1. Laboratory of Synthetic Protein Chemistry, The Rockefeller University, New York, New York 10065, USA
2. Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, New York 10065, USA

Correspondence to: Tom W. Muir¹ Correspondence and requests for materials should be addressed to T.W.M. (Email: muirt@rockefeller.edu).

Numerous post-translational modifications of histones have been described in organisms ranging from yeast to humans¹. Growing evidence for dynamic regulation of these modifications, position- and modification-specific protein interactions, and biochemical crosstalk between modifications has strengthened the 'histone code' hypothesis, in which histone modifications are integral to choreographing the expression of the genome^{1, 2}. One such modification, ubiquitylation of histone H2B (uH2B) on lysine 120 (K120) in humans³, and lysine 123 in yeast⁴, has been correlated with enhanced methylation of lysine 79 (K79) of histone H3 (refs 5–8), by K79-specific methyltransferase Dot1 (KMT4)^{9, 10, 11}. However, the specific function of uH2B in this crosstalk pathway is not understood. Here we demonstrate, using chemically ubiquitylated H2B, a direct stimulation of hDot1L-mediated intranucleosomal methylation of H3 K79. Two traceless orthogonal expressed protein ligation (EPL) reactions were used to ubiquitylate H2B site-specifically. This strategy, using a photolytic ligation auxiliary and a desulphurization reaction, should be generally applicable to the chemical ubiquitylation of other proteins. Reconstitution of our uH2B into chemically defined nucleosomes, followed by biochemical analysis, revealed that uH2B directly activates methylation of H3 K79 by hDot1L. This effect is mediated through the catalytic domain of hDot1L, most likely through allosteric mechanisms. Furthermore, asymmetric incorporation of uH2B into dinucleosomes showed that the enhancement of methylation was limited to nucleosomes bearing uH2B. This work demonstrates a direct biochemical crosstalk between two modifications on separate histone proteins within a nucleosome.

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