Drug-resistant histone deacetylases in filamentous fungi

DNA of the eukaryotic nucleus is organized by histones, small basic proteins which are subject to reversible, postsynthetic modifications, such as phosphorylation, glycosylation, ADP-ribosylation, ubiquitination and acetylation. Histone acetylation is a reversible reaction catalyzed by histone acetyltransferases (HAT) and histone deacetylases (HD). HATs introduce acetyl moieties to specific lysine residues of the N-terminal tails of histories. The reverse reaction is carried out by HDs. HATs have been shown to play an important role in transcriptional activation, whereas HDs maybe involved in transcriptional repression, although the exact flinction is not clear. Butyrate, the antibiotic trichostatin and cyclic tetrapeptides (HC-toxin, trapoxin, apicidin) have been reported to inhibit HDs in several organisms and thereby cause anti-neoplastic effects and lead to cell cycle arrest and differentiation. We could show that extracts of the filamentous fungus Cochliobolus carbonum contain inhibitor- resistant HD activities in contrast to the close relative Aspergillus nidulans which has normal sensitive HDs. Cochliobolus carbonum is a maize pathogen colonizing maize cells presumably by inhibition of host HDs by HC toxin. We will study inhibitor-resistant HDs of C. carbonum and inhibitor susceptible HDs of A. nidulans in order to learn more about the structure of HDs (catalytic center, inhibitor binding site) and their mode of action. We will first establish optimal extraction procedures for subsequent biochemical, immunological and enzymatic characterization and punification of inhibitor-resistant/sensitive HDs of the filamentous fungi Cochliobolus and Aspergillus. As a second step we will analyze the encoding genes, and finally try to identify the structure of catalytic center(s) and toxin binding site(s) of these enzymes. The final goal will be to understand and to clarify the biochemical and molecular basis for resistance of HDs and eventually to design specific inhibitors for distinct HD classes, that may selectively be used as chemotherapeutic agents.

Histone acetylation plays a fundamental role for diverse cellular processes. Histone acetyltransferases and deacetylases (HDACs) in general are leading to activation or silencing of genes, respectively. Specific inhibitors of HDACs as trichotatin A or cyclic tetrapeptides thereby represent important tools for the study of the underlying mechanisms. The cyclic tetrapeptide HC-toxin is produced by the filamentous fungus and maize pathogen Cochliobolus carbonum. Interestingly, this fungus expresses inhibitor resistant HDAC activities as a detoxifying mechanism against endogenous toxin. Since HDACs have been identified as transcriptional regulators, playing a key role in the regulation of gene expression, modified HDACs could have considerable potential for chemotherapy since aberrant histone acetylation is involved in disease. Therefore, the analysis and identification of such undefined HDACs in Cochliobolus carbonum and Aspergillus nidulans as a reference system should contribute to a better understanding of the biological role of histone acetylation in general and for the mechanism of pathogenesis in detail. Divers biochemical experiments and mutational analysis in C. carbonum strongly indicated that an enzyme named HDC4 (Histone Deacetylase Cochliobolus 4) which is homologous to the yeast protein HOS3 is a putative candidate for inhibitor resistance in this filamentous fungus. A deletion mutant of HDC4 lacked resistant activity in protein extracts. Furthermore, molecular sizes of HDC4 and homologous enzymes in A. nidulans and yeast are remarkably different indicating a deviating structure responsible for insensitivity towards HDAC inhibitors. Our results furthermore suggest that a second "extrinsic" factor or factors exist that render sensitive extracts resistant. HC-toxin insensitivity of the HDACs of C. carbonum obviously is an integral part of the trait of HC- toxin production and hence pathogenesis. Therefore expression of an inhibitor resistant HDAC and (not yet identified) external factor(s) represent important self-protection mechanisms against HC-toxin.