Cloning of PRMT, H2A constructs and cell lines
PRMTs were cloned in pcDNA3.1 containing one Flag and two HA tags. Transfected HEK293 cells were selected in Dulbecco modified Eagle's medium (DMEM) containing G418 (PAA Laboratories GmbH, Pasching, Austria). Recombinant human H2A full-length and 4-129 constructs were cloned in pET21a.
Antibodies and peptides
The following antibodies were used: H3K4me3 (Abcam Inc., Cambridge, MA, USA), H3K9me3 (Upstate Laboratories Inc., New York, NY, USA), H3K20me3 (Upstate), H2AK9Ac (Upstate), HA (12CA5) and Flag (Sigma Chemical Co., St Louis, MO, USA). The H2AR29me2 antibody was raised in rabbit against the peptide FPVGR(me2a)VHRLLGC. Additional peptides used were the unmethylated petide FPVGRVHRLLGC, the H2AR29 monomethylated peptide FPVGR(me1a)VHRLLGC, and the H2AR3 di-methylated peptide SGR(me2a)GKAGGC.
Expression of recombinant H2A
Recombinant H2A constructs in pET21a were expressed in the Rosetta Escherichia coli strain, and induced with 0.4 mol/l isopropyl β-D-1-thiogalactopyranoside (IPTG) for 3 hours at 37°C. The preparation of inclusion bodies was performed as described previously . H2A was dialysed against water for subsequent in vitro methylation assays.
Extraction of endogenous histones from cells
Cells from different cell lines and PRMT knockdown cells were lysed in Ex-250 buffer (20 mmol/l HEPES pH 7.5, 250 mmol/l NaCl, 0.5 mmol/l MgCl2, 0.5% NP40) for 15 minutes on ice. After centrifugation the pellet was resuspended in 2% SDS and sonicated.
For the purification of H2A methylating enzymes, approximately 8 × 109 HeLa S3 cells were collected. The cell pellet was resuspended in hypotonic buffer (20 mmol/l HEPES pH 7.5, 20 mmol/l NaCl, 5 mmol/l MgCl2). and incubated for 15 minutes on ice. After homogenization, the cytoplasmic fraction was removed by centrifugation. The nuclei were then lysed in hypotonic buffer + 0.5% NP-40 detergent for 30 minutes, and the soluble nuclear fraction was isolated by centrifugation for 30 minutes. After pre-clearing, the extract was adjusted to 100 mmol/l NaCl, and loaded onto a Q-Sepharose column (Pharmacia, Uppsala). Elution was performed with a stepwise increase of NaCl concentration, from 200 mmol/l to 1000 mmol/l NaCl. The H2A activity-containing fraction was diluted to 50 mmol/l NaCl, and loaded onto a diethylaminoethyl cellulose (DEAE)-Sepharose column (Pharmacia, Uppsala). The active fractions from the DEAE column were loaded onto a heparin-Sepharose column (Pharmacia, Uppsala), and factions from this column were dialysed against 20 mmol/l Tris pH 9 and 20 mmol/l NaCl. An in vitro methylation assay was then performed. The 400 and 600 mmol/l fractions from the heparin-Sepharose column were analysed by MS.
MS analysis of heparin-sepharose fractions
For details see Additional file 5.
Stable cell lines expressing the different Flag-HA-PRMTs were seeded onto poly-L-lysine-treated coverslips, fixed in 4% paraformaldehyde/1% sucrose for 15 minutes, and permeabilised in PBS + 0.6% Triton for 5 minutes. Incubation with rabbit Flag antibody (Sigma, 1:200) and secondary antibody (Fluor 488 anti-rabbit IgG, Jackson Immunoresearch Laboratories, Inc., West Grove, PA, USA) was performed in PBS/0.1% Tween/3% BSA, then the cells were stained with 4',6-diamidino-2-phenylindole (DAPI) and mounted using mounting medium (Vectashield; Vector Laboratories Inc., Burlingame, CA, USA). Image acquisition was performed with a UV confocal microscope (SP2; Leica Microsystems Wetzlar GmbH, Wetzlar, Germany).
Immunoprecipitation of Flag-HA-PRMTs
Stable cell lines, expressing individual PRMTs, were grown to confluency and lysed in Ex-250 buffer (20 mmol/l HEPES, pH 7.5, 250 mmol/l NaCl, 0.5 mmol/l MgCl2, 0.5% NP40), followed by centrifugation and dilution to 150 mmol/l NaCl. After another centrifugation step, HA antibodies (3 μg per 2 × 107 cells) were added, and incubated with 15 μl protein G sepharose. The beads were washed twice with Ex-150 buffer (20 mmol/l HEPES pH 7.5, 150 mmol/l NaCl, 0.5 mmol/l MgCl2, 0.5% NP40), and twice with PBS.
In vitro methylation assays
An aliquot (2 μg) of core histones, recombinant H2A, MBP (Sigma) or glutathione S-transferase (GST)-GAR were incubated with 10 μl of immunoprecipitated PRMTs in PBS in a final volume of 30 μl. To this were added 0.5 μCu S-adenoslyl methionine (Amersham Laboratories, Amersham, Buckinghamshire, UK), then the mixture was incubated for 1 hour at 30°C, and reactions stopped by addition of loading buffer. Samples were loaded onto a 18.7% polyacrylamide gel, blotted onto a nitrocellulose membrane, and subjected to autoradiography.
Reverse transcription and real-time PCR
Total RNA was extracted from cells (Trizol reagent; Invitrogen). cDNA was synthesised by using the MMLV reverse transcriptase (Fermentas) and oligodT primer according to the manufacturer's instructions. One-tenth of the cDNA was amplified by real-time PCR with SYBR Green detection using a thermocycler with fluorescence detection (7300; Applied Biosystems, Foster City, CA, USA) and primers (see Additional file 6, table S1).
ImageJ software was used for quantification of both Ponceau-stained bands and western-blot signals. The values corresponding to the western-blot signals were normalized to the total amount of histones loaded, and the ratios between the normalized signals in wild-type and PRMT1/PRMT6 knockdown cells or PRMT6 overexpressing cells, respectively, were calculated.
Cells were crosslinked with 1% formaldehyde for 2 minutes at room temperature, and the reaction was stopped by the addition of glycine to a final concentration of 0.25 mol/l. The fixed cells were rinsed twice with PBS, and resuspended in 1 ml of Lysis Buffer 1 (50 mmol/l Tris pH 8, 2 mmol/l EDTA pH 8, 0.1% NP40, 10% glycerol) per 4 × 107 cells. After 10 minutes on ice the lysate was centrifuged for 5 minutes at 500 g. The nuclear pellet was resuspended in 1 ml of Lysis Buffer 2 (50 mmol/l Tris pH 8, 10 mmol/l EDTA pH 8, 0.1% NP40, 0.2% SDS) and sonicated in a water-bath sonicator (Diagenode, Liege), then centrifuged at 21000 g for 10 minutes. The cleared supernatant was pre-cleared with 150 ml blocked beads for 1 hour, rotating at 4°C. Chromatin was diluted 10 times in ChIP Dilution Buffer (50 mmol/l Tris pH 8, 150 mmol/l NaCl, 0.25% NP40), and then incubated overnight at 4°C with 4 mg of the relevant antibody. The solution was then incubated for 2 hours with rotation at 4°C with 40 μl blocked beads to recover the bound material. The beads were washed, twice in 10 ml of Low Salt Buffer (20 mmol/l Tris pH 8, 150 mmol/l NaCl, 2 mmol/l EDTA pH 8, 0.25% NP40, 0.02% SDS), once with 10 ml of High Salt Buffer (20 mmol/l Tris pH 8, 250 mmol/l NaCl, 2 mmol/l EDTA pH 8, 0.25% NP40, 0.02% SDS), and eluted by two 15-minute incubations at 30°C with 125 μl elution buffer (1% SDS, 0.1 mol/l NaHCO3). Chromatin was reverse-crosslinked for 4 hours at 65°C in the presence of 10 U RNase (Roche Applied Science, Indianapolis, IN, USA) and 10 mg/ml proteinase K. DNA was extracted by phenol-chloroform, and further purified with a commercial kit (PCR Purification Kit; Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions.
MS/MS analysis of methylated H2A
For details see Additional file 5, additional methods