Acetylome Profiling Reveals Overlap in the Regulation of Diverse Processes by Sirtuins, Gcn5, and Esa1

Michael Downey,Jeffrey R Johnson,Norman E Davey,Billy W Newton,Tasha L Johnson,Shastyn Galaang,Charles A Seller,Nevan Krogan,David P Toczyski

doi:10.1074/mcp.m114.043141

Michael Downey, Jeffrey R Johnson + Show 7 more

Open Access

https://doi.org/10.1074/mcp.m114.043141

Copy DOI

Abstract

Although histone acetylation and deacetylation machineries (HATs and HDACs) regulate important aspects of cell function by targeting histone tails, recent work highlights that non-histone protein acetylation is also pervasive in eukaryotes. Here, we use quantitative mass-spectrometry to define acetylations targeted by the sirtuin family, previously implicated in the regulation of non-histone protein acetylation. To identify HATs that promote acetylation of these sites, we also performed this analysis in gcn5 (SAGA) and esa1 (NuA4) mutants. We observed strong sequence specificity for the sirtuins and for each of these HATs. Although the Gcn5 and Esa1 consensus sequences are entirely distinct, the sirtuin consensus overlaps almost entirely with that of Gcn5, suggesting a strong coordination between these two regulatory enzymes. Furthermore, by examining global acetylation in an ada2 mutant, which dissociates Gcn5 from the SAGA complex, we found that a subset of Gcn5 targets did not depend on an intact SAGA complex for targeting. Our work provides a framework for understanding how HAT and HDAC enzymes collaborate to regulate critical cellular processes related to growth and division.

Highlights

Acetylation of histone tails serves as a regulator of eukaryotic transcription by neutralizing the positive charge on such tails and by serving as a conditional-binding interface for chromatin remodeling enzymes containing acetyllysine-binding bromodomains [8, 9]
The HST1, HST2, and SIR2-regulated Acetylome—Analysis of yeast whole-cell extracts (WCEs) with an antibody generated against acetylated lysine revealed an increase in acetylated species in strains deleted for yeast sirtuins SIR2, HST1, and HST2 (Fig. 1A, left, hereafter referred to as “sirtuins”)
To identify other proteins whose acetylations are regulated by sirtuin enzymes, we compared the acetylation landscape of sir2⌬ hst1⌬ hst2⌬ triple mutant cells to isogenic counterparts using stable isotopic analysis of amino acids in cell culture (SILAC, Fig. 1A, right)

Summary

Introduction

Acetylation of histone tails serves as a regulator of eukaryotic transcription by neutralizing the positive charge on such tails and by serving as a conditional-binding interface for chromatin remodeling enzymes containing acetyllysine-binding bromodomains [8, 9]. In strains lacking multiple sirtuins [1], suggesting that there is some overlap in function for these enzymes in the regulation of non-histone targets.

Results

Conclusion