Regulation of mycotoxin production by chromatin structure

Potentially carcinogenic mycotoxins can appear in food and feed as a result of fungal infection of the crop, or the infection of stored products. Toxins, but also other important metabolites such as antibiotics, accumulate during the entry of a fungus into secondary metabolism and the reproductive cycle. Despite a considerable body of knowledge on the regulation of genes and gene clusters involved in mycotoxin biosynthesis, it is still unclear which signals trigger the transition step from being repressed under conditions of active growth to being activated under conditions of growth arrest. Investigating genes involved in the regulation of chromatin structure in the model fungus Aspergillus nidulans we observed in transcriptome analysis that a mutant with inactivated putative heterochromatin-protein1 (HP1) showed untimely, praecox activation of several genes involved in the biosynthesis of antibiotics and mycotoxins. In collaboration with the group of Nancy Keller (Univ. Wisconsin, USA) we substantiated our preliminary results and found that a deletion in HP1 (designated hepA in A. nidulans), or a deletion in the class-II histone de-acetylase hdaA, suppressed the mycotox-negative phenotype of laeA. Strains defective in LaeA function are unable to activate a number of sterigmatocystin-cluster (ST) genes and are defective in ST production. LaeA is a protein with similarity to methyltransferases and could be involved in the activation of ST production by specific histone (histone H3 lysine 4) methylation. Besides the acetylation status, also the methylation status is known to regulate chromatin accessibility and transitions between repressing heterochromatin and activating euchromatin. In this research proposal we plan to investigate the chromatin structure at the ST-gene cluster in a number of A.nidulans mutants which carry deletions in putative chromatin regulators. From our preliminary evidence we hypothesize that during active growth "facultative heterochromatin" structures might inhibit ST-cluster gene expression whereas the onset of ST production in secondary metabolism is associated with conversion of heterochromatic marks to euchromatic marks. Using Chromatin-Immunoprecipitation (ChIP), transcriptional analysis and nuclease accessibility assays, we will concentrate on A. nidulans HepA and how its function depends on different methylation states of histone H3 lysine 9 (H3K9), and on factors defining these states (methyltransferases, de-methylases, acetylases, deacetylases). The proposed work might lead to new general insights into facultative heterochromatin formation, a process so far only described in metazoans and believed to be essential for heritable gene silencing during development. In collaboration with the group of N. Keller, who is studying LaeA-dependent, activating histone methylation marks, our studies might unravel a mechanism how entry into fungal secondary metabolism and mycotoxin production is triggered.

Fungi produce a complex set of `secondary` metabolites (SMs) which are protecting them against environmental stresses (e.g. UV light) or potential microbial competitors (e.g. bacteria). For humans and animals, these metabolites can be toxic (termed `mycotoxins`) and appear in food and feed as a result of fungal infection of the crop or the stored products. Toxins and other important secondary metabolites such as antibiotics and cholesterol- lowering statins usually are produced when fungi stop growth and enter the reproductive cycle. The analysis of sequenced fungal genomes revealed that in basically all species genes involved in SM biosynthesis are usually clustered in `toxin islands` which are co-ordinately regulated during the onset of secondary metabolism. This research project started with the working hypothesis that regulation of the toxin islands might include epigenetic modification of chromatin components because this would facilitate a coordinated regulation of large segments in the fungal genome. In chromatin DNA is wrapped around specialized proteins (called histones) and specific modifications of histone proteins define the degree of packaging and thus accessibility of the underlying genetic material. When such histone modifications are stable over several cell generations, they are called `epigenetic` modifications. In the course of this project we could show that several toxin islands in Aspergillus nidulans are transcriptionally silenced during active growth (primary metabolism) by the formation of strongly packed, repressive heterochromatin structures. Furthermore, we were able to demonstrate that secondary metabolism disrupts heterochromatic structures specifically within the toxin islands, but not outside of these regions. Strikingly, loss of heterochromatin components not only lead to higher amounts of known metabolites, but also trigger the production of metabolites which were not yet known for A. nidulans. Thus, the results from this project led to the identification of an entirely novel molecular mechanism lying behind the coordinated activation of fungal secondary metabolism and mycotoxin production.