Preparation of Histone H3 with Lys4 Methylation

Human histones were prepared as described previously [19 (link)]. The human linker histone H1.2 was bacterially produced as a recombinant protein, and was purified as described previously [20 (link)]. To obtain histone H3 with the Lys4 methylation, the H3.2 K4C/C110A mutant was prepared. In the H3.2 K4C/C110A mutant, the Lys4 and Cys110 residues were replaced by Cys and Ala, respectively. The Cys residue inserted at position 4 of H3 was then alkylated [21 (link)], and H3.2 with a monomethyl-lysine analog, N-monomethyl-aminoethylcysteine, at position 4 (referred to as H3K4_Cme1 in this report) was prepared [22 (link)]. We analyzed the H3K4_Cme1 peptide by mass spectrometry, and confirmed that the modified peptide containing the H3K4_Cme1 residue, but little unmodified peptide, was detected. This suggested that the H3K4 mono-methylation was nearly complete. The nucleosomes were reconstituted with the 147 base-pair Widom 601 sequence [23 (link),24 (link)] by the salt dialysis method, as described previously [25 (link)]. The resulting nucleosomes were purified by native polyacrylamide gel electrophoresis, using a Prep Cell model 491 apparatus (Bio-Rad).