Identification of a functional hotspot on ubiquitin required for stimulation of methyltransferase activity on chromatin. Author Matthew Holt, Yael David, Sam Pollock, Zhanyun Tang, Jongcheol Jeon, Jaehoon Kim, Robert Roeder, Tom Muir Publication Year 2015 Type Journal Article 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. Keywords Structure-Activity Relationship, Humans, Protein Binding, Mutation, Amino Acid Sequence, Sequence Homology, Amino Acid, Protein Processing, Post-Translational, Methylation, Epigenesis, Genetic, Surface Properties, Histones, Methyltransferases, Nucleosomes, Chromatin, Software, Lysine, Protein Engineering, Ubiquitin, Ubiquitination, Histone-Lysine N-Methyltransferase Journal Proc Natl Acad Sci U S A Volume 112 Issue 33 Pages 10365-70 Date Published 2015 Aug 18 ISSN Number 1091-6490 DOI 10.1073/pnas.1504483112 Alternate Journal Proc Natl Acad Sci U S A PMCID PMC4547310 PMID 26240340 PubMedPubMed CentralGoogle ScholarBibTeXEndNote X3 XML