FACT Disrupts Nucleosome Structure by Binding H2A-H2B with Conserved Peptide Motifs

Abstract

FACT, a heterodimer of Spt16 and Pob3, is an essential histone chaperone. We show that the H2A-H2B binding activity that is central to FACT function resides in short acidic regions near the C termini of each subunit. Mutations throughout these regions affect binding and cause correlated phenotypes that range from mild to lethal, with the largest individual contributions unexpectedly coming from an aromatic residue and a nearby carboxylate residue within each domain. Spt16 and Pob3 bind overlapping sites on H2A-H2B, and Spt16-Pob3 heterodimers simultaneously bind two H2A-H2B dimers, the same stoichiometry as the components of a nucleosome. An Spt16:H2A-H2B crystal structure explains the biochemical and genetic data, provides a model for Pob3 binding, and implies a mechanism for FACT reorganization that we confirm biochemically. Moreover, unexpected similarity to binding of ANP32E and Swr1 with H2A.Z-H2B reveals that diverse H2A-H2B chaperones use common mechanisms of histone binding and regulating nucleosome functions.

Keywords: Spt16-Pob3; crystal structure; histone binding; histone chaperone; nucleosome reorganization.

Figure 1. Spt16-C and Pob3-C contain the H2A-H2B Binding Determinants of FACT

(A) Domain organization of S. cerevisiae Spt16 and Pob3 showing constructs used for H2A-H2B binding assays. (B) Binding affinities (mean K_D and standard deviations) and stoichiometry (N, sites) determined by ITC. Affinities are for full-length H2A-H2B or a variant (*) that lacks the first 29 residues of H2B (NB is no binding). (C) Isotherms for the constructs numbered in panel B. Isotherm 12 represents the heat of dilution for full-length H2A-H2B. (D) Overlay of raw (top panel) and integrated (bottom panel) ITC data for full-length H2A-H2B binding to full-length FACT (black) and Spt16-ΔC, Pob3-ΔC (blue). FACT binding was fit to a two-site binding model with stoichiometry and affinities shown for both sites. The data for full-length FACT also appears in Figures 3C and 4E. (E) EMSA of FACT (200 nM) titrated with H2A-H2B shows that binding requires the Spt16-C and Pob3-C domains and displays shifts that are consistent with a stoichiometry of 2:1 (H2A-H2B: FACT). See also Figure S1 for residue conservation, Table S1 for strains, and Table S2 for plasmids.

Figure 2. Spt16 and Pob3 minimal binding domains

(A) ITC-derived binding affinities (as in Figure 1) for H2A-H2B^30-130 with Spt16-C peptides. The Minimal Binding Domain (MBD) is indicated. (B) Isotherms and integrated heat responses for Spt16-MBD and Pob3-MBD peptides with H2A-H2B^30-130. (C) Binding affinities for full-length H2A-H2B and the indicated Pob3 peptides. (D) Sequence of the Spt16 acidic domain indicating the sites of truncations assayed in vivo. YPAD is rich medium, -lys is synthetic medium lacking lysine (growth indicates the Spt⁻ phenotype resulting from incomplete repression of the lys2-128∂ reporter allele in these strains; (Rando and Winston, 2012), and HU150 is YPAD with 150 mM hydroxyurea.

Figure 3. Specific Tyr/Phe residues make the largest contributions to binding for Spt16 and Pob3

(A) Alanine mutations (red) in the Spt16-MBD peptide with affinity estimated from ITC for H2A-H2B^30-130 (left) and phenotype when introduced into the SPT16 locus (right). Mean K_D is given in μM with the standard deviation indicated. (B) Equivalent analysis for Pob3. (C) Integrated ITC data for H2A-H2B binding to full-length wild type and mutant FACT. The data for full-length FACT also appears in Figures 1D and 4E. (D) Selected Spt16-C and Pob3-C-MBD mutants display synthetic phenotypes. See also Figure S2.

Figure 4. Spt16^MBD:H2A-H2B structure and biochemical validation of binding to Spt16 and Pob3

(A) Fo-Fc omit map (3× RMSD) around Spt16 E967-Y972. The map was phased on an H2A-H2B model that was refined in the absence of the Spt16 peptide. (B) Hydrogen bonding interactions (green dashed lines). (C) ITC-derived binding affinities of Spt16 and Pob3-MBDs to H2A-H2B^30-130. NB is no binding, nd is not determined. (D) Fluorescence anisotropy binding assays. Fluorescein-Spt16 MBD and unlabeled H2A-H2B (black line), competition of Fluorescein-Spt16 bound to H2A-H2B by unlabeled Spt16-MBD (purple) and unlabeled Pob3-MBD (pink). (E) Raw (top) and integrated (bottom) ITC binding data for full-length FACT and H2A-H2B (black) or full-length FACT and H2A-H2B M62E (red). The data for full-length FACT also appears in Figures 1D and 3C. See also Figure S1 and Table S2.

Figure 5. Spt16-C and Pob3-C are required for nucleosome reorganization

(A) Phenotypes of selected Pob3 (pink) or Spt16 (purple) mutations alone or combined with htb1-M62E (black) or htb2-M62E (gray), all integrated at their native loci. (B) The Spt16-C complex superimposed onto H2A-H2B dimers of the canonical nucleosome (PDB 1ID3; (White et al., 2001). One of these sites is labeled Pob3 in accord with the model that Spt16 and Pob3 bind equivalently to the same reorganized nucleosome. (C) Closeup demonstrating the conflict between Spt16 and DNA binding to H2A-H2B. (D) The rate of digestion by DraI was determined for nucleosomes reconstituted with recombinant S. cerevisiae histones in the presence of Nhp6 and the Spt16-Pob3 heterodimers indicated (Xin et al., 2009); see Figure 1). The rates for Nhp6 alone and with full-length Spt16-Pob3 were set to 0% and 100%, respectively. Each construct was tested at least in triplicate, with the standard deviation shown. (E) Model of FACT binding the components of a nucleosome including two H2A-H2B dimers bound by Spt16-C and Pob3-C and H3-H4 histones bound by the Spt16-N and Spt16-M domains (Kemble et al., 2013; Stuwe et al., 2008) and DJK, TF, CPH unpublished). See also Figure S3.

Figure 6. Superposition of Spt16 with ANP32E and Swr1

(A) Spt16 and ANP32E (PDB 4cay; (Obri et al., 2014) following overlap on H2B molecules. (B) Spt16 and Swr1 (PDB 4m6b; (Hong et al., 2014). (C) Alignment of Spt16, Pob3, ANP32E and SWR1 sequences. (D) Ability to maintain transcriptional repression in strains bearing mutations in aromatic anchor and acidic helix-capping residues, integrated at the endogenous POB3 or SPT16 loci as in Figure 2. See also Figure S4.