Cell culture
The cell lines used were described [6],[11] including the human hypopharyngeal squamous carcinoma cell line FADU, human tongue squamous carcinoma cell line SAS, and lung cancer cell lines H1299 and A549 (obtained from ATCC). Human embryonic kidney 293 T cell line was used in transient transfection experiments. SNU398 is a human hepatoma cell line obtained from Dr. YS Jou (Academia Sinica, Taiwan).
Protein extraction, western blot analysis, RNA extraction, quantitative real-time PCR, in vitromigration/invasion assay
For extraction of proteins from cell lines, cell lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS) containing protease inhibitors was used. Cell lysates were clarified by centrifugation at 13,000 rpm, 4°C for 10 min. The protein content was determined by Bradford method (Bio-Rad Laboratories, Hercules, CA, USA). For western blot analysis, 50 μg to 100 μg protein extracts from each clone were loaded to 10% SDS-PAGE gels and transferred to nitrocellulose filters. The filters were probed with different antibodies (Additional file 17), and an anti-ß-actin antibody was selected as a loading control. Signals were developed using an ECL chemiluminescence kit (Millipore Corporation, Billerica, MA, USA). Total RNA was isolated using Trizol reagent (Life Technologies Corporation, Carlsbad, CA, USA) according to manufacturer’s recommendations. Single-stranded cDNA was synthesized by the RevertAid™ First Strand cDNA Synthesis Kit (Fermentas International, Inc., Burlington, Canada). Real-time PCR was performed on a StepOnePlus™ Real-Time PCR System (ABI: Applied Biosystems, Foster City, CA USA) according to the manufacturer’s instructions. The 2-ΔΔCt method of relative quantification was used to estimate the copy number of gene expression, and 18S was selected as an internal control. The oligonucleotides used for real-time PCR were shown in Additional file 18. For in vitro migration/invasion assay, eight-μm pore size Boyden chamber was used. Cells (4 × 104) in 0.5% serum-containing RPMI were plated in the upper chamber and 15% fetal bovine serum was added to RPMI 1640 in the lower chamber as a chemo-attractant. For invasion assay, the upper side of the filter was covered with Matrigel (BD Biosciences, San Jose, CA, USA) (1:2 dilution with RPMI). After 12 h for migration assay or 24 h for invasion assay, cells on the upper side of the filter were removed, and cells that remained adherent to the underside of membrane were fixed in 4% formaldehyde and stained with Hoechst 33342 dye. The number of migrated cells was counted using a fluorescence microscope. Ten contiguous fields of each sample were examined using a 40× objective to obtain a representative number of cells which migrated/invaded across the membrane.
Plasmids, transfection, and luciferase assays
The plasmids used and transfection methods were described using calcium phosphate or lipofection transfection method [6],[11]. The expression vectors of HIF-1α, HIF-2α, CBP, Twist1 promoter-driven luciferase construct, and WDR5 promoter-driven luciferase construct were described [6],[11]. The TET1 and INSIG1 promoter regions were cloned and the reporter constructs were shown in Additional file 2. The oligonucleotides used for plasmis constructions were shown in Additional file 19. The reporter constructs were co-transfected into 293 T cells with different expression vectors and an internal control plasmid. Luciferase assays were performed using the same amount of cell extracts and corrected for transfection efficiency using an internal control (GFP) [6],[11].
Quantitative chromatin immunoprecipitation (Chip)
ChIP assay was performed as described [6]. Briefly, cells were cross-linked with 1% formaldehyde for 10 min and stopped by adding glycine to a final concentration of 0.125 M. Fixed cells were washed twice with TBS (20 mM Tris, pH 7.5, 150 mM NaCl) and harvested in 5 mL of SDS buffer (50 mM Tris, pH 8.0, 0.5% SDS, 100 mM NaCL, 5 mM EDTA, and protease inhibitors). Cells were pelleted by centrifugation and suspended in 2 mL of IP buffer (100 mM Tris, pH 8.6, 0.3% SDS, 1.7% Triton X-100, 5 mM EDTA). Cells were sonicated with a 0.25-inch diameter probe for 15 s twice using an MSE-soniprep 1500 sonicator (setting 18). For each immunoprecipitation, 1 mL of lysate was precleared by adding 50 μL of blocked protein A beads (50% protein A-Sepharose, Amersham Biosciences; 0.5 mg/mL bovine serum albumin, 0.2 mg/mL salmon sperm DNA) at 4°C for 1 h. Samples were spun, and the supernatants were incubated at 4°C for overnight with no antibody, IgG, anti-HIF-2α antibody. Immune complexes were recovered by adding 50 μL of blocked protein A beads and incubated overnight at 4°C. Beads were successively washed with: (1) mixed micelle buffer (20 mM Tris, pH 8.1, 150 mM NaCl, 5 mM EDTA, 5% w/v sucrose, 1% Triton X-100, 0.2% SDS); (2) buffer 500 (50 mM Hepes, pH 7.5, 0.1% w/v deoxycholic acid, 1% Triton X-100, 500 mM NaCl, 1 mM EDTA); (3) LiCl detergent wash buffer (10 mM Tris, pH 8,0.5% deoxycholic acid, 0.5% Nonidet P-40, 250 mM LiCl, 1 mM EDTA); and (4) TE buffer (10 mM Tris, 1 mM EDTA) and then eluted with 1% SDS and 0.1 M NaHCO3. Twenty milliliters of 5 M NaCl was added to the elutes, and the mixture was incubated at 65°C for 5 h to reverse the cross-linking. After digestion with proteinase K, the solution was phenol/chloroform-extracted and ethanol-precipitated. For qChIP analysis [11], DNA fragments were resuspended in 400 μL of water and 5 μL was used by real-time PCR. Each sample was calculated as the percentage of input sample. The sequences of PCR regions and primers used in ChIP assay are listed (Additional file 20). The antibodies used are listed (Additional file 17).
Immunofluorescence staining
For immunofluorescence staining [6],[11], cells on glass coverslips or chamber slides were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. After washing three times for 10 min with PBS, fixed cells or slides were blocked with blocking buffer (PBS, 0.1% Tween-20, with 3% goat serum) for 1 h and incubated with primary antibody diluted in blocking buffer overnight at 4°C. After washing three times with PBS for 10 min, the fixed cells were treated with the appropriate secondary antibody (fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG or rhodamine-conjugated anti-rabbit IgG) (Sigma) that was diluted in blocking buffer for 1 h at room temperature. Finally, the fixed cells were washed three times for 10 min with TBS, and their nuclei were counterstained, mounted, and observed by using fluorescence microscope or confocal microscope.
Lentiviral infection
Lentivirus containing short hairpin RNAs (shRNAs) expressed in a lentiviral vector (pLKO.1-puro) were generated in 293 T cells as previously described [11]. Packaging plasmid pCMVΔR8.91 was obtained from SC Teng (National Taiwan University, Taiwan). Various pLKO plasmids to knockdown HIF-1α, HIF-2α, TET1, INSIG1, and scrambled control were provided by National RNAi Core Facility of Academia Sinica, Taipei, Taiwan. For lentivirus production, 293 T cells were transfected with 15 μg pLKO.1-puro lentiviral vectors expressing different shRNAs along with 1.5 μg of envelope plasmid pMD.G and 15 μg of packaging plasmid pCMVΔR8.91. Virus was collected 48 h after transfection. To prepare various knockdown cells, FADU or H1299 cells were infected with lentivirus for 24 h, and used for various assays after 4 to 5 days. The sequence of the lentiviral siRNA vectors were shown in Additional file 21.
RNA-Seq data analysis
The RNA-Seq libraries were prepared according to the standard Illumina protocol with the mRNAseq Illumina TruSeq and were sequenced using Illumina Hiseq2000 to obtain 100 bp paired-end reads. The reads were aligned to the hg19 reference assembly with the TopHat/Cufflinks [34] alignment package using Ensembl annotations. Transcript abundance was measured in fragments per kb of exon per million fragments mapped (FPKM).
5hmC chemical labeling
5hmC labeling reactions were performed according to the previous protocol [35]. Briefly, sonicated genomic DNA (average 400 bp, 500 ng/μL) was incubated with 50 mM HEPES buffer (pH 7.9), 25 mM MgCl2, 100 mM UDP-6-N3-Glc, and 2 mM βGT for 1 h at 37°C. The labeled DNA was purified by the QIAquick Nucleotide Removal kit (QIAGEN) and eluted in H2O. The click chemistry was performed with the addition of 150 mM of disulfide-biotin, and the mixture was incubated for 2 h at 37°C. The labeled DNA fragments were then purified by the QIAquick Nucleotide Removal kit (QIAGEN) and enriched by Dynabeads Streptavidin C1 (Invitrogen), and subsequently released by DTT treatment. The enriched DNA fragments were first purified by Micro Bio-Spin 6 spin columns (Bio-Rad) followed by MinElute PCR Purification Kit (QIAGEN).
5hmC-Seq data analysis
All the 5hmc-enriched DNAs were sequenced using Illumina Hiseq2000 containing more than 2.5 × 107, 50 bp single-end reads per sample. Raw sequence reads were mapped onto the reference human genome (NCBI Build UCSC hg19) using the Bowtie v0.12.7 algorithm (-m 1 -v 3 --best --strata) [36]. Unique and monoclonal reads were used for further analysis. The distribution of 5hmC reads at promoters or in gene body regions were analyzed by PAVIS [37] (Additional file 6a). Further analysis was done using Bioconductor using packages ChIPpeakAnno, biomaRt, MEDIPS and in-house scripts. The genes with 5hmC peaks at promoter region (approximately -3000 to 0 bp upstream of the transcription start site) were chosen for further analysis.
Selection of overlapping groups of genes from RNA-Seq and 5hmC-Seq data followed by Gene Ontology (GO) analysis
The genes that have differential 5hmC-enriched promoter regions from 5hmC-Seq analysis and are differentially regulated from RNA-Seq analysis are selected for functional enrichment analysis. For 5hmC-Seq data, the criteria for genes with 5hmC-enriched promoter regions are: the peak levels of Hypoxia (scrambled control) >4X Hypoxia (TET1-si), Hypoxia (scrambled control) >2X Normoxia (scrambled control), and Normoxia (scrambled control) >2X Normoxia (TET1-si). The differentially expressed genes from RNA-Seq are identified by the criteria: Hypoxia (scrambled control) >2X Hypoxia (TET1-si), Hypoxia (scrambled control) > Normoxia (scrambled control), and Normoxia (scrambled control) > or = Normoxia (TET1-si). The 98 genes that were chosen through the overlapping criteria by 5hmC-Seq and RNA-Seq analyses were further analyzed by GO-based functional enrichment analysis. GO-based functional enrichment analysis (Fisher’s Exact test) was used to measure the gene-enrichment in annotation terms for selected genes. The genes in the GO terms that passed the criteria of P value <0.01 and at least two genes in each GO term were considered for further analysis.
Detection of 5hmC levels within specific gene locus (hMeDIP)
Genomic DNA was prepared using a genomic DNA extraction kit (QIAGEN, Germany). Detection of 5-hydroxymethylcytosine (5hmC) within specific gene locus was performed by using EpiJET 5-hmC Analysis Kit (Thermo Scientific, USA). The DNA was glucosylated and digested with the Glc-5-hmC sensitive restriction endonuclease Epi MspI. Glucosylation/digestion levels were analyzed by qPCR. The sequences of oligonucleotides used for hMeDIP were shown in Additional file 22.
Methylated DNA immunoprecipitation (MeDIP)
Genomic DNA was prepared using a genomic DNA extraction kit (QIAGEN, Germany) and sonicated with Bioruptor (Diagenode Inc., USA) to produce random fragments ranging in mean size from 300 to 1,000 bp. Two micrograms of fragmented DNA was denatured for 10 min at 95°C and immunoprecipitated for 16 h at 4°C with 5 μL of 5-methylcytidine antibody (Eurogentec, Germany) in a final volume of 250 μL IP buffer (20 mM Tri-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 0.05% Triton X-100). The mixture was incubated with 50 μL protein A beads for another 4 h at 4°C and washed three times with 1 mL of IP buffer. Beads were resuspended with 500 μL digestion buffer (50 mM Tris (pH 8.0), 10 mM EDTA, 1% SDS) containing 10 μL proteinase K (20 mg/mL stock) overnight at 65°C. DNA was phenol/chloroform-extracted, ethanol-precipitated, and resuspended in 200 μL of water for real-time PCR analysis. The sequences of oligonucleotides used for MeDIP were shown in Additional file 22.
Yeast strains, transformations, and media
PJ69-4A (MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4Δ gal80Δ LYS2∷GAL1-HIS3 GAL2-ADE2 met2∷GAL7-lacZ) was used as the yeast strain. Media was prepared as described [38]. All yeast transformations were conducted using the high efficiency method described [39].
Co-immunoprecipitation assays
Co-immunoprecipitation was performed by incubating antibody with 500 μL of whole cell extracts in IP buffer (20 mM Tri-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, and protease inhibitor) with 50 μL of protein A/G mix magnetic beads (Millipore) for 4 h at 4°C. The immunoprecipitates were washed several times with IP buffer to removing any non-bound proteins. Finally, components of the bound immune complex are eluted, and then analyzed by western blot detection to verify the identity of proteins.
Measurement of transcriptional activation strength
The activation strength was measured by two different assays which measure the expression of the reporter genes: HIS3, which encodes imidazoleglycerol-phosphate dehydratase and catalyzes the sixth step in yeast histidine biosynthesis. The pGBDU constructs containing HIF-1α, TET1, or TET1 truncation mutants were generated according to the length and restriction sites mentioned in Additional file 3. Plasmids were transformed into the yeast strain PJ69-4A [40]. Transformed clones were selected on SC-Ura plates and confirmed for HIS3 expression on SC-His plates. Five to 10 freshly transformed colonies were mixed and spotted in five-fold serial dilutions onto plates. Plates were kept at 30°C until colonies formed.
Statistical analysis
The independent Student’s t-test was used to compare the continuous variables between two groups, and the χ 2 test was applied for comparison of dichotomous variables. The control groups of all the statistical analyses were specified in the figure legends. The level of statistical significance was set at 0.05 for all tests.
Accession number
Sequencing data have been deposited to the Gene Expression Omnibus (GEO) under accession number GSE59990.
