Methylation of histone H3 K79 by Dot1L is a hallmark of actively transcribed genes that depends on monoubiquitination of H2B K120 (H2B-Ub) and is an example of histone modification cross-talk that is conserved from yeast to humans. We report here cryo-EM structures of Dot1L bound to ubiquitinated nucleosome that show how H2B-Ub stimulates Dot1L activity and reveal a role for the histone H4 tail in positioning Dot1L. We find that contacts mediated by Dot1L and the H4 tail induce a conformational change in the globular core of histone H3 that reorients K79 from an inaccessible position, thus enabling this side chain to insert into the active site in a position primed for catalysis. Our study provides a comprehensive mechanism of cross-talk between histone ubiquitination and methylation and reveals structural plasticity in histones that makes it possible for histone-modifying enzymes to access residues within the nucleosome core.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR), in most organisms, catalyzes the four-electron reduction of the thioester (S)-HMG-CoA to the primary alcohol (R)-mevalonate, utilizing NADPH as the hydride donor. In some organisms, including the opportunistic lung pathogen Burkholderia cenocepacia, it catalyzes the reverse reaction, utilizing NAD+ as a hydride acceptor in the oxidation of mevalonate. B. cenocepacia HMGR has been previously shown to exist as an ensemble of multiple non-additive oligomeric states, each with different levels of enzymatic activity, suggesting that the enzyme exhibits characteristics of the morpheein model of allostery. We have characterized a number of factors, including pH, substrate concentration, and enzyme concentration, that modulate the structural transitions that influence the interconversion among the multiple oligomers. We have also determined the crystal structure of B. cenocepacia HMGR in the hexameric state bound to coenzyme A and ADP. This hexameric assembly provides important clues about how the transition among oligomers might occur, and why B. cenocepacia HMGR, unique among characterized HMGRs, exhibits morpheein-like behavior.

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Methylation of histone H3, lysine 79 (H3K79), by Dot1L is a hallmark of actively transcribed genes that depends on monoubiquitination of H2B at lysine 120 (H2B-Ub), and is a well-characterized example of histone modification cross-talk that is conserved from yeast to humans. The mechanism by which H2B-Ub stimulates Dot1L to methylate the relatively inaccessible histone core H3K79 residue is unknown. The 3.0 Å resolution cryo-EM structure of Dot1L bound to ubiquitinated nucleosome reveals that Dot1L contains binding sites for both ubiquitin and the histone H4 tail, which establish two regions of contact that stabilize a catalytically competent state and positions the Dot1L active site over H3K79. We unexpectedly find that contacts mediated by both Dot1L and the H4 tail induce a conformational change in the globular core of histone H3 that reorients K79 from an inaccessible position, thus enabling this side chain to project deep into the active site in a position primed for catalysis. Our study provides a comprehensive mechanism of cross-talk between histone ubiquitination and methylation and reveals an unexpected structural plasticity in histones that makes it possible for histone-modifying enzymes to access residues within the nucleosome core.