Changing the Selectivity of p300 by Acetyl-CoA Modulation of Histone Acetylation

Abstract

Determining how histone acetylation is regulated is vital for treating the many diseases associated with its misregulation, including heart disease, neurological disorders, and cancer. We have previously reported that acetyl-CoA levels alter p300 histone acetylation in a site-specific manner in vitro. Here we further investigate how changing acetyl-CoA concentrations alter the histone acetylation pattern by altering p300 specificity. Interestingly, these changes are not a simple global change in acetylation, but rather site specific changes, whereby acetylation at some sites increase while others decrease. We also demonstrate how the p300 inhibitor C646 can pharmacologically alter p300 histone acetylation patterns in vitro and in cells. This study provides insight into the mechanisms regulating p300 residue specificity, a potential means for altering p300 dependent histone acetylation, and an investigation into altering histone acetylation patterns in cells.

Discovering Targets of Non-enzymatic Acylation by Thioester Reactivity Profiling

“…Probe labeling for almost all proteins was concentration-dependent and not saturatable, as expected for a non-enzymatic reaction (Figure 2b) (Olia et al, 2015). Of note, robust labeling was observed even at 100 μM which lies in the range of cellular acetyl-CoA levels (estimated 20–200 μM) (Henry et al, 2015). Using competition studies, we found that labeling of proteins by thioester 1 was strongly impeded by pre-incubating lysates with excess acetyl-CoA (Figure 2c).…”

“…To understand whether malonyl-CoA’s effects on glycolytic enzymes may stem from covalent non-enzymatic malonylation, we performed parallel assessments of GAPDH malonylation and activity as a function of time. Time-dependent enzyme activity loss was minimized by performing these analyses at 200 μM, which likely represents the upper range of physiologically relevant malonyl-CoA concentrations (Henry et al, 2015; Tokutake et al, 2012; Tokutake et al, 2010). Consistent with a covalent inhibition mechanism, malonyl-CoA reduces GAPDH activity in a time-dependent manner (Figure 4b).…”

Procollagen Lysyl Hydroxylase 2 Expression Is Regulated by an Alternative Downstream Transforming Growth Factor β-1 Activation Mechanism

“…Interestingly, both PLOD1 and PLOD3 expression were downregulated when treated with the CBP/P300 inhibitor, but PLOD1 expression was increased in unstimulated fibroblast when P300 and CBP was depleted by esiRNA. In some circumstances C646 can enhance P300-induced acetylation, and also its specificity toward P300/CBP is under debate (59). Therefore, the discrepancy between inhibitor and knockdown effects on PLOD1 and PLOD3 might be related to such issues.…”

Acetyl Coenzyme A: A Central Metabolite and Second Messenger

“…Importantly, the concentration of acetyl-CoA (or the acetyl-CoA/CoA ratio) does not only determine the enzymatic activity of KATs, but also alters their specificity. Thus, KATs like E1A binding protein p300 (EP300) and CREB binding protein (CREBBP, best known as CBP) exhibit distinct Hill values (i.e., different degrees of cooperativity) at varying acetyl-CoA levels, meaning that their selectivity (i.e., their preference to acetylate distinct lysine residues) changes (Denisov and Sligar, 2012;Henry et al, 2015).…”