A Class 1 Histone Deacetylase as Major Regulator of Secondary Metabolite Production in Aspergillus nidulans

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

Histone modifications and chromatin dynamics: a focus on filamentous fungi

The readout of the genetic information of eukaryotic organisms is significantly regulated by modifications of DNA and chromatin proteins. Chromatin alterations induce genome-wide and local changes in gene expression and affect a variety of processes in response to internal and external signals during growth, differentiation, development, in metabolic processes, diseases, and abiotic and biotic stresses. This review aims at summarizing the roles of histone H1 and the acetylation and methylation of histones in filamentous fungi and links this knowledge to the huge body of data from other systems. Filamentous fungi show a wide range of morphologies and have developed a complex network of genes that enables them to use a great variety of substrates. This fact, together with the possibility of simple and quick genetic manipulation, highlights these organisms as model systems for the investigation of gene regulation. However, little is still known about regulation at the chromatin level in filamentous fungi. Understanding the role of chromatin in transcriptional regulation would be of utmost importance with respect to the impact of filamentous fungi in human diseases and agriculture. The synthesis of compounds (antibiotics, immunosuppressants, toxins, and compounds with adverse effects) is also likely to be regulated at the chromatin level

Domain architecture of histone lysine methyltransferases (a, b), protein arginine methyltransferases (c), and histone demethylases (d) of different species

homologs of human proteins (see ) were searched using the Comparative Database from the BROAD Institute (). Corresponding proteins were identified by a search () and domain architectures of proteins were analyzed by the Simple Modular Architecture Research Tool (SMART; ). The number of amino acids of proteins is shown. Accession numbers are given in and .<p><b>Copyright information:</b></p><p>Taken from "Histone modifications and chromatin dynamics: a focus on filamentous fungi"</p><p></p><p>Fems Microbiology Reviews 2008;32(3):409-439.</p><p>Published online 25 Jan 2008</p><p>PMCID:PMC2442719.</p><p>© 2008 The Authors; Journal compilation © 2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd</p

Characterization of Inhibitor-Resistant Histone Deacetylase Activity in Plant-Pathogenic Fungi

HC-toxin, a cyclic peptide made by the filamentous fungus Cochliobolus carbonum, is an inhibitor of histone deacetylase (HDAC) from many organisms. It was shown earlier that the HDAC activity in crude extracts of C. carbonum is relatively insensitive to HC-toxin as well as to the chemically unrelated HDAC inhibitors trichostatin and D85, whereas the HDAC activity of Aspergillus nidulans is sensitive (G. Brosch et al., Biochemistry 40:12855-12863, 2001). Here we report that HC-toxin-resistant HDAC activity was present in other, but not all, plant-pathogenic Cochliobolus species but not in any of the saprophytic species tested. The HDAC activities of the fungi Alternaria brassicicola and Diheterospora chlamydosporia, which also make HDAC inhibitors, were resistant. The HDAC activities of all C. carbonum isolates tested, except one non-toxin-producing isolate, were resistant. In a cross between a sensitive isolate and a resistant isolate, resistance genetically cosegregated with HC-toxin production. When fractionated by anion-exchange chromatography, extracts of resistant and sensitive isolates and species had two peaks of HDAC activity, one that was fully HC-toxin resistant and a second that was larger and sensitive. The first peak was consistently smaller in extracts of sensitive fungi than in resistant fungi, but the difference appeared to be insufficiently large to explain the differential sensitivities of the crude extracts. Differences in mRNA expression levels of the four known HDAC genes of C. carbonum did not account for the observed differences in HDAC activity profiles. When mixed together, resistant extracts protected extracts of sensitive C. carbonum but did not protect other sensitive Cochlibolus species or Neurospora crassa. Production of this extrinsic protection factor was dependent on TOXE, the transcription factor that regulates the HC-toxin biosynthetic genes. The results suggest that C. carbonum has multiple mechanisms of self-protection against HC-toxin

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genomemining of fungal secondary metabolites. Inhibitor screening, however, requires purified enzyme activities. Since class 1 deacetylases exerttheir function as multiprotein complexes, they are usually not active when expressed as single polypeptides in bacteria. Therefore, endogenouscomplexes need to be isolated, which, when conventional techniques like ion exchange and size exclusion chromatography are applied, islaborious and time consuming. Tandem affinity purification has been developed as a tool to enrich multiprotein complexes from cells and thusturned out to be ideal for the isolation of endogenous enzymes. Here we provide a detailed protocol for the single-step enrichment of activeRpdA complexes via the first purification step of C-terminally TAP-tagged RpdA from Aspergillus nidulans. The purified complexes may then beused for the subsequent inhibitor screening applying a deacetylase assay. The protein enrichment together with the enzymatic activity assay canbe completed within two days. The video component of this article can be found at https://www.jove.com/video/59527

Synthesis of Substituted Triazole-Pyrazole Hybrids using Triazenopyrazoles Precursors

A synthesis route to access triazole-pyrazole hybrids via pyrazolotriazenes was developed. Contrary to existing methods, this route allows the facile N-functionalization of the pyrazole before the attachment of the triazole unit via a copper-catalyzed azide-alkyne cycloaddition. The developed methodology was used to synthesize a library of over fifty novel multi-substituted pyrazole-triazole hybrids. We could also demonstrate a one-pot strategy that renders the isolation of potentially hazardous azides obsolete. In addition, the compatibility of the method with solid-phase synthesis was shown exemplarily