1. Volume 63, Issue 6, Pages 965-975 (September 2016)
The NuA4 Core Complex Acetylates Nucleosomal Histone H4 through a Double Recognition Mechanism 
Peng Xu, Chengmin Li, Zhihong Chen, Shuanying Jiang, Shilong Fan, Jiawei Wang, Junbiao Dai, Ping Zhu, Zhucheng Chen 
Molecular Cell 
Volume 63, Issue 6, Pages (September 2016)
DOI: /j.molcel
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2. Molecular Cell 2016 63, 965-975DOI: (10.1016/j.molcel.2016.07.024)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1 Structure of the NuA4 Core Complex
(A) Domain architectures of Esa1, Epl1, Yng2, and Eaf6 colored green, orange, yellow, and cyan, respectively. The Tudor domain of Esa1, the C-terminal domain (CTD) of Epl1, and the plant homeodomain (PHD) of Yng2 are not present in the crystal structures and are colored gray.
(B and C) Two orthogonal views of the structure of the NuA4 core complex. Esa1 is shown as a surface presentation with the catalytic pocket colored red in (B). The CataL and the HBL are labeled in (C). The bound histone peptide is colored magenta.
See also Figures S1 and S2.
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4. Figure 2 Conformational Changes of the HBL of Esa1
(A) Superposition of the structures of the catalytic pocket of Esa1 in the NuA4 core complex and in the free state. Free Esa1 is colored gray (PDB: 3T09). The CataL and the HBL are colored red.
(B) Interactions between the FRR motif of Epl1 and the HBL of Esa1. Hydrogen-bond interactions are shown as dotted lines.
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5. Figure 3 Histone Tail Recognition by the Catalytic Pocket of Esa1
(A) Interactions between the catalytic pocket of Esa1 and the histone Htz1 peptide.
(B) Activity of the NuA4 core complex toward WT and mutant histone H4-K16 peptides. 1 μM NuA4 core complex was mixed with 800 μM histone peptides, and the reactions were monitored using pyruvate dehydrogenase coupling assays. Error bars indicate the SD of three independent measurements.
(C) Sequences of the N-terminal tails of yeast canonical histones and Htz1, with the target lysine residues colored blue and the P-1 positions colored green. The Htz1 peptide used for activity assays and crystallization is underlined in magenta. The H4-K16 peptide used for activity assays is underlined in black.
(D) Relative activities of the WT and four mutants of the NuA4 core complex toward histone H4 (black bars) and Htz1 (yellow bars) peptides and the NCP (white bars). Activities toward nucleosomes were detected by western blotting using 10 nM NuA4 core complex and 1 μM NCP. Error bars indicate SD (n = 3).
(E) MMS sensitivity assays. A 10-fold serial dilution of each culture of yeast was spotted onto medium with or without 0.03% MMS. RR/EE, R318E/R324E; RK/DD, R321D/K322D.
(F) Superposition of the structures of the NuA4 core complex bound with histone peptide (green) and acetyl-CoA (gray).
See also Figures S3 and S4.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
Cryo-EM Structure of the NuA4 Core Complex with the Nucleosome
(A) Side view of the EM map docked with the crystal structures of the NuA4 core complex and the 601 sequence NCP (PDB: 3MVD) and the NMR structure of the Tudor domain of Esa1 (PDB: 2RO0). The spatial locations of the NCP, the catalytic core, and the helical bundle are indicated. The density maps of the nucleosome and the NuA4 core complex are colored gray and green, respectively. One copy of the N-terminal tails of H4, H3, H2A, and H2B are presented as spheres and colored dark green, blue, pink, and purple, respectively. The nucleosome NuA4-contacting site above the H2A-H2B dimer is highlighted with a dashed red circle.
(B) Top view of the EM structure. The catalytic residue (E338) of Esa1 is presented as red spheres.
(C) View into the catalytic pocket. The nucleosome-DFL (R321/K322)-contacting site is highlighted with a circle. The slice shown in (D) is indicated. Asp24 of H4 and Lys13 of H2A are colored red and labeled.
(D) View perpendicular to the slice in (C). The spatial locations of Esa1, Epl1, the helical bundle, and the Tudor domain are indicated.
See also Figures S5 and S6.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
Biochemical Analyses of the Recognition of Nucleosomal H4 by the NuA4 Core Complex
(A) Nucleosome acetylation analyzed by western blotting. WT and H3-4 mutant NCPs (1 μM) were mixed with the NuA4 core complex (10 nM). Acetylation was detected using an antibody against acetylated-K14 of H3 (top), and the positions of different histones migrated in the gels were visualized with Coomassie brilliant blue (bottom). Mutant H3-4 migrates slightly more slowly than WT H4. Omission of the enzyme complex was used as a control (Con). Three independent experiments were performed, and representative gels are shown.
(B) Activities of WT and DFL mutants of the NuA4 core complex. The activities toward nucleosomes were determined as in (A), except that a pan-antibody against acetylated-lysine was used (top). The relative activities toward NCP (bottom, open bars) and histone peptide (solid bars) are reported. The activities toward the H4-K16 peptide were measured as described in Figure 3B. Error bars indicate SD (n = 3).
(C) Western blot analyses of acetylation of WT and two H2A mutant NCPs by the NuA4 core complex. AAA and E61A, H2A with mutations D90A/E91A/E92A and E61A, respectively. The experiments were performed as in (B).
(D) SPR analysis of the binding of the EpcA-N domain of Epl1 to WT (red circle) and mutant (D90R/E91K/E92K, black triangle) H2A-H2B dimers.
See also Figure S4E.
Molecular Cell  , DOI: ( /j.molcel )
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