Identification of a functional hotspot on ubiquitin required for stimulation of methyltransferase activity on chromatin.

TitleIdentification of a functional hotspot on ubiquitin required for stimulation of methyltransferase activity on chromatin.
Publication TypeJournal Article
Year of Publication2015
AuthorsHolt, MT, David, Y, Pollock, S, Tang, Z, Jeon, J, Kim, J, Roeder, RG, Muir, TW
JournalProc Natl Acad Sci U S A
Volume112
Issue33
Pagination10365-70
Date Published2015 Aug 18
ISSN1091-6490
KeywordsAmino Acid Sequence, Chromatin, Epigenesis, Genetic, Histone-Lysine N-Methyltransferase, Histones, Humans, Lysine, Methylation, Methyltransferases, Mutation, Nucleosomes, Protein Binding, Protein Engineering, Protein Processing, Post-Translational, Sequence Homology, Amino Acid, Software, Structure-Activity Relationship, Surface Properties, Ubiquitin, Ubiquitination
Abstract

<p>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.</p>

DOI10.1073/pnas.1504483112
Alternate JournalProc Natl Acad Sci U S A
PubMed ID26240340
PubMed Central IDPMC4547310
Grant ListDK071900 / DK / NIDDK NIH HHS / United States
R01 DK071900 / DK / NIDDK NIH HHS / United States
R01 CA129325 / CA / NCI NIH HHS / United States
R37-GM086868 / GM / NIGMS NIH HHS / United States
CA 129325 / CA / NCI NIH HHS / United States
R37 GM086868 / GM / NIGMS NIH HHS / United States
R01 GM107047 / GM / NIGMS NIH HHS / United States