Title | Identification of a functional hotspot on ubiquitin required for stimulation of methyltransferase activity on chromatin. |
Publication Type | Journal Article |
Year of Publication | 2015 |
Authors | Holt, MT, David, Y, Pollock, S, Tang, Z, Jeon, J, Kim, J, Roeder, RG, Muir, TW |
Journal | Proc Natl Acad Sci U S A |
Volume | 112 |
Issue | 33 |
Pagination | 10365-70 |
Date Published | 2015 Aug 18 |
ISSN | 1091-6490 |
Keywords | Amino 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> |
DOI | 10.1073/pnas.1504483112 |
Alternate Journal | Proc Natl Acad Sci U S A |
PubMed ID | 26240340 |
PubMed Central ID | PMC4547310 |
Grant List | DK071900 / 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 |