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

Significance This article applies a range of protein ligation methods at the level of chromatin to understand the cross-talk mechanism between the well-established biomedical target the human Dot1 (hDot1L) methyltransferase and the ubiquitylation of H2B at lysine 120. Through a systematic structure–activity relationship study of the ubiquitin surface in regard to hDot1L-mediated H3K79 methylation and further investigation with precisely engineered chromatin substrates, a functional hotspot within ubiquitin was identified that is essential to the stimulation of hDot1L activity. More broadly, this work shows how chemical synthesis approaches can be used to precisely tailor protein posttranslational modifications to afford mechanistic insights that would be impossible by other methods. 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.