Cell culture and drug treatment
The NPC cell lines (CNE2, Hone1, C666) and the lung cancer cell lines (H1264, H460) were obtained from Prof. Zhiqiang Xiao and Prof. Rong Tan (Xiangya Hospital, Central South University). The corresponding IR-resistant cell lines were prepared as previously described 27. NPC cell lines were cultured with DMEM/High Glucose medium (Biological Industries) containing 10% FBS (VISTECH, SE100-B), and lung cancer cell lines with RPMI 1640 medium (Biological Industries) containing 10% FBS, in a humidified incubator with 5% CO2 at 37°C. Small molecular inhibitors THZ1 (Selleck, S7549), BS-181 (Selleck, S1572), KU-55933 (MedChemExpress, HY-12016), and PI-103 (Selleck, S1038) were used at 0.25 µM, 10 µM, 10 µM, and 0.4 µM respectively.
Construction and transfection of shRNA plasmids
The shRNA oligos were synthesized by Tsingke (Tsingke Biotechnology) and cloned into pLKO.1-TRC vector. The sequences of shRNA were listed in Supplementary Table S1.
Tumor cell viability analysis after irradiation
To confirm IR-resistance, cells were seeded into 96-well plates at the density of 1000-2000 per well. After 24 hours, cells were irradiated with the indicated X-ray dose at room temperature at 200 cGy/min with a linear accelerator (X-Rad 225, Precision). The viability was either measured daily using MTT (Sigma, M5655) for 6 days, or measured at day 5 after irradiation. For western blotting experiments, cells were harvested at day 2 after IR treatment.
For xenograft tumor model, 5×106 of the indicated cells were suspended in 100 µL DPBS (Biological Industries) and injected subcutaneously into the flank of 6 weeks old female BALB/c nude mice (Hunan SJA Laboratory Animal). The tumors were measured thrice weekly with a digital caliper, and the volume calculated using the formula: 0.5 × (length × width2). Once the tumors reach 200 mm3 in size, the mice were irradiated with a single dose of 4 Gy under 2,2,2-tribromethanol anesthesia (Avertin). Only the tumor was irradiated, and the rest of the body was shielded by lead. All the animal experiments were approved by the Medical Ethics Committee of Central South University, and conducted according to the Guidelines of Animal Handling and Care in Medical Research in Hunan Province, China.
Cells were washed with cold DPBS (Biological Industries) two times and then lysed in sample buffer (2% SDS, 10% glycerol, and 62.5 mM Tris-HCl, pH 6.8) supplemented with 1× protease inhibitor cocktail (Sigma, P8340), sodium fluoride (10 mM, Sigma, 450022), and sodium orthovanadate (1 mM, Sigma, 450243). After sonication, the protein concentration was determined using BCA assay (Beyotime, P0009). The following primary antibodies were used: mouse anti-RNAPII Ab (1:3000, Abcam, ab817), mouse anti-RNAPII phosphor S5 Ab (1:3000, Abcam, ab5408), rabbit anti-RNAPII phosphor S2 Ab (1:5000, Abcam, ab5095), mouse anti-tubulin HRP Conjugate (1:5000, Cell Signaling Technology, 12351S). The corresponding secondary antibodies (Thermo Fisher Scientific) were used at 1:5000. The signal was detected with ECL substrates (Millipore, WBKLS0500).
ChIP-seq and ChIP-qPCR
The cells in a 100 mm dish were cross-linked with 1% formaldehyde for 10 min, quenched with 125 mM glycine for 5 min, rinsed with ice-cold PBS twice, and scraped in 1 mL PBS. After spinning at 1350 g for 5 min at 4℃, the pellet was resuspended in 500 µL Lysis Buffer I (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100), and incubated at 4℃ for 10 min with rotating. After spinning at 4℃, the pellet was then resuspended in 500 µL Lysis Buffer II (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), and incubated for 10 min at room temperature. After spinning at 4℃, the pellet was resuspended in 500 µL Lysis Buffer III (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine). The cell lysate was sonicated till the DNA was sheared to 200 bp. The lysate was then quenched by 1% of Triton X-100, supplemented with 1 × protease inhibitor, and spun at 12000 rpm for 10 min at 4℃. 50 µL supernatant was reserved as input, and the rest was incubated overnight at 4℃ with the magnetic beads bound with 4 µg anti-RNAPII (Abcam, ab817), anti-RNAPII phospho S5 (Abcam, ab5408), or anti-RNAPII phospho S2 (Abcam, ab5095). The beads were washed four times with Wash Buffer (50 mM HEPES-KOH, pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-deoxycholate). The DNA was eluted with 100 µL of Elution Buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS). The cross-links were reversed by incubating in a heating oscillator at 65℃ for 2 hours. The sample was incubated with 4 µL of 25 mg/mL RNase A overnight at 65℃, and then with 2 µL of 10 mg/mL proteinase K at 55°C for 4 hours. The DNA was purified by phenol:chloroform:isoamyl alcohol extraction and resuspended in 20 µL ddH2O. ChIP-seq library was prepared using NEBNext® ChIP-Seq Library Prep Reagent Set (New England Biolab, E7645S). The library was sequenced on Illumina NovaSeq 6000 (Novogene). ChIP-qPCR was performed using the SYBR Green qPCR Master Mix (SolomonBio, QST-100) on the QuantStudio 3 Real-Time PCR system (Applied Biosystems). Primers were listed in Supplementary Table S1.
ChIP-seq raw reads were filtered using trim_galore v0.6.0, aligned to the hg38 genome assembly using Bowtie2 v184.108.40.206 with default parameters. Duplicate reads were removed using MarkDuplicates from the gatk package v.220.127.116.11. The pausing index (PI) was calculated as previously described 12. The RefSeq gene model was downloaded from UCSC. ChIP-seq and input reads were calculated using bedtools coverage v2.26.0, mapped to the TSS regions (TSSR, –50 to +300 bp relative to TSS) and the gene bodies (TSS +300 bp to +3 kb past the TES) for each annotated RefSeq isoform. The reads density was normalized by the region length and by the mapped filtered read numbers multiplied by 1 million (rpm/bp). The input was then subtracted and PI was calculated as the ratio between RNAPII density in the TSSR and the gene body. For multiple RefSeq isoforms of the same gene, the one with the strongest RNAPII ChIP-seq signal in the TSSR, at least 0.001 rpm/bp, was selected. All those genes with PI >2 were defined as paused, and the rest were non-paused. The bigwig tracks were generated using bamCompare from deeptools. Negative values were set to zero. IGV v.2.4.13 was used to visualize the bigwig tracks. ChIP-seq profiles were created by computeMatrix and plotProfile in deeptools.
RNA-seq and RT-qPCR
RNA was extracted using TRIzol (Life Technologies, 87804). Libraries were prepared using mRNA-Seq Sample Preparation Kit (Illumina) and sequenced on an Illumina NovaSeq platform (Novogene). Raw reads were filtered using trim_galore, then mapped to hg38 genome assembly using STAR v2.7.1a. Differential expression analysis was performed using the Bioconductor package DESeq2. For RT-qPCR, RNA was converted to cDNA using the PrimeScript RT reagent Kit (Takara, RR037A).
Immunofluorescence and live cell imaging
Cells were quickly rinsed with pre-warmed DPBS, fixed with pre-cooling methanol for 10 min at -20°C, and permeabilized in 0.5% Triton X-100 for 10 min. Cells were then blocked with 5% Bovine Serum Albumin for 30 min and incubated overnight at 4°C with anti-RNAPII phospho S5 (1:1500, Abcam, ab5408), anti-RNAPII phospho S2 (1:3000, Abcam, ab5095), anti-γ-Tubulin (1:1000, Sigma, T6557), anti-α-Tubulin (1:1000, Abcam, ab18251), or anti-ACA human centromere antibody (1:3000, gifted by Prof. Xuebiao Yao at University of Science and Technology of China). Cells were rinsed with DPBS thrice and incubated at room temperature for 1 hour with Alexa Fluor 488, 546, or 647 secondary antibodies (1:400, Thermo Fisher Scientific). For frozen sections, mouse tumor tissue was immediately embedded in optimum cutting temperature media (SAKURA Tissue-Tek® O.C.T. Compound 4583) and frozen at -80°C. Cryostat sections (12µm) were made and kept at -80°C. Slides were fixed in acetone for 5 minutes at room temperature and then rinsed with DPBS twice before being blocked with 5% BSA for 30 minutes. The slides were then incubated overnight at 4°C with anti-RNAPII phospho S5 (1:1000), rinsed thrice, and incubated for 1 hour at room temperature with Alexa Fluor 488 secondary antibody (1:400). DNA was visualized by DAPI staining (0.5 µg/mL, Sigma, D9542). The samples were mounted with SlowFadeTM Diamond Antifade Mountant (Thermo Fisher Scientific, S36963).
For live imaging, cells in glass bottom dish (NEST, 801001) were transfected with pEGFP-Tubulin and mCherry-H2B using Lipofectamine 3000 (Invitrogen), and synchronized with double thymidine block procedure. Cells were then released into thymidine-free medium for 8 hours and imaged in PeCon environmental microscope incubator (ZEISS) at 37°C and 5% CO2. Images were collected on the LSM 880 confocal microscope using a 63× oil immersion objective lens with Airyscan mode (ZEISS).
All the human tissue related experiments were approved by the Medical Ethics Committee of Central South University, and the informed consent was obtained from the patients. IHC was performed to detect the level of RNAPII pS5 in5 pairs of nasopharynx cancer samples using RNAPII phosphor S5 antibody (1:10000, Abcam, ab5408). The IHC score was evaluated by two independent investigators blinded to the histopathologic features and clinical characteristics using the intensity and proportion of positively stained tumor cells as previously described 18.
The experiments were carried out at least three times. Data were presented as the mean ± standard deviation (SD). Statistical analysis and survival fraction analysis was performed using GraphPad Prism 9. Flow cytometry data were analyzed using ModFit LT 4.1. The statistical details of each experiment were indicated in the respective figure legends. The two-tailed unpaired Student’s t-test or chi-squared test was performed to evaluate significant differences between the two groups. Kolmogorov-Smirnov test was used to compare significant differences between two groups of pausing index. P values are presented as star marks in figures: *p < 0.05, **p < 0.01, ***p < 0.001.
ChIP-seq and RNA-seq datasets were deposited to the NCBI GEO database under the accession number GSE185423. All custom scripts are available from the authors upon request.