Histone deacetylases (HDACs) remove acetyl moieties from lysine residues at histone tails and nuclear regulatory proteins and thus significantly impact chromatin remodeling and transcriptional regulation in eukaryotes. In recent years, HDACs of filamentous fungi were found to be decisive regulators of genes involved in pathogenicity and the production of important fungal metabolites such as antibiotics and toxins. Here we present proof that one of these enzymes, the class 1 type HDAC RpdA, is of vital importance for the opportunistic human pathogen Aspergillus fumigatus. Recombinant expression of inactivated RpdA shows that loss of catalytic activity is responsible for the lethal phenotype of Aspergillus RpdA null mutants. Furthermore, we demonstrate that a fungus-specific C-terminal region of only a few acidic amino acids is required for both the nuclear localization and catalytic activity of the enzyme in the model organism Aspergillus nidulans. Since strains with single or multiple deletions of other classical HDACs revealed no or only moderate growth deficiencies, it is highly probable that the significant delay of germination and the growth defects observed in strains growing under the HDAC inhibitor trichostatin A are caused primarily by inhibition of catalytic RpdA activity. Indeed, even at low nanomolar concentrations of the inhibitor, the catalytic activity of purified RpdA is considerably diminished. Considering these results, RpdA with its fungus-specific motif represents a promising target for novel HDAC inhibitors that, in addition to their increasing impact as anticancer drugs, might gain in importance as antifungals against life-threatening invasive infections, apart from or in combination with classical antifungal therapy regimes.

An outstanding feature of filamentous fungi is their ability to produce a wide variety of small bioactive molecules that contribute to their survival, fitness, and pathogenicity. The vast collection of these so-called secondary metabolites (SMs) includes molecules that play a role in virulence, protect fungi from environmental damage, act as toxins or antibiotics that harm host tissues, or hinder microbial competitors for food sources. Many of these compounds are used in medical treatment; however, biosynthetic genes for the production of these natural products are arranged in compact clusters that are commonly silent under growth conditions routinely used in laboratories. Consequently, a wide arsenal of yet unknown fungal metabolites is waiting to be discovered. Here, we describe the effects of deletion of hosA, one of four classical histone deacetylase (HDAC) genes in Aspergillus nidulans; we show that HosA acts as a major regulator of SMs in Aspergillus with converse regulatory effects depending on the metabolite gene cluster examined. Co-inhibition of all classical enzymes by the pan HDAC inhibitor trichostatin A and the analysis of HDAC double mutants indicate that HosA is able to override known regulatory effects of other HDACs such as the class 2 type enzyme HdaA. Chromatin immunoprecipitation analysis revealed a direct correlation between hosA deletion, the acetylation status of H4 and the regulation of SM cluster genes, whereas H3 hyper-acetylation could not be detected in all the upregulated SM clusters examined. Our data suggest that HosA has inductive effects on SM production in addition to its classical role as a repressor via deacetylation of histones. Moreover, a genome wide transcriptome analysis revealed that in addition to SMs, expression of several other important protein categories such as enzymes of the carbohydrate metabolism or proteins involved in disease, virulence, and defense are significantly affected by the deletion of HosA.

In filamentous fungi, arginine methylation has been implicated in morphogenesis, mycotoxin biosynthesis, pathogenicity, and stress response although the exact role of this posttranslational modification in these processes remains obscure. Here, we present the first genome-wide transcriptome analysis in filamentous fungi that compared expression levels of genes regulated by type I and type II protein arginine methyltransferases (PRMTs). In Aspergillus nidulans, three conserved type I and II PRMTs are present that catalyze asymmetric or symmetric dimethylation of arginines. We generated a double type I mutant (ΔrmtA/rmtB) and a combined type I and type II mutant (ΔrmtB/rmtC) to perform genome-wide comparison of their effects on gene expression, but also to monitor putative overlapping activities and reciprocal regulations of type I and type II PRMTs in Aspergillus. Our study demonstrates, that rmtA and rmtC as type I and type II representatives act together as repressors of proteins that are secreted into the extracellular region as the majority of up-regulated genes are mainly involved in catabolic pathways that constitute the secretome of Aspergillus. In addition to a strong up-regulation of secretory genes we found a significant enrichment of down-regulated genes involved in processes related to oxidation-reduction, transmembrane transport and secondary metabolite biosynthesis. Strikingly, nearly 50% of down-regulated genes in both double mutants correspond to redox reaction/oxidoreductase processes, a remarkable finding in light of our recently observed oxidative stress phenotypes of ΔrmtA and ΔrmtC. Finally, analysis of nuclear and cytoplasmic extracts for mono-methylated proteins revealed the presence of both, common and specific substrates of RmtA and RmtC. Thus, our data indicate that type I and II PRMTs in Aspergillus seem to co-regulate the same biological processes but also specifically affect other pathways in a non-redundant fashion.