Worden EJ et al. (2019), Mechanism of Cross-talk between H2B Ubiquitinat...

XB-ART-56624

Cell 2019 Mar 07;1766:1490-1501.e12. doi: 10.1016/j.cell.2019.02.002.

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

Worden EJ , Hoffmann NA , Hicks CW , Wolberger C .

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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.

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Species referenced: Xenopus laevis
Genes referenced: dot1l h2ac21 h2bc21 prc1 rcc1

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	Graphical abstract
	Figure 1. Structures of Dot1L Bound to H2B-Ubiquitin Nucleosome (A) Cryo-EM reconstruction of the 2:1 active-state complex. (B) Cryo-EM reconstruction of the poised-state complex. (C) Atomic model of the active-state complex between Dot1L and the H2B-Ub nucleosome. The H4 tail, H3K79Nle, and the acidic patch are depicted as spheres, and the SAM cofactor is depicted in stick representation. See also Figures S1, S2, S3, and S4.
	Figure 2. Analysis of Dot1L Binding and Activity (A) Schematic of the Dot1L domain architecture. (B) Michaelis-Menten titrations of two variants of Dot1L. Left: titration with H2B-Ub nucleosome. Right: titration with unmodified nucleosome. Error bars correspond to the standard error of three replicate measurements. The kcat and KM of the fitted data are reported in the graph field. The reported errors of the fitted kcat and KM correspond to the standard error. (C) Electrophoretic mobility shift assay of Dot1L variants binding to different nucleosome substrates and to free DNA.
	Figure 3. Dot1L Transitions between the Poised and Active States (A and B) Dot1L movements from the poised state to the active state shown as from the side (A) and top (B). Poised-state (yellow) and active-state (green) Dot1L are depicted as cylinders, and the histone octamer is shown as a transparent gray surface. H2A/H2B acidic patch residues are shown as red spheres. Arrows indicate movements made by Dot1L in the switch to the active state. See also Figures S3 and S5.
	Figure 4. Dot1L Interactions with Ubiquitin and the Acidic Patch (A) Overview of the interaction between Dot1L, ubiquitin, and the nucleosome in the active-state structure. Residues important for Dot1L interaction with the nucleosome acidic patch residues are depicted as red sticks. (B) Detailed view of the contacts made between Dot1L and ubiquitin. Dot1L is depicted as a semi-transparent green cartoon. Important residues at the Dot1L-ubiquitin interface are shown as sticks. (C) Endpoint H3K79 methylation activity assays using Dot1L mutants with either unmodified or H2B-Ub nucleosomes. Error bars correspond to the standard deviation of three replicate experiments. (D) Detailed view of interactions between Dot1L and the H2B/H2A acidic patch. Residues at the interface are depicted as sticks and the EM density for Dot1L in the active state is shown as a semi-transparent gray surface. A superimposed poised-state Dot1L is depicted in yellow. (E) Multiple-sequence alignment of Dot1L from different species. The alignment was performed with Clustal Omega (Sievers et al., 2011). See also Figures S3 and S5 and Table S1.
	Figure 5. H4 Tail Interactions with Dot1L (A) Overview of the active Dot1L structure. Dot1L is shown as a transparent green surface, ubiquitin as purple ribbon, and H4 tail as red spheres. (B) H4 tail (red) interaction with the Dot1L binding groove. EM density for the H4 tail is shown as a semi-transparent gray surface. The 22–32 loop from the crystal structure of Dot1L alone (PDB: 1NW3) is colored tan. (C) H4 tail (red) interactions between Dot1L (green) and the H3K79 loop (blue). The surface of Dot1L is shown in semi-transparent green. Potential hydrogen bonding or van der Waals interactions are shown as black dashed lines. Red dashed lines illustrate the direction of the H4 mainchain in the Dot1L binding groove. (D) Modeled position of H4 R17 binding in the Dot1L acidic pocket. Conserved residues in the binding pocket are depicted as sticks, and the surface of Dot1L is shown and colored according the electrostatic potential. (E) Close-up view yeast Dot1p (PDB: 1U2Z) showing arginine from a neighboring Dot1p molecule in the crystal bound in the conserved acidic pocket. Electrostatic surface potential shown as in (D). Electrostatic potential in (D) and (E) was calculated using the APBS tool (Baker et al., 2001). (F) Multiple-sequence alignment of Dot1L from different species. The alignment was performed with Clustal Omega (Sievers et al., 2011). See also Figure S6 and Table S1.
	Figure 6. Conformational Change in Histone H3 Reorients K79 (A) Superimposition of the active-state and poised-state nucleosomes. The active-state histone octamer is depicted in surface representation and colored as in Figure 1. The H3K79 loops from the active-state (blue) and poised-state (yellow) structures are shown as cartoons, and H3K79 is shown as spheres. (B) Close-up view of H3K79 in the active state (blue spheres) and poised state (yellow spheres). The H3K79 sidechain moves ∼10 Å (measured from ε amino groups) from the poised state to the active state. (C) Superimposition of the histone H3K79 loop from the active-state (blue) and poised-state (yellow) structures showing the conformational change that occurs in the transition from the poised state to the active state. In the transition from the poised state to the active state, the H3K79 backbone moves up by 3.6 Å (measured from the Cα carbons of H3K79) and rotates by an angle of ∼90°. (D) EM density (gray surface) for the H3K79 loop in the poised state. (E) EM density (gray surface) for the H3K79 loop in the active state. See also Figure S7 and Table S1.
	Figure 7. Formation of the Dot1L Active-Site Enclosure (A) Superimposition of active-state (green) and poised-state (yellow) Dot1L. The H3K79 loop from the active-state structure is shown as a blue cartoon, and the modeled H3K79 sidechain is shown in stick representation. The disordered F131 and W305 loops from the poised-state structure are depicted as yellow dashed lines. The ε amino group of lysine comes within 3 Å of the SAM methyl donor. (B) Close-up view of the Dot1L active-site enclosure with H3 (blue), Dot1L (green), and H4 (red) shown in stick representation. Sharpened experimental EM density is shown as a gray mesh. A van der Waals contact between Dot1L F131 and H3 T80 is shown as thick a dashed black line, and the hydrogen bonds between R19 and the H3K79 loop are shown as thin black dashed lines. (C) Formation of the Dot1L H3K79 lysine-binding channel. Dot1L is depicted as a green cartoon surrounded by a semi-transparent green surface. H3K79Nle is shown as blue sticks. (D) Endpoint H3K79 methylation activity assays using Dot1L mutants with either unmodified or H2B-Ub nucleosomes. Error bars correspond to the standard deviation of three replicates. See also Figure S7 and Table S1.
	Figure S1. Cryo-EM Processing Pipelines, Related to Figure 1 A, EM processing pipeline for the Poised state structure. The displayed micrograph is low-pass filtered to 20Å for improved particle visibility. B, EM processing pipline for the Active state structures. The displayed micrograph is low-pass filtered to 20Å for improved particle visibility. The Relion 3 particle-based CTF-refinement, beamtilt correction and Bayesian polishing tools were applied in an iterative manner until there was no further change in the resolution or maps.
	Figure S2. Cryo-EM Map and Model Validation, Related to Figure 1 A, Left, Fourier Shell Correlation (FSC) and, right, local resolution assessment by Resmap for the poised Dot1L-nucleosome reconstruction. The final resolution as determined by FSC 0.143 criterion is 3.9 Å. The local resolution in Å is displayed on the sharpened full map (middle = surface, right = cross section). B, FSC and local resolution assessment for the active 2-to-1 Dot1L-nucleosome structure depicted as in A. The final resolution is 2.96Å as determined by the FSC 0.143 criterion. C, FSC and local resolution assessment for the active 1-to-1 Dot1L-nucleosome structure depicted as in a and B. The final resolution is 3.5Å as determined by the FSC 0.143 criterion. D-E, Model-Map FSC curves calculated between the refined atomic models and the masked, sharpened half maps used for refinement (Red, FSCwork), the second masked, sharpened half-map not used in refinement (Blue, FSCtest) and the full, sharpened map (Black, FSCFull). Model-Map FSCs are shown for the Poised structure, D, the 2-to-1 active structure, E, and the 1-to-1 active structure, F. The close agreement between the FSCwork and FSCtest curves and the absence of significant correlation beyond the calculated map resolution for all three curves indicates absence of overfitting and model bias.
	Figure S3. Details of the Poised-State Structure, Related to Figures 1, 3, and 4 A, EM density for the poised-state structure. The unsharpened density is shown as a transparent surface and the sharpened density is shown as an opaque surface colored as in the main text. The N- and C-terminal parts of Dot1L are denoted with C and N. B, The poised state structure atomic model colored as in the main text. C, Close up view of Dot1L in the poised state showing that the only direct contact between Dot1L and the nucleosome is through the acidic patch. The F131 and W305 loops do not have defined density in the poised state and are depicted as yellow dashed lines. D, Detailed view of the poised state interaction with the acidic patch superimposed with the active state structure. The poised state structure is colored yellow and the active state structure is colored green. Sharpened EM density for the poised state structure is shown as a transparent gray surface and acidic patch residues of H2A and H2B are shown as sticks. E, Close up view of the poised state Dot1L active site showing that a modeled SAM cofactor is too far for the methyl transfer reaction to occur.
	Figure S4. Example Density of the 2:1 Active-State EM Structure, Related to Figure 1 A, A vertical slice through the 2:1 active state structure centered on H3K79Nle. Atomic models of all chains are shown as sticks and the EM density is shown as a gray mesh. Areas highlighted in the boxes are denoted with letters b, c, d and e. B, Example density of the SAM cofactor. C, Example density of the interaction between ubiquitin and the Dot1L C-terminal helix. D,E, Example EM density of the DNA.
	Figure S5. Structural Comparisons of Dot1L in Different States, Related to Figures 3 and 4 A Superimposition between the active state Dot1L and the Dot1L crystal structure (PDB: 1NW3). B, Superimposition between the active state and poised sate Dot1L. The disordered Dot1L F131 and W305 loops in the poised state are depicted as dashed yellow lines. C, Close up view of loop restructuring that occurs in Dot1L during the transition to the active state. The active state loops are colored red and the loops from the crystal structure of Dot1L (PDB: 1NW3) are colored blue. D, Close up view of the superimposed Dot1L-ubiquitin interaction in the poised and active states showing that the contact surface does not change. The convex surface made up by the Dot1L C-terminal helix and the β8- β9 loop is depicted as a dashed line. E, Surface representation of the nucleosome colored by electrostatic potential. The highly charged acidic patch is indicated by the black square. F-J, Close up views of the arginine anchor interaction with the acidic patch for different protein-nucleosome complexes. F, Dot1L, G, LANA peptide (PDB: 1ZLA), H, RCC1 (PDB: 3MVD), I, Sir3 (PDB: 3TU4), J, PRC1 Ubiquitylation Module (PDB: 4R8P).
	Figure S6. Details of the H4 Tail Interaction with Dot1L, Related to Figure 5 A, Example density of the H4 tail in the poised state structure is depicted as a gray mesh and shows that residues after K20 are not structured. B The active state Dot1L structure (red) is superimposed with several high resolution nucleosome crystal structures (PDB: 1KX3,1KX5, 5YOD, 1S32, 3UTA, 5Y0C, 3C1B, 3UT9 and 3UTB) colored tan showing that the H4 tail is usually unstructured or associated with DNA. C, The Dot1L crystal structure (PDB: 1NW3) is depicted in cartoon representation and colored brown with a semi-transparent gray surface. The groove for H4 tail is closed in this state. D, The active Dot1L structure is depicted in cartoon representation and colored green with a semi-transparent gray surface. The groove for the H4 tail is open in this state.
	Figure S7. Details of the H3K79 Conformational Change, Related to Figures 6 and 7 A, The active state Dot1L structure (blue) is superimposed with the poised Dot1L structure (yellow) and several high resolution nucleosome crystal structures (PDB: 1KX3,1KX5, 5YOD, 1S32, 3UTA, 5Y0C, 3C1B, 3UT9 and 3UTB) colored tan showing that the sidechain of H3K79 is always held in an inaccessible state. B, Superimposition of the active state Dot1L (green) the yeast Dot1p crystal structure (PDB: 1U2Z, gray) showing the relative conformations of the W305 and F131 loops. The yeast Dot1p residue W543 is in a similar position as W305 in Dot1L. C, Overview of the 1-to-1 active state structure. D, Close up view of the H3K79Nle loop on the side of the nucleosome that is bound by Dot1L showing that H3K79Nle is oriented upward. E, Close up view of the H3K79Nle loop on the side of the nucleosome that is not bound by Dot1L, showing that H3K79Nle is oriented in the inaccessible conformation. F, superimposition between active state Dot1L (green) and several structures of inhibitor bound Dot1L (gray, PDB: 4EKG, 4EKI, 3UWP, 4EQZ, 4ER0, 4ER3, 4ER5, 4ER6, 4ER7,4HRA, 5MW3, 4WV1, 5DTM, 5DTQ, 5DTR, 5DRT, 5DRY, 5DSX, 5DT2, 5MVS, 5MW4) showing that the F131 loop (blue) restructures upon inhibitor binding. F131 in the various inhibitor-bound structures is shown in red stick representation.

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