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Structural basis of H2A.Z recognition by SRCAP chromatin-remodeling subunit YL1

Nature Structural & Molecular Biology volume 23, pages 317–323 (2016)Cite this article

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Abstract

Histone variant H2A.Z, a universal mark of dynamic nucleosomes flanking gene promoters and enhancers, is incorporated into chromatin by SRCAP (SWR1), an ATP-dependent, multicomponent chromatin-remodeling complex. The YL1 (Swc2) subunit of SRCAP (SWR1) plays an essential role in H2A.Z recognition, but how it achieves this has been unclear. Here, we report the crystal structure of the H2A.Z-binding domain of Drosophila melanogaster YL1 (dYL1-Z) in complex with an H2A.Z–H2B dimer at 1.9-Å resolution. The dYL1-Z domain adopts a new whip-like structure that wraps over H2A.Z–H2B, and preferential recognition is largely conferred by three residues in loop 2, the hyperacidic patch and the extended αC helix of H2A.Z. Importantly, this domain is essential for deposition of budding yeast H2A.Z in vivo and SRCAP (SWR1)-catalyzed histone H2A.Z replacement in vitro. Our studies distinguish YL1-Z from known H2A.Z chaperones and suggest a hierarchical mechanism based on increasing binding affinity facilitating H2A.Z transfer from SRCAP (SWR1) to the nucleosome.

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Figure 1: Characterization of YL1-Z and overall structure of YL1-Z–H2A.Z–H2B.
Figure 2: Structural details of interactions between YL1-Z and H2A.Z–H2B.
Figure 3: Characterization of specific interactions between YL1-Z and H2A.Z–H2B.
Figure 4: H2A.Z residues recognized by YL1-Z.
Figure 5: The central role of Swc2-Z (YL1-Z) in H2A.Z replacement in budding yeast.
Figure 6: YL1-Z acts as a histone chaperone.
Figure 7: Model for H2A.Z–H2B transactions mediated by YL1.

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Acknowledgements

We thank Y. Bai, B. Zhu, C. Tang and Y. Huang for invaluable suggestions; Shanghai Synchrotron Radiation Facility beamline scientists for technical support with data collection; Y. Chen, H. Wang and J. Wang for advice on ITC analysis; and X. Yu for assistance with AUC analysis. The study was supported by grants from the Chinese Ministry of Science and Technology (2015CB856200 and 2013CB910203 to Z. Zhou) and grants from the Natural Science Foundation of China (31521002, 31270833 and 31123003 to Z. Zhou). C.W. and A.R. are supported by the Janelia Research Campus, Howard Hughes Medical Institute. F.W. and D.W. are supported by the Center for Cancer Research, US National Cancer Institute. Z. Zhou is supported as a CAS-Novo Nordisk Great Wall Professor.

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  1. Xiaoping Liang, Shan Shan, Lu Pan and Jicheng Zhao: These authors contributed equally to this work.

Authors and Affiliations

  1. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Xiaoping Liang, Shan Shan, Lu Pan, Jicheng Zhao, Zhuqiang Zhang, Yingzi Huang, Li Huang, Xuehui Liu, Qiang Zhong, Jizhong Lou, Guohong Li & Zheng Zhou

  2. University of Chinese Academy of Sciences, Beijing, China

    Xiaoping Liang, Lu Pan, Li Huang & Zheng Zhou

  3. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA

    Anand Ranjan & Carl Wu

  4. Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA

    Feng Wang, Hanqiao Feng, Debbie Wei & Carl Wu

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  1. Xiaoping Liang
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Contributions

X. Liang performed the ITC experiments and histone deposition assays. S.S. reconstituted the complex and determined the structure. L.P. and J.Z. performed the ChIP-qPCR experiments and subcellular colocalization assays. A.R., F.W., D.W. and L.P. performed the EMSAs and histone exchange assays. X. Liu and H.F. performed NMR experiments. Q.Z. and J.L. performed binding-energy calculations. Y.H., Z. Zhang, L.H. and G.L. performed biochemical experiments and data analysis. C.W. and Z. Zhou designed the study, analyzed the data and wrote the manuscript. All authors contributed to experimental design and commented on the manuscript.

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Correspondence to Carl Wu or Zheng Zhou.

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Integrated supplementary information

Supplementary Figure 1 Single-chain histone dimers form stable 1:1 complexes with YL1-Z (Swc2-Z).

(a), The single chain H2A.Z–H2B and single chain H2A–H2B used in this study. Shown on top are the sequence alignments of human and yeast single chain H2A.Z–H2B (scZB) and corresponding secondary structures. H2B (red), H2A.Z (yellow), and extended H2A.Z αC-helix (orange). Yeast-specific residues colored in red. (b), Sedimentation velocity experiments show that human YL1-Z (hYL1-Z) and Drosophila YL1-Z (dYL1-Z) mainly form a 1:1 complex with human scZB (hscZB), in both 0.2 M NaCl and 0.5 M NaCl. The svedbergs value (S) and molecular weight (kD) are shown in each figure. (c), Sedimentation equilibrium experiments show that both human YL1-Z residues 1–77 and human YL1-Z residues 12–77 form a 1:1 complex with yeast scZB in 0.1 M NaCl. Removal of 11 residues within hYL1-Z Rn-region shows no effect on stoichiometry of the complex.

Supplementary Figure 2 Human YL1-Z is intrinsically disordered.

(a), 1H–15N heteronuclear single quantum coherence (HSQC) spectra of human YL1-Z in histone-free form. The backbone 1H–15N resonances of hYL1 residue 1–77 were fully assigned and the peaks representing YL1-Z residues were labeled. (b), The hYL1-Z backbone Cα chemical shift deviations corresponding to random coil values are presented. The results show that hYL1-Z largely remains as a disordered polypeptide.

Supplementary Figure 3 Typical ITC data with fitting curves.

(a), Isotherms for titration of hYL1-Z (WT and mutants) to scZB and scAB in 0.2 M NaCl. (b), Isotherms for titration of hYL1-Z (WT and mutants) to scZB or scAB (WT and mutants) in 0.5 M NaCl.

Supplementary Figure 4 Crystal packing of the dYL1-Z–H2A.Z–H2B crystal and the omit map for dYL1-Z.

(a), Cross-section of the crystal lattice for dYL1-Z–H2A.Z–H2B complex. Two complex molecules in the neighbouring asymmetric units are shown in surface diagram or ribbon diagram. Colored in orange are H2A.Z αC-helix and L2-loop showing conformation changes. The crystal packing reveals binary interactions between two H2A.Z–H2B dimers and both dYL1-Z Rn- and Rc- regions. (b), Close view of dYL1-Z Rn-region bound to scZB. The dYL1-Z Rn interacts with H2A.Z residues (E64, E67, K74, D93, E94, E95), which are either identical (E64, E67, D93, E94, E95) or highly conserved (K74) in H2A. (c), The omit map of dYL1-Z generated by PHENIX. The map is contoured at the 1.0- level at 1.9-Å resolution. The figure was generated by Pymol. dYL-1 (magenta), H2A.Z (yellow), H2B (red), and the extended H2A.Z αC-helix (orange).

Supplementary Figure 5 Effect of yeast Swc2-Z regional mutations on H2A.Z exchange activity and H2A.Z occupancy.

(a), Schematic view of Swc2-Z(YL1-Z) family and Mut2 regions subject to mutagenesis. Residues substituted with alanine are labeled "A". (b), Phenotypes of WT, swc2Δ, swc2-ΔZ (Swc2 residues 1–89 are deleted) strains. (c), ChIP-PCR for H2A.Z-HA occupancy in yeast strains containing Swc2 WT, Swc2-Mut2 and Swc2-ΔZ, in which Swc2 residues 1–89 are deleted. (d), Histone exchange assay for SWR1 WT, SWR1 (Swc2 Mut2) and SWR1 (Swc2-ΔZ). The reaction mix contained 5 nM nucleosome, 2 nM SWR1, 5 nM H2A.Z 3×Flag–H2B dimer and 1 mM ATP.

Supplementary Figure 6 Comparative analysis of H2A.Z–H2B recognition modes for H2A.Z or H2A chaperones.

Structures in comparison are H2A.Z chaperone YL1-Z, Anp32e-ZID (4CAY) (Obri, A. et al. Nature 505, 648-53, 2014), Swr1-Z (4M6B) (Hong, J. et al. Mol Cell 53, 498-505, 2014), Chz1-Z (2JSS) (Zhou, Z. et al. Nat Struct Mol Biol 15, 868-9, 2008) and H2A chaperone FACT subunit Spt16 residue 915–943(4KHO) (Hondele, M. et al. Nature 499, 111-4, 2013) and residue 958–999 (4WNN) (Kemble, D.J. et al. Mol Cell 60, 294-306, 2015). The chaperone structures are shown in ribbon mode and histone structures in surface mode. (a), Interaction between H2A.Z-H2B dimer and H2A.Z chaperone YL1-Z (left), Anp32e-ZID (middle) and Swr1-Z (right). Structures of YL1-Z, Anp32e-ZID and Swr1-Z are colored in magenta, cyan and blue. The H2A.Z αC-cleft and H2B pocket recognized by H2A.Z chaperones are highlighted in a dotted rectangle and circle, respectively. (b), Close view of H2A.Z αC-cleft (right) and H2B pocket (left) in contact with H2A.Z chaperones. Structures of YL1-Z, Anp32e-ZID, Swr1-Z are superimposed. While YL1-Z interacts with the H2A.Z αC-cleft using the end of the α1-helix followed by a β-bend, Anp32e-ZID and Swr1-Z both use a short helix for interaction. Importantly, YL1-Z, Anp32e-ZID and Swr1-Z each bury a similar aromatic residue (Y44, F623, Y235, respectively) in the H2B pocket. (c), Structure of yeast FACT subunit Spt16 residues 915–956 (colored in green) interacting with H2A–H2B (left) and close view of the contact with H2B pocket (right). The side chain of F939 is colored in orange for clear view. (d), Structure of yeast FACT subunit Spt16 residues 958–999 (colored in orange) interacting with H2A–H2B (left) and close view of the contact with H2B pocket (right). (e), Structure comparison of YL1-Z and Chz1. Chz1 residues 63–124 (green) are shown in ribbon representation. Binding of YL1-Z and Chz1 at the H2A.Z α1–α2 patch, highlighted in the dotted circle, are mutually exclusive.

Supplementary Figure 7 Binding energy of H2A.Z–H2B in complex with YL1-Z, Swr1-Z, Anp32e ZID, Chz1-Z or the H2A.Z nucleosome.

The structures used for calculation are YL1-Z (this study), Anp32e-ZID (4CAY) (Obri, A. et al. Nature 505, 648-53, 2014), Swr1-Z (4M6B) (Hong, J. et al. Mol Cell 53, 498-505, 2014), Chz1-Z (2JSS) (Zhou, Z. et al. Nat Struct Mol Biol 15, 868-9, 2008), H2A.Z nucleosome (H2A.Z Nucleosome, 1F66) (Suto, R.K. et al. Nat Struct Biol 7, 1121-4, 2000). Highlighted in red are values calculated by using YL1-Z, include YL1 residue 24–46 (YL1-Z Rc-1 and Rc-2) interacting with H2A.Z αC-cleft and H2B pocket, and YL1 residue 46–69 (YL1-Z Rc-3) interacting with H2A.Z α1–α2 patch.

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Liang, X., Shan, S., Pan, L. et al. Structural basis of H2A.Z recognition by SRCAP chromatin-remodeling subunit YL1. Nat Struct Mol Biol 23, 317–323 (2016). https://doi.org/10.1038/nsmb.3190

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