Chaperone-mediated acetylation of histones by Rtt109 identified by quantitative proteomics

Abstract

Rtt109 is a fungal-specific histone acetyltransferase (HAT) that associates with either Vps75 or Asf1 to acetylate histone H3. Recent biochemical and structural studies suggest that site-specific acetylation of H3 by Rtt109 is dictated by the binding chaperone where Rtt109-Asf1 acetylates K56, while Rtt109-Vps75 acetylates K9 and K27. To gain further insights into the roles of Vps75 and Asf1 in directing site-specific acetylation of H3, we used quantitative proteomics to profile the global and site-specific changes in H3 and H4 during in vitro acetylation assays with Rtt109 and its chaperones. Our analyses showed that Rtt109-Vps75 preferentially acetylates H3 K9 and K23, the former residue being the major acetylation site. At high enzyme-to-substrate ratio, Rtt109 also acetylated K14, K18, K27 and to a lower extent K56 of histone H3. Importantly, this study revealed that in contrast to Rtt109-Vps75, Rtt109-Asf1 displayed a far greater site-specificity, with K56 being the primary site of acetylation. For the first time, we also report the acetylation of histone H4 K12 by Rtt109-Vps75, whereas Rtt109-Asf1 showed no detectable activity toward H4. This article is part of a Special Issue entitled: From protein structures to clinical applications.

Figure 1

Overview of the analytical scheme for profiling histone modifications.

Figure 2. LC/MS analyses of intact histone H3 obtained from in vitro HAT assay

(A) Intact mass profiles of histone H3 obtained from in vitro HAT assay with Rtt109-Vps75 and (B) with Rtt109-Asf1. Incubation times (from top to bottom) are 0, 1, 5, 15, 30, 60 and 90 min. (C) Bar graph represent the extent of H3 acetylation after HAT assay with Rtt109-Vps75 and (D) with Rtt109-Asf1.

Figure 3. LC/MS analyses of intact histone H4 obtained from in vitro HAT assay

(A) Intact mass profiles of histone H4 obtained from in vitro HAT assay with Rtt109-Vps75. (B) Bar graph showing the extent of H4 acetylation after assay with Rtt109-Vps75. (C) Intact mass profiles of histone H4 obtained from in vitro HAT assay with Rtt109-Asf1. Incubation times (top to bottom) are 0, 1, 5, 15, 30, 60, 90 min.

Figure 4. Relative proportion of histone acetylation by Rtt109-Vps75 for different enzyme to substrate ratios

Distribution of acetylated histone for (A) H3 and (B) H4. (C) in vitro acetylation of histone H3 acetylation by Rtt109 alone (without chaperone) after an incubation period of 15 min (upper panel) and 60 min (lower panel).

Figure 5. Identification of modified residues and determination of acetylation stoichiometry of H3 incubated with Rtt109-Vps75/Asf1

(A) Extracted ion chromatograms of the doubly charged tryptic peptides corresponding to the acetylated and propionylated forms of ⁹KSTGGKAPR¹⁷. (B) MS/MS spectrum of the acetylated tryptic peptide ⁹KacSTGGKAPR¹⁷. Acetylation stoichiometry of H3 lysine residues following incubation with (C) Rtt109-Vps75 and (D) Rtt109-Asf1. (E) Primary acetylation sites of Rtt109-Vps75 (K9 and K23 located on the N-terminus tail of H3) and Rtt109-Asf1 (K56 located on the H3 fold domain). The cartoon representation of yeast histone H3 (red) and H4 (blue) are taken from nucleosome PDB 1ID3 (prepared using the Pymol Molecular Graphics system, version 1.2r1). (F) Amino acid sequence of yeast histone H3.

Figure 6. Identification of modified residues and determination of acetylation stoichiometry of H4 incubated with Rtt109-Vps75

(A) Extracted ion chromatograms of the doubly charged tryptic peptides corresponding to the acetylated and propionylated forms of ⁴GKGGKGLGKGGAKR¹⁷. (B) MS/MS Spectrum of the acetylated tryptic peptide ⁴GKGGKGLGKacGGAKR¹⁷. (C) Acetylation stoichiometry of H4 lysine residues (K5, K8, K12) following incubation with Rtt109-Vps75. (D) Amino acid sequence of yeast histone H4.