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. 2017 Jan 25;45(2):643-656.
doi: 10.1093/nar/gkw892. Epub 2016 Oct 5.

sNASP and ASF1A Function Through Both Competitive and Compatible Modes of Histone Binding

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Free PMC article

sNASP and ASF1A Function Through Both Competitive and Compatible Modes of Histone Binding

Andrew Bowman et al. Nucleic Acids Res. .
Free PMC article

Abstract

Histone chaperones are proteins that interact with histones to regulate the thermodynamic process of nucleosome assembly. sNASP and ASF1 are conserved histone chaperones that interact with histones H3 and H4 and are found in a multi-chaperoning complex in vivo Previously we identified a short peptide motif within H3 that binds to the TPR domain of sNASP with nanomolar affinity. Interestingly, this peptide motif is sequestered within the known ASF1-H3-H4 interface, raising the question of how these two proteins are found in complex together with histones when they share the same binding site. Here, we show that sNASP contains at least two additional histone interaction sites that, unlike the TPR-H3 peptide interaction, are compatible with ASF1A binding. These surfaces allow ASF1A to form a quaternary complex with both sNASP and H3-H4. Furthermore, we demonstrate that sNASP makes a specific complex with H3 on its own in vitro, but not with H4, suggesting that it could work upstream of ASF1A. Further, we show that sNASP and ASF1A are capable of folding an H3-H4 dimer in vitro under native conditions. These findings reveal a network of binding events that may promote the entry of histones H3 and H4 into the nucleosome assembly pathway.

Figures

Figure 1.
Figure 1.
sNASP and ASF1A bind competitively to histone H3. (A) Crystal structure of ASF1A bound to an H3–H4 dimer (PDB code: 2IO5) with residues from the previously mapped sNASP binding site (25) shown as yellow spheres. (B) Detailed view of H3 residues involved in binding the TPR domain of sNASP in complex with ASF1A. (C) Gel filtration elution profile of free MBP H3 (116–135). (D) Gel filtration elution profile of ASF1A bound to MBP H3 116–135 (ASF1A was kept at a molar excess over the MBP H3 (116–135) peptide to visualise both free and bound ASF1A). (E) Elution profile of equimolar amounts of sNASP, ASF1A and MBP H3 116–135, revealing that sNASP can effectively outcompete ASF1A for binding to the H3 C-terminal peptide. (F) Elution profile of sNASP complexed with full-length histone H3. (G) Elution profile of sNASP, ASF1A and full-length H3. ASF1A is unable to bind to H3 whilst it is associated with sNASP, eluting in its unbound fraction.
Figure 2.
Figure 2.
sNASP solubilises H3–H4–ASF1A by forming a stable sNASP–H3–H4–ASF1A complex. (A) Salt titration showing the solubility of the H3–H4–ASF1A complex is dependent on ionic strength. Soluble and insoluble material were separated by centrifugation before analysis by SDS-PAGE PAGE and coomassie staining. (B) The precipitate formed at lower ionic strength conditions in (A) (200 mM sodium chloride) can be solubilized through titration of sNASP. At an equimolar ratio of sNASP to H3–H4–ASF1A near complete solubilization is observed. (C) Gel filtration elution profile of sNASP bound to the H3–H4–ASF1A complex showing co-elution of all four proteins as visualized by SDS-PAGE and coomassie staining. A molar excess of ASF1A over all other components was used to gauge the stoichiometry of the complex. (D) Gel filtration elution profile of sNASP and ASF1A showing that the two chaperones elute in separate fractions, and therefore do not interact in the absence of their histone cargo. (E) Elution profile of the yeast homolog of sNASP, Hif1, showing that the complex formed between sNASP family of histone chaperones and H3–H4–ASF1A is evolutionary conserved.
Figure 3.
Figure 3.
The effect of sNASP and ASF1A mutants on sNASP–H3–H4–ASF1A complex formation. (A) Diagrammatic representation of the F2H experiment. sNASP is tethered to a LacO array through an mCherry-LacI fusion, whilst ASF1A is expressed as a soluble mEGFP fusion. As the chaperones do not interact directly, interaction between the two chaperones is likely mediated through endogenous cellular H3–H4. Yellow stars represent mutations that disrupt the known histone binding surfaces of the two chaperones. (B) mEGFP-ASF1A does not recruit to the empty mCherry-LacI construct, but does recruit to the sNASP fusion. Disruption of the H3 binding interface of ASF1A by the V94R mutation abrogates recruitment of ASF1A, however, disruption of the sNASP TPR-H3 peptide interaction through the EYL>ASS triple mutation has little effect of ASF1A recruitment. (C) Quantification of images shown in (B). Asterisks represent a P value of <0.001 as determined by the Wilcoxon rank sum test. (D) Reconstitution of the sNASP EYL>ASS-H3–H4–ASF1A in vitro demonstrates that disruption of the TPR–H3 peptide interaction has little effect on the sNASP's ability to form a complex with H3–H4–ASF1A.
Figure 4.
Figure 4.
Isolation and characterization of two sNASP specific monobodies. (A) Domain diagrams of wild-type (WT) sNASP and truncation mutants N-330 and cTPR used in this study showing the TPR motifs in green, the acidic region in yellow and the capping region (CR) in gray. The contiguous TPR mutant (cTPR) is a deletion of the interrupting acidic domain and seamless stitching together of the TPR2 motif. The sNASP N-330 truncation was used as an antigen for monobody library screening. (B) Diagrammatic representation of the sNASP protein with corresponding residues shown for each domain. (C) Outline of monobody generation strategy. Adopted from Figure 1A of (40). (D) Dissociation constants of two sNASP specific binders, mbsNASP_1 and mbsNASP_13 (denoted mb1 and mb13 for brevity), were determined by yeast display. Measurements were also carried out in the presence of HEK293 lysate to probe the specificity of the interaction. The error bars on each data point show SD. from triplicate measurements. The dissociation constant (KD) values shown are the average of KD values determined from triplicate measurements, and the errors shown are the SD. (E) Gel filtration analysis of MBP-mb1 and mb13 binding to full-length sNASP. Both monobodies eluted with sNASP in a single complex (peak 1), demonstrating their non-overlapping binding sites. (F) Gel filtration analysis of MBP-mb1 and mb13 binding to the sNASP cTPR truncation mutant. Whilst mb13 retained its interaction with sNASP in the absence of the central acidic domain (peak 1), MBP-mb13 eluted in its separate fraction (peak 2).
Figure 5.
Figure 5.
Probing the secondary modes of sNASP interaction using monobodies mb1 and mb13. (A) Gel filtration profile of equimolar amounts of sNASP, MBP–H3 116–135, MBP-mb1 and mb13. Whilst mb13 remains predominantly associated with the sNASP-MBP–H3 116–135 complex (peak 1), MBP-mb1 is partially displaced, suggesting that it undergoes binding site competition with the H3 peptide (free MBP–H3 116–135 and MBP-mb1 have partially overlapping elution profile represented by peak 2). (B) Gel filtration profile of sNASP, MBP-mb1, H3 full-length and mb13, showing that MBP-mb1 and mb13 are largely excluded from the sNASP–H3 complex (peak 1), eluting in their separate fractions (peaks 2 and 3, respectively). (C) Elution profile of sNASP, MBP-mb1, mb13, H3–H4 and ASF1A, showing that MBP-mb1 (peak 2) and mb13 (peak 3) are largely displaced from the sNASP–H3–H4–ASF1A complex (peak 1). (D) Diagrammatic representation of the complexes formed between sNASP, its various histone substrates and the monobodies mb1 and mb13. Whilst mb13 retains its binding to sNASP in the presence of the H3 116–135 peptide, it is displaced upon either H3 full-length or H3–H4–ASF1A complex binding. MBP-mb1 is partially displaced by the H3 116–135 peptide, suggesting binding site competition, and is fully displaced upon H3 full-length or H3–H4–ASF1A complex binding.
Figure 6.
Figure 6.
The central acidic domain of sNASP is necessary for H3–H4–ASF1A binding. (A) Domain diagrams of the contiguous (cTPR) truncation mutant compared to wild-type sNASP. (B) Elution profile of the sNASP cTPR mutant and H3–H4–ASF1A separated out by gel filtration chromatography. The conductivity spike representing the higher salt from sample preparation eluting in the bed volume of the column is indicated. (C) Fractions from the elution separated by SDS-PAGE reveal that the cTPR mutant, although retaining its ability to interact with the H3 116–135 peptide, is unable to bind to the H3–H4–ASF1A complex.
Figure 7.
Figure 7.
sNASP and ASF1A are capable of folding an H3–H4 dimer in vitro. (A) Flow chart showing the two different strategies for reconstituting the sNASP–H3–H4–ASF1A complex in vitro. (B) H3 and H4 monomers dissolved in water were mixed with sNASP and ASF1A and the resulting complexes separated by gel filtration chromatography. The positions of aggregates, sNASP–H3–H4–ASF1A complex and free ASF1A are shown. (C) Reconstituting the sNASP–H3–H4–ASF1A complex. sNASP (N), ASF1A (A), H3 (3) and H4(4) were combined in all possible orders of addition and the soluble and insoluble fractions isolated and analyzed by SDS-PAGE. (D) Quantification and ranking of the total relative precipitate (insoluble fraction) formed from each order of addition shown in (C). The experiment was carried out in triplicate with the error representing the standard deviation. (E) Tetrasome assembly assay comparing the sNASP–H3–H4–ASF1A complexes formed from either prefolded histones or unfolded histones. Positions of tetrasomes, disomes and free DNA are shown. 0.5 μM of DNA was combined with 1, 2 or 4 μM of H3 & H4 (lanes 1–3), H3–H4 dimers (lanes 4–6) or sNASP–H3–H4–ASF1A complex made from prefolded H3–H4 (lanes 7–9) or unfolded H3 & H4 (lanes 10–12), representing a 1:1, 1:2 and 1:4 molar ratio of DNA to H3–H4 tetramer in each case. (F) A molecular model for the role of sNASP and ASF1 in H3–H4 chaperoning. The interaction between sNASP and H3 is mediated by the TPR domain binding to the H3 C-terminus, and through additional contacts involving the acidic domain and an interaction site on the TPR domain/capping region that lies outside of the central H3 peptide-binding channel. ASF1A cannot compete for H3 binding when it is bound by sNASP. Folding with H4 causes a conformational change in H3, which results in a transition of the H3 C-terminal region from the TPR domain to its position within the globular core of the histone fold. As ASF1 recognises the folded surface of an H3–H4 dimer, this transition is accompanied by ASF1 binding at the C-terminal region of H3. sNASP is retained within the H3–H4–ASF1 complex through its secondary modes of interaction with the H3–H4 dimer that are compatible with ASF1 binding. In complex with H3–H4–ASF1, sNASP contributes to the solubility of the histones and prevents their aggregation.

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