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

Matthew T. Holt, Yael David, Sam Pollock, Zhanyun Tang, Jongcheol Jeon, Jaehoon Kim, Robert G. Roeder, Tom W. Muir

Research output: Contribution to journalArticlepeer-review

42 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)10365-10370
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number33
StatePublished - Aug 18 2015

All Science Journal Classification (ASJC) codes

  • General


  • Chromatin
  • Dot1L
  • Epigenetics
  • Protein chemistry
  • Ubiquitin


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