Thursday April 1
Plenary Session III
Fungal Physiology and Gene Expression
PL3.1
Chromatin-level regulation of metabolic gene clusters in
Aspergillus
Joseph Strauss
Fungal Genetics and Genomics Unit, Austrian Institute of Technology (AIT) and
joseph.strauss@boku.ac.at
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.
References
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.
PL3.2
Matthew Sachs
msachs@mail.bio.tamu.edu
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.
PL3.3
Jürg Bähler
j.bahler@ucl.ac.uk
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.
PL3.4
Pascale Daran-Lapujade,
Jack T. Pronk
p.a.s.daran-lapujade@tudelft.nl
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.
PL3.5
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.
c.a.m.van.den.hondel@biology.leidenuniv.nl
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.