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HEK293FT cells transfected with the indicated plasmid constructs were lysed in mild-RIPA buffer supplemented with Protease and Phosphatase Inhibitors Cocktails (Merck). Immunoblotting analysis was performed 24 h post-transfection using anti-VP16 (1:50; Santa Cruz Biotechnology, #sc-7545; or 1:1000; Abcam, #ab4808) or anti-GAL4 (DBD) (1:500; Santa Cruz Biotechnology, #sc-510) antibodies. Total soluble protein extracts from HeLa and U2OS cells and immunoblotting were performed as described in Castroviejo-Bermejo et al. (47 (link)). For PALB2 detection, a polyclonal antibody was raised in rabbit and used at a 1:5000 dilution. Mouse monoclonal anti-alpha tubulin (1:200 000; Abcam, #ab7291) served as the loading control. Horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse (1:10 000; Jackson ImmunoResearch) were used as secondary antibodies.

Whole-cell extracts were prepared in IPH lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 5mM ethylenediaminetetraacetic acid (EDTA), 0.5% NP40). Nuclear extracts were prepared as follows: cells were first lysed in buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, protease inhibitors) and centrifuged at 10 000 g for 5 min. Buffer B (20 mM HEPES pH7.9, 0.4 M NaCl, 1 mM EDTA, 10% glycerol, 1 mM DTT, protease inhibitors) was then added to the nuclear pellet fraction, which was shaken vigorously for 2 h and centrifuged at 10 000 g for 10 min. The supernatant (nuclear fraction) was aliquoted and stored at –80°C until used. All procedures were performed at 4°C. For DNAse treatments, nuclear extracts were incubated with an excess of DNAse I for 1 h at 37°C prior to immunoprecipitation. The effectiveness of DNAse treatment was checked on plasmidic DNA (see Supplementary Figure S1).
Standard procedures were used for immunoprecipitation and western blotting (10 (link)). The primary antibodies used in these experiments were directed against the following: control immunoglobulin G (IgG) (sc-2027; Santa Cruz), HA (ab18181; Abcam), GAL4 (sc- 510; Santa Cruz), DNMT3A (sc-20703; Santa Cruz), PADI4 (ab18181; Abcam), Citrulline (ab100932; Abcam), Citrulline H3 (ab1791, Abcam), HDAC1 (pAb-053–050; Diagenode), Actin (Sigma; A5316) and TBP (ab818, Abcam).