SET8 Recognizes the Sequence RHRK20VLRDN within the N Terminus of Histone H4 and Mono-methylates Lysine 20

Abstract

Methylation of lysine 20 in histone H4 has been proven to play important roles in chromatin structure and gene regulation. SET8 is one of the methyltransferases identified to be specific for this modification. In this study, the minimal active SET domain of SET8 has been mapped to the region of amino acids 195-352. This region completely retains the same methylation activity and substrate specificity as the full-length SET8. The SET domain recognizes a stretch of specific amino acid sequence around lysine 20 of H4 for its methylation activity. Methylation assays with N terminus mutants of H4 that contain deletions and single alanine or glutamine substitutions of charged residues revealed that SET8 requires the sequence RHRK 20 VLRDN for methylation at lysine 20. The individual mutation of any charged residue in this sequence to alanine or glutamine abolished or greatly decreased levels of methylation of lysine 20 of H4 by SET8. Interestingly, mutation of lysine 16 to alanine, arginine, glutamine, or methionine did not affect methylation of lysine 20 by the SET domain. Mass spectrometric analysis of synthesized H4 N-terminal peptides modified by SET8 showed that SET8 selectively mono-methylates lysine 20 of H4. Taken together, our results suggested that the coordination between the amino acid sequence RHRK 20 VLRDN and the SET domain of SET8 determines the substrate specificity and multiplicity of methylation of lysine 20 of H4.Chromosomal nucleosomes in eukaryotic cells consist of an octamer core of histones H2A, H2B, H3, and H4, as well as 146 bp of DNA, which wraps around the histone core. The majority of the histone protein residues fold inside the histone core, whereas their unstructured N-terminal tails stretch outside the nucleosome (1). Covalent modifications of histones, such as acetylation, methylation, and phosphorylation, have been demonstrated to play important roles in the regulation of chromatin structure and gene activity (2-8). Acetylation of histone lysines has been proven to be associated with euchromatin formation and maintenance, as well as gene activation (9, 10). Five lysine residues in the N termini of histones H3 and H4 have been identified to be the main target sites of methylation: lysines 4, 9, 27, and 36 of histone H3 and lysine 20 of histone H4. To date, methylation of H3 lysines has been extensively studied, and the role of tri-methylation of lysine 9 in heterochromatin formation and gene repression has been well documented (4, 6, 11-13). Structures of SET domains of several histone H3 methyltransferases have been elucidated (14 -21).Lysine 20 of H4 has been shown to be methylated to the mono-, di-, or tri-methylation forms in vivo (22-25). Human PR-Set7 and SET8 were first reported to be two methyltransferases responsible for methylation of lysine 20 of H4 (26 -28). Suv4-20h1 and Suv-20h2 were then identified to be specific for tri-methylation of lysine 20 of H4 (25). In yeast, Set9 was reported to be able to mono-, di-, and tri-methylate lysine 20 of H4 (29). Most methyltransferas...

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