Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation

Abstract

Numerous post-translational modifications of histones have been described in organisms ranging from yeast to humans. 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. One such modification, ubiquitylation of histone H2B (uH2B) on lysine 120 (K120) in humans, and lysine 123 in yeast, has been correlated with enhanced methylation of lysine 79 (K79) of histone H3 (refs 5-8), by K79-specific methyltransferase Dot1 (KMT4). 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.

H2B monoubiquitylation is a 5′-enriched active transcription mark and correlates with exon–intron structure in human cells

“…Clearly, the bidirectional pattern of H3K4me3 in the regions surrounding the TSS and the broader distribution of H2Bub1 in transcribed regions provide strong evidence that there is a discrepancy between H2Bub1 and H3K4me3. However, the hDOT1L-mediated H3K79 methylations are tightly regulated by H2Bub1 in the same nucleosome (McGinty et al 2008). In agreement with these clustering patterns, the H3K79 methylations and H2Bub1, but not H3K4me3, showed strongly correlated patterns of average modification signals in the gene body regions (Supplemental Fig.…”

“…In Saccharomyces cerevisiae, monoubiquitylation of lysine residue 123 in histone H2B was shown to play a positive role in H3K4 and H3K79 methylations, which are involved in transcription initiation and elongation, respectively (Briggs et al 2002;Sun and Allis 2002). In mammals, H2Bub1 coupled to RAD6-mediated transcription has been reported to directly stimulate H3K4 methylation ), and synthetically ubiquitylated H2B stimulates DOT1L-mediated H3K79 methylations in vitro (McGinty et al 2008;Kim et al 2009;Oh et al 2010). Despite many efforts to infer the roles of histone modifications, however, the crosstalk between H2Bub1 and the multiple methylations of H3K4 and H3K79 is not fully understood, especially in mammals.…”

Catalytic site remodelling of the DOT1L methyltransferase by selective inhibitors

“…Most PKMTs contain a conserved SET domain, with the exception of DOT1L, which has a protein fold that more closely resembles PRMTs 6 . DOT1L is also unique in that it is the only PKMT to methylate histone H3 on Lysine 79 (H3K79) 7,8 , a chromatin mark associated with active chromatin and transcriptional elongation 9 .…”

Synthesis of Heterobifunctional Protein Fusions Using Copper‐Free Click Chemistry and the Aldehyde Tag

“…[3,14,15] An alternative approach to generating protein-protein fusions is through chemical conjugation. Native chemical ligation of C-terminal thioesters with b-amino thiols is a powerful method for generating protein-protein fusions, [16][17][18] but at least one coupling partner must be linked at its terminus. In principle, greater topological diversity can be achieved by introducing bioorthogonal functional groups at specific amino acid side chains of the two proteins.…”