biostudies-literatureHolt MTNIDDK NIH HHSNCI NIH HHSNIGMS NIH HHS10365-70https://www.ebi.ac.uk/biostudies/studies/S-EPMC4547310biostudies-literatureUnknown112(33)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.Proceedings of the National Academy of Sciences of the United States of AmericaIdentification of a functional hotspot on ubiquitin required for stimulation of methyltransferase activity on chromatin.PMC4547310CA 129325R37-GM086868R01 CA129325R37 GM086868R01 DK071900DK071900R01 GM107047David YMuir TWTang ZPollock SRoeder RGHolt MTKim JJeon JfalseIdentification of a functional hotspot on ubiquitin required for stimulation of methyltransferase activity on chromatin.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.2015-01-01T00:00:00Z2015 Aug2024-12-04T11:45:22.136Z2019-03-27T01:57:13ZS-EPMC45473102624034010.1073/pnas.1504483112