Functional role of distinct histone deacetylases in the filamentous fungus Aspergillus nidulans

One amazing feature of eukaryotic cells is the packaging of the genomic DNA of approx. 1-2m in length into a cell nucleus of only few m in size. Basic proteins, so-called core histones, enable the condensation of the DNA. Histones and DNA together form the chromatin with the nucleosome as its basic element. However, permanent rearrangements of chromatin structure are of essential importance in regulating gene expression and replication. Thus chromatin is no "rigid mass" but an extremely dynamic molecule. Responsible for this dynamics are enzymes which can modify histones in a very specific way and thereby change the structure of distinct chromatin sections. Among these modifications, acetylation processes are the most prominent ones, whereby an acetyl group is linked to specific lysins of distinct core histones. Each acetylation reaction neutralizes a positive charge in the nucleosomes and therefore weakens the interaction of histones and DNA or may interfere with the higher order packing of chromatin both allowing transcriptional regulators to gain access to the DNA. On the other hand, acetylation may act as a highly specific signal that enables or at least facilitates the binding of enzymes responsible for replication or transcriptional processes. However, histone acetylation plays a crucial role in gene regulation in eukaryots. Histones can be acetylated by histone acetyltransferases (HATs) and can be deacetylated by a second group of enzymes, the histone deacetylases (HDACs). In contrast to HATs, for which to date no potent inhibitors are known, there is a panel of agents available that affect HDACs in a very selective way. Today, these inhibitors are important tools for the study of histone acetylation processes. Recently, we have identified and partially characterized HDACs of different classes in the filamentous fungus Aspergillus nidulans. A further characterization and the clarification of the function of these enzymes is the goal of this project. Since filamentous fungi are more complex than yeast yet genetic manipulation is relatively simple and easy to perform, they have widely been regarded as model systems to study the basis of eukaryotic gene regulation. Moreover, many of these organisms play an important role in the spoilage of food or represent dangerous pathogenic agents which can cause life- threatening infections in patients. For these reasons the study of these organisms is also from a large economic and medical interest.

Along with other histone modifying enzymes, histone deacetylases (HDACs) are generally accepted as key players in the epigenetic regulation of eukaryotic gene expression removing acetyl groups from specific lysine residues in the amino-terminal tails of the core-histones. In general, acetylation of histones interferes with the higher order packing of chromatin allowing transcriptional regulators to gain access to the DNA. In addition, acetylation may act as a highly specific signal that enables or at least facilitates the binding of enzymes or factors involved in replication or transcription. In recent years we have intensively studied HDACs in different fungi. We were able to identify and characterize all HDACs in the model organism and filamentous fungus Aspergillus nidulans. Our analyses revealed that some of these enzymes exhibit fungal-specific structures and biological functions that make them clearly distinguishable from homologous proteins of other eukaryotic species (e.g. yeast or mammals). The typical class 2 HDAC HdaA for example turned out to be necessary for the protection of the fungus against oxidative stress due to the activation of protective enzymes involved in the antioxidant defense of Aspergillus. This is an interesting aspect in filamentous fungi, since oxidative stress has been implicated to play a distinct role in human defense mechanisms against pathogenic organisms like Aspergillus fumigatus. Deletion of hdaA, however, causes another drastic effect in A. nidulans. In collaboration with the laboratory of Nancy Keller (Wisconsin), we were able to show that loss of HdaA activity leads to an increase of the penicillin production as well as a remarkable upregulation of the expression of other secondary metabolites. This is a basic finding because in addition to important pharmaceuticals such as penicillin or cyclosporine, potent poisons like aflatoxins and ergot-alkaloids are also part of the metabolic portfolio of filamentous fungi. In contrast to HdaA, inactivation of the class 1 enzyme RpdA turned out to be actually essential for the vitality of A. nidulans. However, in addition to its impact for growth and sporulation, RpdA has another characteristic feature. Beside the highly conserved catalytic core domain of class 1 HDACs in all eukaryotes, RpdA type proteins of filamentous fungi contain a remarkable extension of the C-terminus that cannot be deleted without affecting the biological activity of this enzyme. This characteristic feature of RpdA-type proteins and its significance for growth and development turn these proteins into potentially interesting targets for fungal-specific HDAC inhibitors - an attractive aspect with respect to the development of new antifungal drugs. Since several Aspergillus strains are not only well known for infection of food and crop plants but have been also reported as causative agents of infections in immuno-compromised patients, development of novel antimycotic drugs is highly desirable. A further investigation of their mode of action, necessary cofactors and potential target genes of HDACs in A. nidulans and in filamentous fungi in general is a major goal of a current project application.