Antibodies and reagents
Anti-FLAG antibody (Sigma-Aldrich, A8592), anti-GFP antibody (Roche, 11,814,460,001), anti-GAPDH antibody (Ambion, AM4300), anti-H4 (Millipore, 07-108) and oligos (Sigma-Aldrich) were used.
Animal handling and experiments
All the experiments were performed on male Sprague–Dawley rats (spp. Rattus norvegicus) or SCID mice after approval of the Institute Animal Ethics Committee, Advanced Centre for Treatment Research and Education in Cancer and the Committee for the Purpose of Control and Supervision on Animals, India, standards. Protocol to induce the sequential stages of liver carcinogenesis is as previously described .
Core histones were applied horizontally to the top of a 15% AUT-PAGE and sealed using sealing buffer (1% w/v agarose, 0.75 mol/L potassium acetate, pH 4, 20% v/v glycerol and 0.001% pyronin Y). The gel was electrophoresed at a constant voltage of 200 V.
Reversed-phase separation was carried out on a C18 column (1.0 × 250 mm, 5 mm, 300 Å; Phenomenex). Mobile phases A and B consisted of water and acetonitrile, respectively, with 0.05% trifluoroacetic acid. The flow rate was 0.42 ml/min, and the gradient started at 20% B and increased linearly to 30% B in 2 min, to 35% B in 33 min, 55% B in 120 min and 95% B in 5 min. After washing with 95% B for 10 min, the column was equilibrated at 20% B for 30 min, and a blank was run between each sample injection.
Histone spots of interest from AUT-PAGE and the fractions of RP-HPLC were subjected to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) using MALDI-TOF/TOF mass spectrometer (Bruker Daltonics Ultraflex II). In brief, gel pieces were washed, destained, reduced, alkylated and subjected to in-gel digestion, and HPLC fractions were subjected to in solution trypsin digestion. Mass spectra were acquired on reflector ion positive mode. Database searching for protein masses was carried out using MASCOT search engine (version 2.2.03) by comparing the peptide masses with those in the NCBInr protein database (database version: NCBInr_20080812.fasta) in Rattus species. The searches were carried out with trypsin digestion, one missed cleavage, fixed carbamidomethylation of cysteine residues and optional oxidation of methionine with 100 ppm mass tolerance for monoisotopic peptide masses.
Isolation of total RNA and PCR
Total RNA was extracted from cells as per the manufacturer’s (Macherey-Nagel) instructions. It was further treated with DNaseI for 30 min at 72 °C to degrade any possible DNA contamination. RNA (2 µg) was subjected to reverse transcription using M-MLV reverse transcriptase and random hexamer primers according to the manufacturer’s (Fermentas) instructions. cDNAs were then amplified with the corresponding gene-specific primer sets (see Additional file 1: Figure S11). For RT-PCR, PCR was conducted for 24 cycles using the condition of 30 s at 94 °C, 1 min at 58 °C and 1 min at 72 °C. The PCR products were analyzed on a 1% agarose gels containing 0.5 µg/ml ethidium bromide. For real-time PCR Syber-Green from Ambion was used. The reactions were performed and monitored using QuantStudio 12K Flex Real-Time PCR System.
Histone purification and dimerization
Histones were purified and the H2A–H2B dimers were reconstituted as previously described . The dimers were purified by size exclusion chromatography using HiLoad 16/60 Superdex-200 gel filtration column (GE).
Equilibrium unfolding of dimers
The dimers were subjected to equilibrium unfolding which was monitored by observing both secondary and tertiary structure changes.
Secondary structure changes
Unfolding was observed in response to thermal and chemical denaturant by circular dichroism.
Unfolding was carried out starting from 20 up to 80 °C with a 2 °C increment and an equilibration time of 3 min. The CD spectra of only three temperatures are plotted for clarity. Analysis of the thermal unfolding curves suggests that dip at 222 nm can serve as a good spectroscopic probe for monitoring secondary structure unfolding [see Additional file 1: Figure S8a(i)]. Further, the unfolding was completely reversible with no protein aggregation as suggested by the completely overlapping unfolding and refolding curves [see Additional file 1: Figure S8a(ii)]. The data obtained could be fit into two-state unfolding model for dimeric proteins with residual in the range of only ± 2 using IgorPro [see Additional file 1: Figure S8a(iii)].
Urea-induced denaturation was also monitored with CD with an increment of 0.2 M urea concentration starting from 0 M, and like thermal denaturation, the dip at 222 nm in the CD spectra was used to plot the unfolding [see Additional file 1: Figure S8b(i)]. Initially, a titration up to 8 M urea was carried out; however, as the unfolding was complete in 3 M urea, subsequent titrations were performed with up to 5 M concentration of urea. The denaturation was completely reversible [see Additional file 1: Figure S8b(ii)]. Similar to the thermal unfolding data, the chemical denaturation data could be fit into the two-state unfolding model [see Additional file 1: Figure S8c(iii)].
Tertiary structure changes
To follow the tertiary structure unfolding, urea-induced denaturation monitored by fluorescence spectroscopy was performed.
On carrying out urea-induced denaturation, there was a drop in the fluorescence intensity with the unfolding of proteins as expected because of the quenching of fluorescence of the tyrosines previously buried in the dimer interface [see Additional file 1: Figure S8c(i)]. The drop in the intensity of emission maxima at 305 nm could be used for monitoring and plotting denaturation as there was no apparent redshift [see Additional file 1: Figure S8c(i)]. The folding was reversible [see Additional file 1: Figure S8c(ii)]; however, the pre- and post-transition baselines in the urea denaturation curve had a positive slope as observed in previous reports . However, to ensure that transitions were not missed during the unfolding process, denaturation was carried out with GdmCl as well. Similar pre- and post-transition baselines corroborated the urea denaturation data (see Additional file 1: Figure S9). The unfolding also showed a concentration dependence as is expected for a dimeric protein [see Additional file 1: Figure S8c(iii)] and could be fit into the two-state model of unfolding [see Additional file 1: Figure S8c(ii)] substantiating the data obtained for secondary structure unfolding.
For making mutants for the study, site-directed mutagenesis was performed using the kit and guidelines given in the QuickChange™ Site-Directed Mutagenesis Kit from Stratagene. Oligos were procured from Sigma-Aldrich.
The unfolding data were fit into the two-state model of unfolding as described previously .
H2A1H and H2A2A3 coding sequences were cloned into peYFPn1 (YFP at C-terminal) vector and transfected in CL38 cells. LSM510 Meta (Zeiss) microscope equipped with CO2 and temperature maintenance accessories was used to carry out the studies. The nuclei was bleached (in a box of fixed area) using 488-nm laser set at 100% power, and the recovery in the region was monitored for 1 h. Images were taken at 30-s intervals for the first 15 min and then at a 5-min interval for the remaining 45 min to minimize photobleaching. Quantification of the recovery was done as described previously .
Molecular dynamics simulation
All the simulations were performed using the Gromacs-4.6.5 software, with periodic boundary conditions. The particle mesh Ewald method was used to treat the long-range electrostatics, together with a cutoff of 1.2 nm for the short-range repulsive and attractive dispersion interactions, which were modeled via a Lennard–Jones potential. The Settle algorithm was used to constrain bond lengths and angles of water molecules and the P-Lincs for all other bond lengths. The time step of 2 fs was used for the entire system. The temperature was kept constant at 300 K by using the Nose–Hoover thermostat method. To control the pressure at 1 atmosphere, Parrinello–Rahman method was used. The following DNA sequence was used to model nucleosomes: ATCAATATCCACCTGCAGATTCTACCAAAAGTGTATTTGGAAACTGCTCCATCAAAAGGCATGTTCAGCTGAATTCAGCTGAACATGCCTTTTGATGGAGCAGTTTCCAAATACACTTTTGGTAGAATCTGCAGGTGGATATTGAT.
Cell line maintenance and synchronization
The cells from the human origin were maintained in appropriate growth media depending on the line at 37 °C with 5% CO2 supplemented with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM l-glutamine (Sigma). Cell lines CL38 and CL44 from rat liver origin were cultured in MEM (invitrogen) media with 10% FBS and were maintained at 37 °C with 5% CO2.
For overexpression experiments, mammalian expression vectors with CMV promoters (pcDNA3.1, pcDNA3.1 FLAG HA or peYFPn1) were used. The coding sequence of H2A1H (NM_001315492.1) or H2A2A3 (NM_001315493.1) was cloned in frame. For generating stable lines, the CL38 and CL44 cells were transfected with vectors (empty or encoding gene of interest) using TurboFect (ThermoFisher). Stable populations were selected by adding G418 (Sigma-Aldrich) in the growth media.
For cell cycle experiments, cells were enriched in the early G1-phase by serum starvation (0.1% FBS) for 24 h. Cells were released from the arrest by supplementing the media with 10% FBS.
Cell cycle analysis
Ethanol-fixed cells were washed twice with PBS and suspended in 500 µl of PBS with 0.1% Triton X-100 and 100 µg/ml of RNaseA followed by incubation at 37 °C for 30 min. After incubation, propidium iodide (25 µg/ml) was added followed by incubation at 37 °C for 30 min. DNA content analysis was carried out in a FACSCalibur flow cytometer (BD Biosciences, USA). Cell cycle analysis was performed using the ModFit software from Verity house.
Histone isolation and immunoblot analysis
First, nuclei were isolated from cells. For this, the cell pellet was resuspended in 0.1 ml PBS in a microcentrifuge tube. To this suspension, 0.9 ml lysis solution (250 mM sucrose, 50 mM Tris–Cl pH 7.5, 25 mM KCl, 5 mM MgCl2, 0.2 mM PMSF, 50 mM NaHSO3, 45 mM sodium butyrate, 10 mM β-ME and 0.2% v/v Triton X-100) was added. Tube was inverted several times and centrifuged for 15 min at 800 g, 4 °C. For nuclei isolation from tissues, the tissue was homogenized in hypotonic buffer (10 mM HEPES pH 7.5, 10 mM KCl, 0.2 mM EDTA, 0.1% NP40, 10% glycerol, 1 mM DTT) using Dounce homogenizer. The homogenate was overlayed on the same buffer containing 1.8 M sucrose and ultracentrifuged (20,000g for 2 h). The nuclear pellet obtained was subjected to histone extraction by acid extraction method by adding 0.3 ml of 0.2 M H2SO4. The tubes were vortexed thoroughly with intermittent incubation on ice. The tubes were then centrifuged at 13,000g, 4 °C for 30 min. The supernatant was transferred to a fresh tube without disturbing the pellet. The proteins in the supernatant were precipitated by adding 4 volumes of acetone and stored overnight at −20 °C. The tubes were then centrifuged at 13,000g, 4 °C for 10 min. The pellet was washed once in chilled acidified acetone (0.05 M HCl in 100% acetone) and once in chilled 100% acetone. Protein pellet was dried in vacuum centrifuge for 15 min. The pellet was resuspended in 0.1% β-ME at −20 °C. For immunoblotting, histones were resolved on 18% SDS–polyacrylamide gel, transferred to PVDF membrane and probed with antibodies. Signals were detected by using ECL plus detection kit (Millipore; Catalogue no. WBKLS0500).
Cell viability was quantified by its ability to reduce tetrazolium salt 3-(4,5-dimethylthiazole-2ϒ)-2,5-diphenyl tetrasodium bromide (MTT) to colored formazan products. MTT reagent (5 mg/ml in PBS) was added to the cells at 1/10th volume of the medium to stain only the viable cells and incubated at 37 °C for 4 h. MTT solubilization buffer (0.01 M HCl, 10% SDS) of twofold volume was added to the cells, followed by incubation in the dark at 37 °C for 24 h. The absorbance was measured at 570 nm with Spectrostar Nano-Biotek, Lab Tech plate reader. Cell viability was expressed as the percentage of absorbance obtained in the control cultures.
Colony formation assay
The cells (n = 1000) were plated in triplicate in 60-mm tissue culture plates, and they were allowed to grow as a monolayer for 14 days. Cells were incubated in complete culture medium, with media changes after every 2–3 days. After 14 days, the cells were fixed with 4% paraformaldehyde for 1 h. The colonies were stained with 0.5% crystal violet (0.5 in 70% ethanol) for 1 h at room temperature, rinsed and air-dried. Surviving colonies with more than 50 cells were counted, and images were captured using a high-resolution Nikon D70 camera (Nikon, Tokyo, Japan). For quantification of the size of the colonies, ImageJ was used.
Wound healing assay
Cells were seeded at a high density, serum-starved for 16 h and wounded when the cells formed a confluent monolayer. Recovery of the wounds was recorded by using an inverted microscope equipped with CO2 and temperature maintenance accessory for 20 h with images captured at 10-min interval.
MNase digestion assay
Nuclei containing 2 mM CaCl2 were incubated for 2, 4, 6, 8 and 10 min with 5U MNase/mg of DNA at 37 °C in MNase digestion buffer (15 mM Tris–Cl pH 7.4, 15 mM NaCl, 2 mM CaCl2, 60 mM KCl, 15 mM β-ME, 0.5 mM spermidine, 0.15 mM spermine, 0.2 mM PMSF, protease and phosphatase inhibitors). The digestion was stopped by adding equal volume of 2 × lysis buffer (0.6 M NaCl, 20 mM EDTA, 20 mM Tris–Cl pH 7.5, 1% SDS). MNase-digested samples were treated with RNaseA (100 μg/ml) for 30 min at 37 °C followed by proteinase K (80 μg/ml) treatment for 2 h at 50 °C. The samples were extracted sequentially with phenol, phenol/chloroform and chloroform followed by ethanol precipitation at −20 °C. The precipitated DNA was recovered by centrifugation at 10,000g for 20 min. The DNA pellet was washed, air-dried and dissolved in TE buffer, and its concentration was determined by A260/A280 absorbance. MNase-digested samples were resolved on 1.8% 1XTAE agarose gel electrophoresis with 0.5 μg/ml ethidium bromide.