DAXX envelops a histone H3.3-H4 dimer for H3.3-specific recognition

Abstract

Histone chaperones represent a structurally and functionally diverse family of histone-binding proteins that prevent promiscuous interactions of histones before their assembly into chromatin. DAXX is a metazoan histone chaperone specific to the evolutionarily conserved histone variant H3.3. Here we report the crystal structures of the DAXX histone-binding domain with a histone H3.3-H4 dimer, including mutants within DAXX and H3.3, together with in vitro and in vivo functional studies that elucidate the principles underlying H3.3 recognition specificity. Occupying 40% of the histone surface-accessible area, DAXX wraps around the H3.3-H4 dimer, with complex formation accompanied by structural transitions in the H3.3-H4 histone fold. DAXX uses an extended α-helical conformation to compete with major inter-histone, DNA and ASF1 interaction sites. Our structural studies identify recognition elements that read out H3.3-specific residues, and functional studies address the contributions of Gly 90 in H3.3 and Glu 225 in DAXX to chaperone-mediated H3.3 variant recognition specificity.

Figure 1. Structure of the ternary complex of DAXX histone-binding domain (HBD, 178–389) bound to histones H3.3 and H4, and comparison with the ternary HJURP–CENPA–H4 complex

a, Schematics of domain architecture of DAXX, H3.3 and H4. b, A ribbon view of the crystal structure of the DAXX–H3.3–H4 ternary complex, with the segment containing the variant-specific Ala 87-Ala 88-Ile 89-Gly 90 sequence boxed in red and the C-terminal tail of H4 (in yellow) boxed in black. c, A blown-up view of the variant-specific segment. d, Close-up views of interactions made by DAXX residues Ser 220, Tyr 222, Phe 317, Arg 251, Arg 328 and Asp 331 in the complex; see also Supplementary Fig. 5. e, DAXX mutants in the H3.3–H4 interface abrogate binding to endogenous histones in 293T cells. Bottom panels show immunoprecipitated DAXX and histones on the same Coomassie-stained gel. f, g, Striking similarities in coiled-coil interactions between α-helices of the chaperone (contain common interfacial DXXLXXXL(X)₁₃VIXKY segment) and histone in DAXX (f) and HJURP (g) complexes.

Figure 2. DAXX competes with major DNA interactions sites and prevents histone tetramer formation through conformational changes in H3.3

a, b, Alternative views of a model involving replacement of the histone H3–H4 tetramer (in the nucleosomal context) by the DAXX–H3.3–H4 complex. c, Schematic comparing α-helical alignments for H3.3 in the nucleosome with that in the DAXX–H3.3–H4 complex. d, Tetrasome assembly does not occur from a DAXX HBD–H3.3–H4 complex. A (H3.3–H4)₂ tetrasome can be assembled on an 84-bpDNAfragment by salt dialysis (right-most lane), but not from free H3.3–H4 dimers or DAXX HBD–H3.3–H4 trimers by mixing at physiological conditions. e, Interactions between α2 and α3 helices of H3 across the dimeric H3–H3′ interface in the H3–H4 tetramer (Protein Data Bank accession 1AOI). f, Intermolecular hydrogen bonding interactions in the DAXX–H3.3–H4 complex. Side chains that undergo conformational changes on formation of the ternary complex with DAXX are highlighted in yellow and boxed in e and f. g, Test for H3–H3 homodimerization in H3.3–H4 and DAXX–H3.3–H4 complexes by cysteine (Cys 110) crosslinking.

Figure 3. DAXX uses an extended α-helical conformation to compete with ASF1 interaction sites

a, Structure of the ASF1–H3–H4 complex (Protein Data Bank 2HUE), with the C-terminal tail of H4 shown in yellow and boxed in black. b, Relocation of the flexible C terminus of H4 and its anchoring through hydrogen-bonding and hydrophobic interactions in the DAXX–H3.3–H4 complex. c, Co-immunoprecipitation from HeLa cells shows competition of ASF1A/ASF1B and DAXX for H3.3–H4 dimers in vivo. Knockdown (left panels) or overexpression (right panels) of ASF1A and/or ASF1B modulate ASF1A and ASF1B protein levels in nuclear extracts (bottom). Immunoprecipitation of tagged H3.3 shows increased association with DAXX upon co-depletion of ASF1A and ASF1B and, vice versa, decreased association upon ASF1B overexpression. d, Co-immunoprecipitation from293T cells with transiently transfected wild-type H3.3 and mutant incapable of binding ASF1B shows direct competition by ASF1B and DAXX.

Figure 4. A structural role for H3.3 G90 as the major determinant for H3.3 variant specificity of DAXX in vivo

a, Mutagenesis of the H3.3AAIG motif in vivo reveals a dominant role of G90 in directing variant-specific histone chaperones DAXX and CAF-1. Co-immunoprecipitation (top panels) of transiently transfected H3.3 and H3.2 mutants in 293T cells. b, c, Expanded views of the specificity-determining core segment of the DAXX–H3.3–H4 complex for wild-type (panel b) and H3.3(G90M) mutant (panel c, mutant in yellow). Water molecules are shown as orange balls and labelled from 1 to 10, with hydrogen-bonding network in dashed red lines.

Figure 5. The DAXX tower provides H3.3 G90 specificity through direct and water-mediated contacts with H3.3 αN, α1 and α2 helices

a, Hydrogen-bonding interactions at either end of the shifted αN helix of H3.3 in the DAXX–H3.3–H4 complex. b, In vitro pull-down experiment with the DAXX tower helices (α1 and α2) andH3.3 tail deletion showing the importance of its αN helix. c, In vitro pull down with immobilized DAXX tower and mutants of H3.3 Gly 90 orH3.2Met 90. d, In vitro pull down with mutant DAXX tower showing the role of DAXX E225 in discriminating H3.3 G90. e, In vivo transient transfection and pull down of combinations of DAXX and histone H3.3 mutants. DAXX(E225A) allows binding of H3.2 in vivo. Transient expression (bottom panels) and coimmunoprecipitation (top panels) of DAXX mutants and H3.3 or H3.2. E225A confers on DAXX the propensity to bind H3.2 next to H3.3 (top panel). f, Expanded view of the specificity-determining core segment of the DAXX–H3.3–H4 complex containing a DAXX E225A mutant (in yellow).