Park SH et al. (2019), Cryo-EM structure of the human MLL1 core comple...

XB-ART-56806

Nat Commun 2019 Dec 05;101:5540. doi: 10.1038/s41467-019-13550-2.

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Cryo-EM structure of the human MLL1 core complex bound to the nucleosome.

Park SH , Ayoub A , Lee YT , Xu J , Kim H , Zheng W , Zhang B , Sha L , An S , Zhang Y , Cianfrocco MA , Su M , Dou Y , Cho US .

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Mixed lineage leukemia (MLL) family histone methyltransferases are enzymes that deposit histone H3 Lys4 (K4) mono-/di-/tri-methylation and regulate gene expression in mammals. Despite extensive structural and biochemical studies, the molecular mechanisms whereby the MLL complexes recognize histone H3K4 within nucleosome core particles (NCPs) remain unclear. Here we report the single-particle cryo-electron microscopy (cryo-EM) structure of the NCP-bound human MLL1 core complex. We show that the MLL1 core complex anchors to the NCP via the conserved RbBP5 and ASH2L, which interact extensively with nucleosomal DNA and the surface close to the N-terminal tail of histone H4. Concurrent interactions of RbBP5 and ASH2L with the NCP uniquely align the catalytic MLL1SET domain at the nucleosome dyad, thereby facilitating symmetrical access to both H3K4 substrates within the NCP. Our study sheds light on how the MLL1 complex engages chromatin and how chromatin binding promotes MLL1 tri-methylation activity.

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Species referenced: Xenopus laevis
Genes referenced: ash2l dot1l dpy30 kmt2a rbbp5 wdr5

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	Fig. 1. Cryo-EM structure of the MLL1RWSAD-NCP complex. a Schematic domain architectures for the core components of the human MLL1 complex used in the cryo-EM study. b Immunoblot to detect H3K4 methylation in the in vitro histone methyltransferase assay. Antibodies indicated on right. The substrates were free recombinant histone H3 (left) and the NCP (right), respectively. Immunoblots for unmodified H3 as well as RbBP5 and ASH2L included as controls. c Cryo-EM 3D reconstruction of the MLL1RWSAD-NCP complex. The composite map of MLL1RWSAD-NCP was locally filtered to the estimated resolution. The subcomplexes, i.e., RbBP5-NCP and MLL1RWS-NCP, shown in dashed boxes. d Top (left) and front (right) views of the MLL1RWSAD-NCP structure. The S-adenosyl-l-homocysteine (SAH) was represented as a sphere (red) and the MLL1 core components shown in cartoon representation (RbBP5: cyan, WDR5: green, MLL1SET: slate, ASH2L: pink, and DPY30 dimer: cerulean and teal). Widom 601 DNA and four histones were colored as indicated on the bottom. Two black dashed squares highlighted the nucleosome contact points near SHL1.5 and SHL7 by MLL1RWSAD. Illustrations of the protein structure and cryo-EM maps used in all figures were generated with PyMOL (Delano Scientific, LLC) and Chimera67/ChimeraX70.
	Fig. 2. RbBP5 interaction with the NCP. a The cryo-EM structure of the RbBP5-NCP subcomplex (4.2â€‰Ã…). The interaction interface was enlarged and shown on right. Insertion (I)-loop, Anchoring (A)-loop, and Quad-R of RbBP5, as well as the H4 tail highlighted in purple,Â orange, blue, and red, respectively. Histone H3 shown in green. b Interaction of Quad-R, as indicated, with DNA backbone. Red line, histone H4 tail. c Immunoblot to detect in vitro histone methyltransferase activity with the NCP as the substrate. The MLL1RWSAD complex reconstituted with wild-type and Quad-R-mutated RbBP5 indicated on top. d Immunoblot to detect in vitro histone methyltransferase activity with the NCP as the substrate. The MLL1RWSAD complex reconstituted with RbBP5 wild-type and deletion mutant proteins indicated on top. e The interface between RbBP5 and the H4 tail. Key residues on RbBP5 I-/A-loops indicated. The H4 tail (His18 to core) represented by a red line and the extended tail beyond His18 represented by a dash line. f Structural superposition of the H4 tails upon RbBP5 (cyan) and Dot1L (PDB ID: 6NJ9)31 binding. The RbBP5 and Dot1L at the interfaces enclosed by the blue and pink outlines, respectively. g In vitro pull-down assay for RbBP5 and the NCP. Ni-NTA-bound fractions were shown and His-tagged wild-type or mutant RbBP5 proteins shown on top. Immunoblot for H3 used to detect the NCP in the bound fraction. Immunoblot for H4 used as a control.
	Fig. 3. The WDR5-MLL1SET-ASH2LSPRY-NCP subcomplex. a Rigid-body fitting of human WDR5 crystal structure (PDB ID: 2H14)28 into the cryo-EM map of MLL1RWS-NCP. Secondary structures of WDR5 were shown in green. b Rigid-body fitting of MLL1SET and ASH2LSPRY into the Cryo-EM map of MLL1RWSAD-NCP. The MLL1SET-RbBP5330â€“375-ASH2LSPRY crystal structure (PDB ID: 5F6L)16 was used. Characteristic secondary structures of MLL1SET (SET-I, SET-C and SET-N) were shown within black dashed circles. The catalytic active site represented by red sphere. c, Comparison of human MLL1-NCP structure with yeast SET1 crystal structure (K. lactis, dark goldenrod, PDB ID: 6CHG, right top)20 and cryo-EM structure (S. cerevisiae, light slate blue, PDB ID: 6BX3, right bottom)21. Orange circles indicated IDR regions of ASH2L (human), Bre2 (K. lactis), and Cps60 (S. cerevisiae). An extra domain in S. cerevisiae SET1 complex, Cps40, colored grey.
	Fig. 4. ASH2L interacts with the nucleosomal DNA through IDRs. a Structure prediction of ASH2LIDR. The structure of ASH2L IDR regions was not available and thus not assigned in the corresponding cryo-EM map (dashed circle). The structure prediction approach was employed to model ASH2L IDR regions as described in the STAR methods. Linker-IDR colored green and Loop-IDR colored blue in the ASH2LIDR model structure. b Stereo-view of the ASH2L-DPY30 model structure and its contacts with DNA. The structure of ASH2L is a composite from crystal structure of ASH2LSPRY (PDB ID: 5F6L)16 and the modeled ASH2LIDR. The schematics of ASH2L was shown at the bottom and key residues 202â€“207 in ASH2LIDR were highlighted in red.
	Fig. 5. ASH2L Linker-IDR is important for NCP binding and methyltransferase activity. a Multiple sequence alignment of ASH2L Linker-IDR region (residues 202â€“254). The blue box indicated 205-KRK-207, key residues for NCP recognition. b Top, electrophoretic mobility shift assay of ASH2L and ASH2L mutants as indicated on top. Bottom, the unbound NCP in the gel image was quantified by ImageJ and presented after normalization against the NCP alone signal, which was arbitrarily set as 1 (100%). This experiment was repeated separately to confirm the main conclusions. c Immunoblot to detect in vitro histone methyltransferase activity with the NCP as the substrate. Reconstituted MLL1RWSAD complexes containing wild-type and mutant ASH2L, used as indicated on top. Immunoblots of RbBP5 and ASH2L included as controls.
	Fig. 6. The MLL1SET domain aligns at the nucleosome dyad. a MLL1SET (slate) and two copies of histone H3 (green) were highlighted against the faded MLL1RWSAD-NCP structure. The SAH molecule represented as a sphere and marked the catalytic active site of MLL1SET. b Schematic model of NCP recognition mediated by the MLL1 core complex. The I-loop of RbBP5 and the active site of MLL1SET were colored in blue and red, respectively.

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