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Proc Natl Acad Sci U S A. 2015 Aug 18;112(33):10365-70. doi: 10.1073/pnas.1504483112. Epub 2015 Aug 3.

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

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Laboratory of Synthetic Protein Chemistry, The Rockefeller University, New York, NY 10065;
Department of Chemistry, Princeton University, Princeton, NJ 08544;
Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, NY 10065;
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, South Korea.
Department of Chemistry, Princeton University, Princeton, NJ 08544;


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.


Dot1L; chromatin; epigenetics; protein chemistry; ubiquitin

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