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

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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 post-translational modification.
Publisher
NATL ACAD SCIENCES
Issue Date
2015-08
Language
English
Article Type
Article
Keywords

HISTONE H2B; HUMAN-CELLS; H3K4 METHYLATION; UBIQUITYLATION; NUCLEOSOME; DYNAMICS; PROTEIN; LEUKEMOGENESIS; ACTIVATION; EFFICIENT

Citation

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, v.112, no.33, pp.10365 - 10370

ISSN
0027-8424
DOI
10.1073/pnas.1504483112
URI
http://hdl.handle.net/10203/203957
Appears in Collection
BS-Journal Papers(저널논문)
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