Berndsen CE et al. (2008), Molecular functions of the histone acetyltransf...

XB-ART-39122

Nat Struct Mol Biol 2008 Sep 01;159:948-56. doi: 10.1038/nsmb.1459.

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Molecular functions of the histone acetyltransferase chaperone complex Rtt109-Vps75.

Berndsen CE , Tsubota T , Lindner SE , Lee S , Holton JM , Kaufman PD , Keck JL , Denu JM .

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Histone acetylation and nucleosome remodeling regulate DNA damage repair, replication and transcription. Rtt109, a recently discovered histone acetyltransferase (HAT) from Saccharomyces cerevisiae, functions with the histone chaperone Asf1 to acetylate lysine K56 on histone H3 (H3K56), a modification associated with newly synthesized histones. In vitro analysis of Rtt109 revealed that Vps75, a Nap1 family histone chaperone, could also stimulate Rtt109-dependent acetylation of H3K56. However, the molecular function of the Rtt109-Vps75 complex remains elusive. Here we have probed the molecular functions of Vps75 and the Rtt109-Vps75 complex through biochemical, structural and genetic means. We find that Vps75 stimulates the kcat of histone acetylation by approximately 100-fold relative to Rtt109 alone and enhances acetylation of K9 in the H3 histone tail. Consistent with the in vitro evidence, cells lacking Vps75 showed a substantial reduction (60%) in H3K9 acetylation during S phase. X-ray structural, biochemical and genetic analyses of Vps75 indicate a unique, structurally dynamic Nap1-like fold that suggests a potential mechanism of Vps75-dependent activation of Rttl09. Together, these data provide evidence for a multifunctional HAT-chaperone complex that acetylates histone H3 and deposits H3-H4 onto DNA, linking histone modification and nucleosome assembly.

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Species referenced: Xenopus laevis
Genes referenced: asf1a tab3

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	Figure 1. Rtt109-Vps75 acetylates residues within the H3 tail. (a) MS/MS spectrum of histone H3 peptide K(ac)STGGK(ac)APR, residues 9-17, conclusively confirms H3K9 and H3K14 are acetylated within the protein. The observed b and y fragment ions, resulting from collison-induced dissociation of the peptide backbone, are labeled on the peptide sequence and in spectrum with their ion type, position and mass/charge (m/z). (b,c) MATa, bar1 strains of the indicated genotypes were arrested in G1 phase with Î± factor mating pheromone and released into a synchronous cell cycle in pheromone-free media for the indicated times at 30 Â°C. Whole-cell extracts were analyzed by immunoblotting with the indicated antibodies. (d) Immunoblot signal from anti-H3K9ac from the mid-S phase 30-min time point in b or c were quantified by chemiluminescence on a LAS-300 (Fujifilm) and calculated relative to the total H3 signal. The average percent acetylation relative to the wild-type (WT) strain for three independent experiments is depicted. (e) Immunoblot signal from anti-H3K14ac. Samples were treated and analyzed as in d. (f) Immunoblot signal from anti-H3K23ac. Samples were treated and analyzed as in d for two independent experiments. Error bars in d and e indicate s.d., and error bars in f indicate average error.
	Figure 2. Rtt109 stimulates histone-deposition activity of Vps75. Histone H3 and H4 deposition by Vps75 in the presence or absence of Rtt109 was monitored by supercoiling assay. DNA was unwound before adding to the deposition reaction, and deposition activity was quantified by measuring the density as a percentage of that of the total DNA that was supercoiled in each lane, which corrects for possible variations in loading. Values shown are averages of three independent experiments Â± s.d. Shown at the right are representative control assays of no Vps75 or Rtt109 control, Vps75 only or Rtt109-Vps75 from the same gel. The zero time point for each assay used the same stock of relaxed DNA and is shown in the âNo chaperoneâ control. Gel images were inverted for clarity.
	Figure 3. Structure of Vps75. (a) Front and back views of the X-ray crystal structure of native monomer of Vps75. The domain I helix (residues 8-53) is shown in dark blue and domain II (residues 57-221) is shown in green. (b) Structure of Nap1 (PDB 2AYU)36. Domains I and domain II are colored as in a. Structural figures of Vps75 and Nap1 were made using MacPyMOL40.
	Figure 4. Dimerization between domain I of Vps75 monomers. (a) Vps75 monomers interact with symmetry mates in adjacent asymmetric units creating a dimer through domain I helix (residues 8-53). Residues are colored to indicate those involved in forming the dimer interface and type of interaction with hydrophobic interactions in brown, hydrogen-bonding in green and Ï-stacking in blue. Domain II is shown in gray. (b) Cartoon of interaction surface created by domain I of Vps75. Residues are colored according to interaction type as in a. Gray sections are not resolved in the current structural models of Vps75. Residues labeled in bold are altered in the Vps75ESEE construct (containing C21E V25S I28E V32E substitutions). (c) Spectra from analytical ultracentrifugation of wild-type Vps75 (black squares) and Vps75ESEE (blue circles).
	Figure 5. Comparison of surface electrostatics of the Nap1 family of histone chaperones. (a) Native Vps75 (left) and SeMet Vps75 (right). The acidic cavity and basic patch are indicated for reference. (b) Nap1 (ref. 36; left) and INHAT37 (right). Areas of negative charge are shown in red, and areas of basic charge are shown in blue.
	Figure 6. Characterization of Vps75 acidic cavity mutants. Depictions of the surface electrostatics of the acidic cavity for native Vps75 (a), Vps75p with alanine substitutions at residues 198, 199 and 202 (b) and for Vps75q with alanine substitutions at residues 205, 206 and 207 (c). Negative charge is shown in red and positive charge in blue. (d) Saturation curves of H3-H4 for Vps75o (boxes), Vps75p (open circles) and Vps75q (filled circles). Reactions were performed as described in the Supplementary Methods with full steady-state kinetics shown in Supplementary Table 1. For Rtt109-Vps75q, the kcat/KM value was (8.3 Â± 3) Ã 103 M-1 s-1. For Rtt109-Vps75o, the kcat/KM value was (3.8 Â± 1) Ã 104 M-1 s-1. For Rtt109-Vps75p, the kcat/KM 4.2 Â± 2 Ã 104 M-1 s-1. Assays were performed in duplicate with the average rate from the two experiments shown. Error bars indicate average error. (e) H3 binding curves for Vps75 and Vps75q. Kd was calculated from four independent experiments, and the average Kd Â± s.d. is shown. (f) Representative western blot of H3K9ac in wild-type (WT) and Vps75q yeast cells. Cells were treated, prepared for immunoblotting and analyzed as described in Figure 1b. The average percentage of acetylation relative to that of the wild-type strain for each of two independent experiments is shown (designated 1 and 2). P-values were calculated from three measurements of the chemiluminescence in each experiment using the ANOVA function in Kaleidagraph.

References [+] :

Adkins, The histone chaperone anti-silencing function 1 stimulates the acetylation of newly synthesized histone H3 in S-phase. 2007, Pubmed