293T cells (ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St. Louis, MO USA) supplemented with 10% fetal bovine serum (FBS; HyClone) and 1% penicillin and streptomycin. NIKS cells were maintained at subconfluence on mitomycin C-treated J2 3T3 feeder cells in F medium (0.66 mM Ca2+) composed of three parts F-12 medium and one part DMEM supplemented with 5% FBS, insulin (5 μg/mL), cholera toxin (8.4 ng/mL), adenine (24 μg/mL), epidermal growth factor (10 ng/mL), and hydrocortisone (0.4 μg/mL), as previously described22. J2 3T3 feeder cells were removed from the culture dish by treatment with 0.02% EDTA. To remove the epithelial cells from the culture dish, the monolayer was rinsed with phosphate-buffered saline (PBS) followed by the addition of 0.1% trypsin/0.5 mM EDTA. The cells were then recovered from the suspension by multiple dilutions with E medium and cold PBS, followed by centrifugation.
Generation of NIKS cell lines containing HPV genomes
Plasmids containing HPV16 and HPV18 genomes were digested with BamHI or EcoRI to release the whole viral genome. The linearized HPV16 and HPV18 genomes were then re-circularized and purified as described previously23. Cells (2×105) were seeded in each well of a 6-well plate with incomplete F-media (no EGF). On the following day, the cells were transfected with 1600 ng re-circularized HPV DNA and 400 ng pcDNA6 encoding a blasticidin resistance gene (Invitrogen) using FuGENE HD (Promega). The next day, the cells were seeded onto a 75 cm2 flask over blasticidin-resistant feeders with incomplete F-media. Cells were selected with 4 μg/ml blasticidin S with complete F-media (with 10 ng/ml EGF) for 4 days and cultured for another 2–3 days in the absence of blasticidin23.
The confluency of NIKS cells in culture strongly induces commitment to terminal differentiation24, 25. Therefore, to induce differentiation, NIKS cells harboring HPV16 or HPV18 were cultured to post-confluent conditions and then collected for further analysis.
For analysis of the chromatin organization of the HPV16 and HPV18 genomes, cultured NIKS cells harboring HPV16 and HPV18 were washed twice with ice-cold PBS and harvested with a rubber policeman. The pellet was centrifuged at 3000×g for 5 min and washed with a buffer containing 10% sucrose, 60 mM KCl, 35 mM HEPES (pH 7.4), 5 mM KH2PO4, 5 mM MgCl2, and 0.5 mM CaCl2. The pellet was then suspended in a cold buffer containing 10% sucrose, 60 mM KCl, 35 mM HEPES (pH 7.4), 5 mM KH2PO4, 5 mM MgCl2, 3 mM CaCl2, and 15 mM NaCl. After the addition of Nonidet P-40 (final concentration, 0.1%), the samples were pipetted 10 times. To obtain genomic DNA, NIKS cells harboring HPV16 and HPV18 were washed twice with ice-cold PBS and harvested with a rubber policeman. The DNA was then purified using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany).
Micrococcal nuclease (MNase) digestion of chromatin and purified DNA
The DNA was digested with 1–5 U MNase at 25°C for 5 min. The reactions were stopped by the addition of 10 mM EDTA, 1% SDS, and proteinase K (800 μg/mL) at 37°C overnight. The DNA was then purified using phenol and phenol-chloroform extraction and ethanol precipitation.
MNase-treated chromatin was examined using Southern blotting. The blot was probed with full-length HPV16 or HPV18 using a Phototope Kit (New England Biolabs, Ipswich, MA, USA). The DNA was loaded onto a 1.5% agarose gel in 0.5× TBE, and the gel was electrophoresed at 50V for 3 h. The electrophoretically separated DNA was transferred from the agarose gel to a nylon membrane (Hybond-N+; General Electric, CT, USA) at room temperature overnight. Hybridization was conducted at 42°C overnight. The bands were detected by chemiluminescent detection and the Phototope-Star Detection Kit (New England Biolabs).
Nucleosome scanning analysis
MNase-treated chromatin and purified DNA samples were electrophoretically separated on a 1.5% agarose gel; mononucleosome-sized fragments were excised from the gel and purified. The resulting material was analyzed on the ViiA 7 Real-Time PCR System (Life Technologies) using Power SYBR Green/ROX master mix (Thermo Fisher Scientific) with 15 min denaturation at 95°C, followed by 45 cycles of 95°C for 15 s and 60°C for 60 s. The PCR amplicons were set every 30 bp of the HPV genome, spanning the LCR and the E6 gene. Each amplicon was 140 bp long, including 30mer primers, and overlapped with its neighboring amplicons by 110 bp (Figure 2).