title: Identification of a functional hotspot on ubiquitin required for stimulation of methyltransferase activity on chromatin.
abstract: Ubiquitylation of histone H2B at lysine 120 (H2B-Ub) plays a critical role in transcriptional elongation, chromatin conformation, as well as the regulation of specific histone H3 methylations. Herein, we report a strategy for the site-specific chemical attachment of ubiquitin to preassembled nucleosomes. This allowed expedited structure-activity studies into how H2B-Ub regulates H3K79 methylation by the methyltransferase human Dot1. Through an alanine scan of the ubiquitin surface, we identified a functional hotspot on ubiquitin that is required for the stimulation of human Dot1 in vitro. Importantly, this result was validated in chromatin from isolated nuclei by using a synthetic biology strategy that allowed selective incorporation of the hotspot-deficient ubiquitin mutant into H2B. The ubiquitin hotspot additionally impacted the regulation of ySet1-mediated H3K4 methylation but was not required for H2B-Ub-induced impairment of chromatin fiber compaction. These data demonstrate the utility of applying chemical ligation technologies to preassembled chromatin and delineate the multifunctionality of ubiquitin as a histone posttranslational modification.
authors.affiliation: Laboratory of Synthetic Protein Chemistry, The Rockefeller University, New York, NY 10065;
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Holt
authors.affiliation: Department of Chemistry, Princeton University, Princeton, NJ 08544;
authors.forename: Yael
authors.initials: Y
authors.lastname: David
authors.affiliation: Department of Chemistry, Princeton University, Princeton, NJ 08544;
authors.forename: Sam
authors.initials: S
authors.lastname: Pollock
authors.affiliation: Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, NY 10065;
authors.forename: Zhanyun
authors.initials: Z
authors.lastname: Tang
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, South Korea.
authors.forename: Jongcheol
authors.initials: J
authors.lastname: Jeon
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, South Korea.
authors.forename: Jaehoon
authors.initials: J
authors.lastname: Kim
authors.affiliation: Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, NY 10065;
authors.forename: Robert G
authors.initials: RG
authors.lastname: Roeder
authors.affiliation: Department of Chemistry, Princeton University, Princeton, NJ 08544; muir@princeton.edu.
authors.forename: Tom W
authors.initials: TW
authors.lastname: Muir
mesh_terms: D000595:Amino Acid Sequence; D002843:Chromatin / Q000737:chemistry*; D044127:Epigenesis, Genetic; D011495:Histone-Lysine N-Methyltransferase; D006657:Histones / Q000737:chemistry*; D006801:Humans; D008239:Lysine / Q000737:chemistry; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry*; D009154:Mutation; D009707:Nucleosomes / Q000737:chemistry; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods; D011499:Protein Processing, Post-Translational; D017386:Sequence Homology, Amino Acid; D012984:Software; D013329:Structure-Activity Relationship; D013499:Surface Properties; D025801:Ubiquitin / Q000737:chemistry*; D054875:Ubiquitination
chemical_list: D002843:Chromatin; D006657:Histones; D009707:Nucleosomes; D025801:Ubiquitin; C473477:DOT1L protein, human; D008780:Methyltransferases; D011495:Histone-Lysine N-Methyltransferase; D008239:Lysine
keywords: Dot1L; chromatin; epigenetics; protein chemistry; ubiquitin