Mechanism of Cross-talk between H2B Ubiquitination and H3 Methylation by Dot1L

Abstract

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.

Structural basis for COMPASS recognition of an H2B-ubiquitinated nucleosome

“…Several structures now exist of H2B-Ub-activated 427 methyltransferases bound to ubiquitinated nucleosomes which reveal highly divergent 428 strategies for ubiquitin recognition (Figure 5). The distinct ubiquitin binding modes 429 employed by COMPASS (this study), Dot1L (Anderson et al, 2019;Valencia-Sanchez 430 et al, 2019;Worden et al, 2019) and the MLL1 core complex (Xue et al, 2019) reveal a 431 striking plasticity in how ubiquitin is able to interact with and activate these different 432 histone methyltransferases. The high complexity of the ubiquitin mark likely enables 433 H2B-Ub to communicate with these different enzymatic complexes and template the 434 deposition of "activating" marks during transcription.…”

“…To drive tight association between COMPASS and 84 the nucleosome, we utilized a variant of histone H3 in which K4 was substituted with the 85 non-native amino acid, norleucine (Nle) (Worden et al, 2019). Lysine-to-norleucine 86 mutations have been shown to greatly increase the affinity of SET-domain 87 methyltransferases for their substrates in a S-adenosylmethionine (SAM)-dependent 88 manner (Jayaram et al, 2016;Lewis et al, 2013;Worden et al, 2019). To assess the 89 gain in affinity imparted by the H3K4Nle substitution, we used gel mobility shift assays 90 to measure binding of COMPASS to different nucleosome variants in the presence of 91 SAM ( Figure S1).…”

“…Our structure reveals the basis of crosstalk between H2B-ubiquitination and H3K4 404 methylation by Saccharomyces cerevisiae COMPASS and shows that it methylates its 405 target lysine in an asymmetric manner by recognizing the H2B-Ub and H3K4 on 406 opposite sides of the nucleosome (Figure 1d). This asymmetric recognition is distinct 407 from the H3K79 methyltransferase, Dot1L, which is also stimulated by H2B-Ub but 408 which methylates H3 on the same, cis-H3, side of the nucleosome (Figure 5c) 409 (Anderson et al, 2019;Valencia-Sanchez et al, 2019;Worden et al, 2019). To our 410 knowledge, the asymmetric recognition of H2B-Ub and H3K4 by COMPASS and 411 COMPASS-related complexes Xue et al, 2019) is the first example of 412 trans-nucleosome histone crosstalk.…”

“…It is notable that, for both COMPASS and 446 Dot1L, the presence of ubiquitin conjugated to H2B does not appear to increase affinity 447 of the enzyme for the nucleosome (Figure S1) (Worden et al, 2019). The lack of a 448 discernable effect on binding energy suggests that ubiquitin binding primarily affects 449 catalytic activation and, in the case of Dot1L, has been shown to increase kcat but not 450 KM (McGinty et al, 2009;Worden et al, 2019). Alternatively, It has been suggested that 451 ubiquitin activates Dot1L by using its binding energy to pay the energetic cost of 452 inducing a conformational change in the globular core of histone H3, thereby inserting 453 K79 into the Dot1L active site (Worden et al, 2019).…”

“…Compared to the nucleosome-free Saccharomyces cerevisiae 442 COMPASS structure (Qu et al, 2018) and the structure of Kluyveromyces lactis 443 COMPASS bound to an H3 peptide and SAM (Hsu et al, 2018), there are no 444 discernable conformational changes in the Set1 catalytic domain that could explain how 445 H2B-Ub stimulates methylation ( Figure S5). It is notable that, for both COMPASS and 446 Dot1L, the presence of ubiquitin conjugated to H2B does not appear to increase affinity 447 of the enzyme for the nucleosome (Figure S1) (Worden et al, 2019). The lack of a 448 discernable effect on binding energy suggests that ubiquitin binding primarily affects 449 catalytic activation and, in the case of Dot1L, has been shown to increase kcat but not 450 KM (McGinty et al, 2009;Worden et al, 2019).…”