Lysine residues in N-terminal and C-terminal regions of human histone H2A are targets for biotinylation by biotinidase

Abstract

In eukaryotic cell nuclei, DNA associates with the core histones H2A, H2B, H3, and H4 to form nuclosomal core particles. DNA binding to histones is regulated by posttranslational modifications of N-terminal tails, e.g., acetylation and methylation of histones. These modifications play important roles in the epigenetic control of chromatin structure. Recently, evidence has been provided that biotinidase and holocarboxylase synthetase catalyze the covalent binding of biotin to histones. Primary aim of this study was to identify biotinylation sites in histone H2A and its variant H2AX. Secondary aims were to determine whether acetylation and methylation of histone H2A affect subsequent biotinylation, and to determine whether biotinidase and holocarboxylase synthetase localize to the nucleus in human cells. Biotinylation sites were identified using synthetic peptides as substrates for biotinidase. These studies provided evidence that K9 and K13 in the N-terminus of human histone H2A and H2AX are targets for biotinylation, and that K125, K127, and K129 in the C-terminus of histone H2A are targets for biotinylation. Biotinylation of lysine residues was decreased by acetylation of adjacent lysines, but was increased by dimethylation of adjacent arginines. The existence of biotinylated histone H2A in vivo was confirmed by using modificationspecific antibodies. Antibodies to biotinidase and holocarboxylase synthetase localized primarily to the nuclear compartment, consistent with a role for these enzymes in regulating chromatin structure. Collectively, these studies have identified five novel biotinylation sites in human histones; histone H2A is unique among histones in that biotinylation sites include amino acid residues from the Cterminus.

Nonenzymatic biotinylation of histone H2A

“…Biotin attachment sites have been reported for histones H2A, H3, and H4. [13][14][15] Five sites have been identified on histone H2A at lysines 9, 13, 125, 127, and 129, with the first two confirmed by detection with antibodies to biotinylated peptides. 14 None of these sites are found in sequences related to the well recognized target sequence of biotin-dependent carboxylases, Ala/Val-Met-Lys-Met/Leu.…”

“…Curiously, H2A is reported to contain biotin attachment sites on lysines 9 and 13 in vivo, suggesting that these sites may in fact be chemically favored in a similar manner when using biotinidase. 14 Comparatively, the target lysine in BCCP is over 10 Å in distance from the nearest basic residue, suggesting pKa alteration to be minimal [ Fig. 7(B)].…”

“…[13][14][15] Histones are highly basic proteins responsible for packaging DNA into chromatin. [16][17][18] Roughly 25% of the amino acids in histones are arginines and lysines.…”

“…The detection of biotin as a histone modification comes from the use of antibodies made to biotinylated histone peptides. [13][14][15] These synthetic peptides, corresponding to the N-and C-terminal tails of histones, were biotinylated through the action of biotinidase using biocytin as an exchange substrate. Antibodies made to these structures have detected biotin on histones H2A, H3, and H4.…”

“…Using this method, histone H2A was found to be biotinylated at lysines 9 and 13. 14 Recently, it has been shown that the biotinyl moiety of bio-5 0 -AMP can be transferred to a lysine residue in protein in the absence of a catalytic enzyme. 19 These experiments derive from the initial observation that a mutant form of BirA could promiscuously biotinylate lysine residues in various proteins.…”