title: Trithorax-group protein ASH1 methylates histone H3 lysine 36.
abstract: Drosophila discs absent, small, or homeotic-1 (ASH1) is a member of trithorax-group proteins that play essential roles in epigenetic regulation of Hox genes. Drosophila ASH1 genetically interacts with trithorax and has been reported to methylate histone H3 lysine 4 (K4) as well as H3 K9 and H4 K20. The function of mammalian ASH1, by contrast, has remained largely unknown. Here we report a histone lysine scanning mutation assay using recombinant core histones and in vitro reconstituted nucleosomes to identify targets of mammalian methyltransferases by fluorographic, Western blot, and mass spectrometric analyses. The assay reproduced specificities of previously known histone methyltransferases and further revealed unexpectedly that mammalian ASH1 mono- or di-methylates histone H3 K36 but not any other lysine residues of recombinant unmodified mammalian histones. Under the same experimental condition, lysine to arginine substitution of histone H3 at position 36 abolished the methyltransferase activity of Drosophila ASH1, suggesting that K36 is their specific target. We also demonstrate that native ASH1 proteins, consisting of the carboxy-terminal domains including the catalytic site, retain the specificity for K36. Taken together, our data suggest that ASH1 subfamily of SET domain proteins have K36-specific methyltransferase activities evolutionarily conserved from flies to mammals.
authors.affiliation: Genome Structure and Expression, School of Biomedical Science, and Biochemical Genetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyoku, Tokyo, Japan. ytanaka.bgen@mri.tmd.ac.jp
authors.forename: Yujiro
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Zen-Ichiro
authors.initials: Z
authors.lastname: Katagiri
authors.forename: Koji
authors.initials: K
authors.lastname: Kawahashi
authors.forename: Dimitris
authors.initials: D
authors.lastname: Kioussis
authors.forename: Shigetaka
authors.initials: S
authors.lastname: Kitajima
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004330:Drosophila / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D017344:Genes, Insect; D006657:Histones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D008239:Lysine / Q000737:chemistry; D008745:Methylation; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009707:Nucleosomes / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: C490910:ASH1 protein, Drosophila; D017931:DNA Primers; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D006657:Histones; D009707:Nucleosomes; D011994:Recombinant Proteins; D014157:Transcription Factors; D008239:Lysine