Thursday April 1


Plenary Session III


Fungal Physiology and Gene Expression



Chromatin-level regulation of metabolic gene clusters in Aspergillus

Joseph Strauss

Fungal Genetics and Genomics Unit, Austrian Institute of Technology (AIT) and BOKU University Vienna, Austria


In fungi primary and secondary metabolism genes involved in the same metabolic pathway are often clustered in the genome. This arrangement may facilitate co-regulation of these genes in response to environmental or developmental signals. Chromatin represents both the physiological substrate and a physical barrier for transcription factors to access regulatory elements on gene promoters. By adopting different levels of compaction and the correct architecture of the underlying nucleosomal subunits chromatin regulates gene expression and chromosome function. An epigenetic code defined by covalent modifications of the nucleosomal histone proteins is conserved throughout eukaryotes and defines the state of chromatin. In Aspergillus and other fungi, these modifications have been shown to influence metabolic and developmental processes. The examples presented here show that nitrogen metabolism and the production of secondary metabolites are regulated at the level of chromatin structure and accessibility. In the nitrate assimilation and proline degradation gene clusters the activity of the bi-directional promoters are influenced by nucleosome positioning (1). The major transcription factors play a decisive role in this process, e.g. the GATA factor AreA mediates histone acetylation and, in cooperation with the pathway-specific transcription factor NirA, subsequent nucleosome remodelling (2).


The transition from primary to secondary metabolism (SM) in different Aspergillus species is also associated with drastic chromatin rearrangements. A. nidulans mutants lacking components involved in the formation of strongly repressive heterochromatin (Heterochromatin-protein-1, H3-K9 methyltransferase) show over-expression of genes involved in biosynthesis of several secondary metabolites. LaeA, a conserved principal regulator of SM, is counteracting the decoration of histones by repressive marks (3). Moreover, the inactivation of a COMPASS-complex component (CclA) leads to reduction of repressive H3K9 marks in gene clusters for which metabolites have not been identified before (4). Chromatin restructuring upon entry into SM may be a conserved mechanism in fungi and modification of the chromatin landscape may thus lead to a more complete picture of the secondary metabolome in fungi.



1.            Reyes-Dominguez, Y. et al. (2008) Eukaryot Cell 7, 656-63.

2.            Berger, H. et al. (2008) Mol Microbiol 69, 1385-98.

3.            Reyes-Dominguez, Y. et al. (2010) Mol Microbiol (in press).

4.            Bok, J. W., et al. (2009) Nat Chem Biol 5, 462-4.












Gene regulation through the control of ribosome movement

Matthew Sachs

Texas A&M University, USA

The regulated translation of mRNA can affect both rates of protein synthesis and mRNA stability. The expression of the small subunit of fungal arginine-specific carbamoyl phosphate synthetase is controlled by the translation of an upstream open reading frame (uORF) present in the 5’-leader region of its mRNA.  Translation of the uORF, which specifies the arginine attenuator peptide (AAP), leads to reduced gene expression in response to elevated levels of arginine.  Our data, based mainly on studies of Neurospora crassa arg-2 and Saccharomyces cerevisiae CPA1, indicate that, first, translation of this coding region causes ribosomes to stall at the uORF termination codon when the level of the amino acid arginine is high.  The stalled ribosome blocks the access of scanning ribosomes to the downstream start codon that is used to initiate synthesis of the biosynthetic enzyme, thus reducing gene expression. We have direct evidence from cell-free translation systems that arginine and related molecules cause stalling by interfering with the activity of the ribosome peptidyl transferase center and indications that these molecules induce a conformational change in the nascent AAP within the ribosome.  Second, stalling of the ribosome at the uORF termination codon destabilizes the mRNA through the nonsense-mediated mRNA decay (NMD) pathway.  Analyses of mRNA stability through pulse-chase studies in wild-type and nmd- N. crassa strains provide direct evidence that the stability of the arg-2 mRNA is controlled by NMD. mRNA transcriptomes of wild-type and mutant cells have provided additional insights into NMD-control in N. crassa.





Dynamic repertoire of the fission yeast transcriptome surveyed at single-nucleotide resolution

Jürg Bähler

University College London


Recent data from several organisms indicate that the transcribed portions of genomes are larger and more complex than expected, and many functional properties of transcripts are not based on coding sequences but on regulatory sequences in untranslated regions or non-coding RNAs.  Alternative start and polyadenylation sites and regulation of intron splicing add additional dimensions to the rich transcriptional output.  This transcriptional complexity has been sampled mainly using hybridization-based methods under one or few conditions.  We applied direct high-throughput sequencing of cDNAs (RNA-seq), complemented with data from high-density tiling arrays, to globally sample transcripts of S. pombe, independently from available gene annotations.  We interrogated transcriptomes under multiple conditions, including rapid proliferation, meiotic differentiation and environmental stress, and in RNA processing mutants, to reveal the dynamic plasticity of the transcriptional landscape as a function of environmental, developmental, and genetic factors.  High-throughput sequencing proved to be a powerful and quantitative method to deeply sample transcriptomes at maximal resolution.  In contrast to hybridization, sequencing showed little, if any, background noise and was sensitive enough to detect widespread transcription in >90% of the genome, including traces of RNAs that were not robustly transcribed or rapidly degraded.  The combined sequencing and array data provided rich condition-specific information on novel, mostly non-coding transcripts, untranslated regions and gene structures, thus improving the existing genome annotation.  Sequence reads spanning exon-exon or exon-intron junctions gave unique insight into a surprising variability in splicing efficiency across introns and genes.  Splicing efficiency was largely coordinated with transcript levels, and increased transcription led to increased splicing in test genes.  Hundreds of introns showed regulated splicing during cellular proliferation or differentiation.




Regulation of the glycolytic activity in Saccharomyces cerevisiae: a systems biology approach

Pascale Daran-Lapujade, Jack T. Pronk

Delft University of Technology


The Embden-Meyerhof-Parnas pathway of glycolysis is the key pathway of sugar metabolism in many living organisms, including man. Glycolysis is a central pathway for carbon assimilation and is directly linked to all industrial applications of Saccharomyces cerevisiae from biomass to biofuel production. Despite this large economical impact and decades of investigation, the regulation of yeast glycolysis remains elusive and to date, attempts at controlling the glycolytic flux by genetic engineering have consistently failed.
To improve our understanding on the mechanisms governing the glycolytic flux in baker’s yeast, a systems biology approach was undertaken. Yeast was grown under various conditions at steady-state in chemostat and in dynamic conditions using tightly controlled cultivation tools. The glycolytic system was investigated in a quantitative manner using a systems approach integrating all levels in the gene expression cascade from gene to in vivo flux (i.e. transcripts, proteins, active enzymes, metabolites, fluxes). This multi-level approach showed that, at steady-state as in dynamic environment, the local fluxes in glycolysis are largely governed by metabolic regulation, i.e. by in vivo activation/inhibition of enzyme activities by metabolites. Conversely, hierarchical regulation (i.e. regulation of protein concentration) is only marginally involved in the regulation of the local glycolytic fluxes. In all tested conditions baker’s yeast displays a ‘glycolytic overcapacity’ that may be regarded as a waste of energy (glycolytic proteins can represent up to 20% of the whole cell protein) but could represent a selective advantage for yeast cells evolving in natural environments exposed to circadian cycles and a to variety of stressful conditions. The potential selective advantage during diurnal temperature oscillations will be discussed.





Filamentous-fungal biotechnology:  Veni, Vidi, Vici?

Cees A.M.J.J. van den Hondel

Department of Molecular Microbiology and Biotechnology, Institute of Biology, Leiden University, Kluyver Centre for Genomics of Industrial Fermentation, Sylviusweg 72, 2333 BE, Leiden, The Netherlands.


The establishment of genetic transformation of Aspergillus nidulans has been the basis for a new set of developments in filamentous-fungal biotechnology. Almost thirty years later, it is interesting to have a short look back at the expectations at that time, the technology development which occurred and the progress which has been made up until now.

Although an enormous increase in understanding basic biological processes which occur in our (biotechnologically important) model filamentous fungi has taken place, clearly a number of bottlenecks remain which hamper the yields which are theoretically achievable. This lecture will focus on the progress made and future directions for research addressing three important bottlenecks: 1. protein folding and secretion of (heterologous) proteins; 2. degradation of secreted proteins in the culture fluid; 3. optimal morphology of filamentous fungi in the bioreactor.

Finally the prospects of filamentous-fungal biotechnology in the future will be discussed.






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