Two factor authentication: Asf1 mediates crosstalk between H3 K14 and K56 acetylation

Abstract

The ability of histone chaperone Anti-silencing factor 1 (Asf1) to direct acetylation of lysine 56 of histone H3 (H3K56ac) represents an important regulatory step in genome replication and DNA repair. In Saccharomyces cerevisiae, Asf1 interacts functionally with a second chaperone, Vps75, and the lysine acetyltransferase (KAT) Rtt109. Both Asf1 and Vps75 can increase the specificity of histone acetylation by Rtt109, but neither alter selectivity. However, changes in acetylation selectivity have been observed in histones extracted from cells, which contain a plethora of post-translational modifications. In the present study, we use a series of singly acetylated histones to test the hypothesis that histone pre-acetylation and histone chaperones function together to drive preferential acetylation of H3K56. We show that pre-acetylated H3K14ac/H4 functions with Asf1 to drive specific acetylation of H3K56 by Rtt109-Vps75. Additionally, we identified an exosite containing an acidic patch in Asf1 and show that mutations to this region alter Asf1-mediated crosstalk that changes Rtt109-Vps75 selectivity. Our proposed mechanism suggests that Gcn5 acetylates H3K14, recruiting remodeler complexes, allowing for the Asf1-H3K14ac/H4 complex to be acetylated at H3K56 by Rtt109-Vps75. This mechanism explains the conflicting biochemical data and the genetic links between Rtt109, Vps75, Gcn5 and Asf1 in the acetylation of H3K56.

Figure 1.

Time course site-specific plots for H3/H4 acetylation (A:WT; B: H3K9ac; C: H3K14ac; D: H3K23ac; E: H3K27ac; and F: H3K56ac) by Rtt109–Vps75 with and without Asf1. Reactions were monitored for ∼90 min without Asf1; as Asf1 increases the reaction rate, Asf1-containing reactions were monitored for ∼190 s. Individual pre-acetylated lysine's are not shown in (B–F) and remain ∼100% at any time point of assays. The error bar represents the standard error in acetylation percentage. The inset in (G) is to scale down the y-axis for H3K56ac to illustrate the acetylation details of each site.

Figure 2.

Analysis of Asf1-dependent changes to residue selectivity as a function of pre-acetylated states of histone H3. (A) Comparison of site-specific k_cat(app) of H3/H4 (with and without singly acetylated mark) by Rtt109–Vps75 acetylation in the absence or presence of Asf1. The error bar represents the standard error in k_cat(app). The apparent k_cat are summarized in Supplementary Tables S1 and 2. (B) Free difference for residue acetylation as compared to non-enzymatic acetylation with and without Asf1. Changes in the apparent free energy of residue selectivity due to Asf1 with varying states of histone H3. (C) The apparent free energy changes to selectivity due to different acetylation states of histone H3 with and without Asf1. In these heat maps, the right side of the diagonal is the selectivity between residues in the absence of Asf1 and the left side is in the presence of Asf1, and the horizontal and vertical black bars represent the sites of pre-acetylation. If Asf1 has no impact on selectivity, then both sides of the diagonal will be mirror images of each other as is the case with no pre-acetylation and H3K27ac. All other changes represent changes in selectivity due to Asf1.

Figure 3.

Structural analysis of Asf1 reveals acidic patch. (A) Electronic potential surface for Asf1 H3/H4 complex without histone tail (PDB ID: 2io5), calculated using PyMOL plugin APBS electrostatics and default settings. (B) Structure of Asf1 H3/H4 complex with proposed Asp and Glu mutation to the acidic patch highlighted in cyan.

Figure 4.

Mutants of Asf1 can suppress the Asf1-mediated cross-talk between H3K14ac and H3K56ac. Comparison of site-specific k_cat(app) of (A) H3/H4 or (B) H3K14ac/H5 by Rtt109–Vps75 acetylation in the presence of Asf1 mutants. The error bar represents the standard error in k_cat(app). The apparent k_cat are summarized in Supplementary Tables S3 and 4. (C) Free energy differences due to the addition of K14ac (right column) with various mutations to Asf1, and the changes to free energy due to mutations to Asf1 with H3/H4 (middle column) and H3K14ac/H4 (left column).

Figure 5.

Analysis of E105 mutations impact of Asf1 acidic patch. (A) Electronic potential surface for Asf1 (PDB ID: 2io5), calculated using PyMOL plugin APBS electrostatics and default settings. Asf1 E105A and E105R mutations were computationally made in PyMol using mutagenesis wizard and APBS electrostatics were calculated as for WT. (B) Free energy changes to selectivity due to mutation to Asf1, where the right side of the diagonal is the selectivity between residues with WT Asf1 and the left side is in the presence of Asf1 with E105A or R mutations.

Figure 6.

Analysis of E105A/R-dependent changes to residue selectivity as a function of pre-acetylated states of histone H3. (A) Comparison of site-specific k_cat(app) of H3/H4 (with and without singly acetylated H3) by Rtt109–Vps75 acetylation in the presence of Asf1 E105A/R mutants. The error bar represents the standard error in k_cat(app). The apparent k_cat are summarized in Supplementary Table S5. (B) Free energy changes to selectivity due to different acetylation states of histone H3 with E105A or R mutations to WT Asf1. Right is the free energy difference between Asf1(E105A) and Asf1 and left is the difference between Asf1(E105R) and Asf1.

Figure 7.

Analysis of fluorescence titration of histones H3/H4 (white squares or white circles) or H3K14ac/H4 (dark gray circles or dark gray squares) into 1 nM Alexa Fluor 546 labeled WT Asf1 (A) or Asf1 mutant E105A (B). Data were fit using non-linear regression in GraphPad Prism. Error bars represent triplicate datasets. These data suggest acetylation of H3K14 impacts binding to WT Asf1 but not E105A Asf1.

Figure 8.

Decision tree for Asf1-dependent changes in Rtt109–Vps75 acetylation. The rectangles represent the acetylation output by Rtt109Vps75, and the difference in font represent the relative selectivity for that starting point. The red rectangles represent those substrate whose rate is as fast or faster than the acetylation of H3/H4 by Rtt109–Vps75.