Volume 53, Issue 3, Pages 498-505 (February 2014) The Catalytic Subunit of the SWR1 Remodeler Is a Histone Chaperone for the H2A.Z- H2B Dimer  Jingjun Hong, Hanqiao Feng, Feng Wang, Anand Ranjan, Jianhong Chen, Jiansheng Jiang, Rodolfo Ghirlando, T. Sam Xiao, Carl Wu, Yawen Bai  Molecular Cell  Volume 53, Issue 3, Pages 498-505 (February 2014) DOI: 10.1016/j.molcel.2014.01.010 Copyright © 2014 Elsevier Inc. Terms and Conditions

Molecular Cell 2014 53, 498-505DOI: (10.1016/j.molcel.2014.01.010) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 Structure of the Swr1589–650-scH2B-H2A.Z Complex (A) Illustration of the location of the ATPase domains and the amino acid sequence in region 599–627 of the Swr1 subunit. The letters in bold indicate the amino acids that are folded in the complex, whereas the regular letters indicate the disordered loop. (B) Deviation of Cα chemical shifts of Swr1589–650 from random coil values: Swr1589–650 in the free form (top) and in complex with scH2B-H2A.Z (bottom). The regions in red are not observable, given that they form folded structures in the complex and the complex is larger than 25 kDa. (C) Velocity sedimentation result of the Swr1589–650-scH2B-H2A.Z complex, showing a 2.54S complex with a molecular weight of 27.7 kDa. (D) Overall structure of the Swr1589–638-scH2B-H2A.Z complex. Only regions 599–605 and 614–627 in the Swr1-Z domain are folded in the complex. See also Figure S1. Molecular Cell 2014 53, 498-505DOI: (10.1016/j.molcel.2014.01.010) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 Specific Interactions between the Swr1-Z Domain and scH2B-H2A.Z (A–C) Hydrophobic and electrostatic interactions between the Swr1-Z domain and scH2B-H2A.Z. (D–F) Effects of mutations on the binding affinity between the Swr1-Z domain and scH2B-H2A.Z. (G) Comparison of the H2A.Z αC helix in the Swr1-Z domain-scH2B-H2A.Z complex and the corresponding region in the H2A model. (H) Effects of replacement of the αC residues in H2A.Z by those of H2A or vice versa on the binding affinity. Here, Kd(WT) is the dissociation equilibrium constant between the Swr1-Z domain and scH2B-H2A.Z. See also Figure S2 and Table S1. Molecular Cell 2014 53, 498-505DOI: (10.1016/j.molcel.2014.01.010) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 Chaperone Function of the Swr1-Z Domain (A) The structural difference between the C-terminal region of H2A.Z in the crystal structure of the Swr1-Z domain-scH2B-H2A.Z complex and that of H2A in the nucleosome (PDB ID: 1ID3). (B) The Swr1-Z domain and Chz1 bind to opposite surfaces on H2A.Z-H2B. (C) Velocity sedimentation of a mixture of Swr1589–650, scH2B-H2AZ, and Chz163–124, showing an S20,w of 2.92 S (36.0 kDa), indicating that Chz1, H2A.Z-H2B, and Swr1 form a heterotetrameric complex. (D) The Swr1-Z domain blocks sites on H2A.Z-H2B for DNA and H3 binding in the nucleosome. (E) Salt elution of the mixture of DNA-(H3-H4)2 tetrasome and H2A.Z-H2B with (solid line) and without (dashed line) the presence of the Swr1-Z domain. (F) Comparison of the amount of histones H3 and H4 in the tetrasome input with those in the nucleosome. The top panel shows an SDS gel of the input tetrasome and the reconstituted nucleosome produced in the presence of the Swr1-Z domain. The bottom panel shows the cross-section of the H4 bands in the top panel. See also Figure S3. Molecular Cell 2014 53, 498-505DOI: (10.1016/j.molcel.2014.01.010) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 The Swr1-Z Domain Is important for SWR1 Function and Is Conserved (A) Mutations in the Swr1-Z domain impair H2A.Z-H2B incorporation into the nucleosome in vitro. In this assay, the mononucleosome was reconstituted with the Widom 601 DNA plus a 40 bp linker DNA at one end (Ranjan et al., 2013). The octameric nucleosome with two H2A or one H2A and one H2A.Z or two H2A.Z is represented with AA, ZA, or ZZ, respectively. (B) Mutations in the Swr1-Z domain led to slower cell growth in the presence of caffeine. K727G mutation in Swr1 completely inactivates the ATPase activity of SWR1. The SD of three experiments for each point is close to the size of the symbol. (C) Mutations in the Swr1-Z domain reduce the incorporation of H2A.Z at promoter regions of some genes. ARS610 is the control. The error bars are SD of three ChIP experiments. (D and E) NMR spectra of p400945–1,017 and SRCAP501–607 in complex with H2A.Z-H2B. Peaks from free p400945–1,017 and SRCAP501–607 are shown in red. Peaks from p400 and SRCAP in 1:1 ratio with H2A.Z-H2B are shown in blue. The disappeared p400945–1,017 and SRCAP501–607 peaks upon the addition of H2A.Z-H2B indicate they are associated with the histones and becomes part of the folded region of the complex. (F and G) ITC results of titrations of p400945–1,017, SRCAP501–607, and their mutants to the H2A.Z-H2B dimer and the H2A-H2B dimer. See also Figure S4 and Table S2. Molecular Cell 2014 53, 498-505DOI: (10.1016/j.molcel.2014.01.010) Copyright © 2014 Elsevier Inc. Terms and Conditions