that the expression levels of nvd were significantly reduced in Su(var)2-10 knockdown animals compared to control animals (Fig. 2c). We also confirmed that Nvd protein levels were clearly reduced in the PG of Su(var)2-10 knockdown animals (Supplementary Fig. 2). These results indicate that Su(var)2-10 is required for nvd upregulation. Expression levels of other ecdysone biosynthetic gene were not significantly reduced in Su(var)2-10 knockdown animals (Fig. 3c), while sro expression was slightly reduced in phm>Su(var)2-10-RNAi-1 animals (Fig. 2c), suggesting the possibility that Su(var)2-10 also regulates sro expression. However, immunostaining revealed that there was no significant difference in Sro protein levels between control and Su(var)2-10 knockdown animals (Supplementary Fig. 2). In addition, spok transcription and Spok protein levels were not reduced in Su(var)2-10 knockdown animals (Fig. 2c and Supplementary Fig. 2). These results suggest that Su(var)2-10 is dispensable for upregulation of spok, sro, and other ecdysone biosynthetic genes.

Fig. 2.

Fig. 2.

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Knockdown of Su(var)2-10 and Su(var)205 causes impaired pupariation due to reduced nvd expression. a and b) Knockdown of Su(var)2-10 and Su(var)205 in the PG causes developmental resulted in third instar arrest. Percentages of pupariated animals and larvae died at first/second and third instar (shown as “L1/L2 lethal” and “L3 lethal,” respectively) (a) and percentages of pupariated animals at indicated time points (b) in control animals possessing only phm-Gal4 (phm>+) or UAS-RNAi construct (+>gene-of-interest-RNAi), Su(var)2-10 knockdown [phm>Su(var)2-10-RNAi-1 and phm>Su(var)2-10-RNAi-2], and Su(var)205 knockdown animals [phm>Su(var)205-RNAi-1 and phm>Su(var)205-RNAi-2] are shown. Sample sizes are 90–100 for each genotype. c) nvd expression levels are reduced in Su(var)2-10 and Su(var)205 knockdown animals. Expression levels of ecdysone biosynthetic genes (nvd, spok, sro, phm, dib, and sad) were measured using qPCR at 96 h after hatching. Box and scatter plots of 5 biological replicates are shown in each experimental group. Statistically significant differences between groups are indicated by different lowercase letters (P < 0.05; Tukey’s test), while there is no statistically significant difference between groups with the same lowercase letter (P > 0.05). d) Overexpression of nvd rescues developmental arrest in Su(var)2-10 and Su(var)205 knockdown animals. Percentages of pupariated animals among knockdown animals (phm>gene-of-interest-RNAi) (circle), Bm-nvdWT-expressing knockdown animals (phm>gene-of-interest-RNAi Bm-nvdWT) (triangle), and Bm-nvdH190A-expressing knockdown animals (phm>gene-of-interest-RNAi Bm-nvdH190A) (square) are shown at indicated time points. Sample sizes are 55–80 for each group. Asterisks indicate a statistically significant difference between phm>Su(var)2-10-RNAi-2 and phm>Su(var)2-10-RNAi-2 Bm-nvdWT (** P < 0.01, *** P < 0.001; Fisher’s exact test).

Fig. 3.

Fig. 3.

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nvd expression is reduced in Su(var)2-10 and Su(var)205 mutants. a) Su(var)2-10 and Su(var)205 mutants show developmental defects. Percentages of pupariated animals and larvae died at 1st/2nd and 3rd instar (shown as “L1/L2 lethal” and “L3 lethal,” respectively) in wild-type animals (+/+), heterozygous Su(var)2-10 mutants [Su(var)2-102/+ and Su(var)2-10zimp−2/+], homozygous Su(var)2-10 mutants [Su(var)2-102/Su(var)2-102 and Su(var)2-10zimp−2/Su(var)2-10zimp−2], trans-heterozygous Su(var)2-10 mutants [Su(var)2-102/Su(var)2-10zimp-2], heterozygous Su(var)205 mutants [Su(var)2052/+ and Su(var)2054/+], homozygous Su(var)205 mutants [Su(var)2052/Su(var)2052 and Su(var)2054/Su(var)2054], and trans-heterozygous Su(var)205 mutants [Su(var)2052/Su(var)2054] are shown. Sample sizes are 100 for each genotype. b) Percentages of pupariated animals among wild-type and mutants are shown at indicated time points. c) nvd expression levels are reduced in Su(var)2-10 and Su(var)205 trans-heterozygous mutants. Expression levels of ecdysone biosynthetic genes (nvd, spok, sro, phm, dib, and sad) were measured using qPCR at 96 h after hatching. Box and scatter plots of 5 biological replicates are shown in each experimental group. Statistically significant differences between groups are indicated by different lowercase letters (P < 0.05; Tukey’s test), while there is no statistically significant difference between groups with the same lowercase letter (P > 0.05). n. s., not significant. d) Administration of 20E rescues developmental arrest in Su(var)205 mutant animals. Percentages of pupariated trans-heterozygous Su(var)2-10 and Su(var)205 mutants cultured on a control medium (circle) and 7-DHC- (triangle) or 20E-supplemented media (square) are shown at indicated time points. Sample sizes are 60 for each group.

We next sought to determine whether developmental arrest in Su(var)2-10 knockdown animals was due to downregulation of nvd expression. Wild-type Bombyx nvd (Bm-nvdWT) or a mutated type of Bombyx nvd possessing H190A mutation (Bm-nvdH190A), which causes a loss of the Nvd enzymatic activity (Yoshiyama-Yanagawa et al. 2011), were expressed under the control of UAS in the PG of Su(var)2-10 knockdown animals. The larval-to-prepupal transition was blocked in phm>Su(var)2-10-RNAi-1 and RNAi-2 animals, as well as in Bm-nvdH190A-expressing Su(var)2-10 knockdown animals [phm>Su(var)2-10-RNAi-1/2 Bm-nvdH190A], whereas pupariation was induced in Bm-nvdWT-expressing Su(var)2-10 knockdown animals [phm>Su(var)2-10-RNAi-1/2 Bm-nvdWT] (Fig. 2d). Furthermore, administration of 7-DHC, the first ecdysone intermediate produced by Nvd, or 20E rescued developmental arrest in phm>Su(var)2-10-RNAi-1 and RNAi-2 animals (Supplementary Fig. 3). These results indicate that developmental arrest in Su(var)2-10 knockdown animals is caused by the downregulation of nvd.

Su(var)2-10 knockdown animals showed an increase in DNA content in PG cells and PG tissue size (Supplementary Fig. 4). This hypertrophic PG phenotype has been observed in mutant Su(var)205 animals (Spierer et al. 2005), which encodes a major component of heterochromatin called HP1a (Eissenberg et al. 1992; Eissenberg and Elgin 2014; Schoelz and Riddle 2022). Because both Su(var)205 and Su(var)2-10 belong to the Su(var) genes (Eissenberg et al. 1992; Hari et al. 2001), which are essential for heterochromatin formation and normal expression of heterochromatin-residing genes (Elgin and Reuter 2013; Ninova, Godneeva, et al., 2020; Schoelz and Riddle 2022), we hypothesized that Su(var)205 is also involved in the regulation of nvd. First, we confirmed that knockdown of Su(var)205 in the PG caused an increase in DNA content and tissue size (Supplementary Fig. 4). Furthermore, approximately half of the Su(var)205 knockdown animals [phm>Su(var)205-RNAi-1/2] were arrested at the third instar, and those animals that did develop further showed a significant delay in pupariation (Fig. 2, a and b). In addition, the expression levels of nvd mRNA and Nvd protein levels were reduced in Su(var)205 knockdown animals (Fig. 2c and Supplementary Fig. 2), and their developmental defects were rescued by forced expression of Bm-nvdWT and the administration of 7-DHC and 20E (Fig. 2d and Supplementary Fig. 3). These results indicate that Su(var)205-mediated nvd upregulation is required for proper onset of larval-to-prepupal transition.

nvd expression is reduced in Su(var)2-10 and Su(var)205 mutants

To confirm the importance of Su(var)2-10 and Su(var)205 in ecdysone biosynthesis, we observed the phenotype of animals carrying mutant alleles of Su(var)2-10, Su(var)2-102, and Su(var)2-10zimp−2, which possess a point mutation in the coding region (W260Stop) (Hari et al. 2001) and the insertion of a transposon in the Su(var)2-10 gene region (Mohr and Boswell 1999), respectively. Most of the wild type (+/+) and heterozygous Su(var)2-10 mutants [Su(var)2-102/+ and Su(var)2-10zimp−2/+] underwent pupariation by 120 h after hatching, whereas homozygous Su(var)2-102 mutants [Su(var)2-102/Su(var)2-102] did not hatch, and homozygous Su(var)2-10zimp−2 mutants [Su(var)2-10zimp−2/Su(var)2-10zimp−2] died at the first (41%), second (29%), and third instar (28%) (Fig. 3a). These results indicate that Su(var)2-10 is required for embryonic and larval development. Trans-heterozygous Su(var)2-10 mutants [Su(var)2-102/Su(var)2-10zimp−2], on the other hand, showed milder phenotypes: 28%, 40%, and 19% of Su(var)2-102/Su(var)2-10zimp−2 animals died at the first, second, and third instar, respectively. The other 13% of the animals underwent pupariation (Fig. 3a), although they showed a significant delay in pupariation (Fig. 3b). These results suggest that ecdysone biosynthesis is downregulated in Su(var)2-102/Su(var)2-10zimp−2 animals. Indeed, nvd mRNA expression levels were reduced in Su(var)2-102/Su(var)2-10zimp−2 animals compared to wild type and heterozygous Su(var)2-10 mutants at 96 h after hatching (Fig. 3c). In addition, the expression levels of spok, sro, phm, and sad were reduced in Su(var)2-102/Su(var)2-10zimp−2 animals (Fig. 3c). Considering that phm>Su(var)2-10-RNAi-1 animals showed a slight decrease in sro expression, it is likely that Su(var)2-10 is also involved in the transcriptional regulation of sro in the PG. However, because neither 7-DHC nor 20E administration rescued developmental defects in Su(var)2-102/Su(var)2-10zimp−2 (Fig. 3d), developmental delay in Su(var)2-102/Su(var)2-10zimp−2 animals is probably caused by multiple mechanisms.

We further investigated the phenotypes of animals carrying Su(var)205 mutant alleles, Su(var)2052 and Su(var)2054, possessing point mutations that cause amino acid substitutions (V26M and K169Stop, respectively) (Eissenberg et al. 1992; Shaffer et al. 1993). Approximately half of the heterozygous Su(var)205 mutants [Su(var)2052/+ and Su(var)2054/+] underwent larval-to-prepupal transition at the same time as wild-type animals, whereas the other half died at the first or second instar (Fig. 3, a and b). Homozygous Su(var)205 mutants [Su(var)2052/Su(var)2052 and Su(var)2054/Su(var)2054] did not hatch (Fig. 3a). These results indicate that Su(var)205 is required for embryonic and early larval development. In contrast, most of the trans-heterozygous Su(var)205 mutant animals [Su(var)2052/Su(var)2054] developed into the third instar, but they could not undergo pupariation (Fig. 3, a and b). This suggests the possibility that Su(var)2052/Su(var)2054 animals were arrested at the third instar because of a decrease in ecdysone biosynthetic activities. This possibility is supported by the fact that expression levels of nvd, spok, and sro were low in Su(var)2052/Su(var)2054 animals (Fig. 3c), and administration of 20E, but not 7-DHC, partially rescued their developmental defects (Fig. 3d). These results suggest that Su(var)2052/Su(var)2054 animals died at the third instar due to reduced ecdysone biosynthetic activities. Considering that nvd and sro expression levels were reduced in phm>Su(var)205-RNAi-1 (Fig. 2c), it is probable that Su(var)205 is involved in not only nvd but also sro transcriptional upregulation in the PG. However, another possibility is that Su(var)205 expressed in other tissues is involved in sro expression indirectly. Altogether, the above results serve as evidence that Su(var)2-10 and Su(var)205 are required for nvd upregulation.

In addition, we found that Su(var)2-10 and Su(var)205 mutants did not show defects in DNA content and cell number in the PG (Supplementary Fig. 4). This result indicates that Su(var)2-10 and Su(var)205 are dispensable for mitosis and endocycle progression in the PG. However, we could not confirm the hypertrophic PG phenotype in Su(var)2-10 and Su(var)205 mutants (Supplementary Fig. 4), which suggests that Su(var)2-10 and Su(var)205 mutations might affect systemic growth signaling, such as the insulin signaling pathway.

Su(var)2-10 protein is localized near the pericentromeric heterochromatin

We investigated the localization patterns of Su(var)2-10 and Su(var)205 gene products, Su(var)2-10 and HP1a proteins, respectively, in PG cells using immunohistochemistry. We utilized 2 transgenic lines carrying GFP-fused Su(var)2-10 to visualize Su(var)2-10 protein distribution: Su(var)2-10-GFP, in which Su(var)2-10 carrying C-terminal GFP tag is expressed under the control of endogenous upstream sequences of Su(var)2-10 gene [generated by the model organism Encyclopedia of Regulatory Network (modERN) Project; related information is available in FlyBase (http://flybase.org/reports/FBtp0111904.html)], and Su(var)2-10CC02013, in which GFP-coding sequence franked by splicing acceptor and donor sites was inserted within the first intron between 2 coding exons of Su(var)2-10 gene (Buszczak et al., 2007) (https://flybase.org/reports/FBal0211741.html). In these strains, GFP-fused Su(var)2-10 was localized in the nucleus, whereas HP1a was localized in a part of the nucleus that represents the heterochromatic region (Supplementary Fig. 5). Interestingly, GFP-fused Su(var)2-10 puncta were detected near HP1a-positive and DNA-dense core heterochromatic regions in PG cells (Supplementary Fig. 5). These results suggest that Su(var)2-10 is juxtaposed with the heterochromatic region.

Su(var)2-10-GFP was then introduced in control [phm>mCherry.nls, Su(var)2-10-GFP] and Su(var)2-10 knockdown animals [phm>mCherry.nls Su(var)2-10-RNAi-1/2, Su(var)2-10-GFP], as well as Su(var)205 knockdown animals [phm>mCherry.nls Su(var)205-RNAi-1/2, Su(var)2-10-GFP]. We confirmed that Su(var)2-10-GFP expression was diminished in PG cells of Su(var)2-10 knockdown animals (Fig. 4, a and b). In contrast, the HP1a protein localization pattern was unchanged in PG cells of Su(var)2-10 knockdown animals (Fig. 4, a and b), suggesting that Su(var)2-10 does not control HP1a localization within PG cells. Likewise, HP1a protein levels were significantly reduced in PG cells of Su(var)205 knockdown animals; however, Su(var)2-10-GFP expression level and the percentage of Su(var)2-10-GFP-puncta-positive cells in the PG of Su(var)205 knockdown animals were comparable to those of control animals (Fig. 4, a and b). These results suggest that Su(var)2-10 and HP1a proteins do not mutually regulate their protein levels or their subcellular localization.

Fig. 4.

Fig. 4.

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Su(var)2-10 and HP1a protein localization patterns in PG cells. a) Confocal sections of the PGs (upper 2 panels) and PG cells in higher magnification (lower 3 panels) of control [phm>mCherry.nls, Su(var)2-10-GFP], Su(var)2-10 knockdown [phm>mCherry.nls Su(var)2-10-RNAi-1/2, Su(var)2-10-GFP], and Su(var)205 knockdown animals [phm>mCherry.nls Su(var)205-RNAi-1/2, Su(var)2-10-GFP] at 72 h after hatching. Su(var)2-10-GFP and HP1a proteins were visualized by antibodies against GFP (green) and HP1a (magenta), respectively. DNA was detected by Hoechst (blue), and the nuclei of PG cells were labeled by mCherry.nls (white). The PGs are indicated by dotted lines. Arrows indicate Su(var)2-10-GFP-positive puncta juxtaposed to the heterochromatic region. Scale bars: 50 µm (upper panels) and 10 µm (lower panels). b) Scatter and box plots showing the percentages of Su(var)2-10-GFP puncta-positive PG cells in control and knockdown animals at 72 (left) and 96 h after hatching (right). Statistically significant differences between groups are indicated by different lowercase letters (P < 0.05; Steel–Dwass test), while there is no statistically significant difference between groups with the same lowercase letter (P > 0.05). Sample sizes are 14–17 for each group.

Insulin and PTTH signal-mediated nvd upregulation and developmental acceleration is abrogated by Su(var)2-10 and Su(var)205 knockdown

The PG receives various neuropeptides, such as prothoracicotropic hormone (PTTH) and insulin-like peptides (ILPs), which leads to the activation of ecdysone biosynthesis (McBrayer et al. 2007; Walkiewicz and Stern 2009; Yamanaka et al. 2013; Niwa and Niwa 2016; Pan et al. 2021). We investigated whether Su(var)2-10 and Su(var)205 act downstream of PTTH and insulin signaling in the PG. As shown in previous studies, forced expression of an active form of insulin receptor (InR.A1325D; indicated as InRCA) in the PG (phm>InRCA) accelerates the timing of pupariation (Ohhara et al. 2017) (Fig. 5a). In contrast, Su(var)2-10 knockdown in InRCA-expressing animals [phm>InRCA Su(var)2-10-RNAi-1] completely blocked this acceleration, and the animals were arrested at the larval stage, similar to what occurred in the case of the Su(var)2-10 knockdown animals (Fig. 5, a and b). Furthermore, knockdown of Su(var)205 in InRCA-expressing animals [phm>InRCA Su(var)205-RNAi-1] also abrogated the acceleration of pupariation, and the percentage of pupariated animals among InRCA-expressing Su(var)205 knockdown animals was comparable with that among Su(var)205 knockdown animals (Fig. 5, a and c). These results suggest that Su(var)2-10 and Su(var)205 are required for insulin signaling-mediated acceleration of pupariation.

Fig. 5.

Fig. 5.

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Insulin and PTTH signal-mediated developmental acceleration and transcriptional upregulation of nvd are abrogated by S(var)2-10 and Su(var)205 knockdown. a–c) Percentages of pupariated animals among control (phm>+) (a), Su(var)2-10 knockdown [phm>Su(var)2-10-RNAi-1] (b), and Su(var)205 knockdown animals [phm>Su(var)205-RNAi-1] (c) with UAS-InRCA and UAS-RasV12 (triangle and square, respectively) or without any additional transgenes (circle) are shown at indicated time points. Sample sizes are 50–65 for each genotype. d) Upregulation of nvd transcription caused by InRCA- and RasV12-overexpression is cancelled by Su(var)2-10 and Su(var)205 knockdown. Expression levels of ecdysone biosynthetic genes (nvd, spok, sro, phm, dib, and sad) were measured using qPCR at 72 h after hatching. Box and scatter plots of 5 biological replicates are shown in each experimental group. Statistically significant differences between groups are indicated by different lowercase letters (P < 0.05; Tukey’s test), while there is no statistically significant difference between groups with the same lowercase letter (P > 0.05).

In contrast, forced expression of a constitutively active form of Ras (RasV12), a downstream effector of PTTH (Rewitz et al. 2009), partially rescued developmental arrest in Su(var)2-10 knockdown animals [phm>RasV12 Su(var)2-10-RNAi-1] (Fig. 5b), suggesting that Su(var)2-10 is independent of PTTH signaling in the PG. However, the percentage of pupariated animals was low among RasV12-expressing Su(var)2-10 knockdown animals (34%) compared to the corresponding percentage among control (95%) and RasV12-overexpressing animals (phm>RasV12) (82%) (Fig. 5, a and b). Thus, we could not exclude the possibility that Su(var)2-10 acts downstream of PTTH signaling. In contrast, pupariation in RasV12-expressing Su(var)205 knockdown animals [phm>RasV12 Su(var)205-RNAi-1] was significantly delayed compared with that in RasV12-expressing animals (Fig. 5, a and c), suggesting that Su(var)205 is required for PTTH signal-mediated acceleration of larval-to-prepupal transition.

The above results raise the possibility that Su(var)2-10 and Su(var)205 are required for insulin and PTTH signal-mediated upregulation of nvd transcription. To test this possibility, we examined the expression levels of ecdysone biosynthetic enzymes in InRCA- and RasV12-expressing Su(var)2-10 and Su(var)205 knockdown animals at 72 h after hatching. InRCA overexpression led to transcriptional enhancement of nvd and other ecdysone biosynthetic genes (Fig. 5d), indicating that insulin signaling accelerates the transcription of ecdysone biosynthetic genes. In contrast, nvd upregulation caused by InRCA overexpression was cancelled in Su(var)2-10 and Su(var)205 knockdown animals (Fig. 5d). These results suggest that insulin and PTTH signal-mediated enhancement of nvd expression requires Su(var)2-10 and Su(var)205. In addition, spok, sro, phm, and sad upregulation were blocked in InRCA-expressing Su(var)2-10 and Su(var)205 knockdown animals (Fig. 5d), suggesting that Su(var)2-10 and Su(var)205 were also involved in insulin signal-mediated upregulation of these ecdysone biosynthetic genes. However, considering that Su(var)2-10 and Su(var)205 knockdown caused selective downregulation of nvd expression, Su(var)2-10 and Su(var)205 are not essential for transcription of spok, sro, phm, and sad but required for InRCA-induced ectopic upregulation of these genes.

RasV12 overexpression also caused transcriptional upregulation of nvd, while RasV12-induced enhancement of nvd expression was cancelled by Su(var)2-10 and Su(var)205 knockdown (Fig. 5d), suggesting that Su(var)2-10 and Su(var)205 act downstream of PTTH signal to induce nvd expression. The expression levels of other ecdysone biosynthetic genes were enhanced in RasV12-expressing animals (Fig. 5d), but this upregulation was not abrogated by Su(var)2-10 and Su(var)205 knockdown (Fig. 5d). These results suggest that Su(var)2-10 and Su(var)205 are dispensable for PTTH signal-mediated upregulation of other ecdysone biosynthetic genes.

Discussion

In Drosophila, ecdysone biosynthetic enzymes show a significant increase in expression levels in the late third instar (Ou et al. 2016; Ohhara et al. 2017), and this surge is essential for larval-to-prepupal transition. The transcriptional regulatory mechanisms of ecdysone biosynthetic enzymes by transcription factors have been investigated over the last 20 years. In contrast, comparatively little is known about the chromatin regulators supporting the gene expression of these enzymes. In the present study, we demonstrated that Su(var)2-10 and Su(var)205 are required for upregulation of nvd. Knockdown of Su(var)2-10 and Su(var)205 in the PG caused a defect in larval-to-prepupal transition and a decrease in nvd expression (Figs. 1 and 2), and nvd overexpression or administration of 7-DHC, a biosynthetic precursor of ecdysone produced by Nvd, rescued developmental defects in Su(var)2-10 and Su(var)205 knockdown animals (Fig. 2 and Supplementary Fig. 2). The expression level of nvd was also reduced in Su(var)2-10 and Su(var)205 mutants (Fig. 3). These results indicate that Su(var)2-10 and Su(var)205 promote pupariation through the regulation of nvd expression.

An important question that is yet to be resolved is how Su(var)2-10 and Su(var)205 regulate the expression of heterochromatic nvd gene. Both Su(var)2-10 and Su(var)205 belong to the Su(var) gene group, a set of genes required for heterochromatin formation (Eissenberg et al. 1992; Hari et al. 2001; Elgin and Reuter 2013). At the molecular level, Su(var)2-10 encodes a SUMO E3 ligase to modify chromatin state and gene expression via SUMOylation of target proteins (Rytinki et al. 2009; Ninova, Godneeva, et al. 2020), whereas HP1a, the product of Su(var)205 gene, is a nonhistone chromosomal protein deposited on heterochromatin to ensure normal heterochromatin formation (Eissenberg and Elgin 2014; Schoelz and Riddle 2022). Several studies have shown that heterochromatin-residing genes, such as rolled and light, require a heterochromatin environment and Su(var)205 for their normal expression in Drosophila (Wakimoto and Hearn 1990; Eberl et al. 1993; Lu et al. 1996, 2000; Yasuhara and Wakimoto 2006). Furthermore, a recent study showed that Su(var)2-10 supports proper expression of heterochromatic genes through the regulation of histone H3 lysine 9 trimethylation (H3K9me3) (Ninova, Godneeva, et al. 2020). Given that binding of HP1a to H3K9me3 is responsible for structural properties of the heterochromatic region (Canzio et al. 2011; Eissenberg and Elgin 2014; Larson et al. 2017; Strom et al. 2017; Schoelz and Riddle 2022), one possibility is that Su(var)2-10-mediated histone modification and subsequent HP1a recruitment in the nvd gene locus is required for the establishment of an inherent chromatin structure that is suitable for its normal expression. In contrast, SUMOylation promotes the targeting of HP1a to pericentromeric heterochromatin in mammals (Maison et al. 2011). Hence, another possible mechanism is that Su(var)2-10 SUMOylates HP1a in the PG to confer a proper heterochromatic structure that is required for nvd transcriptional activation, although it remains unclear whether HP1a is SUMOylated in a Su(var)2-10-dependent manner. It would be interesting to investigate whether H3K9me3 level and HP1a accumulation in the nvd locus is dependent on Su(var)2-10 activity.

Although we could not obtain the data showing the direct interaction between Su(var)2-10/HP1a and the nvd gene locus, we observed that HP1a was localized to the heterochromatic region in PG cells and that Su(var)2-10 protein is juxtaposed with the pericentromeric heterochromatin region in PG cells (Fig. 4 and Supplementary Fig. 5). These results support the hypothesis that Su(var)2-10 and HP1a interact with the nvd gene locus to allow its normal expression. Importantly, heterochromatin-juxtaposed Su(var)2-10 puncta were observed in some, but not all, PG cells and the percentages of Su(var)2-10-pisitive puncta were divergent among PGs (Fig. 4), raising the possibility that Su(var)2-10 localization near the heterochromatic region is affected by heterogeneous cellular physiologies, such as cell cycle. Given that endocycle progression is asynchronous among PG cells (Ohhara et al., 2017; Ohhara et al., 2019) and that the distribution of chromatin-associated proteins including histone H1 and proliferating cell nuclear antigen in the polytene chromosome is dynamically changed in accordance with the endocycle progression (Andreyeva et al., 2017), we put forward a hypothesis that Su(var)2-10 localization near the heterochromatic regions is coupled with endocycle progression in PG cells. For example, a possible mechanism is that Su(var)2-10 protein is recruited near the heterochromatic region when heterochromatic structure is loosed before or after DNA replication.

Su(var)2-10 belongs to a conserved PIAS protein family. PIAS proteins were originally identified as suppressors of transcription factors called signal transducer and activator of transcription (STAT) proteins (Rytinki et al. 2009). Importantly, the newest study demonstrated that Su(var)2-10 promotes larval-to-prepupal transition through the negative regulation of STAT92E (a sole STAT protein in Drosophila) in the PG (Cao et al. 2022). The study also showed that the autocrine Unpaired3 (Upd3)-induced JAK (Janus kinase)/STAT signaling in the PG negatively controls the timing of pupariation (Cao et al. 2022). Thus, one possible mechanism is that Su(var)2-10 upregulates nvd expression via the negative regulation of Upd3-induced JAK/STAT signaling. In addition, Su(var)2-10 proteins positively or negatively regulate various proteins, including transcription factors other than STATs (Rytinki et al. 2009; Rabellino et al. 2017). In Drosophila PG, βFtz-F1, a nuclear receptor/transcription factor regulating ecdysone biosynthesis, is the substrate of SUMOylation (Talamillo et al. 2013). Thus, βFtz-F1 is likely to be a prominent candidate for the Su(var)2-10 target protein. However, because inhibition of βFtz-F1 causes a decrease in Phm and Dib expression (Parvy et al. 2005), other SUMO E3 ligases are likely involved in the regulation of βFtz-F1. Alternatively, we speculate that Su(var)2-10 modulates the activity of other transcription factors, such as Séance and Mld, to promote nvd expression (Uryu et al. 2018). Further studies are needed to elucidate the target proteins and pathways of Su(var)2-10 in the PG.

In addition to nvd, the expression levels of spok and sro were also reduced in Su(var)2-102/Su(var)2-10zimp−2 and Su(var)2052/Su(var)2054 mutants (Fig. 3c). This suggests that Su(var)2-10 and Su(var)205 are also involved in the regulation of spok and sro expression. Knockdown of Heterogeneous nuclear ribonucleoprotein at 87F (Hrb87F), which encodes an RNA-binding protein and belongs to the Su(var) gene group (Piacentini et al. 2009), resulted in a decrease in nvd and spok expression (Fig. 1). Considering that Hrb87F promotes gene expression through interaction with HP1a (Piacentini et al. 2009), one possible mechanism is that Hrb87F acts in concert with HP1a to promote nvd and spok expression.

Ecdysone biosynthesis in the PG is controlled by neuropeptides, such as ILPs and PTTH (McBrayer et al. 2007; Walkiewicz and Stern 2009; Yamanaka et al. 2013; Niwa and Niwa 2016; Pan et al. 2021). ILPs and PTTH stimulate insulin-mTOR and Ras/MAPK signaling pathways in the PG, respectively, which in turn activate ecdysone biosynthesis and subsequent larval-to-prepupal transition through the regulation of the endocycle (Ohhara et al. 2017; Shimell et al. 2018). Because the acceleration of larval-to-prepupal transition and nvd transcriptional upregulation by forced activation of insulin and Ras/MAPK signaling in the PG was abrogated by Su(var)2-10 and Su(var)205 knockdown (Fig. 5), it can be surmised that Su(var)2-10 and Su(var)205 allow the ILP/PTTH-endocycle pathway to stimulate nvd expression. In addition, considering that DNA content and tissue size did not decrease but, rather, increased in the PGs of Su(var)2-10 and Su(var)205 knockdown animals (Supplementary Fig. 3), Su(var)2-10 and Su(var)205 seem to negatively modulate the activity of endocycling in the PG. One possible mechanism is that Su(var)2-10 and Su(var)205 permit nvd transcriptional upregulation while acting in a negative feedback loop to downregulate endocycle progression to switch from endocycling to the ecdysteroidogenic phase in the PG.

In summary, our results indicate that Su(var)2-10 and HP1a are novel regulators of nvd expression and larval-to-prepupal transition (Fig. 6). The genetic evidence in our study shows that Su(var)2-10 and HP1a positively regulate the transcription of the heterochromatic gene nvd, supporting the idea that a heterochromatic environment is required for certain heterochromatin-residing genes to be normally expressed. This study provides the basis for understanding the transcriptional upregulation mechanisms of heterochromatic genes and their significance in steroidogenesis and development.

Fig. 6.

Fig. 6.

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A model for Su(var)2-10- and Su(var)205-mediated regulation of nvd transcription and developmental transition. Drosophila PG expresses a heterochromatin-residing gene, neverland (nvd), which encodes an enzyme that catalyzes the initial step of ecdysteroid biosynthesis. Our genetic evidence shows that Su(var)2-10 and Su(var)205, encoding Su(var)2-10 and HP1a proteins, respectively, positively regulate the transcription of neverland and that Su(var)2-10- and Su(var)205-mediated upregulation of neverland is required for ecdysone biosynthesis and subsequent larval-to-prepupal transition. We propose that Su(var)2-10- and HP1a-dependent regulation of inherent heterochromatin structure is essential for appropriate expression of the neverland gene.

Supplementary Material

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Acknowledgments

We thank the National Institute of Genetics (Japan), the Vienna Drosophila RNAi Center, and the Bloomington Drosophila Stock Center (supported by NIH P40 OD018537) for fly stocks. The monoclonal anti-HP1 antibody deposited by L. L. Wallrath was obtained from the Developmental Studies Hybridoma Bank, created by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) of the National Institutes of Health (NIH) and maintained at The University of Iowa, Department of Biology, Iowa City, Iowa. We also thank M. B. O’Connor for anti-Spok and anti-Dib antibodies and phm-Gal4, R. Niwa and N. Yamanaka for critical reading of the manuscript, and K. Tamura for providing technical support. T.K. was a recipient of a fellowship from the Japan Society for the Promotion of Science. We thank Editage (www.editage.com) for English language editing.

Funding

This work was supported by grants to YO from the Japan Society for the Promotion of Science (JSPS) KAKENHI (19K16180 and 21H02521), the Uehara Memorial Foundation, and the Takeda Science Foundation. This work was also supported by grants from JSPS KAKENHI (21J10894) to TK.

Conflicts of interest

None declared.

Contributor Information

Yuya Ohhara, School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka, Shizuoka 422-8526, Japan; Graduate School of Integrated Pharmaceutical and Nutritional Sciences, University of Shizuoka, Shizuoka, Shizuoka 422-8526, Japan.

Yuki Kato, Graduate School of Integrated Pharmaceutical and Nutritional Sciences, University of Shizuoka, Shizuoka, Shizuoka 422-8526, Japan.

Takumi Kamiyama, College of Biological Sciences, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan; Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan.

Kimiko Yamakawa-Kobayashi, School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka, Shizuoka 422-8526, Japan; Graduate School of Integrated Pharmaceutical and Nutritional Sciences, University of Shizuoka, Shizuoka, Shizuoka 422-8526, Japan.

Data Availability

All numerical data (except for the data obtained in RNAi screens) and R scripts for figures and statistical analysis are available in Figshare (https://doi.org/10.6084/m9.figshare.20626095). Confocal images are also shared in Figshare (https://doi.org/10.6084/m9.figshare.20646858). The data obtained in the first and second RNAi screens are shown in Supplementary Tables 3 and 5, respectively. The numerical data visualized in Supplementary Fig. 1a are available in Ohhara et al., 2019.

Supplemental material is available at GENETICS online.

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iyac137_Supplementary_Figure_S1
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iyac137_Supplementary_Figure_S2
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iyac137_Supplementary_Figure_S1
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iyac137_Supplementary_Figure_S4
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iyac137_Supplementary_Figure_S5
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iyac137_Supplementary_Table_S1
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iyac137_Supplementary_Table_S2
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iyac137_Supplementary_Table_S3
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iyac137_Supplementary_Table_S4
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iyac137_Supplementary_Table_S5
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Data Availability Statement

All numerical data (except for the data obtained in RNAi screens) and R scripts for figures and statistical analysis are available in Figshare (https://doi.org/10.6084/m9.figshare.20626095). Confocal images are also shared in Figshare (https://doi.org/10.6084/m9.figshare.20646858). The data obtained in the first and second RNAi screens are shown in Supplementary Tables 3 and 5, respectively. The numerical data visualized in Supplementary Fig. 1a are available in Ohhara et al., 2019.

Supplemental material is available at GENETICS online.

Articles from Genetics are provided here courtesy of Oxford University Press

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