Western blot
Histone protein was extracted using the Histone Extraction Kit (Active Motif, California, USA) based on the manufacturer’s instructions. The concentration of extracted proteins was assayed using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Santa Clara, CA, the USA). Dithiothreitol at a final concentration of 2.5 mM was added to the extracted proteins and warmed in a heat block at 95°C for 5 min. The adjusted proteins were then applied to the sodium dodecyl sulfate (SDS)–polyacrylamide gel for electrophoresis. Next, the proteins were transferred into the polyvinylidene difluoride membrane (GE Healthcare, the UK). The membrane was blocked with 5% skim milk for 30 min at room temperature and was incubated with the primary antibodies (anti-H3K4me3 antibody [MABI0304, diluted 1:500; GeneTex, Funakoshi Co., Ltd.] and anti-H3 antibody [9715S, diluted 1:1000; Cell Signaling, Danvers, MA, the USA]) overnight at 4°C. The membranes were then incubated with secondary antibodies for 30 min at room temperature. Pierce ECL Plus Substrate (Thermo Fisher Scientific, Santa Clara, CA, the USA) was applied to detect signals. The band intensity was quantitated using ImageJ (NIH, Bethesda, the USA). The densities of the H3K4me3 bands were corrected for H3 and then quantified by comparing with those of the bands under normoxic condition without Dznep treatment.
RNA isolation, reverse transcription, and qPCR
RNA was isolated using RNAiso Plus (Takara Bio Inc., Shiga, Japan) based on the manufacturer’s instructions. Reverse transcription was performed using the PrimeScript RT Master Mix (Takara Bio Inc.). First-strand cDNA was used to determine the relative mRNA expression with THUNDERBIRD SYBR QPS-201 (Toyobo Co., Ltd., Osaka, Japan) on the CFX96 System (Bio-Rad Laboratories, Inc., Hercules, CA, the USA). The expression level of each gene was normalized by β-actin. All measurements were performed in triplicate, and three independent experiments were conducted. Table 1 shows the primer sequences used in qPCR.
Cell culture
HK-2 cells were used in in vitro experiments (CRL-2190, ACTT, Manassas, VA, the USA). The cells were cultured in Dulbecco’s Modified Eagle Medium (Sigma Aldrich) with 10% fetal bovine serum (Sigma Aldrich) and 1% penicillin/streptomycin (Thermo Fisher Scientific). Next, they were cultured in a humidified atmosphere with 5% CO2 at 37°C. Hypoxic condition was established via exposure to 1% O2 in a hypoxic cultivation incubator (APM-30D, ASTEC, Fukuoka, Japan).
siRNA
HK-2 cells were passaged in six wells with 1×105 cells. After 24 h, siRNAs with lipofectamine RNAiMAX (Thermo Fisher Scientific) were transfected into HK-2 cells. Stealth RNAi siRNA targeting human Ezh2 and negative control nucleotide (Thermo Fisher Scientific) were used. After 24 of transfection, hypoxic stimulation, Dznep treatment, and assays were initiated.
Chip and FAIRE
HK-2 cells stimulated via hypoxia for 24 h and treated with Dznep were collected with trypsin and fixed with 1% formaldehyde for 10 min at room temperature. The fixed cells were lysed in SDS lysis buffer (10-mM Tris-HCL [pH 8.0], 150-mM NaCl, 1% SDS, and 1-mM EDTA), sonicated by Branson Sonifier Cell Disruptor 350 (Branson Ultrasonics Corp., Danbury, CT, the USA), and electrophoresed on agarose gel to validate the DNA fragmentation status.
In Chip, the primary antibodies were anti-H3K4me3 antibody (MABI0304, diluted 1:100) (GeneTex, Funakoshi Co., Ltd.) and anti-HIF1α antibody (NB100-479, diluted 1:50) (Novus Biologicals, Centennial, CO, the USA). Next, they were reacted with Dynabead M-280 Sheep Anti-Mouse IgG or Dynabead M-280 Sheep Anti-Rabbit IgG (Thermo Fisher Scientific) at 4°C overnight. The reacted beads and the sonicated samples were mixed and rotated at 4°C overnight. The reacted beads were washed and then de-crosslinked at 65°C overnight. After treatment with Pronase (Roche) and RNase A (QIAGEN, Venlo, the Netherlands), the DNAs were extracted via phenol chloroform extraction and quantified via qPCR as the ratio of input (%input). All measurements were performed in triplicate, and three independent experiments were conducted.
In FAIRE, the input samples were de-crosslinked at 65°C overnight and treated with Pronase. In all samples including input samples, DNAs were extracted via phenol chloroform extraction after RNase A treatment. The purified DNAs were quantified via qPCR as %input. Then, the value of %input was standardized as the ratio to the negative control area. Table 1 shows the sequences of the primers used in qPCR. All measurements were performed in triplicate, and three independent experiments were conducted.
Reporter assay for the HIF1α-binding site of the TIMP2 gene
The HIF1α-binding site of the TIMP2 gene was extracted from our previous HIF-1α Chip-seq data [15]. The gene area of this region was amplified via PCR with the primers shown in Table 1. The PCR product was electrophoresed in agarose gel, and the HIF1α-binding DNA was extracted with the QIAquick Gel Extraction Kit (QIAGEN). The HIF1α-binding DNA was subcloned to the Zero Blunt TOPO vector using the Zero Blun TOP PCR Cloning Kit (Thermo Fisher Scientific). Competent high JM109 (TOYOBO) transformed by the TOPO vector was cultivated on the Kanamycin plate. Miniprep was performed on the cultured colonies using the PureYield™ Plasmid Miniprep System (Promega, Madison, WI, the USA), and the correctly cloned plasmid was confirmed based on the DNA sequences. Then, the TOPO vector and pGL3-promotor vector (Promega) were cut using restriction enzymes (KpnI and XhoI; Takara Bio Inc.) and ligated with Mighty Mix (Takara Bio Inc.). The ligated vector was transformed into JM109. Next, JM109 transformed by the ligated vector was cultivated on the ampicillin plate. After performing colony PCR, midiprep was performed on the cultured colonies using the PureYield™ Plasmid Midiprep System (Promega). Then, we obtained the TIMP2-HIF1α-binding DNA inserted into the pGL3-promotor vector (TH vector).
HK-2 cells were passaged in 12 wells with 5×104 cells. After 24 h, the TH vector or the pGL3-promotor vector with Fugene HD (Promega) were transfected into HK-2 cells. After 24 h of transfection, hypoxic stimulation was initiated. After 24 h, the assay was started using the Dual-Luciferase Reporter Assay System (Promega) based on the manufacturer’s instructions. Fluorescence values were used as the ratio of Firefly and Renilla measurements. All measurements were performed in triplicate, and three independent experiments were conducted.
Statistical Analysis
The unpaired two-tailed t-test was conducted to analyze data between two groups. P-values of < 0.05 were considered statistically significant. All analyses were performed with GraphPad Prism version 9.3.1 (GraphPad Software Inc.).