Insects and cell line
Bombyx mori larvae strain P50, obtained from the Research and Development Center of the Sericulture Research Institute of the Academy of Agricultural Sciences of Guangdong Province at China, were reared on fresh mulberry leaves at 27 °C with a 12 h light/12 h dark cycle. Under this condition for 6 days, the fifth instar silkworm larvae started wandering, followed by the beginning of larval–pupal transition. After pupation, the newly molted pupae (P0 stage) were transferred to a clean and dry paper box and reared at 27 °C for 7 days before enclosing into adult moth.
Bombyx mori Bm12 (DZNU-Bm-12) cell line, originally derived from the ovarian tissues [50], was cultured in Grace medium (Invitrogen, California, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Utah, USA) at 28 °C.
5-Aza-dC treatment experiment
For DNA methylation inhibitor treatment in silkworm pupae, 1 mg 5-aza-dC (5-aza-2′-deoxycytidine) (Sigma, California, USA) was dissolved in deionized distilled water (ddH2O) with a 10 μg/μL final concentration. Newly molted pupae (P0 stage) were injected into the abdomen with freshly prepared 5-aza-dC (20 μg per pupa) or ddH2O as control. Three replicates of about 30-40 pupae per replicate were carried out. In addition, for inhibitor 5-aza-dC treatment in the cultured Bm12 cells, the freshly prepared 5-aza-dC was diluted 10 times to 1 μg/μL final concentration, and 1 μg 5-aza-dC was applied to cells.
Genomic DNA preparation and dot blot assay
For genomic DNA preparation, wing from silkworm pupae after 5-aza-dC or ddH2O treatment for 48 h and 72 h were homogenized with 600 μL digestive solution (5 mL 1 M Tris–HCl (pH 8.0), 4 mL 0.5 M EDTA (pH 8.0), 2 mL 5 M NaCl, 5 mL 10% (W/V) SDS, 150 μL 60 ng/μL Protease K), respectively, and the samples were digested overnight at 50 °C. Then, the genomic DNA was extracted with hydroxybenzene–chloroform–isopentanol, precipitated with absolute ethanol and washed with 75% ethanol.
For dot blot assay, genomic DNA samples were diluted into 100 ng/μL concentration and were digested by RNaseA (Promega) to rule out RNA contaminations. For each sample, 500 ng genomic DNA was denatured at 95 °C for 5 min and immediately cooled down on ice. Then, the genomic DNA was spotted on the nitrocellulose blotting membrane (PVDF, GE healthcare) and the membrane was dried on a heater, followed by UV-crosslinking for 10 min. The membrane was then blocked with 3% (w/v) BSA in TBST (20 mM Tris–HCl, 150 mM sodium chloride, 0.05% Tween-20, pH 7.4) for 2 h at room temperature, and incubated with rabbit anti-5mC antibody (diluted 1:1000; Abcam) overnight at 4 °C. After three washes in TBST for 10 min each, the membrane was incubated with a horse radish peroxidase (HRP)-linked secondary goat anti-rabbit IgG antibody (diluted 1:10,000; Dingguo Biotechnology) for 60 min at 37 °C. Meanwhile, the input genomic DNA samples were spotted on the PVDF membrane as mentioned above, and then directly stained with GoldView™ (Dingguo Biotechnology).
Chitin staining assay
The pupal wing discs or adult wings from silkworm treated with 5-aza-dC or ddH2O were collected and fixed in 4% paraformaldehyde overnight. Pupal wing discs or adult wings were dehydrated by concentration gradients of alcohol and xylene, and embedded in paraffin. The embedded wing was sectioned (5 μm) using a microtome (Leica, Germany), affixed to slides, deparaffinized in xylene, and rehydrated with an ethanol gradient for chitin staining [51]. For histology structure observation, the wing slices were dyed with 0.01 mg/mL Fluorescent Brightener 28 (Sigma-Aldrich) for 90 s. After three washes in water for one min each, the tissue slices were counterstained with 0.01 mg/mL propidium iodide (Sigma-Aldrich) for 90 s and then rinsed three times in water for one min each. Finally, 50% glycerol was added to the tissue and then the slides were covered. Fluorescence signals were captured using a FV3000 confocal microscope (Olympus, Japan) at the excitation wavelengths of 365 nm and 535 nm.
Chitin content assay
The pupal wing discs or adult wings from silkworm treated with 5-aza-dC or ddH2O were collected for chitin content assay. For each sample, four wings (two forewing and two hindwing wings) from one silkworm pupa were homogenized in 200 μL of 3% SDS (sodium dodecyl sulfate), incubated at 100 °C for 15 min and centrifuged at 1800g for 10 min at room temperature after cooling. After each pellet was washed with 500 μL ddH2O, it was resuspended in 300 μL of 120% KOH (w/v). To deacetylate chitins, the samples were incubated at 130 °C for 1 h followed by cooling them on ice for 5 min. Then, the sample was mixed with 800 μL of ice-cold 75% (v/v) ethanol, and incubated on ice for 15 min followed by centrifuging them at 1800g for 5 min at 4 °C. After each pellet containing insoluble chitosan (i.e., glucosamine polymer), then was washed with 500 μL of 40% (v/v) ice-cold ethanol and 500 μL of ice-cold ddH2O, the chitosan in each tube was re-suspended in 500 μL of ddH2O.
For chitin content assay, 500 μL of the chitosan solution was mixed with 50 μL of 10% NaNO2 (w/v) and 50 μL of 10% KHSO4 (w/v), and incubated at room temperature for 15 min followed by centrifuging them at 1800g for 15 min at 4 °C. Then, 60 μL of the supernatant of each sample was transferred to a new Eppendorf tube and mixed with 20 μL of NH4SO3NH2, and incubated at room temperature for 5 min. Each sample was added with 20 μL of freshly prepared MBTH (3-methyl-2-benzothiazolone hydrazone hydrochloride hydrate, Sigma-Aldrich), and incubated at 100 °C for 5 min. After cooling to room temperature, 100 μL of each sample was transferred to a well of a 96-wells plate and mixed with 20 μL FeCl3·6H2O. Absorbance of each sample was determined at a wavelength of 650 nm using a FlexStation3 microplate reader (Molecular Devices, USA) and the chitin content of each sample was described as a glucosamine equivalent according to a standard curve constructed using known concentration gradients of glucosamine (Sigma-Aldrich). All reported data were based on three biological replicates.
RNA extraction and qRT-PCR
For RNA extraction, pupal wing discs, adult wings or Bm12 cells were homogenized with 1 mL of RNAiso Plus (TaKaRa, Dalian, China), and total RNA was extracted with chloroform, precipitated with isopropanol and washed with 75% ethanol. Pellets were suspended in the 30 μL RNase-free water and stored at − 80 °C. For cDNA synthesizing, 2 μg RNA was treated with Recombinant DNase I (RNase-free) (TaKaRa, Dalian, China) to remove the genome DNA and was then transcribed to cDNA using the First Strand cDNA Synthesis Kit (TaKaRa, Dalian, China) following the manufacturer’s instructions. The gene expression levels were analyzed using qRT-PCR with the Hieff™ qPCR SYBR Green Master Mix Kit (Yeasen, Guangzhou, China). The PCR conditions were as follows: initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 31 s. The relative mRNA level of gene expression was normalized to the expression level of a housekeeping gene encoding ribosomal protein 49 (rp49) (GenBank accession no. 778453) and analyzed by the 2−ΔΔCt method [52]. All reported data were based on three biological replicates and three technical replicates. All primers used for qRT-PCR listed in Additional file 1: Table S5.
RNA-seq analysis
Total RNA was extracted from silkworm pupal wing discs after 5-aza-dC or ddH2O treatment in newly molted pupae for 48 h using RNAiso Plus (TaKaRa, Dalian, China) as described above. RNA-seq was done on an Illumina HiSeq™ 2500 sequencing platform by the Biomarker Technologies and paired-reads with an average length of approximately 200 nt were generated. The clean reads that were filtered from the raw reads were used for mapping to the reference B. mori genomes. After mapping, the assembled transcripts were generated, and gene expression level of each gene was normalized using FPKM values (fragments per kilobase of exon per million fragments mapped) by the Cufflinks software [53]. All reported data were based on three biological replicates.
Expression, purification of recombinant proteins and polyclonal antibody preparation
The ORF (open reading frame) of select genes were amplified by PCR. The cDNAs were subcloned into the pPET-28a/32a vector infused with a 6× His to generate the recombinant expression vectors, respectively. The recombinant proteins were expressed in Escherichia coli cells (BL21). All primers used for PCR listed in Additional file 1: Table S5.
For purification of proteins with His tag, the transformed E. coli cells were collected by centrifugation and re-suspended in the binding buffer (0.5 M NaCl, 20 mM Tris–HCl, 5 mM Imidazole, pH 7.9 and 1 mM PMSF). The suspension was centrifuged after being lysed by sonication and then purified with Ni-chelating affinity chromatography using the His-Bind® 12 Kit according to the manufacturer’s protocol (Novagen, Wisconsin, USA).
Mouse anti-BmCHT10 and mouse anti-Bmara antibodies were prepared, respectively. In brief, mouse strain BALB/c males, obtained from Guangdong Medical Laboratory Animal Center (GDMLAC), were used for polyclonal antibody preparation by intraperitoneal injection. Mixed the antigens (purified recombinant proteins) with an equal volume of the Freund’s Adjuvant Complete or Incomplete (Sigma-Aldrich) was mixed to form an emulsion. The Complete Freund’s Adjuvant was used for the initial injections and the Incomplete Freund’s Adjuvant was used in the later injections for the boosts. After four times injections with a 200 μL of emulsion (once a week), the mouse serums were isolated and incubated at 37 °C for 2 h. After gentle centrifugation, the supernatants were stored at − 80 °C and used as the primary antibody in the following Western blot analysis and immunohistochemistry experiment.
Western blotting
The pupal wing discs or Bm12 cells were homogenized in Cell Lysis Buffer (Beyotime, Shanghai, China). For Western blot, 40-100 μg proteins extracted from tissues or Bm12 cells were denatured and then separated in 12% SDS-PAGE gel, followed by transferring to a nitrocellulose blotting membrane (GE healthcare). The membrane was blocked with 3% (w/v) BSA in TBST for 2 h at room temperature, followed by hybridization overnight at 4 °C in TBST containing 1% BSA and primary antibody (diluted 1:1000, polyclonal antibodies as described above). The secondary antibody was a horse-radish peroxidase (HRP)-conjugated goat anti-rabbit IgG (diluted 1:10,000, Dingguo Biotechnology). Antibody against tubulin (diluted 1:5000, Dingguo Biotechnology) was used to verify equal loading of the proteins on the gel.
Construction of the reporter luciferase vector
Genomic DNA was extracted from the Bm12 cells. The promoter upstream the ATG of BmCHT10 was amplified by PCR according the sequence of its genome DNA sequence (Gene ID: 101736080) and cloned into pMD-18T vectors (TaKaRa, Dalian, China). After the digestion by double restriction enzymes, Sma I and BgI II, the promoter fragments were cloned into the luciferase reporter plasmid, pGL3-basic vector (Promega, Madison, USA). The primers used for constructing the vectors are listed in Additional file 1: Table S5.
Cell culture, transfection and transcriptional activity assay
Bm12 cells at their logarithmic growth phase were inoculated in culture media in 12- or 24-well culture plates (Corning, New York, NY, USA) and cultured for 12 h. Cell transfection and co-transfection were conducted when the cells were at approximately 80% density. Plasmid DNAs were mixed with Fugene HD transfection reagent (Promega, Madison, USA) and added to cells in each well of 12- or 24-well culture plates with Grace medium (Invitrogen). To normalize the firefly luciferase activity, the renilla luciferase vector, pRL-SV40, was co-transfected with each of the pGL3-derived vectors containing tested promoters. The cells were cultured for additional 48 h at 28 °C, followed by the luciferase activity assay, protein or RNA isolation.
For luciferase activity measurement, the cells were washed twice with filtered PBS and then lysed in 100 μL Passive Lysis Buffer (Promega, Madison, WI, USA). The samples were centrifuged at 800g for 5 min at room temperature. The supernatant was used to analyze the luciferase activity using the Dual- Luciferase Assay System according to the manufacturer’s protocol with a luminometer (IBA7300, Veritas, Turner Biosystems). The luciferase activity was normalized to the renilla luciferase activity. All assays included three biological replications and three technical replicates. The luciferase activity was represented as mean ± standard error (SE).
RNA interference (RNAi)
For RNAi in the Bm12 cell line, a 400–600 bp unique fragment in the ORF of target genes was chosen as a template for synthesizing gene-specific dsRNA using the T7 RiboMAXTM Express RNAi System (Promega, Wisconsin, USA). dsRNA (1 μg) was used to transfect the Bm12 cells with 4 μl Fugene HD transfection reagent in the Opti-MEM Reduced Serum Medium. The cells were collected 48 h after transfection. All assays included three biological replications. The sequences of primers are listed in Additional file 1: Table S5.
Immunohistochemistry
The newly dissected silkworm pupal wing discs or adult wings were fixed in 4% paraformaldehyde for 30 min at room temperature. For BmCHT10 protein and chitin staining, pupal wing discs or adult wings were blocked in PBS containing 5% BSA and 0.5% Triton-X (PBT) for 1–2 h, and then incubated with the primary antibody (mouse anti-BmCHT10, diluted 1:200) at 4 °C overnight. After being washed three times for 10 min each in 0.2% PBT, the samples were then incubated with Alexa Fluor™ 488 goat anti-mouse IgG (diluted 1:200; Invitrogen) for 2 h. Fluorescent Brightener 28 (Sigma-Aldrich) was added to stain chitin. The wings stained with anti-BmCHT10 and Fluorescent Brightener 28 were observed and imaged using a FV3000 confocal microscope (Olympus).
For 5mC staining, the fixed pupal wing discs or adult wings were first incubated with 2 M HCl solution for 20 min and then neutralized with 100 mM Tris–HCl (pH 8.5) for 10 min at room temperature. After blocking, the pupal wing discs or adult wings were incubated with the primary antibody (rabbit anti-5mC, diluted 1:1000, Abcam) at 4 °C overnight. After 3 washes, the samples were incubated at room temperature for 2 h with the goat anti-rabbit IgG secondary antibody conjugated with Alexa Fluor™ 594 (diluted 1:200, Invitrogen). The nuclei were stained with DAPI (Beyotime, Shanghai, China) for 20 min. Then the wings stained with anti-5mC and DAPI were observed and imaged using a FV3000 confocal microscope (Olympus).
BS-seq analysis
Genomic DNA of B. mori was extracted from pupal wing discs as described above. After treated with bisulfite, unmethylated cytosines were converted into uracil using MethylDetectorTM (Active Motif, Carlsbad, CA, USA), whereas methylated cytosines remain unchanged. Polymerase chain reaction (PCR) was then performed and PCR products were cloned into pMD18-T vector for following sequencing. By aligning with the sequence of unconverted gDNA using DNAMAN software (Lynnon Biosoft), a single-base-resolution DNA methylation distribution can be quantified. All assays included three biological replications. The results of BS-seq are listed in Additional file 1: Table S2.
Nuclear protein preparation and DNA pull-down
Nuclear proteins were extracted from Bm12 cells according to the instruction of NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, Waltham, USA). For DNA pull-down assay, the oligonucleotides conjugated with biotin at 5′ end were synthesized by Qingke Biotechnology, and the single-stranded oligo-probes were heated at 95 °C for 10 min and then slowly cooled to room temperature to obtain the double-stranded probes. Then, the oligo-probes were linked to the streptavidin-coated beads. To minimize non-specific interactions, the oligo-bead complexes were incubated for 30 min with a blocking buffer (2.5 mg/mL albumin from bovine serum (BSA), 10 mM HEPES pH 7.6, 10 mM glutamate potassium, 2.5 mM DTT, 10 mM magnesium acetate, 5 mM EGTA, 3.5% glycerol with 0.003% NP-40 and 5 mg/mL polyvinylpyrrolidone). Immobilized double-stranded probes were incubated with 20 μg of nuclear extract for 4 h at 4 °C with constant rotation in a 400 μL of protein binding buffer (10 mM HEPES pH 7.6, 100 mM glutamate potassium, 80 mM potassium chloride, 2.5 mM DTT, 10 mM magnesium acetate, 5 mM EGTA, 3.5% glycerine with 0.001% NP-40). Protein-DNA complexes were then washed three times with a wash buffer (10 mM HEPES pH 7.6, 100 mM glutamate potassium, 2.5 mM DTT, 10 mM magnesium acetate, 5 mM EGTA, 3.5% glycerol, 0.5 mg/mL BSA, 0.05% NP-40). Proteins bound to the probe were eluted with 20 μL of a denaturing Laemmli sample loading buffer (50 mM Tris–HCl, 100 mM DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol) at 37 °C for 15 min. The target proteins in the supernatant were identified by Western blot with anti-FLAG antibody (#14793, Cell Signaling Technology, MA, USA) at 1:2000 dilution.
Electrophoretic mobility shift assay (EMSA)
The recombinant Bmara protein was prepared as described above. EMSA was conducted using the LightShift Chemiluminescent EMSA Kit (Thermo Scientific). The oligonucleotide probes conjugated with biotin at 5′ end were heated at 95 °C for 10 min and then slowly cooled to room temperature. Binding assays were performed according to the manufacture’s protocol. Briefly, the recombinant Bmara proteins were incubated for 20 min at room temperature with 20 μL binding buffer containing 50 ng of poly (dI-dC), 2.5% glycerol, 0.05% NP-40, 50 mM potassium chloride, 5 mM magnesium chloride, 4 mM EDTA and 20 fmol of a biotinylated end-labeled double-stranded probe. Different concentrations of cold probes (unlabeled) were added into the binding mixture as competitors. Polyacrylamide gels (6%) were run at 100 volts for 1.5 h on ice. After electrophoresis, the proteins were blotted onto positively charged nylon membranes (Hybond Nþ; Amersham Biosciences) and the bands were visualized using the EMSA Kit according to the manufacturer’s protocol.
Chromatin immunoprecipitation (ChIP)
ChIP was performed in the Bm12 cells following the instruction of Pierce™ Magnetic ChIP Kit (Thermo Scientific). Briefly, approximately 4 × 106 cells were set up, cross-linked with 1% formaldehyde for 10 min after transfected with overexpression vectors of Bmara-FLAG for 48 h, and then de-cross-linked with glycine. The cells were broken up with extraction buffer containing protease/phosphatase inhibitors. The nuclei were treated with the MNase diluted in MNase Digestion Buffer for 15 min at 37 °C, and the nuclei were released from the cells using several ultrasonic pulses and 20 s ice-cold interval. The protein-DNA complexes were immunoprecipitated using anti-FLAG antibody (#14793, Cell Signaling Technology, MA, USA) or normal rabbit IgG (as a control) (Thermo Fisher Scientific, Massachusetts, USA) for 8 h at 4 °C with constant mixing in a Mini Rotating Incubator (Qilinbeier, Haimen, China), and then enriched by Protein A/G Magnetic Beads for 2 h at 4 °C with mixing before being reversely cross-linked at 65 °C for 30 min with vigorous rotation in thermomixer comfort (Eppendorf, Hamburg, Germany). DNA was purified using the column method (Thermo Scientific), and detected by reverse transcription PCR. All assays included three biological replications.
Bioinformatics analysis
The amino acid sequences were downloaded from the NCBI protein database. The conserved domain of the protein amino acid sequences was predicted using SMART [54]. The multiple sequence alignment of the protein amino acid sequences was aligned with Clustal Omega [55]. The results of conserved domain prediction and multiple sequence alignment were listed in Additional file 1: Figure S1.
The cis-regulation elements (CRE) were predicted with the JASPAR 2020 [46]. The protein molecular weight of the candidate transcription factors was analyzed with The Sequence Manipulation Suite [56]. The results of CRE prediction and protein molecular weight analysis were listed in Additional file 1: Table S3.
Statistical analysis
Data are presented as mean ± SE. p values for the purpose of group comparisons were calculated using student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001).
