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Secondary metabolite biosynthesis genes in fungi are usually physically linked and organized in large gene clusters. The physical linkage of genes involved in the same biosynthetic pathway minimizes the amount of regulatory steps necessary to regulate the biosynthetic machinery and thereby contributes to physiological economization. Regulation by chromatin accessibility is a proficient molecular mechanism to synchronize transcriptional activity of large genomic regions. Chromatin regulation largely depends on DNA and histone modifications and the histone code hypothesis proposes that a certain combination of modifications, such as acetylation, methylation or phosphorylation, is needed to perform a specific task. A number of reports from several laboratories recently demonstrated that fungal secondary metabolite (SM) biosynthesis clusters are controlled by chromatin-based mechanisms and histone acetyltransferases, deacetylases, methyltransferases, and proteins involved in heterochromatin formation were found to be involved. This led to the proposal that establishment of repressive chromatin domains over fungal SM clusters under primary metabolic conditions is a conserved mechanism that prevents SM production during the active growth phase. Consequently, transcriptional activation of SM clusters requires reprogramming of the chromatin landscape and replacement of repressive histone marks by activating marks. This review summarizes recent advances in our understanding of chromatin-based SM cluster regulation and highlights some of the open questions that remain to be answered before we can draw a more comprehensive picture.[PUBLICATION ABSTRACT]

Fungal species have the capability of producing an overwhelming diversity of bioactive substances that can have beneficial but also detrimental effects on human health. These so-called secondary metabolites naturally serve as antimicrobial “weapon systems”, signaling molecules or developmental effectors for fungi and hence are produced only under very specific environmental conditions or stages in their life cycle. However, as these complex conditions are difficult or even impossible to mimic in laboratory settings, only a small fraction of the true chemical diversity of fungi is known so far. This also implies that a large space for potentially new pharmaceuticals remains unexplored. We here present an overview on current developments in advanced methods that can be used to explore this chemical space. We focus on genetic and genomic methods, how to detect genes that harbor the blueprints for the production of these compounds (i.e., biosynthetic gene clusters, BGCs), and ways to activate these silent chromosomal regions. We provide an in-depth view of the chromatin-level regulation of BGCs and of the potential to use the CRISPR/Cas technology as an activation tool.

Cells respond to environmental fluctuations by regulating multiple transcriptional programs. This response can be studied by measuring the effect of environmental changes on the transcriptome or the proteome of the cell at the end of the response. However, the dynamics of the response reflect the working of the regulatory mechanisms in action. Here, we utilized a fluorescent stress reporter gene to track the dynamics of protein production in yeast responding to environmental stress. The response is modulated by changes in both the duration and rate of transcription. We probed the underlying molecular pathways controlling these two dimensions using a library of ~1,600 single‐ and double‐mutant strains. Dissection of the effects of these mutants and the interactions between them identified distinct modulators of response duration and response rate. Using a combination of mRNA‐seq and live‐cell microscopy, we uncover mechanisms by which Msn2/4, Mck1, Msn5, and the cAMP/PKA pathway modulate the response of a large module of stress‐induced genes in two discrete regulatory phases. Our results and analysis show that transcriptional stress response is regulated by multiple mechanisms that overlap in time and cellular location. Synopsis Genetic screens of yeast transcription response dynamics identify partially uncoupled parameters (response intensity and duration) and their differential regulation. Epistasis analysis of individual parameters uncovers two distinctly regulated response phases. The duration of the transcriptional response to stress and its rate are partially decoupled parameters regulated by different pathways. Epistasis analysis of factors affecting the rate and duration of the response uncovers new interactions and sheds light on the underlying mechanisms. Osmotic stress response involves two phases of Msn2/4 nuclear localization, first a synchronous nuclear phase, followed by export to cytoplasm, and then a Mck1‐dependent asynchronous nuclear phase. Graphical Abstract Genetic screens of yeast transcription response dynamics identify partially uncoupled parameters (response intensity and duration) and their differential regulation. Epistasis analysis of individual parameters uncovers two distinctly regulated response phases.

Sequence analyses of fungal genomes have revealed that the potential of fungi to produce secondary metabolites is greatly underestimated. In fact, most gene clusters coding for the biosynthesis of antibiotics, toxins, or pigments are silent under standard laboratory conditions. Hence, it is one of the major challenges in microbiology to uncover the mechanisms required for pathway activation. Recently, we discovered that intimate physical interaction of the important model fungus Aspergillus nidulans with the soil-dwelling bacterium Streptomyces rapamycinicus specifically activated silent fungal secondary metabolism genes, resulting in the production of the archetypal polyketide orsellinic acid and its derivatives. Here, we report that the streptomycete triggers modification of fungal histones. Deletion analysis of 36 of 40 acetyltransferases, including histone acetyltransferases (HATs) of A. nidulans, demonstrated that the Saga/Ada complex containing the HAT GcnE and the AdaB protein is required for induction of the orsellinic acid gene cluster by the bacterium. We also showed that Saga/Ada plays a major role for specific induction of other biosynthesis gene clusters, such as sterigmatocystin, terrequinone, and penicillin. Chromatin immunoprecipitation showed that the Saga/Ada-dependent increase of histone 3 acetylation at lysine 9 and 14 occurs during interaction of fungus and bacterium. Furthermore, the production of secondary metabolites in A. nidulans is accompanied by a global increase in H3K14 acetylation. Increased H3K9 acetylation, however, was only found within gene clusters. This report provides previously undescribed evidence of Saga/Ada dependent histone acetylation triggered by prokaryotes.

Histone posttranslational modifications (HPTMs) are involved in chromatin-based regulation of fungal secondary metabolite biosynthesis (SMB) in which the corresponding genes-usually physically linked in co-regulated clusters-are silenced under optimal physiological conditions (nutrient-rich) but are activated when nutrients are limiting. The exact molecular mechanisms by which HPTMs influence silencing and activation, however, are still to be better understood. Here we show by a combined approach of quantitative mass spectrometry (LC-MS/MS), genome-wide chromatin immunoprecipitation (ChIP-seq) and transcriptional network analysis (RNA-seq) that the core regions of silent A. nidulans SM clusters generally carry low levels of all tested chromatin modifications and that heterochromatic marks flank most of these SM clusters. During secondary metabolism, histone marks typically associated with transcriptional activity such as H3 trimethylated at lysine-4 (H3K4me3) are established in some, but not all gene clusters even upon full activation. KdmB, a Jarid1-family histone H3 lysine demethylase predicted to comprise a BRIGHT domain, a zinc-finger and two PHD domains in addition to the catalytic Jumonji domain, targets and demethylates H3K4me3 in vivo and mediates transcriptional downregulation. Deletion of kdmB leads to increased transcription of about ~1750 genes across nutrient-rich (primary metabolism) and nutrient-limiting (secondary metabolism) conditions. Unexpectedly, an equally high number of genes exhibited reduced expression in the kdmB deletion strain and notably, this group was significantly enriched for genes with known or predicted functions in secondary metabolite biosynthesis. Taken together, this study extends our general knowledge about multi-domain KDM5 histone demethylases and provides new details on the chromatin-level regulation of fungal secondary metabolite production.

Plant pathogenic fungi provoke devastating agricultural losses and are difficult to control. How these organisms acquire micronutrients during growth in the host environment remains poorly understood. Here we show that efficient regulation of copper acquisition mechanisms is crucial for plant colonization and virulence in the soilborne ascomycete Fusarium oxysporum , the causal agent of vascular wilt disease in more than 150 different crops. Using a combination of RNA-seq and ChIP-seq, we establish a direct role of the transcriptional regulator Mac1 in activation of copper deficiency response genes, many of which are induced during plant infection. Loss of Mac1 impaired growth of F . oxysporum under low copper conditions and abolishes pathogenicity on tomato plants and on the invertebrate animal host Galleria mellonella . Importantly, overexpression of two Mac1 target genes encoding a copper reductase and a copper transporter was sufficient to restore virulence in the mac1 mutant background. Our results establish a previously unrecognized role of copper reduction and uptake in fungal infection of plants and reveal new ways to protect crops from phytopathogens.

Screening for new bioactive microbial metabolites, we found a novel okaramine derivative, for which we propose the trivial name lemmokaramine, as well as two already known okaramine congeners – okaramine H and okaramine J - responsible for antimicrobial activity of the recently described microscopic filamentous fungus, Keratinophyton lemmensii BiMM-F76 (= CCF 6359). In addition, two novel substances, a new cyclohexyl denominated lemmensihexol and a new tetrahydroxypyrane denominated lemmensipyrane, were purified and characterized. The compounds were isolated from the culture extract of the fungus grown on modified yeast extract sucrose medium by means of flash chromatography followed by preparative HPLC. The chemical structures were elucidated by NMR and LC-MS. The new okaramine (lemmokaramine) exerted antimicrobial activity against Gram-positive and Gram-negative bacteria, yeasts and fungi and anticancer activity against different mammalian cell lines (Caco-2, HCT116, HT29, SW480, MCM G1, and MCM DLN). Furthermore, we found a significant antioxidant effect of lemmokaramine following H 2 O 2 treatment indicated by activation of the Nrf2 pathway. This is the first report describing analysis and structural elucidation of bioactive metabolites for the onygenalean genus Keratinophyton .

Two new Keratinophyton species, K. kautmanovae sp. nov. and K. keniense sp. nov., isolated from soil samples originating from two different geographical and environmental locations (Africa and Europe) are described and illustrated. Phylogenetically informative sequences obtained from the internal transcribed spacer (ITS) region and the nuclear large subunit (LSU) rDNA, as well as their unique phenotype, fully support novelty of these two fungi for this genus. Based on ITS and LSU combined phylogeny, both taxa are resolved in a cluster with eight accepted species, including K . alvearium , K . chongqingense , K . hubeiense , K. durum , K. lemmensii , K . siglerae , K. submersum , and K. sichuanense . The new taxon, K. kautmanovae , is characterized by clavate, smooth to coarsely verrucose conidia, absence of arthroconidia, slow growth at 25 °C, and no growth at 30 °C, while K. keniense is morphologically unique with a high diversity of conidial shapes (clavate, filiform, globose, cymbiform and rhomboid). Both species are described based on their asexual, a chrysosporium-like morph. While the majority of hitherto described Keratinophyton taxa came from Europe, India and China, the new species K. keniense represents the first reported taxonomic novelty for this genus from Africa.

Filamentous fungi naturally grow on solid surfaces, yet most genetic and biochemical analyses are still performed in liquid cultures. Here, we report a multiplexing platform using high-throughput photometric continuous reading that allows parallel quantification of hyphal growth and reporter gene expression directly on solid medium, thereby mimicking natural environmental conditions. Using this system, we have quantified fungal growth and expression of secondary metabolite GFP-based reporter genes in saprophytic Aspergillus and phytopathogenic Fusarium species in response to different nutrients, stress conditions and epigenetic modifiers. With this method, we provide not only novel insights into the characteristic of fungal growth but also into the metabolic and time-dependent regulation of secondary metabolite gene expression.