The human neuroblastoma cells SH-SY5Y (ATCC, Manassas, VA, USA) were cultured in 1:1 MEM and Ham’s F12 media (Mediatech, Manassas, VA, USA) supplemented with 15% (v/v) fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA, USA) and 1% penicillin/streptomycin (P/S; Mediatech). Cells were maintained at 37°C with 5% CO2, and split 1:2 every 3 to 4 days when the cells reached approximately 80% confluency. For harvesting, the cells were treated with trypsin-ethylenediaminetetraacetic acid (EDTA) (Mediatech) for 2 to 3 minutes to release them from the surface of the culture flask. Complete growth medium was then added to the flask containing suspension cells to inactivate trypsin. Cells were transferred to a sterile centrifuge tube and pelleted by spinning at 800 rpm for 5 minutes at 4°C and gently washed twice with ice-cold PBS. This study did not involve any human subjects or biomaterials taken from human subjects, and thus no ethical approval was required.
For hormone treatment, SH-SY5Y cells were cultured in culture flasks until the cultures became approximately 80% confluent. Confluent cells were carefully washed twice with phenol red-free 1:1 DMEM/F12 media (Mediatech) supplemented with 15% charcoal dextran-treated serum (Atlanta Biologicals) and 1% P/S, and then cultured in the phenol red-free medium for 24 hours. Lyophilized 4,5α-dihydrotestosterone (DHT; Sigma-Aldrich, St. Louis, MO, USA) and 17β-estradiol (E2; Sigma-Aldrich) were diluted with molecular biology grade absolute ethanol (Fisher Scientific, Pittsburgh, PA, USA) to make 1 μM DHT or 1 μM E2 stock solutions. The stock solutions were further diluted with prewarmed complete phenol red-free culture medium to the desired final concentrations for hormone treatment and carefully added to the confluent cells. Cells were incubated in the hormone-supplemented phenol red-free medium at 37°C with 5% CO2 for 2 hours.
siRNA-mediated knockdown of AR, ERα, SUMO1, or NCOA5 was conducted using Lipofectamine RNAiMAX transfection agent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Briefly, SH-SY5Y cells were cultured in complete growth medium without antibiotics in a 6-well culture plate. When cells were approximately 50% confluent, the medium was substituted with phenol red-free culture medium without antibiotics and the cells were further incubated for 24 hours. siRNA (Santa Cruz Biotechnology, Dallas, TX, USA) targeting AR, ERα, SUMO1, or NCOA5 (150 pmol) was diluted in 250 μl phenol red-free Opti-MEM I Reduced-Serum Medium (Invitrogen). Lipofectamine RNAiMAX (7.5 μl) was diluted in 250 μl phenol red-free Opti-MEM I reduced-serum medium in a separate tube. Then, the diluted siRNA and the diluted Lipofectamine RNAiMax were combined. The siRNA-Lipofectamine complex was incubated at room temperature for 5 minutes and added to the cells to a final siRNA concentration of 10 nM. The cells were incubated for 24 hours and then treated with 1 nM DHT, 1 nM E2, or ethanol (vehicle), for 2 hours according to the aforementioned hormone treatment procedure before harvesting for subsequent analysis. The list of siRNAs is shown in Additional file 1. Transfection efficiency was assessed by qRT-PCR analysis (Additional file 2).
Quantitative RT-PCR analysis
Quantitative RT-PCR analyses were performed as described . Total RNA from the cells was isolated using TRIzol (Invitrogen) and purified using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) following the manufacturers’ instructions. Human brain tissues were homogenized in the Bullet Blender Homogenizer (Next Advance, Averill Park, NY, USA) using nuclease-free glass beads, and total RNA was isolated from homogenized tissues using the RNeasy Mini Kit (Qiagen). RNA concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). A total of 1 μg of purified total RNA was used for cDNA synthesis using the iScriptcDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocols. The reaction was incubated at 25°C for 5 minutes, followed by 42°C for 30 minutes, and terminated at 85°C for 5 minutes. After reverse transcription, the cDNA reaction mixture was diluted to a volume of 50 μl with nuclease-free water and used as a template for qPCR analyses. Real-time PCR analyses were conducted using the Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Primers for qRT-PCR analyses (listed in Additional file 3) were designed using the Primer3 software  and synthesized by the Integrated DNA Technologies (Coralville, IA, USA). The relative quantity of transcripts in each sample was calculated using standard curves based on the relative quantity of 18S RNA transcript in 10-fold serial dilutions of that sample.
DNA was isolated from SH-SY5Y cells using the DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer’s protocols. The promoter regions of RORA were then amplified by a PCR method tailored for long stretches of nucleotides using the GoTaq Long PCR Master Mix (Promega, Madison, WI, USA) and DNA primers tagged with SfiI restriction sites at the 5′ end (Integrated DNA Technologies) according to the manufacturer’s protocols. Primers for PCR cloning are listed in Additional file 3. Briefly, a total of 0.5 μg of purified human genomic DNA isolated from SH-SY5Y cells was combined with the GoTaq Long PCR Master Mix and 10 μM (final concentration) of each DNA primer. The thermal cycling condition was set as follows: 95°C for 2 minutes, 30 cycles of 92°C for 30 seconds and 65°C for 15 minutes, followed by 72°C for 10 minutes. PCR products were analyzed by gel electrophoresis using 1% agarose. The bands with expected sizes were excised from the gel and purified using the Wizard SV Gel and PCR Clean-Up System (Promega).
Purified PCR products with different sizes were then separately inserted into pGEM-T Easy Vector (Promega) containing lacZ and ampicillin-resistant genes following the manufacturer’s instructions. The vector containing each PCR product was transformed into JM109 High-Efficiency Competent E.coli cells (Promega) by heat-shocking at exactly 42°C in a water bath for 50 seconds. Transformed bacteria were spread on duplicate Luria-Bertani LB agar plates containing 100 μg/ml ampicillin, 0.5 mM isopropyl-β-D-thio-galactoside (IPTG), and 80 μg/ml 5-bromo-4-chloro-indolyl-β-D-galactopyranoside (X-Gal), and incubated at 37°C overnight for blue-white screening. Well-isolated white colonies were selected and further cultured in LB medium supplemented with 125 μg/ml ampicillin at 37°C for 12 to 16 hours with shaking at 250 rpm.
Plasmid DNA was purified from bacteria using the Wizard Plus SV Minipreps DNA Purification System (Promega) according to the manufacturer’s protocol. Presence of RORA promoter in the plasmid was validated by long PCR analysis using GoTaq Long PCR Master Mix (Promega), followed by gel electrophoresis. RORA promoter inserts were then released from the pGEM-T Easy plasmids using SfiI restriction enzyme and purified by gel electrophoresis. The luciferase vector pGL4.20[luc2/Puro] (Promega) containing firefly luciferase, puromycin-resistant, and ampicillin-resistant genes was prepared by digestion with the SfiI restriction enzyme and dephosphorylation using TSAP Thermosensitive Alkaline Phosphatase (Promega) to prevent self-recircularization of the linearized vector during ligation. RORA promoter inserts were then ligated into the dephosphorylated luciferase vector using LigaFast Rapid DNA Ligation System (Promega) and transformed into the JM109 High-Efficiency Competent E.coli cells. Transformed bacteria were cultured on LB agar plate containing 125 μg/ml ampicillin. Colonies of bacteria were harvested and further cultured in LB medium containing 125 μg/ml ampicillin overnight. Luciferase plasmids containing the RORA promoter regions were then purified from the transformed bacteria using Wizard Plus SV Minipreps DNA Purification System (Promega). Presence of RORA promoter insert was confirmed by long PCR analysis.
Dual-luciferase reporter assays
The pGL4.20[luc2/Puro] vector containing a specific RORA promoter region was mixed with the pGL4.74[hRluc/TK] vector containing Renilla reniformis luciferase gene with a ratio of 50:1 in phenol red-free Opti-MEM I reduced-serum medium (Invitrogen). The FuGENE HD Transfection Reagent (Promega) was then added to the medium containing the vectors to obtain a ratio of 3:1 (that is, 3 μl transfection agent for 1 μg DNA). The mixture was added to a 96-well plate containing SH-SY5Y cells approximately 2 × 104 cells/well) and incubated at 37°C, 5% CO2, for 48 hours. The transfected cells were treated with 10 nM DHT, 10 nM E2, or ethanol control for 2 hours, then dual-luciferase reporter assays (duplicates) were performed using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol. Briefly, lysis buffer was added to the 96-well plates containing hormone-treated transfected cells and complete lysis of cells was assessed under an inverted microscope. Cell lysates were collected and transferred to Cellstar 96-well plate (BioExpress, Kaysville, UT, USA). A Veritas Microplate Luminometer (Turner Biosystems, Sunnyvale, CA, USA) was used for detection of firefly and Renilla luminescence as well as for measurement of both firefly and Renilla luciferase activity signals. Firefly luciferase luminescence in each well was normalized by Renilla luciferase luminescence in the same well.
Prediction of transcription factor binding elements
Putative binding sites of AR and ERα in the human RORA1 promoter region and putative binding sites of RORA in the promoter regions of CYP19A1 were predicted using PROMO 3.0 [45, 46], JASPAR , and SABiosciences EpiTect ChIP Search Portal (SABiosciences, Valencia, CA, USA) programs. For each gene, a total of three to four predicted transcription factor binding sites (listed in Additional file 4) were selected for chromatin immunoprecipitation analyses.
Chromatin was isolated from SH-SY5Y cells and sheared using the ChIP-IT Express Enzymatic Kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s instructions. Briefly, confluent SH-SY5Y cells (approximately 1.5 × 107 cells in a T-175 flask) were fixed with 10% formaldehyde for exactly 5 minutes and the fixation reaction was stopped by adding 10% glycine. The cells were washed with 10 ml ice-cold PBS for 5 seconds, then 6 ml ice-cold PBS supplemented with 0.5 mM (final concentration) phenylmethylsulfonyl fluoride (PMSF) supplied in the kit was added to the culture flask to wash and chill the cells. The crosslinked cells were transferred from the flask to a pre-chilled centrifuge tube by scraping gently with a cell scraper. Crosslinked cells were homogenized by douncing 40 to 50 times on ice using a dounce homogenizer with a tight pestle to release the nucleus. Optimal cell lysis was assessed under a phase contrast microscope using a hemacytometer. The cell lysate was transferred to a 1.7 ml microcentrifuge tube and centrifuged for 10 minutes at 5,000 rpm (2,400 RCF) in a 4°C microcentrifuge to pellet nuclei. Chromatin was then isolated from the nuclear pellets and sheared into 150 to 1,000 bp fragments by incubating with 10 U/ml (final concentration) Enzymatic Shearing Cocktail (Active Motif) at 37°C for exactly 10 minutes. The enzymatic shearing reaction was stopped by adding EDTA to a final concentration of 10 mM EDTA and chilling the reaction tube on ice for 10 minutes. To assess shearing efficiency and determine DNA concentration in the sheared chromatin, a 50 μl aliquot of each sheared chromatin sample was reverse-crosslinked by mixing with 150 μl nuclease-free water and 10 μl 5 M NaCl. The reaction was incubated at 65°C in a water bath overnight. After incubation, 1 μl RNaseA (10 μg/μl) was added to each tube and the reaction was incubated at 37°C for 15 minutes. The reaction was then mixed with 10 μl Proteinase K (0.5 μg/μl) and further incubated at 42°C for 1.5 hours. The reverse-crosslinked DNA was isolated using standard phenol/chloroform extraction technique and purified using the Chromatin IP DNA Purification Kit (Active Motif). DNA concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Optimal shearing was assessed by agarose gel electrophoresis. For chromatin immunoprecipitation reaction, the remaining enzymatically sheared, non-reverse-crosslinked chromatin was aliquoted into multiple tubes, each of which contained approximately 25 μg chromatin DNA.
Each aliquot of chromatin was then used as input chromatin for sequential immunoprecipitation according to the manufacturer’s protocol for the Re-ChIP-IT Kit (Actif Motif). For each reaction, sheared chromatin (approximately 25 μg per reaction) was first immunoprecipitated by mixing with 1 μg of anti-AR, anti-ERα, anti-RORA, or IgG antibody and 25 μl Protein G Magnetic Beads (Active Motif). The reaction was then incubated on an end-to-end rotator overnight at 4°C. After incubation, the immunoprecipitated chromatin was eluted from the magnetic beads using the Re-ChIP-IT Elution Buffer (Active Motif) and desalted using the Active Motif Desalting Columns to remove the first antibody on the chromatin. Then, 1 μg of the second antibody (anti-NCOA1, anti-NCOA5, anti-SUMO1, anti-FHL2, or IgG antibody) and 25 μl of the LSV Protein G Magnetic Beads (Active Motif) were added to the desalted chromatin (approximately 90 μl) and incubated on an end-to-end rotator overnight at 4°C to re-immunoprecipitate the chromatin. To validate that successful re-immunoprecipitation was caused by the second antibody and not by carried over first antibody, a re-immunoprecipitation reaction without the second antibody (that is, no-second-antibody control) was also performed in parallel and included in subsequent qPCR analysis. After incubation, DNA from re-immunoprecipitated chromatin was isolated and purified using the ChIP DNA Purification Kit (Active Motif). The list of antibodies for sequential ChIP is shown in Additional file 1.
Real-time, quantitative PCR analysis of immunoprecipitated DNA
Real-Time qPCR analysis was conducted using Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems) to determine the enrichment of each AR/ERα/RORA binding element in immunoprecipitated or sequentially immunoprecipitated DNA. Primers for qPCR analysis were designed using Primer3 software  and synthesized by Integrated DNA Technologies (IDT). Input DNA was diluted into five 10-fold serial dilutions and included in the qPCR analysis. Relative enrichment values of AR/ERα/RORA binding elements in each sequentially immunoprecipitated chromatin were calculated using standard curves obtained from the enrichment of respective AR/ERα/RORA binding elements in the 10-fold serial dilutions of input DNA. The list of primers is shown in Additional file 3.
The SH-SY5Y cells were cultured in complete growth medium until the confluency was approximately 70 to 80% and hormone treatment was conducted as mentioned above. Co-immunoprecipitation (co-IP) assays were then conducted using Pierce Crosslink Magnetic IP/Co-IP Kit (Thermo Scientific) according to the manufacturer’s protocol, using antibodies against four coregulator proteins that were found to be differentially expressed in LCL from individuals with ASD relative to that of unaffected controls (; see Additional file 5). Briefly, the medium was removed from the flask containing cells. Then, cells were washed with ice-cold PBS containing phosphatase and deacetylase inhibitors and whole-cell lysis buffer was added directly into the flask. Protein A/G magnetic beads for immunoprecipitation were treated with anti-NCOA1, anti-NCOA5, anti-SUMO1, anti-FHL2, or nonspecific IgG antibody, and the antibody-bound magnetic beads were crosslinked with 20 μM disuccinimidyl suberate. The list of antibodies used is shown in Additional file 1. The crosslinked magnetic beads were mixed with SH-SY5Y whole-cell extract and incubated overnight at 4°C. The magnetic beads were then collected and protein complexes bound to the beads were eluted. Eluted immunoprecipitated proteins were used for subsequent western blot analysis as described below to determine the enrichment of AR, ERα, or RORA protein.
Protein concentration was determined by BCA assays using Pierce BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer’s directions for microplate assays. Briefly, the sample was mixed with BCA reagent containing bicinchoninic acid (BCA) and cupric sulfate and incubated at 37°C for 30 minutes. To determine protein concentration in an unknown sample, serial dilutions of bovine serum albumin (25 to 2,000 μg/ml) were included in the analysis and used for creating a standard curve. The absorbance of each sample was measured at 562 nm using a Synergy HT Multi-Mode microplate reader (BioTek, Winooski, VT, USA). The protein concentration in each unknown sample was calculated using standard curves obtained from absorbance values of the serial dilutions of albumin standards.
Western blot analysis
A total of 30 μg of protein was mixed with 5× Thermo Scientific Lane Marker Non-Reducing Sample Buffer (Thermo Scientific) containing 0.3 M Tris–HCl, 5% SDS, 50% glycerol, and pink tracking dye. The sample was boiled for 5 minutes and loaded onto a Mini-PROTEAN TGX precast polyacrylamide gel (Bio-Rad, Hercules, CA, USA). Electrophoresis was conducted at 200 V using 1× Tris-glycine buffer containing 25 mM Tris base, 190 mM glycine, and 0.1% SDS, as a running buffer. Proteins on the gel were then transferred to polyvinylidene fluoride (PVDF) membrane and blocking was performed for 1 hour at 4°C using 5% (w/v) non-fat dry milk (Bio-Rad) in Tris-buffered saline and Tween 20 (TBST) buffer containing 2.42 g Trizma-HCl, 8 g NaCl, and 1× Tween 20. Protein detection was conducted by incubating the PVDF membrane with anti-AR, anti-ERα, anti-RORA, or anti-RORA1 antibody (1:200 in 1% milk/TBST) at 4°C overnight. The membrane was washed and treated with donkey secondary antibody conjugated with horseradish peroxidase (HRP; Santa Cruz Biotechnology; 1:2,500) for 1 hour at room temperature. Protein visualization was performed using a chemiluminescence method by incubating the membrane in chemiluminescence substrates (PerkinElmer, Waltham, MA, USA). Protein signals on the membrane were detected using a ChemiDoc XRS+ Imager (Bio-Rad).
The two-tailed Student t test was used to determine the statistical significance of differences in the means of two groups. A P value of less than 0.05 was considered statistically significant. For comparisons of the means of three or more groups, ANOVA followed by post hoc t tests were conducted using the StatPac (Pepin, WI, USA) statistics calculator. A P value of less than 0.05 was considered statistically significant.