Site specificity of histone H4 methylation by wheat germ protein-arginine N-methyltransferase

Abstract

CNBr treatment of calf thymus [methyl-14C]histone H4, methylated in vitro with S-adenosyl-L-[methyl-14C]methionine by a highly histone-specific wheat germ protein methylase I (S-adenosyl-L-methionine:protein-L-arginine N-methyltransferase, EC 2.1.1.23), produced two peptide fragments corresponding to residues 1-83 and 84-102, with the former being radioactive. Two-dimensional peptide mapping of the chymotryptic and tryptic digest of [methyl-14C]histone H4 and analysis of the chymotryptic digest on HPLC have shown that only a single peptide is radiolabeled. In order to define the exact site of methylation (arginine residue), the radioactive peptide from the chymotryptic digest of [methyl-14C]histone H4 was further purified on HPLC by linear and then isocratic elution. The purified chymotryptic peptide was then digested with trypsin and purified on HPLC, and its amino acid composition was determined on HPLC. These results indicate that the peptide corresponding to residues 24-35 of histone H4 is radiolabeled. Since this peptide contains a single arginine residue at position 35, we have concluded that the enzyme is specific not only to the protein substrate but also to the methylation site.

Genome-wide localization of histone 4 arginine 3 methylation in a differentiation primed myeloid leukemia cell line

“…Methylations were described early in the 1970s, but the characterization in the 1980s of the enzymes involved in methylation gave a new impact to the study of processes mediated by methylation (Disa et al, 1986;Gallwitz, 1971). Recently a plethora of substrates were identified (Lee and Bedford, 2002;Wada et al, 2002) and the enzymes involved in demethylation were also described (Cuthbert et al, 2004;Shi et al, 2004;Wang et al, 2004).…”