CHD7 represses the retinoic acid synthesis enzyme ALDH1A3 during inner ear development (original) (raw)

All reagents and culture media were purchased from Life Technologies/Invitrogen (Carlsbad) unless otherwise indicated.

Mice. Chd7Gt/+ (47) (JAX stock 030659) mice were maintained by backcrossing B6129SF1/J (JAX stock 101043) mice to N7. Aldh1a3+/– mice (20) (JAX stock 018846) were maintained by backcrossing with B6129SF1/J (JAX stock 101043) and intercrossing with Chd7Gt/+. Chd7+/– mice were generated by crossing the Chd7flox/flox mice (53) (JAX stock 030660) with EIIa-cre mice (JAX stock 003724). A homozygous breeding pair of _RARE_-lacZ mice was obtained from JAX (stock 008477) for maintenance of the _RARE_-lacZ line. Chd7+/– mice were crossed with _RARE_-lacZ mice to generate Chd7+/–;(_RARE_-lacZ) mice, which were then backcrossed to the homozygous _RARE_-lacZ strain in order to generate Chd7+/–;(_RARE_-lacZ × 2) mice. Chd7+/– mice were maintained by backcrossing with B6129SF1/J mice (JAX stock 101043) and intercrossing with Chd7+/– mice. Chd7flox/flox mice (53) (JAX stock 030660) and Chd7Gt/+ mice (47) (JAX stock 030659) have been recently archived at Jackson Laboratory.

Cell culture. Human SH-SY5Y neuroblastoma cells and 293T cells were purchased from ATCC and cultured in DMEM with 20% FBS. E12.5 forebrain neural stem cell cultures were established from Chd7Gt/+ or Chd7+/+ littermate embryos, as previously described (54). Left and right forebrains were dissected under a microscope, digested with Accutase (Invitrogen) to single-cell suspension for 15 minutes at 37°C, and cultured into individual wells of a 12-well plate in 5% CO2 at 37°C. Yolk sacs were collected for genotyping, as previously described (47), and neural stem cells were cultured in NSCM (DMEM/F, B27 N2, 20 ng/ml EGF, 20 ng/ml bFGF).

Neural progenitor cell differentiation from mouse ESCs. Neural progenitor cell differentiation from ESCs followed a previously published protocol (55). Mouse Chd7+/+ and Chd7Gt/Gt ESCs were harvested and dissociated into single cells using 0.05% Trypsin, seeded at a density of 3 × 105 cells/ml in embryoid body medium (EBM) (ESCM without LIF) onto petri dishes to form embryoid bodies, and cultured for 4 days in EBM. Embryoid bodies were then cultured with 5 μM RA for 4 days, dissociated, and plated into cell culture dishes with DMEM/F12 supplemented with B27, EGF, and bFGF to form neurospheres and cell lines.

Inner ear dissections. Chd7Gt/+ male mice were crossed with Chd7Gt/+ female mice to generate timed pregnancies. Crosses were also established between Aldh1a3+/– and Aldh1a3+/– male and female mice in order to generate timed pregnancies. The day of vaginal plug identification was designated as E0.5. Pregnant dames were euthanized by cervical dislocation at E10.5, and embryos were removed for further dissection. The yolk sac and caudal third of each embryo was saved for DNA isolation and genotyping as previously described (47). Inner ears were dissected from E10.5 embryos into cold PBS. After brief centrifugation at 200 g, PBS was removed and samples stored at –80 °C.

RNA-seq. RNA-seq was performed on E10.5 Chd7+/+, Chd7Gt/+, and Chd7Gt/Gt ears (n = 2 pairs of ears per genotype). Polyadenylated RNA was extracted from each microdissected ear sample using the RNAqueous-Micro RNA Isolation Kit (Ambion). RNA was reverse transcribed, and cDNA libraries were prepared with the Illumina TruSeq RNA-seq v2 kit. Libraries were sequenced using the Illumina HiSeq 2500 platform with paired-end 100-bp reads. Raw reads were aligned to the mouse reference genome (NCBI mm9) using Bowtie2 (v.2.0.6) (56). Unique reads were retained and normalized to the total of mapped reads for each sample. Expression levels of mapped reads were calculated in units of fragments per kilobase of transcript per million mapped reads (FPKM). Genes with low FPKM ( < 0.3) were removed from analysis. Differential expression between genotypes was calculated using DESeq (v.1.22.1), and genes were considered statistically significant with a Benjamini-Hochberg P < 0.05 (57).

Gene ontology analysis. Gene ontology analysis was performed using GOTermFinder on differentially expressed genes identified in the E10.5 RNA-seq analyses (58). Gene ontology terms associated with process, function, and component were obtained using a significance threshold of P < 0.05 and FDR < 0.05.

Bioinformatic analysis. RARE motifs were identified within the mouse Chd7 gene sequence (mm10), including 10,000 bp upstream and downstream inclusive of relevant regulatory regions, using the FIMO script contained within the MEME Suite (59). Using the Galaxy tool suite (60), RAR-bound regions contained within the mouse Chd7 gene sequence were identified by reanalysis of pan-RAR ChIP-seq data (31). ChIP-seq sites were uploaded into the UCSC Genome Browser, and RAR-bound sites at the Chd7 locus were cross-referenced with RAREs.

BED files from a previously published CHD7 ChIP data set (30) were obtained from the NCBI GEO database (accession GSE22341). Coordinates of the BED files were uploaded to custom tracks within the UCSC Genome Browser to allow mapping of the putative CHD7 protein localization site both within the coding sequence of RA-related genes and within a 10-kb window surrounding each gene.

Quantitative real-time PCR. For quantitative RT-PCR in cells, total RNA was extracted from cell pellets with chloroform (ACROS Organics) and isopropanol (ACROS Organics, NJ) followed by DNAse I treatment (Roche) to avoid genomic DNA contamination. For qPCR of inner ears, total RNA was isolated from frozen otic tissue samples using the RNAqueous-Micro Total RNA Isolation Kit (AM1931, ThermoFisher Scientific). Total RNA was quantified by Nanodrop 2000 (ThermoFisher Scientific), and 2 μg RNA per sample was reverse transcribed using SuperScript First-Strand Synthesis System. Quantitative real-time PCR analysis was performed using SYBR master mix (ABI) on an ABI 7500 qPCR machine (ABI) using a standard protocol. Primers are listed in Table 2.

To measure gene expression in E10.5 otocysts, embryos were harvested into 1X PBS on ice. Immediately following collection, embryos were bisected along the sagittal midline. Otic tissue was microdissected bilaterally (with care taken to remove periotic mesenchyme) into 100 μl ×1 PBS, spun at 200 g for 10 seconds, micropipette aspirated, and flash frozen on dry ice. The remaining tissue was collected for genotyping. Total RNA was isolated using the RNAqueous-Micro RNA Isolation Kit (Ambion) and treated with DNaseI prior to cDNA synthesis. cDNA was generated using the SuperScript First-Strand cDNA Synthesis System with oligo(dT) primers (Invitrogen). Gene expression levels were assayed using the TaqMan system (Applied Biosystems). Expression was assayed using TaqMan probes for Chd7 (product Mm01219527_m1) and Aldh1a3 (product Mm00474056_m1). Atp5b (product Mm01160389_g1) was used as a reference control. Each sample was run in triplicate using a StepOne-Plus Real-Time qPCR System (Applied Biosystems). Fold change of the assayed gene relative to Gapdh was defined as 2–ΔΔCT. An unpaired t test (for comparisons between two genotypes) or an ordinary 1-way ANOVA (for comparisons among three genotypes) was performed on _Atp5b_-normalized fold change values to determine statistical significance (P < 0.05).

In situ hybridization. In situ hybridization was performed on sections from paraffin-embedded embryos using digoxigenin-labeled riboprobes as previously described (61), at annealing temperature 65°C. Aldh1a3 in situ probe was generously provided by Kenneth Campbell (Cincinnati Children’s Hospital) (62). Multiple sections from at least 4 ears of each genotype were tested.

Immunofluorescence. Immunofluorescence was performed on sections from paraffin-embedded embryos as previously described (61). Anti-CHD7 (D3F5 6505) (Cell Signaling) and anti-ALDH1A3 (ab129815) (Abcam) were used at final concentrations of 1:7,500. Secondary biotin-conjugated donkey anti-Rabbit antibody (Jackson Immunochemicals) was processed using tyramide signal amplification 555 (T30955) (ThermoFisher) and then visualized on a Leica DMB upright fluorescence microscope.

CHD7 overexpression. Flag-HA-pcDNA3.1 (Addgene) was used as a control vector. pcDNA3.1-Flag-6xHis v.1 hCHD7 WT and pcDNA3.1-Flag-6xHis v.1 hCHD7 K998R vectors, donated by Joanna Wysocka (Stanford University, Stanford, California, USA), were as previously published (39). For mRNA detection, 5 × 106 HEK293T cells were plated in 10-cm2 dishes the day before transfection. Vectors were transfected with Lipofectamine 2000 Transfection Reagent (ThermoFisher Scientific). Empty Flag-HA-pcDNA3.1 was used as a control. Forty-eight hours after transfection, cells were collected and lysed with Trizol. mRNA was extracted with chloroform (ACROS Organics) and isopropanol (ACROS Organics) followed by DNAse I treatment (Roche) to avoid genomic DNA contamination.

ChIP-qPCR. Putative human CHD7-binding sites were identified by analysis of CHD7 ChIP-seq data in hESCs using the UCSC genome browser. Primers were designed to target 4 sites around ALDH1A3: two upstream of the transcription start site and two in the first intron (63) (ENCODE accession ENCSR000AVA) (primer sequences are listed in Table 3). For chromatin preparation, SH-SY5Y cells were plated in 10-cm2 dishes and cultured in DMEM and 10% FBS. Confluent cells were washed twice with PBS while still adherent. Cells were cross-linked in 1% paraformaldehyde for 30 minutes at room temperature and quenched by 250 mM glycine. Cells were then collected in 15 ml conical tubes and harvested by centrifugation. All cell pellets were washed with PBS and frozen on dry ice. Cell pellets were treated with 100 mM Tris (pH 7.5), 10 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, and 10% glycerol and then sheared by sonication. Chromatin was then immunoprecipitated by incubating with 8 μl CHD7 antibody (Cell Signaling) or 4 mg rabbit normal IgG antibody (Jackson ImmunoResearch) prebound to DYNAbeads M-280 (Life Technologies) overnight at 4°C. CHD7-bound beads were washed 3 times with 150 mM NaCl in IP wash buffer, 3 times with 500 mM LiCl (MilliporeSigma) in IP wash buffer, and once with TE. Harvested chromatin was then eluted from the beads, crosslinks were reversed, and DNA was purified with the Minelute PCR purification kit (Life Technologies/Invitrogen). qPCR was carried out with SYBR Master mix on a 7500 ABI qPCR machine.

For mouse E10.5 otocysts, 4 CD-1 timed pregnant females were obtained from Charles River Breeding Labs. A total of 50 embryos were dissected after cervical dislocation and hysterectomy. Both (left and right) otocysts (total of 100) were microdissected from each embryo, collected in 200 μl PBS, and gently spun and the supernatant was discarded. The otocysts were frozen on dry ice and stored at –80°C. Crosslinking and nucleus preparation was performed as described previously (30) (truChIP Chromatin Shearing Reagent Kit, Covaris). Chromatin was sheared by sonication (Covaris S220 Focused-ultrasonicator) to generate 400- to 800-bp chromatin fragments. Chromatin samples were incubated with anti-CHD7 antibody (catalog 6505, Cell Signaling) and immunoprecipitated with Protein A Magnetic Beads (Fisher Scientific). Reversal of cross-linking was performed overnight followed by elution of DNA fragments. BLAST was used to search for regions with sequence similarity between the identified human CHD7-bound sites and mouse upstream regions of Aldh1a3 (23). For qPCR, SYBR Green Master Mix (Applied Biosciences) was used on a StepOne-Plus Real-Time PCR machine (Applied Biosciences), and results were analyzed using the percent input method. Technical triplicates with standard errors were reported, and statistical differences were determined by Student’s t test.

Cell growth and differentiation assays. SH-SY5Y cells (5 × 105 or 1 × 106; n = 3 wells per condition) were plated in 6-well plates and allowed to grow for 2, 4, or 6 days; digested in 0.05% Trypsin-EDTA; resuspended in culture media; and collected and counted in trypan blue with a hemacytometer. Cell numbers were analyzed by Student’s t test, and results were graphed in Excel. For cell differentiation assays, anti-Tuj1 (neuron-specific class III β-tubulin) was used as a marker of differentiated neurons. SH-SY5Y cells were plated onto coverslips in 12-well plates at a density of 5 × 105 cells per well. After 7 days of treatment with DMSO (vehicle), RA (10 μM), or citral (10 μM), cells were washed with PBS and then fixed in 4% paraformaldehyde (MilliporeSigma) for 30 minutes. Cells were then washed with PBS and blocked in 2% BSA blocking solution (MilliporeSigma) for 2 hours at room temperature. Cells were then incubated with anti-Tuj1 antibody (1:100, catalog MRB-435P, Berkeley Antibody Company) overnight at 4°C, washed in PBS, and incubated with goat anti-rabbit Alexa Fluor 555 (1:1,000, catalog A21137, Invitrogen ) for 1 hour at room temperature. Cells were then washed with PBS, incubated with DAPI (1:1,000) for 5 minutes, washed with PBS, and mounted on slides with 50% glycerol. Cells were imaged on a Leica DM5000 B microscope (Leica). For counting, 10 visual fields (×20) per well were selected at random, and the number of Tuj1-positive cells was counted and expressed as a percentage of the total number of DAPI-positive cells.

Treatment of cells with RA or RA inhibitors. For RA-induced SH-SY5Y cell differentiation, 3 × 106 cells were plated into 10-cm2 dishes and cultured in DMEM and 10% FBS. Cells were treated with 1 μM, 5 μM, or 10 μM ATRA (MilliporeSigma) or 13-_ci_s-RA (MilliporeSigma) for 1–12 days. Cells were also treated with the RA inhibitors citral (10 μM) or _N, N-_diethylaminobenzaldehyde (DEAB) (10 μM, MilliporeSigma). Culture media were changed every 2 days. ATRA, 13-_cis_-RA, citral, and DEAB were diluted in DMSO. E12.5 neural progenitor cells were treated with 10 μM ATRA or citral for 3 days, and cell pellets were collected for CHD7 level analysis by Western blotting.

For Western blotting, total nuclear protein was quantified with the BCA Protein Assay Kit (Life Technologies) (10–20 μg per sample), electrophoresed in 4%–12% Bis-Tris gels, and transferred to nitrocellulose membrane at 4°C overnight. Primary antibodies used for Western blotting were anti-CHD7 antibody (catalog 6505, Cell Signaling Technology), anti-CHD5 (catalog H-185, Santa Cruz), anti-FLAG (catalog F7425, MilliporeSigma), and anti-HDAC2 antibody (Santa Cruz). Secondary antibodies were goat anti-rabbit IgG Horseradish Peroxidase (catalog 31461, Thermo Fisher Scientific) and rabbit anti-goat IgG Horseradish Peroxidase (catalog 31403, Thermo Fisher Scientific).

Coimmunoprecipitation. SH-SY5Y cells (5 × 106) were plated in 10-cm2 dishes and grown in DMEM, 10% FBS. For nuclear protein extraction, culture media were removed and washed with cold PBS for 3 times on ice. Cells were then incubated on ice in 3 ml cold Buffer I (15 mM HEPES, 10 mM KCl [MilliporeSigma]), 0.1 mM EDTA (MilliporeSigma), 1 mM MgCl2 (MilliporeSigma), and 350 mM sucrose (MilliporeSigma) and collected with a cell scraper. Cell lysates were then centrifuged at 8,000 g for 5 minutes. Supernatants were discarded, and cell pellets were lysed with Buffer II (15 mM HEPES, 110 mM KCl [MilliporeSigma], 250 mM NaCl [MilliporeSigma], 0.1 mM EGTA [MilliporeSigma]), 5% glycerol (MilliporeSigma), and PMSF (MilliporeSigma). After centrifugation for 10 minutes, supernatants were collected, flash frozen in liquid nitrogen, and stored at –80°C.

For coimmunoprecipitation, nuclear extracts (350 μg per sample) were incubated with specific antibodies overnight and then incubated with protein A/G plus-Agarose Beads (Santa Cruz) for 1 hour at 4°C (see complete unedited blots in the supplemental material). Anti-rabbit normal IgG (Jackson ImmunoResearch) was used as a control antibody for coimmunoprecipitation. Beads were then washed with buffer II (0.01% Tween 20 [MilliporeSigma]) and added to ×4 LDS sample buffer. Samples and bound proteins were detected by Western blotting. Antibodies used for coimmunoprecipitation were anti-CHD7 (catalog 6505, Cell Signaling) and anti-RAR (catalog M-454, Santa Cruz).

Analysis of CHD7 and RAR localization in mouse ESCs. DNA sequences associated with CHD7 and RAR were compared with determine commonly bound regions within the mouse ESC genome. CHD7 ChIP-seq reads were obtained from Schnetz et al. (30) (GEO, GSE22341; SRA, SRP002695) while pan-RAR ChIP-seq reads were obtained from Mahony et al. (31) (GEO, GSE19409; SRA, SRP002214). All data were aligned to the NCBI36 (mm8) build of the mouse genome. BED files containing significant reads were uploaded to Galaxy (v.17.05). For CHD7 ChIP-seq, the high-threshold peak set was used for downstream analysis. For pan-RAR ChIP-seq, the data set corresponding to the +RA treatment group was used. These two data sets were intersected using BEDTools (64) with the following commands: “-wa” and “-u” to enable identification of all CHD7 reads that overlapped at least once with pan-RAR reads; the required minimum overlap (“-f”) between data set reads was set to 50%. Results were plotted as a quantitative Venn diagram using the Venn Diagram Generator available from the Whitehead Institute for Biomedical Research (http://jura.wi.mit.edu/bioc/tools/venn.php) and edited in Illustrator (Adobe).

X-gal staining. Timed pregnancies were established between Chd7+/–;(RARE-lacZ)×2 and Chd7+/–;(RARE-lacZ)×2 or (RARE-lacZ ×2) to ensure similar lacZ copy number between embryos. Litters were collected at E10.5, E11.5, E12.5, and E13.5 following cervical dislocation and hysterectomy. Embryos were fixed and either (a) processed for cryosections, sectioned and stained as previously described (65), or fixed and processed for whole-mount staining, as described previously (66).

RARE-lacZ cell and embryo assays. F9 RARE-lacZ cells were provided by James H. Millonig (Robert Wood Johnson Medical School, New Brunswick, New Jersey, USA) (34), and cultured in 0.4 mg/ml G418 culture medium (10% FBS, Atlas Biologicals]) with 1% penicillin/streptomycin DMEM/F12). For coculture assays, 1 day prior to coculture, F9 cells (105 cells/well) were plated on 0.2% gelatin-coated (MilliporeSigma) 96-well plates in culture media without G418. Separate wells were plated in triplicate for standard ATRA concentration response curves (0, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM), as previously described (34). ATRA was dissolved in DMSO. Timed pregnancies were established between Chd7+/– male and female mice. E8.5 mouse embryos were dissected, washed in ice-cold PBS, and incubated in 1.5-ml Eppendorf tubes containing 10 μl 0.05% Trypsin-EDTA on ice. Yolk sacs were saved and processed for Chd7+/+, Chd7+/–, and Chd7–/– genotyping, as previously described (47). Embryos were digested for 5 minutes at 37°C. Single-cell suspensions were then transferred to the F9 cell plated 96-well plate (1 embryo per well). Plates were cultured overnight at 37°C and 5% CO2 and then processed for β-gal histochemistry and detection using a SpectraMax M2 multimode microplate reader (Molecular Devices). ATRA-treated F9 RARE cells were used to generate a standard curve for comparison with experimental samples.

Luciferase assays. HEK-293T cells (3 × 104) were plated in each well of a 96-well plate the day before transfection. For each combination of plasmids, 8 replicate wells were plated. Flag-HA-pcDNA3.1, pcDNA3.1-Flag-CHD7, and pcDNA3.1-Flag-CHD7 K998R were separately cotransfected with pGL3-RARE-luciferase vector (Addgene) and pRL-SV40P (Addgene) using Lipofectamine 2000 transfection reagent and opti-MEM (Life Technologies). After 24 hours, media were changed to include vehicle (DMSO) with or without 1 μM ATRA and treated overnight before luciferase detection.

For siRNA transfection, DharmaFECT Duo transfection reagent (GE Healthcare Dharmacon Inc.) was used. siGENOME human CHD7 siRNA-SMARTpool (GE healthcare) and siGENOME Cyclophilin B control siRNA were separately cotransfected with pGL3-RARE-luciferase and pRL-SV40P vectors. Culture media were changed 24 hours after transfection followed by addition of DMSO with and without 1 μM ATRA. Luciferase assays were carried out 48 hours after transfection of CHD7 expression or siRNA plasmids. Luciferase and Renilla signals were detected with the Dual Luciferase reporter assay system (Promega) and GloMax multi detection system (Promega). Data were analyzed by Student’s t test.

Inner ear paint-filling. Timed pregnancies were established between Chd7Gt/+;Aldh1a3+/– and Aldh1a3+/– mice. Embryos were collected at E14.5 after cervical dislocation and hysterectomy. Embryos were washed in PBS and fixed in Bodian’s fixative (5% glacial acetic acid, 1.85% formaldehyde, and 75% ethanol) and then cleared using methylsalicylate. Heads were bisected and the brain was removed prior to injection of 3% Whiteout (BIC) solution as previously described (47).

Statistics. Two-tailed Student’s t tests were used to determine significant differences between 2 data sets. The resulting P values were corrected for multiple comparisons using the Bonferroni method. Ordinary 1-way ANOVA tests were used to determine statistical significance among 3 or more data sets. A P value of less than 0.05 was considered significant. All graphs show the mean ± SD, unless otherwise indicated.

Study approval. All mouse husbandry practices and procedures were approved by the University of Michigan Institutional Animal Care and Use Committee.