Structural basis for the interaction of Asf1 with histone H3 and its functional implications

Abstract

Asf1 is a conserved histone chaperone implicated in nucleosome assembly, transcriptional silencing, and the cellular response to DNA damage. We solved the NMR solution structure of the N-terminal functional domain of the human Asf1a isoform, and we identified by NMR chemical shift mapping a surface of Asf1a that binds the C-terminal helix of histone H3. This binding surface forms a highly conserved hydrophobic groove surrounded by charged residues. Mutations within this binding site decreased the affinity of Asf1a for the histone H3/H4 complex in vitro, and the same mutations in the homologous yeast protein led to transcriptional silencing defects, DNA damage sensitivity, and thermosensitive growth. We have thus obtained direct experimental evidence of the mode of binding between a histone and one of its chaperones and genetic data suggesting that this interaction is important in both the DNA damage response and transcriptional silencing.

Fig. 1.

The structure of the human Asf1a N-terminal domain is well conserved. (a) Bundle of 20 structures of human Asf1a (1-156) calculated as indicated in Methods. The coloring scheme indicates the relative position of the backbone atoms with the warmest colors being closest to the carboxyl terminus. (b) Schematic ribbon diagram (generated with pymol, DeLano Scientific, South San Francisco, CA) of the human Asf1a (1-156) structure closest to the mean (colored as in a) superimposed on the crystallographic structure of the homologous domain of S. cerevisiae Asf1 (16) (gray).

Fig. 2.

Interaction of human Asf1a (1-156) with histone H3 (122-135). (a) Superposition of the HSQC spectrum of human Asf1a (1-156) (80 μM) with (red) and without (black) an excess of histone H3 (122-135) (160 μM) at 298 K. (b) Mean-square chemical shift variation calculated as described in Methods for human Asf1a (1-156) upon binding of H3 (122-135) as a function of the sequence. Horizontal lines at 0.2 and 0.3 show the cutoff for the color-coded representation of the protein surface in Fig. 3. The chemical exchange rate was rapid for most of the residues except the highly affected residues 45, 94, and 111, indicated with a vertical gray bar, that exhibited an intermediate exchange rate and severe line broadening due to chemical exchange that led to their absence from the ¹⁵N-HSQC spectrum of the complex. Stars indicate missing values (proline residues or unassigned because of signal overlaps).

Fig. 3.

Structure mapping of the chemical shift variation of Asf1a upon H3 (122-135) binding. Two orientations of the protein are presented in Upper and Lower.(Left) Ribbon representation of the protein. (Left Center) Surface color-coded representation of the mean-square chemical shift variation of Asf1a upon H3 (122-135) binding. The three line-broadened residues are shown in red, residues with Δδ > 0.3 in orange, residues with 0.2 <Δδ < 0.3 in yellow, residues with Δδ < 0.2 in white and undetermined residues in gray. (Right Center) Surface color-coded representation of the residue conservation of Asf1. The conservation was determined by using the rate4site program (38) based on an alignment of 17 Asf1 sequences. The color code is a gradient from red (fully conserved residues) to white (unconserved residues). (Right) Color-coded representation of the electrostatic potential of human Asf1a. The potential was calculated with the software apbs (39). The color code is red for negative values, white for near zero values, and blue for positive values. Figures were generated with pymol, and residues mutated in this work are labeled.

Fig. 4.

H3 (122-135) folds into the native structure upon Asf1a binding. (a) Superposition of the ¹⁵N-HSQC spectrum of ¹⁵N-labeled H3 (122-135) (200 μM) with (red) and without (black) an excess of unlabeled human Asf1a (1-156) (250 μM) at 283 K. At this temperature, the chemical exchange rate was slow compared with the chemical shift variations. G₀ and M_-1 indicate the two amino acids N-terminal to the H3 (122-135) sequence that were required for cloning the sequence in the E. coli expression vector as described in Methods. (b) Hα chemical shift index [δ_Hα^exp-δ_Hα^RC, where exp and RC refer to the experimental and random coil values, respectively, (40)] of the free (black) and bound (red) H3 (122-135). The orange cylinder shows the position of the native C-terminal α3 helix of H3 as observed in the structure of the nucleosome (1). (c) Mean square chemical shift variation calculated as described in Methods of ¹⁵N-H3 (122-135) upon binding of human Asf1a (1-156) as a function of the sequence. (d) Heteronuclear ¹⁵N{¹H}-nuclear Overhauser effect of the bound H3 (122-135).

Fig. 5.

In vitro and in vivo analysis of Asf1 mutants. (a) V94R, D54R, and R108E are involved in the binding of Asf1 to the histone H3/H4 complex. Equal amounts of purified recombinant (His)₆-GST, (His)₆-GST-Asf1a (1-156), and the indicated mutants were bound to glutathione agarose beads. Lower is an anti-GST immunoblot showing the equal quantities of fusion protein bound to the beads. A small quantity of the (His)₆-GST control protein appears as a dimerized species that migrates near the position of the (His)₆-GST-Asf1a proteins (indicated with a star). Binding of histone H3 within H3/H4 complexes to the GST proteins is shown in Upper. H3 was visualized by immunoblotting with anti-H3 antibodies. Input shows the amount of histone used for each binding reaction. (b) Coomassie blue-stained gel of wild-type Asf1-TAP and Asf1-V94R-TAP purified from yeast cell extracts by using the tandem affinity purification protocol. (c) Wild-type and mutant Asf1-13myc proteins are expressed at similar levels in yeast. The UCC6562 asf1Δ strain was transformed with pRS314 (CEN-TRP1) or pRS314 plasmids containing DNA fragments, allowing expression from the endogenous ASF1 promoter of wild-type (WT) or mutant Asf1-13myc proteins as indicated. Protein extracts from exponentially growing cells at 30°C or from cells transferred to 37°C for 4 or 20 h as indicated were analyzed by immunoblotting with anti-myc antibodies to visualize the tagged Asf1 proteins. Asf1-13myc migrated as a closely spaced doublet of bands at ≈75 kDa. The quantity of the faster-migrating band varied between experiments, but no reproducible difference in the ratio of the two forms was seen between the wild type and any of the mutants. After developing the anti-myc blot, membranes were incubated with anti-Cim3/Rpt6 antiserum directed against a 45-kDa subunit of the 26S proteasome to verify that equal quantities of protein extract were loaded in each lane. (d) Sensitivity of asf1 mutants to genotoxic stress and to growth at 37°C. UCC6562 asf1Δ strains transformed with the pRS314 plasmids allowing the expression of wild-type and mutant Asf1-13myc proteins described in a were grown to early stationary phase in synthetic complete medium without tryptophan to maintain selection for the pRS314 plasmids. The 10-fold serial dilutions of cultures were then spotted on yeast extract/peptone/dextrose plates, containing where indicated 5 μg/ml camptothecin (CPT) or 50 mM hydroxyurea (HU) and incubated at 30°C to test for sensitivity to genotoxic stress or at 37°C to test for thermosensitive growth. (e) The same pRS314 plasmids described above were used to test the transcriptional silencing function of the indicated wild-type and mutant Asf1-13myc proteins. These plasmids were transformed into four different silencing reporter strains: UCC6562 (asf1 CAC+ TEL::URA3) tests telomeric silencing in an asf1 CAC+ background, CMY1317 (asf1 cac2 TEL::URA3) tests telomeric silencing in an asf1 cac2 double-mutant background, CMY1314 (asf1 cac2 HMRa::URA3) tests silencing at HMRa in an asf1 cac2 background, and CMY1312 (asf1 cac2 HMLα::URA3) tests silencing at HMLα in an asf1 cac2 background. Strains were grown to early stationary phase in synthetic complete medium without tryptophan to select for the plasmid, and 10-fold dilutions of the cultures were then spotted on plates containing 5-fluoroorotic acid (5-FOA). Repression of the URA3 reporter gene by transcriptional silencing allows growth on the FOA plates (24).