Dynamic methylation of alfalfa histone H3

Dynamic lysine methylation of histone H3 in alfalfa tissue culture cells was studied by labeling with tritiated methionine, purification of variants H3.1 and H3.2 by reversed-phase high pressure liquid chromatography and amino acid analysis. Mono- and dimethyl-L-lysine were the major labeled amino acids. Within 100 h of continued growth conversion from N epsilon-monomethyl-L-lysine (MML) to N epsilon-dimethyl-L-lysine (DML) and N epsilon-trimethyl-L-lysine (TML) was observed, consistent with steady-state histone methylation. During the same time 20% of the methylation label was lost from major variant H3.1 protein and more than 50% from the more highly labeled minor variant H3.2. A similar pattern of label incorporation and loss was observed during a study of histone synthesis and turnover. This conforms with the general observation in animal cells that lysine methylation is limited to newly synthesized histone. Increased methylation of the more highly acetylated forms of histone H3 protein indicates limited accessibility of chromatin for histone methylation. After loss of the labile fraction of newly synthesized H3 variants, stably methylated proteins with 30% of the label in MML, 40% in DML, and 25% in TML remain. Turnover of methyl modification groups independent of histone turnover was not detected.