In vitro biochemical assays

The PAD4 citrullination assays were performed in the buffer (pH 7.5) containing 50 mM Tris-HCl, 5 mM CaCl₂ and 2 mM DTT (freshly added). For free histone H3 citrullination, 50 μM H3 were treated with 5 μM PAD4 at 37 °C for 2 h, and were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by western blot analysis. For nucleosome core particle (NCP) citrullination, 1 μM NCPs were treated with 0.1 μM PAD4 at 37 °C for 2 h. The citrullinated NCPs were analyzed by SDS-PAGE or native page electrophoresis followed by western blot analysis. Nucleosomal array citrullination assays were similarly prepared using 1 μM dodecameric arrays and 0.1 μM PAD4 with slight modification, that is, the concentration of CaCl₂ was reduced to 100 μM to prevent the arrays from precipitation.

For the NCP citrullination-glycation assays, the wild-type or citrullinated NCPs (1 μM) were treated with 5 mM MGO (Sigma) in 1 × PBS buffer (pH 7.4) at 37 °C for 6 h (short treatment) or overnight (long treatment)¹⁸. The buffer exchange between citrullination and glycation assays was performed using 0.5 mL Centrifugal Filter (3 K, Millipore) with a 120-fold v/v for the removal of MGO or Ca²⁺ from the old reaction buffer systems. The co-incubation of NCPs, MGO and PAD4 (or deglycase DJ-1) were also performed under the same conditions at 37 °C overnight. The modified NCPs were analyzed by SDS-PAGE (without boiling or lyophilizing the sample) or native gel followed by western blot analysis. For the SDS-PAGE analysis, H3 was used as loading control, while ‘601’ DNA was used as loading control (by ethidium bromide staining) in the native gel analysis.

For peptide deglycation assays, 2 mM of the peptide substrate was incubated with 10 mM MGO in 1× PBS buffer (pH 7.4) at 37 °C for 30 min and then enriched by magnetic streptavidin beads (Thermo Fisher Scientific, 65602). After being washed by 1× PBS buffer, the glycated peptide was eluted with 100 mM glycine buffer (pH 2.5) and then diluted by using citrullination buffer (Tris-HCl), followed by the incubation with 100 μM PAD4 at 37 °C for 2 h. The elution and reaction buffers used in peptide deglycation were made with H₂¹⁸O (Sigma–Aldrich, 329878). The reactions were analyzed by dot blot and LC-MS.

For PRMT1 methylation assays, 50 μM full-length histone H4 was first incubated with (or without) 1 mM MGO in 1 × PBS buffer at 37 °C for 2 h, and then treated with human recombinant 5 μM PRMT1 heterologously expressed from Sf9 insect cells (Sigma, SRP0141) together with 100 μM SAM (Sigma) at 37 °C for 2 h. The products were separated by SDS-PAGE followed by western blot analysis using the corresponding antibodies.