Inhibition of Maize Histone Deacetylases by HC Toxin, the Host-Selective Toxin of Cochliobolus carbonum

Abstract

HC toxin, the host-selective toxin of the maize pathogen Cochliobolus carbonum, inhibited maize histone deacetylase (HD) at 2 microM. Chlamydocin, a related cyclic tetrapeptide, also inhibited HD activity. The toxins did not affect histone acetyltransferases. After partial purification of histone deacetylases HD1-A, HD1-B, and HD2 from germinating maize embryos, we demonstrated that the different enzymes were similarly inhibited by the toxins. Inhibitory activities were reversibly eliminated by treating toxins with 2-mercaptoethanol, presumably by modifying the carbonyl group of the epoxide-containing amino acid Aeo (2-amino-9,10-epoxy-8-oxodecanoic acid). Kinetic studies revealed that inhibition of HD was of the uncompetitive type and reversible. HC toxin, in which the epoxide group had been hydrolyzed, completely lost its inhibitory activity; when the carbonyl group of Aeo had been reduced to the corresponding alcohol, the modified toxin was less active than native toxin. In vivo treatment of embryos with HC toxin caused the accumulation of highly acetylated histone H4 subspecies and elevated acetate incorporation into H4 in susceptible-genotype embryos but not in the resistant genotype. HDs from chicken and the myxomycete Physarum polycephalum were also inhibited, indicating that the host selectivity of HC toxin is not determined by its inhibitory effect on HD. Consistent with these results, we propose a model in which HC toxin promotes the establishment of pathogenic compatibility between C. carbonum and maize by interfering with reversible histone acetylation, which is implicated in the control of fundamental cellular processes, such as chromatin structure, cell cycle progression, and gene expression.

Purification of histone deacetylase HD1‐A of germinating maize embryos

“…The four histone deacetylases of maize are distinct with respect to their biochemical and kinetic properties [29], to modification by phosphorylation [20], pattern of enzyme activity during the cell cycle and differentiation [16][17][18], substrate specificity [16,20,29] and subnuclear localization [22]. It is puzzling that despite their different properties, all deacetylases of maize are equally inhibited by different unspecific and specific inhibitors, like butyrate, Trichostatin and cyclic tetrapeptides [21,29]. The fact that multiple deacetylases exist and are subject to complex regulation emphasizes their importance for chromatin structure and function [21,43].…”

“…It is puzzling that despite their different properties, all deacetylases of maize are equally inhibited by different unspecific and specific inhibitors, like butyrate, Trichostatin and cyclic tetrapeptides [21,29]. The fact that multiple deacetylases exist and are subject to complex regulation emphasizes their importance for chromatin structure and function [21,43].…”

“…Evidence for a high molecular weight deacetylase complex has also been presented in HeLa cells [23], yeast [27] and chicken erythrocytes [30]. Recently, a mammalian histone deacetylase has been isolated by its strong affinity to trapoxin [35], a cyclic tetrapeptide inhibitory to all historic deacetylases analyzed so far [21,34]. Interestingly, that approach led to the isolation of just one histone deacetylase with a molecular weight of 55 kDa [35].…”

“…The steady state of histone acetylation is maintained by two enzyme activities, histone acetyltransferase and histone deacetylase. These enzyme activities are present as multiple enzyme forms whose activities, properties, intracellular/intranuclear location and substrate specificities are regulated in a complex, cell cycle-related manner [16][17][18][19][20][21][22].…”

“…A genetic approach was used to identify a B-type histone acetyltransferase of yeast (HAT1) with limited sequence homology to N-terminal acetyltransferases [31]; our laboratory has purified a cytoplasmic histone acetyltransferase B of maize to homogeneity [32]; a nuclear A-type histone acetyltransferase of Tetrahymena has been identified as a homolog of the yeast transcriptional coactivator Gcn5p [33]. By utilizing the high affinity of histone deacetylases to cyclic tetrapeptides [21,34], a mammalian histone deacetylase was recently identified [35].…”