Tip60 acetylates six lysines of a specific class in core histones in vitro

Authors

  • Akatsuki Kimura,

    1. Laboratory of Developmental Biology, Department of Cellular Biology, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
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  • Masami Horikoshi

    Corresponding author
    1. Laboratory of Developmental Biology, Department of Cellular Biology, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
    2. Horikoshi Gene Selector Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Corporation (JST), 5-9-6 Tokodai, Tsukuba, Ibaraki 300-2635, Japan
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  • Communicated by: Akira Ishihama

*: Laboratory of Developmental Biology, Department of Cellular Biology, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bukyo-ku, Tokyo 113-0032, Japan; E-mail: horikosh@imcbns.iam.u-tokyo.ac.jp

Abstract

Background: Tip60, an HIV-1-Tat interactive protein, is a nuclear histone acetyltransferase (HAT) with unique histone substrate specificity. Since the acetylation of core histones at particular lysines mediates distinct effects on chromatin assembly and gene regulation, the identification of lysine site specificity of the HAT activity of Tip60 is an initial step in the analysis of its molecular function.

Results: Tip60 significantly acetylates amino-terminal tail peptides of histones H2A, H3 and H4, but not H2B, consistent with substrate preference on intact histones. Preferred acetylation sites for Tip60 are the Lys-5 of histone H2A, the Lys-14 of histone H3, and the Lys-5, -8, -12, -16 of histone H4, determined by a method which combined matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) measurements and Lys-C endopeptidase digestion, or a method detecting the incorporation of radiolabelled acetate into synthetic peptides.

Conclusion: The lysine site specificity of Tip60 in histone amino-terminal tail peptides in vitro has been characterized by an assay measuring the molecular mass of endopeptidase digested peptides, or a previously described assay. These results agree well with our proposed classification of lysines in core histones. The classification may be useful for an analysis of the relationships between HATs and the substrates of other uncharacterized HATs.

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