H4k12ac

ASF1a/b (11 (link)), CAF-1/p150 (Novus Biologicals #NB500–207A1), DAXX (Santa Cruz #sc-7152), HAT1 (Abcam #ab12164), HIRA (Abcam #ab20655), Histone H3 (Abcam #ab7834), H3K9me1 (Millipore #07–450), H3K9me2 (Millipore, #07–212), H4K12ac (Millipore #07–595), Hsc70 (Abcam #ab19136), Hsp90 (Santa Cruz #sc-7947), Importin4 (Abcam #ab28387), MCM2 (BD Transduction Lab #610700), MCM5 (Bethyl A300–195A), NASP (donated by Dr Almouzni), RPL5 (Abcam #ab74744), RPS3a (Abcam, ab171742), SetDB1 (Abcam #ab12317). For Western blot analysis the primary antibodies were detected with a horseradish peroxidase-conjugated secondary antibody, developed with enhanced chemiluminescence (ECL, Pierce) and exposed onto an X-ray film.

The following antibodies were used in this study: H3K27me3 (Millipore 07-449), H3K27ac (Abcam ab4729, Western blot), H3K27ac (Cell signaling, 8173BC, ChIP), H3K4me1 (homemade EDL), H4K12ac (Millipore, 07-595), P300 (Clone NM11, Active Motif 61401), BRD4 (Bethyl A301-985A50, ChIP), BRD4 (Abcam ab128874, Western blot), Cas9 (Millipore MAC133), H2A.Z (Abcam ab150402), mH2A1 (Abcam ab37264, ChIP), mH2A1 (Millipore 07-219, Western blot), mH2A2 (Homemade, Bernstein Lab^{23 (link)}), H3 (Abcam Ab1791), H4 (Abcam, ab177840), GFP (Roche 11814460001), Beta-Actin (Sigma, A5441), Flag (Sigma, F1804), Mouse IgG – DyLight 680 (Invitrogen SA5-10170), Rabbit IgG DyLight 800 (Invitrogen SA5-10044). The antibodies used in this study are listed in Supplementary Table 2.

ChIP assays were carried out using a commercially available kit according to the manufacturer’s instructions (Upstate, Millipore). Briefly, human chondrocytes were fixed, harvested, and sonicated. The sonicated supernatants were diluted with ChIP dilution buffer and precleared with protein G agarose. After removing 1% supernatants as input, the supernatants were incubated with an antibody against Brd3 (Abcam), Brd4 (Abcam), cyclin-dependent kinase 9 (Cdk9) (Santa Cruz Biotechnology, INC), Ser2-phosphorylated RNA polymerase II C-terminal domain (S2P Pol II) (Abcam), H4K5Ac (Millipore), H4K8Ac (Millipore), H4K12Ac (Millipore), and nonspecific IgG (Upstate, Millipore) antibodies overnight at 4 °C with rotation. The protein/DNA complexes and the input were eluted, reversed, and purified. The quantitative analysis of targeted promoter regions was determined by real-time PCR using specific primers (Additional file 1: Table S1), and the results were normalized to the level of input by using the same primers. Each sample for real-time PCR was evaluated by three tests.

Lysates were analysed by SDS–PAGE using the following antibodies: polyclonal antibodies (AV94 and AV100) respectively against yeast histone H4 and a C-terminal peptide of H3 (a gift from Dr Alain Verreault, Université de Montréal), Monoclonal anti-H3K9ac (Cell Signaling, C5B11, Cat. No 9649), Phospho-p38 MAPK (Thr180/Tyr182) (Cell Signaling, D3F9, Cat. No 4511) and p44/42 MAPK (Thr202/Tyr204) phospho (Cell Signaling, 9101S), Histone H4K5ac (Active Motif, 39583, 39584), H4K12ac (Active motif, 39165, 39166), Anti-acetyl-histone H4K16Ac (Millipore, Cat. No 07-329), anti-acetyllysine (Immunechem, ICP0380), Anti-TAP (CBP) (Fisher, cab1001). Anti-tubulin (YOL1/34) (Abcam, ab6161), Anti-V5 (Medimab, ab27671). ECL scans of whole membranes are in Supplementary Fig. S10