Investigation of the acetylation mechanism by GCN5 histone acetyltransferase

The histone acetylation of post-translational modification can be highly dynamic and play a crucial role in regulating cellular proliferation, survival, differentiation and motility. Of the enzymes that mediate post-translation modifications, the GCN5 of the histone acetyltransferase (HAT) proteins family that add acetyl groups to target lysine residues within histones, has been most extensively studied. According to the mechanism studies of GCN5 related proteins, two key processes, deprotonation and acetylation, must be involved. However, as a fundamental issue, the structure of hGCN5/AcCoA/pH3 remains elusive. Although biological experiments have proved that GCN5 mediates the acetylation process through the sequential mechanism pathway, a dynamic view of the catalytic process and the molecular basis for hGCN5/AcCoA/pH3 are still not available and none of theoretical studies has been reported to other related enzymes in HAT family. To explore the molecular basis for the catalytic mechanism, computational approaches including molecular modeling, molecular dynamic (MD) simulation and quantum mechanics/molecular mechanics (QM/MM) simulation were carried out. The initial hGCN5/AcCoA/pH3 complex structure was modeled and a reasonable snapshot was extracted from the trajectory of a 20 ns MD simulation, with considering post-MD analysis and reported experimental results. Those residues playing crucial roles in binding affinity and acetylation reaction were comprehensively investigated. It demonstrated Glu80 acted as the general base for deprotonation of Lys171 from H3. Furthermore, the two-dimensional QM/MM potential energy surface was employed to study the sequential pathway acetylation mechanism. Energy barriers of addition-elimination reaction in acetylation obtained from QM/MM calculation indicated the point of the intermediate ternary complex. Our study may provide insights into the detailed mechanism for acetylation reaction of GCN5, and has important implications for the discovery of regulators against GCN5 enzymes and related HAT family enzymes.     

CAT in the HAT: catabolic inhibition by the histone acetyltransferase GCN5

The nuclear hormone receptor coactivator PGC-1alpha is a key regulator of gluconeogenic genes during fasting. In this issue of Cell Metabolism, Puigserver and colleagues (Lerin et al., 2006) report that the histone acetyltransferase GCN5 inhibits gluconeogenesis by acetylating and sequestering PGC-1alpha in nuclear foci.     

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.     

Fluorescence analysis of a dynamic loop in the PCAF/GCN5 histone acetyltransferase

PCAF and GCN5 are histone acetyltransferase (HAT) paralogs which play roles in the remodeling of chromatin in health and disease. Previously, a conformationally flexible loop in the catalytic domain had been observed in the X-ray structures of GCN5 in different liganded states. Here, the conformation and dynamics of this PCAF/GCN5 alpha5-beta6 loop was investigated in solution using tryptophan fluorescence. A mutant human PCAF HAT domain (PCAF(Wloop)) was created in which the natural tryptophan (Trp-514) remote from the alpha5-beta6 loop was replaced with tyrosine and a glutamate within the loop (Glu-641) was substituted with tryptophan. This PCAF(Wloop) protein exhibited catalytic parameters within 3-fold of those of the wild-type PCAF catalytic domain, suggesting that the loop mutation was not deleterious for HAT activity. While saturating CoASH induced a 30% quenching of Trp fluorescence in PCAF(Wloop), binding of the high-affinity bisubstrate analogue H3-CoA-20 led to a 2-fold fluorescence increase. These different effects correlate with the different alpha5-beta6 loop conformations seen previously in X-ray structures. On the basis of stopped-flow fluorescence studies, binding of H3-CoA-20 to PCAF(Wloop) proceeds via a rapid association step followed by a slower conformational change involving loop movement. Time-resolved fluorescence measurements support a model in which the alpha5-beta6 loop in the H3-CoA-20-PCAF(Wloop) complex exists in a narrower ensemble of conformations compared to free PCAF(Wloop). The relevance of loop dynamics to PCAF/GCN5 catalysis and substrate specificity are discussed.     

PFKFB4 negatively regulated the expression of histone acetyltransferase GCN5 to mediate the tumorigenesis of thyroid cancer

Thyroid cancer (TC) is the most common malignant endocrine tumor, and its incidence has progressively increased over several decades. Accumulating evidence has suggested that PFKFB4, a critical regulatory enzyme of glycolysis, has been implicated in various solid cancers. However, the exact effect of PFKFB4 on TC remains unclear. Hence, the objective of this work was to investigate the role of PFKFB4 in TC and explore the underlying regulatory mechanisms. Here, we provide evidence that mRNA levels of PFKFB4 were upregulated in TC patients’ thyroids and cell lines. Downregulation of PFKFB4 reduced TC cell viability and inhibited colony formation. In addition, the migration and invasion of TC cells were suppressed by PFKFB4 knockdown, suggesting that PFKFB4 is positively correlated with tumorigenesis of TC. Molecularly, knockdown of PFKFB4 significantly inhibited expression of GCN5 and phosphorylation of PI3K/AKT. Moreover, the suppressive role of shPFKFB4 in TC cell growth was reversed by upregulation of GCN5. Finally, the in vivo experiment indicated that downregulation of PFKF4B suppressed tumor growth in xenografts TC model mice. In total, our results suggested that PFKFB4-mediated TC tumorigenesis by positively regulating GCN5 and PI3K/AKT signaling. These findings provide new research directions and therapeutic options considering PFKF4B as a novel diagnosis marker and therapeutic target.     

Characterization of a phosphatase 2C protein as an interacting partner of the histone acetyltransferase GCN5 in Arabidopsis

Arabidopsis GCN5 is a major histone acetyltransferase. The mutation of the gene induces pleiotropic effects on plant development, and affects the expression of a large number of genes. The mechanism of action of this protein in controlling plant chromatin structure and genome expression is not understood. In this work, we report the identification of a number of potential protein interacting partners of GCN5 in Arabidopsis. In particular, GCN5 was shown to interact specifically with a phosphatase 2C protein (AtPP2C-6-6). GCN5 phosphorylated by activities in cellular extracts could be dephosphorylated by AtPP2C-6-6 in vitro. Analysis of T-DNA insertion mutants revealed a positive role of AtPP2C-6-6 in salt induction of stress-inducible genes, while the gcn5 mutation seemed to have no effect on the induction but showed up-regulation of a subset of the stress-inducible genes under non-induced conditions. In addition, the gcn5 mutation seriously reduced acetylation of histone H3K14 and H3K27, whereas the T-DNA insertions of the AtPP2C6-6 gene enhanced the acetylation of these lysine residues. Taken together, the present data suggest that AtPP2C-6-6 may function as a negative regulator of GCN5 activity in Arabidopsis.     

Histone acetyltransferase GCN5 contributes to cell wall integrity and salt stress tolerance by altering the expression of cellulose synthesis genes

Excess soluble salts in soil are harmful to the growth and development of most plants. Evidence is emerging that the plant cell wall is involved in sensing and responding to salt stress, but the underlying mechanisms are not well understood. We reveal that the histone acetyltransferase General control non‐repressed protein 5 (GCN5) is required for the maintenance of cell wall integrity and salt stress tolerance. The levels of GCN5 mRNA are increased in response to salt stress. The gcn5 mutants exhibited severe growth inhibition and defects in cell wall integrity under salt stress conditions. Combining RNA sequencing and chromatin immunoprecipitation assays, we identified the chitinase‐like gene CTL1, polygalacturonase involved in expansion‐3 (PGX3) and MYB domain protein‐54 (MYB54) as direct targets of GCN5. Acetylation of H3K9 and H3K14 mediated by GCN5 is associated with activation of CTL1, PGX3 and MYB54 under salt stress. Moreover, constitutive expression of CTL1 in the gcn5 mutant restores salt tolerance and cell wall integrity. In addition, the expression of the wheat TaGCN5 gene in Arabidopsis gcn5 mutant plants complemented the salt tolerance and cell wall integrity phenotypes, suggesting that GCN5‐mediated salt tolerance is conserved between Arabidopsis and wheat. Taken together, our data indicate that GCN5 plays a key role in the preservation of salt tolerance via versatile regulation in plants.