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Ubinuclein-1 confers histone H3.3-specific-binding by the HIRA histone chaperone complex.
Ricketts MD
,
Frederick B
,
Hoff H
,
Tang Y
,
Schultz DC
,
Singh Rai T
,
Grazia Vizioli M
,
Adams PD
,
Marmorstein R
.
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Histone chaperones bind specific histones to mediate their storage, eviction or deposition from/or into chromatin. The HIRA histone chaperone complex, composed of HIRA, ubinuclein-1 (UBN1) and CABIN1, cooperates with the histone chaperone ASF1a to mediate H3.3-specific binding and chromatin deposition. Here we demonstrate that the conserved UBN1 Hpc2-related domain (HRD) is a novel H3.3-specific-binding domain. Biochemical and biophysical studies show the UBN1-HRD preferentially binds H3.3/H4 over H3.1/H4. X-ray crystallographic and mutational studies reveal that conserved residues within the UBN1-HRD and H3.3 G90 as key determinants of UBN1-H3.3-binding specificity. Comparison of the structure with the unrelated H3.3-specific chaperone DAXX reveals nearly identical points of contact between the chaperone and histone in the proximity of H3.3 G90, although the mechanism for H3.3 G90 recognition appears to be distinct. This study points to UBN1 as the determinant of H3.3-specific binding and deposition by the HIRA complex.
Figure 1. A region of UBN1 containing the Hpc2-related domain (HRD) is able to specifically bind to histone H3.3.(a) Sequence alignment comparing two highly conserved domains in Homo sapiens UBN1, Drosophila melanogaster Yemanuclein and Saccharomyces cerevisiae Hpc2. (b) GST pull-down histone-binding assay. Binding to H3.3/H4 and H3.1/H4 is tested separately in a single input H3/H4 pull-down (left), and in competition by subjecting an equimolar mixture of H3/H4 and His-H3/His-H4 to pull down (right). The H3.3-specific binding activity of GST-UBN1(41–175)/HIRA(1–405) (top), GST-UBN1(92–175) (middle) and free GST (bottom) is compared. (c) Isothermal titration calorimetry was used to quantitatively compare UBN1(92–175) binding with H3.3/H4 (left) and H3.1/H4 (right), ± values represent the standard error of the ITC fit using Origin 7.0. ND, not determined.
Figure 2. Conserved residues in the UBN1-HRD are required for binding with H3.3/H4.(a) Sequence alignment comparing HRD residues in Homo sapiens UBN1, Drosophila melanogaster Yemanuclein and Saccharomyces cerevisiae Hpc2, strictly conserved residues are highlighted in red. (b) GST pull-down assay comparing the H3.3-binding activity of UBN1 wild-type and 27 alanine scanning point mutants (A144 was mutated to I). (c) The IC50 for the binding with H3.3/H4 was quantified for each UBN1(92–175) variant using fluorescence polarization, ± values represent the standard error of the curves fit using GraphPad Prism 5.0a. (d) Four representative fluorescence polarization histone-binding curves are shown error bars represent the s.e.m. of three independent replicates. IC50, half-maximal inhibitory concentration; ND, not determined.
Figure 3. H3.3 residue G90 mediates UBN1 specificity.(a) Sequence alignment comparing human histones H3.1 and H3.3 with the five amino-acid differences highlighted in grey (b) GST pull-down assay comparing the ability of UBN1(122–148) to select between histone mutants where critical residues have been swapped between H3.1 and H3.3. UBN1 binding with wild-type H3.3/H4 and H3.1/H4 was analysed in addition to H3/H4 carrying several H3 mutants: H3.3(G90M), H3.1(M90G), H3.3(SAVM) and H3.1(AAIG). (c) Isothermal titration calorimetry quantification of the UBN1(122–148) interaction with wild-type and mutant H3/H4 complexes. Dissociation constant (Kd) and stoichiometry of UBN1 binding to H3/H4 are reported, ±values represent the standard error of the ITC fit using Origin 7.0. ND, not determined.
Figure 4. Crystal structure of the UBN1/H3.3/H4/Asf1 complex at 2.3-Å resolution.(a) Overall architecture of the UBN1/H3.3/H4/Asf1 complex with the position of H3.3 G90 highlighted in yellow. The structure is composed of UBN1 122–148, H3.3 60–133, H4 21–102 and Asf1 1–154. (b) UBN1 has an intimate association with the H3.3/H4 surface with close proximity to H3.3 G90 (yellow). UBN1 residues with sensitivity to alanine mutation are labelled. (c–d) Detailed stereo view of UBN1 and DAXX interactions with H3.3/H4 showing conserved interaction surfaces. (e) Alignment of the UBN1/H3.3/H4/Asf1 and DAXX/H3.3/H4 structures illustrating structural similarity between UBN1 and DAXX. (f) Surface representation of UBN1 shows that residues 128–132 rest closely over H3.3 G90 (top) and would likely sterically occlude binding with H3.1 M90 (bottom). M90 is represented by substitution of G90 with the highest percentage methionine rotamer. (g) The same view of the DAXX/H3.3/H4 structure shows that DAXX has a cavity where UBN1 128–131 resides. The comparable DAXX site fills with water molecules to sterically occlude binding with H3.1/H4.
Figure 5. UBN1-HRD/H3.3 interface is required for H3.3-binding specificity in intact complexes.FLAG pull-down histone-binding assay. Recombinant complexes of HIRA-FLAG(FL)/UBN1(FL) and HIRA-FLAG(FL)/UBN1(FL)/His-CABIN1(FL) with wild-type and FID138-140AAA mutant UBN1 were analysed for binding to both H3.3/H4 and H3.1/H4.
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