A continuous, nonradioactive assay for histone acetyltransferases

Histone acetyltransferases (HATs) catalyze the acetyl-group transfer from acetyl-CoA to the epsilon-amino group of specific lysine residues within core histone proteins. HATs and other chromatin-remodeling enzymes have been recently shown to regulate gene activation within specific loci. To facilitate mechanistic studies, we have developed two continuous, nonradioactive assays for the prototypical GCN5 HAT. The CoASH generated in the HAT reactions was continuously measured by using a coupled enzyme system with either alpha-ketoglutarate dehydrogenase or pyruvate dehydrogenase. The CoASH-dependent oxidation of alpha-ketoglutarate or pyruvate is accompanied by the reduction of NAD to NADH, which was measured spectrophotometrically at 340 nm. The steady-state rate constants with substrates acetyl-CoA and a synthetic peptide (corresponding to the first 20 amino acids of H3 histone) were determined. The resulting rate constants were not significantly different between the two coupled assays, providing strong validation of these methods. Rate constants were also determined using the commonly employed radioactive filter-binding assay and compared. The 1.5- to 5-fold lower values obtained in the radioactive end-point assay are discussed in terms of the technical problems and limitations of this assay. The coupled assays should be widely applicable since the production of CoASH is common to all HAT enzymes, regardless of protein substrate.     

The catalytic mechanism of the ESA1 histone acetyltransferase involves a self-acetylated intermediate

Yeast ESA1 is a member of the MYST subfamily of histone acetyltransferases (HATs), which use acetyl-coenzyme A (CoA) to acetylate specific Lys residues within histones to regulate gene expression. The structure of an ESA1-CoA complex reveals structural similarity to the catalytic core of the GCN5/PCAF subfamily of HAT proteins. Here we report additional structural and functional studies on ESA1 that demonstrate that histone acetylation proceeds through an acetyl-cysteine enzyme intermediate. This Cys residue is strictly conserved within the MYST members, suggesting a common mode of catalysis by this HAT subfamily. However, this mode of catalysis differs dramatically from the GCN5/PCAF subfamily, which mediate direct nucleophilic attack of the acetyl-CoA cofactor by the enzyme-deprotonated substrate lysine of the histone. These results demonstrate that different HAT subfamilies can use distinct catalytic mechanisms, which have implications for their distinct biological roles and for the development of HAT-specific inhibitors.     

Bisubstrate analogue structure-activity relationships for p300 histone acetyltransferase inhibitors

p300 and CBP are important histone acetyltransferases (HATs) that regulate gene expression and may be anti-cancer drug targets. Based on a previous lead compound, Lys-CoA, we have used solid phase synthesis to generate a series of 11 new analogues and evaluated these compounds as HAT inhibitors. Increased spacing between the CoA moiety and the lysyl moiety generally decreases inhibitory potency. We have found two substituted derivatives that show about 4-fold increased potency compared to the parent compound Lys-CoA. These structure-activity studies allow for a greater understanding of the optimal requirements for potent inhibition of HAT enzymes and pave the way for a novel class of anti-cancer therapeutics.     

Improved inhibition of the histone acetyltransferase PCAF by an anacardic acid derivative

Several lines of evidence indicate that histone acetyltransferases (HATs) are novel drug targets for treatment of diseases like, for example, cancer and inflammation. The natural product anacardic acid is a starting point for development of small molecule inhibitors of the histone acetyltransferase (HAT) p300/CBP associated factor (PCAF). In order to optimize the inhibitory potency, a binding model for PCAF inhibition by anacardic acid was proposed and new anacardic acid derivatives were designed. Ten new derivatives were synthesized using a novel synthetic route. One compound showed a twofold improved inhibitory potency for the PCAF HAT activity and a twofold improved inhibition of histone acetylation in HEP G2 cells.     

Investigation of MYST4 histone acetyltransferase and its involvement in mammalian gametogenesis

Background  

Various histone acetylases (HATs) play a critical role in the regulation of gene expression, but the precise functions of many of those HATs are still unknown. Here we provide evidence that MYST4, a known HAT, may be involved in early mammalian gametogenesis.     

Inhibition of histone acetyltransferase by glycosaminoglycans

Histone acetyltransferases (HATs) are a class of enzymes that participate in modulating chromatin structure and gene expression. Altered HAT activity has been implicated in a number of diseases, yet little is known about the regulation of HATs. In this study, we report that glycosaminoglycans (GAGs) are potent inhibitors of p300 and pCAF HAT activities in vitro, with heparin and heparan sulfate proteoglycans (HSPGs) being the most potent inhibitors. The mechanism of inhibition by heparin was investigated. The ability of heparin to inhibit HAT activity was in part dependent upon its size and structure, as small heparin-derived oligosaccharides (>8 sugars) and N-desulfated or O-desulfated heparin showed reduced inhibitory activity. Heparin was shown to bind to pCAF; and enzyme assays indicated that heparin shows the characteristics of a competitive-like inhibitor causing an approximately 50-fold increase in the apparent Km of pCAF for histone H4. HSPGs isolated from corneal and pulmonary fibroblasts inhibited HAT activity with similar effectiveness as heparin. As evidence that endogenous GAGs might be involved in modulating histone acetylation, the direct addition of heparin to pulmonary fibroblasts resulted in an approximately 50% reduction of histone H3 acetylation after 6 h of treatment. In addition, Chinese hamster ovary cells deficient in GAG synthesis showed increased levels of acetylated histone H3 compared to wild-type parent cells. GAGs represent a new class of HAT inhibitors that might participate in modulating cell function by regulating histone acetylation.     

Inhibition of histone acetylation and deacetylation enzymes affects longevity,development, and fecundity in the pea aphid (Acyrthosiphon pisum)

Histone acetylation is an evolutionarily conserved epigenetic mechanism of eukaryotic gene regulation which is tightly controlled by the opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). In insects, life-history traits such as longevity and fecundity are severely affected by the suppression of HAT/HDAC activity, which can be achieved by RNA-mediated gene silencing or the application of chemical inhibitors. We used both experimental approaches to investigate the effect of HAT/HDAC inhibition in the pea aphid (Acyrthosiphon pisum) a model insect often used to study complex life-history traits. The silencing of HAT genes (kat6b, kat7, and kat14) promoted survival or increased the number of offspring, whereas targeting rpd3 (HDAC) reduced the number of viviparous offspring but increased the number of premature nymphs, suggesting a role in embryogenesis and eclosion. Specific chemical inhibitors of HATs/HDACs showed a remarkably severe impact on life-history traits, reducing survival, delaying development, and limiting the number of offspring. The selective inhibition of HATs and HDACs also had opposing effects on aphid body weight. The suppression of HAT/HDAC activity in aphids by RNA interference or chemical inhibition revealed similarities and differences compared to the reported role of these enzymes in other insects. Our data suggest that gene expression in A. pisum is regulated by multiple HATs/HDACs, as indicated by the fitness costs triggered by inhibitors that suppress several of these enzymes simultaneously. Targeting multiple HATs or HDACs with combined effects on gene regulation could, therefore, be a promising approach to discover novel targets for the management of aphid pests.