Correction: Quantitating the Specificity and Selectivity of Gcn5-Mediated Acetylation of Histone H3

Abstract

Lysine acetyltransferases (KATs) play a unique role in regulating gene transcription as well as maintaining the epigenetic state of the cell. KATs such as Gcn5 and p300/CBP can modify multiple residues on a single histone; however, order and specificity of acetylation can be altered by factors such as histone chaperones, subunit proteins or external stimulus. While the importance of acetylation is well documented, it has been difficult to quantitatively measure the specificity and selectivity of acetylation at different residues within a histone. In this paper, we demonstrate a label-free quantitative high throughput mass spectrometry-based assay capable of quantitatively monitoring all known acetylation sites of H3 simultaneously. Using this assay, we are able to analyze the steady-state enzyme kinetics of Gcn5, an evolutionarily conserved KAT. In doing so, we measured Gcn5-mediated acetylation at six residues (K14.K9 < K23. K18. K27 < K36) and the catalytic efficiency (k cat /K m ) for K9, K14, K18, and K23 as well as the nonenzymatic acetylation rate. We observed selectivity differences of up to 24 kcal/mol between K14 and K18, the highest and lowest measurable k cat /K m . These data provide a first look at quantitating the specificity and selectivity of multiple lysines on a single substrate (H3) by Gcn5.

Histone Acetylation Dynamics Integrates Metabolic Activity to Regulate Plant Response to Stress

“…The global change of acetylation at specific lysine residues suggests that activity of particular enzymes involved in histone acetylation is induced by the stress. However, despite that acetylation enzymes may have preferential lysine targets in histone H3 or H4 (Henry et al, 2013; Kuo and Andrews, 2013; Zhou et al, 2017), acetylation at a particular site can be spread to neighboring lysine residues (Earley et al, 2007). On the other hand, as primary metabolism is greatly altered by stress or growth conditions, a metabolic control of histone acetylation is not excluded (see below).…”

Islet-1 induces the differentiation of mesenchymal stem cells into cardiomyocyte-like cells through the regulation of Gcn5 and DNMT-1

“…The major function of HATs is to perform acetylation modification of the lysine residue at the amino terminus of the chromatin core histones, thereby loosening the chromatin structure and increasing the gene transcription activities (32). The first discovered histone acetyltransferase, Gcn5, primarily modifies nucleosomal histones and the free histones H3 and H4 (33–35). P300 is a coactivator and HAT that modifies 4 histones (H2A, H2B, H3 and H4) (33,36,37).…”

Discordant Effects of Putative Lysine Acetyltransferase Inhibitors in Biochemical and Living Systems

“…UPLC-MS/MS analysis —A Waters Acquity H-class UPLC (Milford, MA, USA) coupled to a Thermo TSQ Quantum Access (Waltham, MA, USA) triple quadrupole (QqQ) mass spectrometer was used to quantify acetylated H3/H4 peptides as previously described [20,21].…”

“…QqQ MS data analysis —Each acetylated and/or propionylated peak was identified by retention time and specific transitions, as previously reported [20,21]. The resulting peak integration was done using Xcalibur software (version 2.1, Thermo, Waltham, MA, USA).…”

“…The resulting peak integration was done using Xcalibur software (version 2.1, Thermo, Waltham, MA, USA). The fraction of a specific peptide ( F p ) is calculated by equation 1, where I s is the intensity of a specific peptide state and I p is the intensity of any state of that peptide, and analyzed as previously described [21,22]. FP = IS/false(true∑IPfalse)…”