Structure of the variant histone H3.3-H4 heterodimer in complex with its chaperone DAXX

Abstract

Mammalian histone H3.3 is a variant of the canonical H3.1 essential for genome reprogramming in fertilized eggs and maintenance of chromatin structure in neuronal cells. An H3.3-specific histone chaperone, DAXX, directs the deposition of H3.3 onto pericentric and telomeric heterochromatin. H3.3 differs from H3.1 by only five amino acids, yet DAXX can distinguish the two with high precision. By a combination of structural, biochemical and cell-based targeting analyses, we show that Ala87 and Gly90 are the principal determinants of human H3.3 specificity. DAXX uses a shallow hydrophobic pocket to accommodate the small hydrophobic Ala87 of H3.3, whereas a polar binding environment in DAXX prefers Gly90 in H3.3 over the hydrophobic Met90 in H3.1. An H3.3-H4 heterodimer is bound by the histone-binding domain of DAXX, which makes extensive contacts with both H3.3 and H4.

Figure 1

Overall structure of the DAXX HBD-H3.3-H4 complex. (A) A ribbon representation showing DAXX HBD, H3.3 and H4, with secondary structure elements labeled in black, red and blue font, respectively. Helices grouped in subdomains are indicated. (B) DAXX-histone interactions. H3.3 and H4 are shown in a surface representation with electrostatic potential distribution. DAXX residues involved in interactions with DAXX are shown as stick models superimposed on the ribbon diagram of DAXX; certain key residues are labeled.

Figure 2

Conformational differences between the DAXX HBD-bound and nucleosomal H3.3-H4 complexes. (A) An H3.3-H4 dimer from the NCP structure (PDB 3AV2), with H3.3 and H4 colored in wheat and light blue, respectively, is superimposed with the DAXX HBD-H3.3-H4 complex. (B) Superimposed structures viewed from a direction perpendicular to that in Figure 2A (looking up from the bottom). (C) A model replacing one nucleosomal H3.3-H4 heterodimer with that from the DAXX HBD complex (ribbon only). The other nucleosomal H3.3-H4 complex is shown in a semi-transparent surface representation superimposed onto a ribbon model. The coloring scheme is the same as in (A) and (B), except that DAXX is colored yellow here. Regions of spatial hindrance for the formation of an H3.3-H4 tetramer are indicated with red dashed-line circles.

Figure 3

H3.3 residues responsible for DAXX binding specificity. (A) A stereo view of H3.3-specific interactions with DAXX HBD. The three H3.3-specific residues are highlighted in magenta, and the interacting DAXX residues are shown (DAXX-yellow; H3.3-green or magenta; H4-cyan). Helix α2 of a canonical H3 (gray; PDB 1KX5) is superimposed and the three residues different from H3.3 are shown for comparison. (B) A schematic diagram showing the lac operator-repressor (LacO-LacI) targeting system for assaying DAXX-H3.3 interactions. (C) Co-localization of DAXX with H3.3, H3.1 and indicated mutants. (D) Top panel: co-IP of full-length Flag-tagged DAXX with HA-tagged histones H3.1 and H3.3. IPs were analyzed by western blotting with indicated antibodies. Bottom panel: GST pull-down of wild-type or the C338S L340N double mutant of DAXX HBD with the H3.1-H4, H3.1(S87A)-H4, or H3.3-H4 complexes as indicated. The pull-downs were analyzed by Coomassie-stained SDS-PAGE.

Figure 4

DAXX residues important for recognition of H3.3. (A) The involvement of Cys338 and Leu340 in the recruitment of H3.3 or H3.1 and their derivatives as indicated (left). The C338S L340N mutant of DAXX interferes with recognition of residue 87 in H3.3 and H3.1 (right). (B) Analyses of DAXX residues important for recognition of Gly90 of H3.3. Top panel: co-IP of DAXX or its E225M K229M double mutant with histones H3.1 and H3.3. Bottom panel: GST pull-down with the wild-type or mutant DAXX HBD (WT, E225M K229M, and Y222E) with H3.1-H4 and H3.3-H4 complexes as indicated. (C) Co-localization of E225M K229M and E225Q K229Q mutants with both H3.3 and H3.1.