Cell culture and reagents
The mouse fibroblast-like cell line C3H10T1/2 and mouse teratocarcinoma cell line ATDC5 were purchased from the RIKEN Cell Bank (Ibaraki, Japan). These cells were cultured at 37°C in a humidified 5% CO2 incubator with Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS) and a 1:1 mixture of DMEM and Ham’s F-12 medium (Sigma-Aldrich) containing 10% FBS. Insulin-transferrin-sodium selenite supplement (ITS; Roche, Basel, Switzerland) was used to induce chondrocyte differentiation in ATDC5 cells. Primary chondrocytes were isolated in accordance with a protocol described by Gartland et al. 39. Briefly, rib cartilage was dissected from newborn mice and soft tissue was removed. Then, rib cartilage was digested with 0.1% collagenase D (Roche) and 0.5% trypsin (Life Technology, Carlsbad, CA, USA) for 6 h at 37°C. Primary chondrocytes were collected by centrifugation and resuspended with DMEM containing 10% FBS and antibiotics. Cells within two passages were used for experiments as primary chondrocytes.
Generation of adenoviruses
cDNAs of Flag-tagged-Dmrt2, HA-tagged-Sox9, Sox5, Sox6, and Runx2 were amplified using Pfu DNA polymerase and subcloned into pAXCAwt vectors (TAKARA Bio, Shiga, Japan). Recombinant adenoviruses were generated using the COS-TPC method by transfection of a recombinant cosmid and the DNA-TPC adenovirus genome into 293 cells 31. C3H10T1/2 cells, ATDC5 cells, and primary chondrocytes were infected with adenoviruses at a multiplicity of infection (MOI) of 20 unless indicated otherwise.
RNA-seq
Total RNA was extracted from primary chondrocytes in which control vector or Sox5/6/9 were adenovirally expressed using NucleoSpin RNA II (Macherey-Nagle, Duren, Germany). Total RNA library preparation was performed using a TruSeq stranded mRNA sample prep kit (Illumina, San Diego, CA, USA), in accordance with the manufacturer’s protocol. Sequencing was performed on an Illumina HiSeq 2500 platform in 75-base single-end mode. Illumina Casava1.8.2 software was used for base-calling. Sequenced reads were mapped to the mouse reference genome sequence (mm10) using TopHat v2.0.13 in combination with Bowtie2 ver. 2.2.3 and SAMtools ver. 0.1.19.
RNA-seq data were analyzed using iDEP (integrated Differential Expression and Pathway analysis) 40. Briefly, read count data of three replicates for control and Sox5/6/9 were generated and uploaded to the iDEP website (http://bioinformatics.sdstate.edu/idep/). Differentially expressed genes (DEGs) were identified using a threshold of false discovery rate (FDR) < 0.05 and minimal fold change > 2. The raw data have been deposited in the NCBI Gene Expression Omnibus database (GEO GSE155118).
Reverse-transcription polymerase chain reaction (RT-PCR) and RT-qPCR
Total RNA was isolated using a Nucleo Spin RNA Plus kit. cDNA was synthesized using ReverTra Ace® qPCR RT Master Mix (TOYOBO, Osaka, Japan). For RT-PCR analysis, cDNA was amplified using KOD FX (TOYOBO) and then the PCR products were electrophoresed in a 1.6% agarose gel with ethidium bromide. Primer pairs used for RT-PCR analysis are listed in Supplemental Table S2. For RT-qPCR analysis, cDNA was amplified with EagleTaq Universal Master Mix (ROX) using a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Primers and TaqMan probes used for cDNA amplification are listed in Supplementary Table S2. The mRNA expression was normalized to β-actin expression levels.
Epigenetic datasets and analysis
ChIP-seq and ATAC-seq datasets were downloaded from the GEO database. We obtained GSE69109 ChIP-seq profiles of newborn mouse rib chondrocytes for Sox9 and H3K27ac 21. ChIP-seq profiles of E12.5 limb buds for Sox9 were obtained from GSE73225 23 and H3K27ac from GSE45456 22. ATAC-seq datasets of growth plate chondrocytes were downloaded from GSE100585 20.
FASTQ data of ChIP-seq and ATAC-seq were aligned to the mouse genome (mm10) using Bowtie and peak calling was performed using MACS software with the default settings (p-value cut-off = 1.00e-05). ChIP-seq and ATAC-seq data were visualized using the Integrative Genomics Viewer (IGV).
Reporter assay
Dmrt2 minimal promoter (–100 to +10) and Sox9 binding region located 18 kb upstream of the Dmrt2 TSS were introduced upstream of the luciferase gene. Reporter genes were co-transfected with the expression vectors and Renilla into HEK293 cells using the FuGENE6 reagent. After 48 h of transfection, the cells were lysed and luciferase activity was measured using specific substrates in a luminometer (Promega, Fitchburg, WI, USA), in accordance with the manufacturer’s protocol. Luciferase activity was normalized by Renilla.
Mice
Dmrt2-/- mice were originally generated by Dr. Randy L. Johnson (Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center) 18. They were maintained and kindly provided by Dr. David Zarkower (Developmental Biology Center and Department of Genetics, Cell Biology, and Development, University of Minnesota) for the experiments. To determine the genotypes of the mice, genomic DNA was purified from mouse tails and examined by PCR using KOD FX Neo (Toyobo). Primer pairs for genotyping were as follows: WT, sense 5′-CTGGACCCGAGTACAGTTCC-3′ and antisense 5′-AATGGTGCGTTCAACTCAGG-3′; KO, sense 5′-TGCGGAGGGCTGGATCTTAAGGAG-3′ and antisense 5′-AGGGGGTGGGGATTTGACACCATC-3′. The PCR product of WT mice was 830 bp and that of KO mice was 270 bp.
All mice were maintained on the C57BL/6 background. Littermate embryos were used for histological analysis. All animal experiments were performed using protocols approved by the Animal Committee of Osaka University Graduate School of Dentistry.
Skeletal preparation
The skin of the mice was removed and fixed in 95% ethanol overnight. Cartilage tissues were stained with 1.5% Alcian blue followed by staining of bone tissues with 0.02% Alizarin red S. Skeletal samples were photographed under a stereoscopic microscope.
Immunohistochemistry
Samples were fixed with 4% buffered paraformaldehyde, embedded in paraffin, and cut into 4-μm-thick sections. Paraffin-embedded sections were deparaffinized and rehydrated, followed by hematoxylin and eosin (H&E) staining. For immunohistochemical analysis, antigen retrieval was performed by incubation in DAKO REAL target retrieval solution for 10 min at 90°C, followed by blocking with 1% bovine serum albumin in phosphate-buffered saline (PBS). Immunohistochemistry was performed using the following antibodies: anti-Dmrt2 (#ARP32224_P050; AVIVA System Biology, CA, USA) at 5 μg/ml, anti-Col2 (#7050; Chondrex, WA, USA) at 1:500 (vol/vol) dilution, and anti-Col10 (LSL LB-0092; Cosmo Bio, Tokyo, Japan) at 1:500 (vol/vol) dilution. Immunoreactivity was visualized with Alexa Fluor® 555 dye-conjugated anti-rabbit IgG (Invitrogen, CA, USA), and counterstaining was performed using 4¢,6-diamidino-2-phenylindole, in accordance with the manufacturer’s protocol.
In situ hybridization
The protocol for in situ hybridization has been described in a previous report 41. Briefly, tissues harvested from WT and Dmrt2-/- littermate mice were fixed with 4% paraformaldehyde and then embedded in paraffin. The tissue blocks were cut into 4-μm-thick sections. Digoxigenin (DIG)-11-UTP-labeled, single-stranded RNA probes were prepared using a DIG RNA labeling kit (Roche), in accordance with the manufacturer’s instructions. We used a 0.4 kb fragment of mouse Col2a1 cDNA, a 0.65 kb fragment of mouse Col10a1 cDNA, and a 0.57 kb fragment of mouse Ihh cDNA to generate antisense and sense probes. Signals were detected with an alkaline phosphatase-conjugated anti-DIG antibody (Roche). All probes were kindly provided by Dr. Noriyuki Tsumaki (Kyoto University, Kyoto, Japan).
BrdU assay
Pregnant mice were anesthetized and administered 1 ml of BrdU Labeling Reagent (Life Technologies) per 100 g body weight by intraperitoneal injection. The mice were sacrificed 2 h later, after which E17.5 WT and Dmrt2-/- littermate mouse tibiae were collected and fixed with 4% paraformaldehyde. Paraffin-embedded sections were subjected to a BrdU assay using a BrdU Staining kit (Invitrogen). For quantitative analysis, BrdU-positive nuclei in round and hypertrophic chondrocytes were excluded.
Western blotting
Cells were rinsed twice with PBS and solubilized in lysis buffer [20 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1.5 mM MgCl2, 10% glycerol, 1% Triton X-100, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, and 0.2 mM sodium orthovanadate]. The lysates were centrifuged at 4°C for 10 min at 15,000 × g and then boiled in sodium dodecyl sulfate (SDS) sample buffer containing 0.5 M β-mercaptoethanol for 5 min. The supernatant was separated by SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, immunoblotted with a primary antibody, and then visualized with horseradish peroxidase-coupled anti-mouse or -rabbit IgGs using an enhanced chemiluminescence detection kit (Immunostar LD; WAKO, Osaka, Japan). An anti-Flag (M2) antibody was purchased from Sigma-Aldrich. Anti-HA (Y-11, #sc-805) was purchased from Santa Cruz Biotechnology (CA, USA). Antibodies against β-actin (M177-3), and Runx2 (D1L7F) were purchased from MBL (Nagoya, Japan) and CST (MA, USA), respectively.
Whole-mount in situ hybridization
DIG-labeled antisense RNA probes for Col2a1 and Sox9 were generated with SP6 or T7 RNA polymerase using the DIG RNA Labeling kit, in accordance with the manufacturer’s instructions. Embryos of WT and Dmrt2-/- littermate mice at E13.5 were fixed in 4% paraformaldehyde/PBS overnight at 4°C. The samples were then hybridized overnight with gene-specific DIG-labeled RNA probes at 70°C overnight. Then, the samples were washed and incubated with an anti-DIG antibody (Antidigoxigenin-AP; Roche). Nitroblue tetrazolium and BCIP (5-bromo-4-chloro-3′-indolyphosphate p-toluidine) were used for signal detection.
ChIP assays
ChIP analysis was performed using a ChIP-IT express kit (Active Motif), in accordance with the manufacturer’s instructions. Briefly, primary chondrocytes and ATDC5 cells were washed with PBS, and chromatin was fixed with formaldehyde to crosslink the proteins to chromatin. Crosslinked chromatin was sonicated with Covaris M220. Sonicated chromatin was immunoprecipitated with an anti-H3K27ac antibody (D5E4, CST), anti-Flag (M2) antibody, and anti-Runx2 antibody (D1L7F, CST). DNA fragments were precipitated with protein-A magnetic beads and amplified by PCR using primer pairs specific for the Sox9-bound enhancer of the Dmrt2 gene (sense primer, 5¢-TTCCAGATGGGCTGAAAC-3¢; anti-sense primer, 5¢-CTGTGCATTGTGGGAGAG-3¢), Ihh gene promoter containing the Dmrt2-binding element (sense primer, 5¢-TGGCCTTTCTCCCTTGTTTTT-3¢; anti-sense primer, 5¢-CAATGTAGCCTTGTCAGGAGTCA-3¢), and Ihh gene promoter containing the Runx2-binding element (sense primer, 5¢-AGCTTTCGGGTTTGCTTCTC-3¢; anti-sense primer, 5¢-GTCTCTCCTTCCCGTTCCTT-3¢). Quantitative analysis of ChIP assays was performed by real-time PCR using SYBR Green.
DNA pulldown assays
Cells were lysed in lysis buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1.5 mM MgCl2, 10% glycerol, 1% Triton X-100, 10 μg/ml leupeptin, 1 mM PMSF, and 0.2 mM sodium orthovanadate). Lysates pre-incubated with streptavidin agarose beads were incubated with 1 μg of a biotinylated double-stranded oligonucleotide probe containing the Dmrt2-binding element in the Ihh gene promoter (sense primer, 5′-AAATAACCAAGATACAATTTGCAAAACACA-3′; anti-sense primer, 5′-TGTGTTTTGCAAATTGTATCTTGGTTATTT-3′) for 3 h. Precipitated oligonucleotides were collected with streptavidin magnetic beads (Dynabeads My One Streptavidin T1, Invitrogen) and washed with lysis buffer. The magnetic beads were resuspended with SDS sample buffer, boiled for 5 min, and then subjected to western blotting.
Statistical analysis
Randomization and blinding were not performed in the animal studies. Sample sizes were estimated based on previous studies of endochondral bone formation 31, 41. Data were statistically analyzed by Student’s t-test for comparison between two groups. For more than two groups, we used one-way analysis of variance (ANOVA) or two-way ANOVA followed by the Tukey test. At least five mice (n = 5–6) were used for the phenotypic analysis. P-values of less than 0.05 were considered statistically significant.