Patient samples, microarray, gene-ontology analysis
Primary oligodendroglioma samples were obtained with approval from the institutional review board at the University of Pennsylvania and were de-identified for the study. For microarray analysis, tumour sample RNA was extracted with Trizol and purified with Qiagen RNeasy, and then assayed on an Affymetrix Human Gene 1.0ST array. Significance Analysis of Microarrays (http://www-stat.stanford.edu/~tibs/SAM/sam.pdf
) was applied to find differentially expressed genes (q
value <10% and fold change >2). Functional analysis of differentially expressed genes was done using the DAVID tool (http://david.abcc.ncifcrf.gov/home.jsp
) using all human genes as a background set.
3T3-L1 cell differentiation, Oil-Red-O staining
3T3-L1 cell differentiation and Oil-Red-O staining were carried out as described previously24
. In brief, confluent 3T3-L1 cells were stimulated with a cocktail containing 0.5 mM isobutylmethylxanthine, 1 μM dexamethasone, 5 μg/ml insulin and 5 μM troglitazone (all from Sigma) to induce differentiation. Cells were maintained in medium with insulin after 2 days of differentiation until ready to be harvested. For Oil-Red-O staining, cells were washed in PBS and then fixed for 20 min at room temperature (25 °C) with 3% paraformaldehyde. Cells were then washed with de-ionized water and stained with Oil-Red-O solution. For quantification, Oil-Red-O staining was dissolved in isopropanol and absorbance was measured at 500 nm.
In vitro histone demethylase assay
The histone demethylase assay was carried out as described previously25
. In brief, 4 μg bulk calf thymus histones (Sigma) were incubated with GST-tagged KDM4C (1.42 μg; BPS Bioscience) in a reaction mix containing 50 mM Tris-HCl pH 8.0, protease inhibitors cocktail, 1 mM αKG, 100 μM FeSO4
and 2 mM ascorbic acid at 37 °C for 4 h, in the absence or presence of various concentrations of d
-2HG or l
-2HG (Sigma). Reaction mixtures were analysed by western blotting using specific antibodies.
Cell culture, transfection and transduction, generation of cell lines
293T cells, NHA cells immortalized by E6/E7/hTERT (provided by R. Pieper26
) and 3T3-L1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) with 10% fetal bovine serum (FBS; CellGro). For expression of wild-type and mutant IDH1/2 in 293T cells, transfection was performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. For generation of IDH2 retrovirus and transduction of 3T3-L1 cells, supernatant from 293T cells transfected with pCL-Eco helper virus and plasmids was collected after 72 h, filtered and applied to cells overnight. For generating 3T3-L1 cell lines with stable expression of wild-type or mutant IDH2, cells were grown in 2.5 μg ml−1
puromycin for 7 days after retroviral transduction. Pooled populations of puromycin-resistant cells were obtained, and then continuously cultured in puromycin. For generation of IDH1 retrovirus and transduction of NHA cells, GP2-293 cells (Clontech, 631458) were calcium phosphate transfected with equal amounts of pVSV-G (Clontech, 631512) and plasmids. Virus was harvested at day 2 and day 3 after transfection and placed on logarithmically growing cells. After infection, cells were placed in 800 μmg ml−1
G418 (Invitrogen) to generate stable cell lines. For siRNA knockdown of KDM4C, transfections were performed with Lipofectamine RNAiMAX (Invitrogen), using siRNAs targeting KDM4C (#1: sense, 5′-GCUUGAAUCUCCCAAGAUATT-3′; antisense, 5′-UAUCUU GGGAGAUUCAAGCTT-3′; #2: sense, 5′-CAAAGUAUCUUGGAUCAAATT-3′; antisense, 5′-UUUGAUCCAAGAUACUUUGCC-3′; #3: sense, 5′-GAGGAGUU UCGGGAGUUCAACAAAU-3′; antisense, 5′-AUUUGUUGAACUCCCGAA ACUCCUC-3′) or a non-targeting control (Dharmacon, #D-001810-01-20) at a concentration of 40 nM.
For IDH mutation analysis, tumour genomic DNA was extracted and the regions surrounding IDH1 codon 132 and IDH2 codons 140 and 172 were amplified by PCR followed by sequencing. IDH1 analysis used forward primer 5′-ACCAAATGGCACCATACGA-3′ and reverse primer 5′-TTCATACC TTGCTTAATGGGTGT-3′ for amplification, and primer 5′-CGGTCTTCAGAG AAGCCATT-3′ for sequencing1
. IDH2 analysis used forward primer 5′-CAG AGACAAGAGGATGGCTAGG-3′ and reverse primer 5′-GTCTGCCTGTG TTGTTGCTTG-3′ for amplification, and the same forward primer for sequencing27
. Out of the 42 tumours analysed, 41 had sufficient high quality genomic DNA for discerning IDH mutation status. The one sample unable to be classified as either IDH wild type or mutant was excluded from further analysis.
The cDNA clone of human IDH1 (BC012846.1) was purchased from the American Type Culture Collection in pCMV-Sport6, and human IDH2 (BC009244) was purchased from Invitrogen in pOTB7. Standard site-directed mutagenesis techniques were used to generate IDH1 R132H by introducing a G395A base-pair change in the IDH1 open reading frame (ORF). IDH2 R172K was made by introducing a G515A change in the IDH2 ORF, while IDH2 R140Q was made with a G419A alteration. Wild-type and mutant sequences were then subcloned into LPC vector. All sequences were confirmed by direct sequencing before expression in 293T cells and retrovirus generation. Retroviral constructs used for neurosphere infection were generated by excising wild-type IDH1 and IDH1 R132H with NotI and PacI restriction enzymes from the previously made vectors and incorporating into the pQCXIH (Clontech, 631516) retroviral vector.
Histone extraction and western blotting
For histone acid extraction, cells were lysed in hypotonic lysis buffer (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, protease inhibitors) for 1 h. H2SO4 was added to 0.2 N overnight at 4 °C with rotation. After spinning down and collecting supernatant, proteins were precipitated in 33% TCA, washed with acetone, and resuspended in de-ionized water. For whole-cell lysates, cells were lysed and sonicated in standard RIPA buffer (1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 0.01 M Tris pH 8.0 and 0.14 M NaCl), and lysates were then centrifuged at 14,000g at 4 °C for 10 min. Supernatants were collected and measured for total protein concentration. For western blotting, lysates were separated by SDS–PAGE, transferred to nitrocellulose membrane, blocked in 5% non-fat milk in PBS containing 0.5% Tween-20, probed with primary antibodies and detected with horseradish-peroxidase-conjugated anti-rabbit or anti-mouse antibodies (GE Healthcare, NA934V and NA931V). Primary antibodies used were: anti-IDH1 (Proteintech, 12332-1-AP), anti-IDH2 (Abcam, ab55271), anti-GST tag (Millipore, 05-311), anti-H3K9me2 (Cell Signaling Tech, 9753), anti-H3K9me3 (Abcam, ab8898), anti-H3K36me3 (Abcam, ab9050), anti-H3K27me3 (Millipore, 17-622), anti-H3K4me3 (Millipore, 17-614), anti-H3K79me2 (Cell Signaling Tech, 9757), anti-KDM4C (Abcam, ab85454), anti-acetyl H3 (Upstate, 06-599), anti-H3 (Cell Signaling Tech, 4499), anti-tubulin (Sigma, T9026), anti-GFAP (Cell Signaling Tech, 3670), anti-β3-tubulin (Cell Signaling Tech, 5666), anti-p85 (Millipore, 06-195), anti-nestin (Millipore, MAB5326), anti-β-actin (Sigma, A5316). Anti-IDH1 R132H mutant antibody was a gift from Agios Pharmaceuticals. Quantification of western blot band intensity was performed using Image J software according to the manufacturer’s instructions.
Metabolite extraction, GC–MS
After gentle removal of culture medium, cells were rapidly quenched with ice-cold 80% methanol and incubated at −80 °C for 20 min. After sonication, extracts were then centrifuged at 14,000g for 20 min at 4 °C to remove precipitated protein and the aqueous metabolites in the supernatant layer were dried under nitrogen gas. For 293T cells, organic acids were further purified by redissolving the dried extract in de-ionized water, followed by elution from an AG-1 X8 100–200 anion exchange resin (Bio-Rad) in 3 N HCl after washing with five column volumes.
For GC–MS analysis, dried extracts were redissolved in a 1:1 mixture of acetonitrile and N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide (MTBSTFA; Regis) and heated for 75 min at 70 °C to derivatize metabolites. Samples were then injected into an Agilent 7890A GC with an HP-5MS capillary column, connected to an Agilent 5975 C mass selective detector operating in splitless mode using electron impact ionization with ionizing voltage of −70 eV and electron multiplier set to 1,060 V. GC temperature was started at 100 °C for 3 min, ramped up to 230 °C at 4 °C min−1 and held for 4 min, then ramped up to 300 °C and held for 5 min. Mass range of 50–500 amu was recorded at 2.71 scans per second. Identification of the 2HG metabolite peak was confirmed using standards obtained from Sigma. The 2HG and glutamate signal intensities were quantified by integration of peak areas.
Quantitative real-time PCR
RNA was isolated using Trizol (Invitrogen). After incubating with DNase, cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen). Quantitative PCR was performed on a 7900HT Sequence Detection System (Applied Biosystems) using Taqman Gene Expression Assays (Applied Biosystems). Gene expression data was normalized to 18S rRNA.
ChIP was performed with the Millipore Magna ChIP G kit (Millipore, 17-611). In brief, 2,000,000 cells were cross-linked with 1% formaldehyde for 10 min at room temperature. After washing with cold PBS, cells were centrifuged and lysed in 500 μl SDS lysis buffer for 10 min on ice. Lysate was then sonicated using Bioruptor sonicator (Diagenode) to shear DNA to approximately 200–600 bp. Samples were spun down and 50 μl of the supernatant was used for each immuno-precipitation overnight with magnetic beads after 10× dilution. Primary antibodies (3 μg per ChIP) used were: anti-H3K9me3 (Abcam, ab8898) and anti-H3K27me3 (Millipore, 17-622). Normal rabbit IgG (Millipore, 12-370) was used as control and showed minimal enrichment. The next day, samples were washed in low-salt immune complex buffer, high-salt immune complex buffer, LiCl immune complex buffer and TE buffer. Histone complexes were eluted in elution buffer plus proteinase K for 2 h at 65 °C. DNA was recovered using columns. Quantitative PCR was performed on purified DNA samples. Primers used are: Adipoq forward, 5′-ATGGCTGAACCACACAGCTTCA-3′; reverse, 5′-AGGGGTCAGGAGA CCTCCCTTT-3′; Cebpa forward, 5′-CTGGAAGTGGGTGACTTAGAGG-3′; reverse, 5′-GAGTGGGGAGCATAGTGCTAG-3′. Data points (Ct) are converted to percentage of input.
Quantitative DNA methylation analysis
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using EpiTyper by MassARRAY (Sequenom) was performed on bisulphite-converted DNA extracted from 3T3-L1 cells. MassARRAY primer design was done as previously described28,29
Immunohistochemistry detection was performed using Discovery XT processor (Ventana Medical Systems). The tissue sections were blocked for 30 min in 10% normal goat serum in 0.2% BSA/PBS, followed by incubation for 5 h with 0.1 μg ml−1 of the rabbit polyclonal anti-H3K9me3 (Abcam, ab8898) or 1 μg ml−1 rabbit polyclonal anti-H3K27me3 (Millipore, 07-449) antibodies and incubation for 60 min with biotinylated goat anti-rabbit IgG (Vector labs, PK6101) at 1:200 dilution. The detection was performed with the DAB-MAP kit (Ventana Medical Systems). The entire slides were scanned by Zeiss Mirax Scan (Carl Zeiss) using a X20/0.8 objective. The scanned image was exported into image analysis software, Metamorph (Molecular Devices). The colour threshold for DAB-positive nuclei was determined and set for all images. Areas above the threshold for the DAB signal and for haematoxylin-counterstained total nuclei were measured in an automated fashion. The ratio between the two parameters were calculated and analysed for statistical significance.
Synthesis of 1-octyl-d-2-hydroxyglutarate
Commercial R(-)-tetrahydro-5-oxofuran-2-carboxylic acid (140 mg, 1.076 mmol) was dissolved in H2O (1 ml), cooled to 0 °C and treated with 1 N KOH (2.16 ml, 2.15 mmol). The resulting solution was stirred at this temperature for 5 min and at ambient temperature for 2 h. It was then concentrated to dryness under reduced pressure and dried. The residue was dissolved in trifluoroacetic anhydride (8 ml) at 0 °C, stirred for 30 min at 0 °C, for 2 h at room temperature, then the volatiles were evaporated under reduced pressure. The residue was dried and dissolved in anhydrous tetrahydrofuran (6 ml). Octanol (0.3 ml, 2.1 eq.) was added to the solution at 0 °C and the mixture was stirred for an overnight period at ambient temperature. Water was added to quench the reaction, and the mixture extracted with EtOAc. The combined extracts were dried over MgSO4, concentrated and purified by Flash chromatography (EtOAc:hexane 1:3 and 1:1) to give 1-octyl-d-2-hydroxyglutarate (110 mg, 39%).
Neurosphere isolation, culture and differentiation
Six days postpartum Ink4a/Arf null (p16/p19−/−) mice were killed, with the isolated subventricular zones subjected to chemical (Pronase E, Calbiochem 7433-2) and mechanical dissociation to obtain a single-cell suspension in full neurobasal medium (Neurobasal medium, GIBCO 21103; B27 supplement without retinoic acid, GIBCO 12587-010; Glutamax, GIBCO 35050; 20 ng ml−1 EGF, R&D Systems 236-EG; 20 ng ml−1 basic FGF, Millipore GF003). On the next day the cells were spun down and re-suspended in fresh medium, and once neurospheres had formed in culture, the spheres were collected and chemically dissociated (Accumax, Innovative Cell Technologies AM105) back into single cells in fresh medium.
One day after final infection, infected neurospheres and a non-infected control were placed in 400 mg μl−1 Hygromycin B (InvivoGen, ant-hg-1). Once selection was complete, isogenic cell lines maintained in full neurobasal medium were chemically dissociated into single cells and plated at the same density in full neurobasal medium with increasing concentrations of retinoic acid (Sigma-Aldrich, R2625). Seventy-two hours later cells were harvested, and expression of proteins was analysed by western blotting.
Measurement of total CpG methylation
DNA methylation was assessed as previously described30
. In brief, 1 × 106
NHA cells were washed with PBS and fixed with 2% paraformaldehyde for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 10 min. Cells were then treated with 2 N HCl for 20 min at room temperature and subsequently neutralized with 100 mM Tris-HCl, pH 8.0. Cells were incubated with anti-5-methylcytosine antibody (Calbiochem, NA 81) at 1:100 dilution for 30 min at room temperature. After washing with PBS, cells were incubated with secondary antibody coupled with ALEXA FLUOR 488 (Invitrogen) for 30 min in the dark. Flow cytometry was done using Becton Dickinson Calibur flow cytometer and analysed using FlowJo software.
All statistical analysis was performed using Student’s t-test (two-sample equal variance; two-tailed distribution).