Metabolic Regulation of Histone Acetyltransferases by Endogenous Acyl-CoA Cofactors

Abstract

SUMMARY The finding that chromatin modifications are sensitive to changes in cellular cofactor levels potentially links altered tumor cell metabolism and gene expression. However, the specific enzymes and metabolites that connect these two processes remain obscure. Characterizing these metabolic-epigenetic axes is critical to understanding how metabolism supports signaling in cancer, and developing therapeutic strategies to disrupt this process. Here, we describe a chemical approach to define the metabolic regulation of lysine acetyltransferase (KAT) enzymes. Using a novel chemoproteomic probe, we identify a previously unreported interaction between fatty acyl-CoAs and KAT enzymes. Further analysis reveals that palmitoyl-CoA is a potent inhibitor of KAT activity and that fatty acyl-CoA precursors reduce cellular acetylation levels. These studies implicate fatty acyl-CoAs as endogenous regulators of histone acetylation, and suggest novel strategies for the investigation and metabolic modulation of epigenetic signaling.

Discovering Targets of Non-enzymatic Acylation by Thioester Reactivity Profiling

“…For both ex situ and in situ samples, 20 μg of labeled proteome was used for SDS-PAGE target analysis, and 2000 μg of labeled proteome was used for LC-MS/MS target analysis. Proteins labeled by 1 were visualized by SDS-PAGE via Cu(I)-catalyzed [3 + 2] cycloaddition with a fluorescent azide as previously reported (Montgomery et al, 2015a; Speers and Cravatt, 2004). Gels were fixed and destained in a solution of 50% MeOH/40% H 2 O/10% AcOH overnight to remove excess probe fluorescence, rehydrated with water, and visualized using an ImageQuant Las4010 (GE Healthcare) with green LED excitation (λ max 520–550 nm) and a 575DF20 filter.…”

“…For competition experiments, proteomes were pretreated with acetyl-CoA (0–800 μM) for 1 hour prior to incubation with thioester 1 . For proteomic studies, labeled proteomes were enriched via Cu(I)-catalyzed [3 + 2] cycloaddition with a biotin azide, desalted, and analyzed by LC-MS/MS as previously reported (Montgomery et al, 2015a). Extended protocols for fluorescence analyses and enrichments are provided in the Supplemental Information.…”

Impact of a High-fat Diet on Tissue Acyl-CoA and Histone Acetylation Levels

“…Both crotonyl-CoA and ACSS2 levels were suppressed by HFD in WAT; further investigation into the impact of diet on histone crotonylation may thus point toward new mechanisms of gene regulation in response to diet. In addition, it has been shown in principle that acyl-CoAs such as palmitoyl-CoA and butyryl-CoA can inhibit KATs (24), although in vivo circumstances in which this definitively occurs have yet to be identified. This study provides evidence for this possibility by pointing to a correlation between the acetyl-CoA:(iso)butyryl-CoA ratio and histone acetylation in the liver.…”

Chemical Biology Approaches for Investigating the Functions of Lysine Acetyltransferases

“…Thep reinstalled desthiobiotin affinity handle on the probe allowed downstream pull-down experiments,which was demonstrated by successful labeling of two representative acetyltransferases,E SA1 and p300. [117] Biotin-labeled bisubstrate inhibitors (5)w ere immobilized on streptavidin-agarose resins.I ncubation of these resinimmobilized bisubstrate inhibitors with cell lysates sensitively enriched several low-abundance nuclear KATs (GCN5, PCAF,a nd MOF), together with several KATc omplexes and NATs.M ore recently,N HS-Sepharose,w hich exhibits ah igher dense surface functionalization than streptavidinagarose,w as applied as an ew type of resin to couple with bisubstrate inhibitors such as Lys-CoA (6), H3K14-CoA (7), and H4K16-CoA (8). Fourier-transform MS demonstrated that Cys-1621 of p300 was an interactive site for acetyl-CoA, mutation of this site to alanine led to 15.6fold increase in the K m value of acetyl-CoA.…”