Structural basis of nucleosome recognition and modification by MLL methyltransferases

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Abstract

Methyltransferases of the mixed-lineage leukaemia (MLL) family—which include MLL1, MLL2, MLL3, MLL4, SET1A and SET1B—implement methylation of histone H3 on lysine 4 (H3K4), and have critical and distinct roles in the regulation of transcription in haematopoiesis, adipogenesis and development1,2,3,4,5,6. The C-terminal catalytic SET (Su(var.)3-9, enhancer of zeste and trithorax) domains of MLL proteins are associated with a common set of regulatory factors (WDR5, RBBP5, ASH2L and DPY30) to achieve specific activities7,8,9. Current knowledge of the regulation of MLL activity is limited to the catalysis of histone H3 peptides, and how H3K4 methyl marks are deposited on nucleosomes is poorly understood. H3K4 methylation is stimulated by mono-ubiquitination of histone H2B on lysine 120 (H2BK120ub1), a prevalent histone H2B mark that disrupts chromatin compaction and favours open chromatin structures, but the underlying mechanism remains unknown10,11,12. Here we report cryo-electron microscopy structures of human MLL1 and MLL3 catalytic modules associated with nucleosome core particles that contain H2BK120ub1 or unmodified H2BK120. These structures demonstrate that the MLL1 and MLL3 complexes both make extensive contacts with the histone-fold and DNA regions of the nucleosome; this allows ease of access to the histone H3 tail, which is essential for the efficient methylation of H3K4. The H2B-conjugated ubiquitin binds directly to RBBP5, orienting the association between MLL1 or MLL3 and the nucleosome. The MLL1 and MLL3 complexes display different structural organizations at the interface between the WDR5, RBBP5 and MLL1 (or the corresponding MLL3) subunits, which accounts for the opposite roles of WDR5 in regulating the activity of the two enzymes. These findings transform our understanding of the structural basis for the regulation of MLL activity at the nucleosome level, and highlight the pivotal role of nucleosome regulation in histone-tail modification.

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Fig. 1: Overview of the structure of the human MLL1–ubNCP complex.
Fig. 2: Interfaces between MLL1 complex and ubNCP.
Fig. 3: Cryo-EM structures of MLL1 in complex with an unmodified nucleosome.
Fig. 4: Structural comparison of human MLL1 and MLL3 complexes.

Data availability

The electron microscopy density maps have been deposited in the Electron Microscopy Data Bank with accession codes EMDB-9998, EMDB-9999, EMDB-0693, EMDB-0694 and EMDB-0695. The final models have been submitted to the PDB with accession codes 6KIU, 6KIV, 6KIW, 6KIX and 6KIZ. All other data are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank the staff of the Electron Microscopy System and the Database and Computation System of the National Facility for Protein Science in Shanghai (NFPS), Zhangjiang Laboratory for their assistance with the electron microscopy instruments and data pre-processing. We thank members of the NFPS Large-scale Protein Preparation System and Mass Spectrometry System for instrument support and technical assistance. This work was supported by the National Key R&D Program of China (2017YFA0504504 and 2016YFA0501803), the National Natural Science Foundation of China (31570766 and U1632130), the Shanghai Municipal Education Commission–Gaofeng Clinical Medicine Grant Support (2017YZ004), the SHIPM-sigma fund No. BJ1-7009-18-1303 and JY201805 from Shanghai Institute of Precision Medicine, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine and Chinese Academy of Sciences Facility-based Open Research Program. J.H. is a recipient of the Thousand Young Talents Program of China and a recipient of the Hundred Talents Program of Shanghai Jiao Tong University School of Medicine.

Author information

H.X. performed sample preparation and biochemical analyses; T.Y. performed electron microscopy data collection; M.C. and Y.L. helped with electron microscopy data collection and analyses; G.Z. and G.Y. helped with nucleosome preparation; M.L. and Y.C. provided valuable advice on the project and the manuscript; and J.H. designed and supervised all the research, and wrote the manuscript with help from all other authors.

Correspondence to Jing Huang.

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Peer review information Nature thanks Brian Stahl and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 Biochemical and structural characterizations of human MLL1–ubNCP complex.

a, Gel filtration (left) and SDS–PAGE analysis (right) of the assembly of the recombinant MLL1 complex, composed of full-length WRAD proteins and MLL1 (residues 3754–3969). Experiments were repeated at least three times with similar results. b, EMSAs of the recombinant human MLL1 complex with either unmodified or H2BK120ub1 NCPs at molar ratios of 1:1, 2:1 and 4:1. Top, input of the EMSA mixtures. Bottom, native PAGE analysis of the gel shifting of NCP and ubNCP by the MLL1 complex. Each assay was repeated at least three times with similar results. c, Flow chart of cryo-EM data processing of the MLL1–ubNCP dataset (resolution of 3.2 Å). Masked 3D classifications without realignment were applied to MLL1–WDR5 (left branch), ASH2L (middle branch) and RBBP5–ubiquitin (right branch).

Extended Data Fig. 2 Cryo-EM analysis of human MLL1–ubNCP complex.

a, Representative micrograph of the cryo-EM dataset of the MLL1–ubNCP complex. b, Representative 2D class averages of cryo-EM particles of the MLL1–ubNCP complex. c, Angular distribution of particle projections of the MLL1–ubNCP reconstruction. d, The gold-standard FSC curve calculated between two halves of the MLL1–ubNCP dataset. e, Local-resolution estimates of the structure of the MLL1–ubNCP complex. f, Representative electron microscopy density maps of the MLL1 complex. g, Representative electron microscopy density maps of the H2BK120ub1–NCP.

Extended Data Fig. 3 Flow chart of cryo-EM data processing of the MLL1–ubNCP dataset, and overall structural comparison of human MLL1 complex with yeast COMPASS complexes.

a, Flow chart of cryo-EM data processing of the MLL1–ubNCP dataset (resolution of 4.0 Å) that reveals the electron microscopy density of the pre-SPRY domain of ASH2L and the dynamic conformations of H2BK120-conjugated ubiquitin. Masked 3D classifications without realignment were applied to the entire MLL1 complex (left branch), the MLL1–ASH2L region (middle branch) and the RBBP5–ubiquitin region (right branch). b, Overall structural comparison of human MLL1 complex with yeast COMPASS complexes. Each MLL1 component and its yeast orthologue are denoted with the same colour.

Extended Data Fig. 4 RBBP5-mediated interactions with NCP, ubiquitin and other subunits of MLL1.

a, b, Density maps of the RBBP5WD40 loops that interact with the histone H2B–H4 cleft, shown in stereo mode. c, Density maps of the H2BK120-conjugated ubiquitin from different 3D classes of the MLL1–ubNCP dataset, shown in stereo mode. The major and minor conformations of RBBP5WD40–H2BK120ub1 are classified on the basis of the percentage of each 3D class from the masked 3D classification of the RBBP5WD40–H2BK120ub1 region. The masked 3D classification was repeated at least three times with similar results. d, Different binding modes of the H2BK120-conjugated ubiquitin to RBBP5WD40 in the MLL3–ubNCP dataset. The major and minor conformations of H2BK120ub1 are classified on the basis of the percentage of each 3D class from masked 3D classification of the RBBP5WD40–H2BK120ub1 region. The masked 3D classification was repeated at least three times with similar results. e, Superposition of all the identified RBBP5WD40–ubiquitin structures from the MLL1–ubNCP and MLL3–ubNCP datasets reveals the movement of H2BK120ub1 between the α-helix-containing loop of RBBP5WD40 and the junction between WDR5 and RBBP5WD40 junction. f, The input of the HMT reactions related to Fig. 2d. The input amounts of the wild-type and mutant MLL1 complexes were quantified according to the band intensities of WDR5. The gel band that results from the incomplete protease 3C digestion of 6×His3C–ubiquitin–H2B is denoted with an asterisk. g, Overview of the structure of the MLL1 complex, showing the cryo-EM density maps of the RBBP5 N- and C-terminal loop regions. The N terminus of RBBP5 (RBBP5N-ter), activation segment and ASH2L-binding motif of RBBP5 (RBBP5AS-ABM), WDR5-binding motif of RBBP5 (RBBP5WBM) and C terminus of RBBP5 (RBBP5C-ter) are coloured in orange, green, cyan, and yellow, respectively. Loop regions of RBBP5 assemble the subunits of the MLL enzyme into an integral complex, through multiple interactions.

Extended Data Fig. 5 Specific recognition between MLL1–ASH2L and the nucleosome.

a, The active site of MLL1SET within the MLL1–ubNCP complex, shown with the density maps of the cofactor-product SAH and the substrate residue H3K4 in stereo mode. b, The recognition interface between MLL1SET and the C-terminal region of histone H2A, shown with the electron microscopy densities of the interacting N-terminal region of MLL1SET in stereo mode. c, The input of the HMT reactions related to Fig. 2g. The input amounts of the wild-type and mutant MLL1 complexes were quantified according to the band intensities of WDR5. d, The input of the EMSAs related to Fig. 2h. e, The input of the HMT reactions related to Fig. 2i. The input amounts of the wild-type and mutant MLL1 complexes were quantified according to the band intensities of MLL1. f, Sequence alignment of the auxiliary β-sheet of ASH2L, with the conserved residues from yeast to human coloured in red and the identical residues from Drosophila to human coloured in blue. ASH2L residues that are important to MLL1 activities are boxed. The auxiliary β-sheet structure of yeast Bre2 (from Kluyveromyces lactis) is denoted with yellow arrows. g, Structural superposition of yeast COMPASS complex (from K. lactis, PDB 6CHG) with the human MLL1–ubNCP complex, aligned between the WD40 domains of human RBBP5 and yeast Swd1 proteins. The auxiliary β-sheet of Bre2 (the yeast counterpart of human ASH2L) is coloured in yellow. h, Enlargement of the region adjacent to the auxiliary β-sheet from g, showing the direct contact of the auxiliary β-sheet of Bre2 with the SHL7 DNA of the nucleosome.

Extended Data Fig. 6 Structural characterization of human MLL1–NCP complex.

a, Flow chart of cryo-EM data processing of the MLL1–NCP dataset. Electron microscopy map for binding mode 2 was not applied with solvent mask (post-processing) because this procedure severely dampened the electron microscopy density of the WDR5–MLL1–ASH2L regions. b, Representative micrograph of the cryo-EM dataset of the MLL1–NCP complex. c, Representative 2D class averages of cryo-EM particles of the MLL1–NCP complex. d, Angular distribution of particle projections from different binding modes of the MLL1–NCP complex. e, The gold-standard FSC curves calculated between two halves of the MLL1–NCP dataset from different binding modes. f, Local-resolution estimates of the MLL1–NCP complex structure from different binding modes.

Extended Data Fig. 7 Biochemical and structural characterizations of human MLL3–ubNCP complex.

a, Gel filtration (left) and SDS–PAGE analysis (right) of the assembly of the recombinant MLL3 catalytic module, composed of full-length WRAD proteins and MLL3 (residues 4707–4911). Experiments were repeated at least three times with similar results. b, EMSAs of the recombinant human MLL3 complex with either unmodified or H2BK120ub1 NCPs at molar ratios of 1:1, 2:1 and 4:1. Top, input of the EMSA mixtures. Bottom, native PAGE analysis of the gel shifting of NCP and ubNCP by the MLL3 complex. Each assay was repeated at least three times with similar results. c, Time course of the H3K4 methylation catalysed by the recombinant human MLL3 complex on unmodified and H2BK120ub1 NCPs. Error bars denote the s.d. from the mean of three replicates. d, Representative micrograph of the cryo-EM dataset of the MLL3–ubNCP complex. e, Representative 2D class averages of cryo-EM particles of the MLL3–ubNCP complex. f, Angular distribution of particle projections of the MLL3–ubNCP reconstruction. g, The gold-standard FSC curve calculated between two halves of the MLL3–ubNCP dataset. h, Local-resolution estimates of the structure of the MLL3–ubNCP complex.

Extended Data Fig. 8 Structural organization of the interface between WDR5, MLL1 and RBBP5 subunits.

a, Detailed view of the interface between WDR5, MLL1SET and RBBP5AS. The key residues of RBBP5AS are shown in stick representation with electron microscopy densities. b, Structural comparison of human MLL1 complex with a group of SET-domain histone methyltransferases—including PRC2 (PDB 5KJH), NSD1 (PDB 3OOI) and DIM5 (PDB 1PEG)—shows that MLL1AS and RBBP5AS correspond to the activation segment motifs (coloured in blue) of these methyltransferases. c, The input of the HMT reactions related to Fig. 4e. The input amounts of the wild-type and mutant MLL1 complexes were quantified according to the band intensities of MLL1. d, End-point HMT assays of mutant MLL1 complex that carries a deletion of MLL1AS (residues 3775–3786). Top, the input of the HMT reactions. The input amounts of the wild-type and mutant MLL1 complexes were quantified according to the band intensities of MLL1. Bottom, end-point HMT assays of the wild-type and mutant MLL1 complexes on unmodified nucleosomes. Each assay was repeated at least three times with similar results. n = 3 independent experiments. Data are mean ± s.d. e, End-point HMT assays of mutant MLL1 complex with a deletion of RBBP5AS (residues 329–336). Top, the input of the HMT reactions. The input amounts of the wild-type and mutant MLL1 complexes were quantified according to the band intensities of MLL1. Bottom, end-point HMT assays of the wild-type and mutant MLL1 complexes on unmodified nucleosomes. Each assay was repeated at least three times with similar results. n = 3 independent experiments. Data are mean ± s.d.

Extended Data Fig. 9 Characterization of the interface between WDR5, MLL3 and RBBP5 in the MLL3–ubNCP complex.

a, Sequence alignment of the catalytic domains of human methyltransferases of the MLL family. The WIN motif, the N-terminal, insertion and C-terminal regions of SET, and the post-SET regions are highlighted with different colours. The MLL1AS region is denoted with a blue line. b, The input of the HMT reactions related to Fig. 4g. The input amounts of the wild-type and mutant MLL1 or MLL3 complexes were quantified according to the band intensities of MLL1 or MLL3, respectively. c, HMT assays of human MLL1 complex, and human MLL3 complexes with or without WDR5, on nucleosome substrates. Top, mono-, di-, and tri-methylation levels of histone H3K4, as determined with antibodies of H3K4me1, H3K4me2 and H3K4me3. Bottom, the input of the HMT reactions. Each assay was repeated at least three times with similar results.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics.

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Xue, H., Yao, T., Cao, M. et al. Structural basis of nucleosome recognition and modification by MLL methyltransferases. Nature 573, 445–449 (2019) doi:10.1038/s41586-019-1528-1

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