Cloning and production of recombinant adenovirus
The zinc finger protein which binds a 9-bp sequence (GGGGGTGAC) in the VEGF-A promoter [26], ZF fused to Dnmt3a-CD (amino acids 608-908, UniProt no. O88508), or Dnmt3a-CD alone were amplified and sub-cloned into the pAdTrackCMV vector using the BglII and XhoI restriction sites. The DNA fragments containing the catalytic domain, H3K9 methyltransferase G9a-like protein (amino acids 1002-1295, UniProt no. Q96KQ7), were cloned in empty or zinc finger-containing pAdTrackCMV vector using the SalI and HindIII restriction sites. The production of adenoviral vectors and adenovirus was based on [31]. Briefly, the pAdTrackCMV vector containing the gene of interest was linearized with PmeI and was co-transformed with pAdEasy-1 vector into Escherichia coli BJ5183. Successful recombination was confirmed by restriction digestion and Sanger sequencing. Six micrograms of PacI linearized vector was transfected into HEK293 cells in T-25 flask. HEK293 are E1 positive, thus allowing virus production. After transfection, the HEK293 cells were maintained in DMEM supplemented with 5% fetal bovine serum in a CO2 incubator at 37°C for 14 to 21 days, with addition of 2 ml medium, every 4 days. The adenoviral vector also expressed GFP to allow for the FACS analysis of infection yields and follow the expression levels of virus-encoded genes. The expression of GFP and the targeted zinc finger-fused methyltransferases was both driven by a CMV promoter. Total infection was confirmed microscopically, and viral lysates were prepared for high-titer virus production. In the end, the mature virus was collected using cesium chloride (CsCl) density gradient centrifugation and gel filtration (Nap™ columns, GE Healthcare, Pewaukee, WI, USA), and it was used for further infections. The optimal viral titer for infection of SKOV3 cells was determined by serial dilutions.
Infection of SKOV3 cells with recombinant adenoviral vectors
SKOV3 cells were obtained from ATCC (American Type Cell culture Collection) and cultured in DMEM supplement with 10% fetal bovine serum, l-glutamine, and penicillin/streptomycin. SKOV3 cells were seeded in a density of 2 × 105 cells per well in a six-well plate, and the following day the cells were infected with the adenoviral vectors. Virus dilutions were selected to yield >95% of infection without affecting the cell viability. The infection yield was determined by measuring GFP fluorescence (FACSCalibur, BD Biosciences, San Jose, CA, USA), where uninfected cells were used as a control. One day post-infection, the free virus was removed and the cells were washed with warm PBS. The samples were collected by trypsinization at day 3 or day 5 post-infection. Half of the cells harvested at day 5 was propagated until day 10 or day 15. For chromatin isolation, the whole protocol was up-scaled to 2 × 107 cells. Corresponding samples were always used for bisulfite sequencing, chromatin immunoprecipitation, and gene expression experiments. The generation of adenoviral particles and infection of SKOV3 was done in compliance with Biosafety Level 2 regulations.
Analysis of methylation by bisulfite conversion
Genomic DNA was isolated using the QIAmp® DNA mini kit (Qiagen, Limburg, The Netherlands). Four hundred nanograms of genomic DNA were digested with BamHI overnight at 37°C, and bisulfite conversion was carried out using sodium bisulfite and sodium hydroxide as described [38]. After bisulfite conversion the genomic DNA was concentrated and purified using Amicon filters (Millipore, Billerica, MA, USA) and amplified using the following primers: FP 5′-GTT TGT TAT TTT TTA TTT GAA T-3′ and RP 5′-AAT CAC TCA CTT TAC CCC TAT C-3′ [20]. The PCR product was subcloned using the Strataclone PCR cloning kit (Agilent Technologies, Santa Clara, CA, USA) and several individual clones were sequenced.
Native chromatin immunoprecipitation (nChIP) and gene expression
Native mononucleosomes were prepared from around 20 million SKOV3 cells by micrococcal nuclease digestion of nuclei as described [39] with minor modifications. More precisely, after MNase treatment, the nuclei were spun down at 13,000g for 10 min, and the soluble nucleosomal supernatant was collected and snap frozen. We used 10 to 15 μg (based on DNA absorbance) of pre-cleared native chromatin per ChIP with anti-H3K9me2 (ab1220, Abcam plc, Cambridge, UK), anti-H3K9me3 (ab8898, Abcam plc, Cambridge, UK), or pan-H4-acetyl (AM 39243) (go to Additional file 1: Figure S5 to see their peptide array specificity profiles). After immobilization on protein G-coated magnetic Dynabeads (Invitrogen, Waltham, MA, USA), the antibody-chromatin complexes were washed with: 1× low salt buffer (20 mM Tris-Cl, 150 mM NaCl, 1% Triton × -100, 0.1% SDS, and 2 mM ethylenediaminetetraacetic acid (EDTA)), 1× high salt buffer (20 mM Tris-Cl, 500 mM NaCl, 1% Triton × -100, 0.1% SDS, and 2 mM EDTA), 1× LiCl buffer (10 mM Tris-Cl, 250 mM LiCl, 1% NP-40, 1% DOC, and 1 mM EDTA), and 2× TE buffer. After each wash, the beads were rotated for 10 min at +4°C. The bound nucleosomes were eluted in elution buffer (50 mM Tris-Cl, 50 mM NaCl, 1 mM EDTA, 1% SDS), for 45 min at room temperature with rotation. DNA was recovered using ChIP DNA purification columns (Active Motif, Carlsbad, CA, USA).
The quantitative PCR assays were performed on a CFX96 Connect Real-Time detection system (Bio-Rad, Hercules, CA, USA) using SsoFast EvaGreen supermix (Bio-Rad, Hercules, CA, USA). In the nChIP experiments, a standard curve was generated to calculate percent of precipitated DNA and test the efficiency of each primer set covering the VEGF-A locus. The primer sequences can be found in Additional file 1: Table S1. To correct for technical quality between the different samples, each amplicon signal was normalized to an internal positive control which carries the corresponding mark and is not affected by the reprogramming (satellite alpha or gene desert-12 amplicons in the case of H3K9me2/3 or PABPC1 amplicon in the case of pan-H4ac). To this end, the percent of precipitated DNA was calculated for each amplicon and then divided by the percent of precipitated DNA obtained with the amplicons from the internal positive control. For nucleosomal mapping, mononucleosomal DNA was compared to a standard curve from genomic DNA and normalized for technical variability to an internal control (HOX11).
For gene expression analyses, total RNA was isolated for each time-point using the Purelink™ RNA mini kit (Ambion, Life Technologies, Carlsbad, CA, USA) and cDNA was prepared with oligo d(T)18 primers (New England Biolabs, Ipswich, MA, USA) from 1 to 2 μg of RNA. After this, qPCR was carried out using VEGF-A specific primers (FP 5′-AGA AGG AGG AGG GCA GAA TCA-3′ and RP 5′-ATG GCT TGA AGA TGT ACT CG-3′), normalized to the housekeeping gene SDHA (FP 5′-TGG GAA CAA GAG GGC ATC TG-3′ and RP 5′-CCA CCA CTG CAT CAA ATT CAT-3′). Non-RT controls were included in all experiments, and the total VEGF-A expression was quantified using the 2−ΔΔCT method (threshold cycle (CT)) [40].
Data analysis
Data are reported as means of biologically independent experiments as indicated. Error bars indicate the corresponding standard error of the mean. P values were determined by Excel using two tailed T tests with equal variance.
