Abstract
H2BK120Ub stabilizes nucleosome-bound DOT1L, thus increasing its histone methyltransferase activity.
Major Finding: H2BK120Ub stabilizes nucleosome-bound DOT1L, thus increasing its histone methyltransferase activity.
Concept: Ubiquitin binding stabilizes the conformation of DOT1L on the nucleosome, resulting in increased catalysis.
Impact: Structures of nucleosome-bound DOT1L may provide a framework for improved DOT1L inhibitor design.
Misregulation of DOT1L, a non–SET domain histone methyltransferase that catalyzes methylation of histone H3K79, is observed in numerous cancers—especially MLL-rearranged leukemias—and DOT1L inhibition has therapeutic promise in these cancers. Although it is observed that DOT1L's H3K79 methylation activity is stimulated by the presence of the H2BK120Ub modification, there is no detailed mechanistic information available regarding the cross-talk between these modifications. Valencia-Sánchez, De Ioannes, Wang, and colleagues report cryo-EM structures of human DOT1L bound to both unmodified and H2BK120 ubiquitinated nucleosome substrates. Notably, their structures captured DOT1L in a “post-catalysis” state on H2BK120Ub nucleosomesH3K79me1 and H3K79me2 could be detected by mass spectrometry in their samples, and SAH was present. DOT1L covers a large interaction surface, making direct contact with histone H4, the acidic patch residues of histones H2A and H2B, and DNA. The structure also clearly identified the regions responsible for direct contact with ubiquitin, with the most prominent interface occurring between the hydrophobic face of helix αK and the hydrophobic patch on ubiquitin. To understand the role of H2BK120Ub interactions in stimulation of DOT1L, they performed histone methyltransferase assays using various interface point mutants in DOT1L. Although these mutants could bind nucleosomes equally well as unmodified DOT1L, they showed impaired stimulation by H2BK120Ub, resulting in a reduction of H3K79me2 and loss of H3K79me3. This suggests that H2BK120Ub does not stimulate DOT1L by increasing its binding affinity and led the authors to hypothesize that H2B ubiquitination stimulates DOT1L activity by optimizing its position for catalysis. They compared the structures of DOT1L bound to unmodified and H2B-ubiquitinated nucleosomes and observed that although interactions with the acidic patch were still visible, DOT1L generally appeared much more mobile in the presence of unmodified H2B. This suggested that H2BK120Ub stimulates DOT1L catalysis by reducing its sampling space on the nucleosome by conformational restriction. Collectively, this work provides a framework for future studies of histone modification cross-talk and for the development of improved DOT1L inhibitors.
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