Tuesday March 30
Parallel session 3: Regulation of Gene Expression at the Genome Level
PS3.1
Heinz D. Osiewacz,
Diana Brust, Andrea Hamann, Karin Luce
Department of Molecular Developmental Biology & Cluster of Excellence
‘Macromolecular Complexes’. Johann Wolfgang Goethe-University. Max-von-Laue-Str.
9, 60438
osiewacz@bio.uni-frankfurt.de
The ascomycete
Podospora anserina is characterized by a limited lifespan. After a
strain specific period of growth the growth rate of a colony decreases, the
morphology of the culture changes and finally the hyphal tips die. This
‘senescence syndrome’ is under control of environmental factors and genetic
traits. Recent investigations uncovered a hierarchical network of pathways
influencing life and healthspan of this eukaryotic ageing model. These pathways
counteract molecular damage which results from natural metabolic processes (e.g.
respiration). In particular, pathways involved in the control of a functional
population of mitochondria were found to play a major role. At the molecular
level different mitochondrial proteases are effective. Among others, PaLON, a
matrix protease was found to be important in protecting cultures against the
consequences of oxidative stress.
Overexpression of
PaLon leads to an increased heathspan, the period in the lifespan,
in which no vital functions are impaired. The corresponding transgenic strains
are more resistant against oxidative stress, are characterized by reduced
protein damage and improved mitochondrial function.
Although, this and other quality control pathways are effective,
P. anserina cultures finally turn to senescence. During this last
step in the fungal life cycle pathways leading to programmed cell death are
induced. Data about investigations intervening into these pathways will be
discussed.
PS3.2
Frequency-modulated nucleo-cytoplasmic shuttling cycles are the basis for
circadian activity and abundance rhythms of the
Neurospora clock transcription factor
WCC
Tobias Schafmeier
and Michael Brunner
tobias.schafmeier@bzh.uni-heidelberg.de
In Neurospora crassa the clock
transcription factor White collar complex (WCC) controls the rhythmic expression
of a large number of genes. The clock protein Frequency (FRQ) regulates WCC
activity in a negative feedback loop by mediating its CK-1a dependent
phosphorylation. In a positive loop, FRQ-dependent phosphorylation reduces the
turnover rate of the WCC, resulting in accumulation of inactive and stable WCC.
Rapid degradation of active WCC is a regulatory mechanism preventing an
overshoot of WCC dependent transcription. WCC undergoes rapid, sub-circadian
cycles of nucleo-cytoplasmic shuttling. These are linked to cycles of FRQ
dependent inactivation by phosphorylation and reactivation by PP2A dependent
dephosphorylation of WCC in the cytosol. Rhythmically expressed FRQ modulates
the kinetics of WCC phosphorylation and shuttling cycles in a circadian manner,
producing a daily rhythm of WCC activity and abundance. Hence, phosphorylation
of the WCC is the molecular basis underlying both, negative feedback of FRQ on
WCC activity and positive feedback of FRQ on WCC stability.
PS3.3
Functional characterization of LaeA
Graeme S. Garvey1,
Jonathan M. Palmer2, Jin Woo
Bok1, Alex J. La Reau1, Nancy P. Keller1,3
1Department
of Medical Microbiology and Immunology, University of Wisconsin-Madison
2Department
of Plant Pathology, University of Wisconsin-Madison
3Department
of Bacteriology, University of Wisconsin-Madison
gsgarvey@wisc.edu
Here we present the initial findings of a biochemical and genetic investigation
into the mechanism of LaeA, a
putative methyltransferase that functions as a global regulator of secondary
metabolism in Aspergillus nidulans.
LaeA has been found to be part of a large nuclear velvet complex that is
required for secondary metabolite production as well as light regulated
morphological development. Preliminary data suggests LaeA may control secondary
metabolite gene clusters through chromatin remodeling. However, there is no
direct evidence linking the velvet complex to chromatin remodeling. We have
initiated a study to functionally characterize LaeA. Several LaeA orthologs were
recombinantly expressed in E. coli
and assayed for solubility. The full length LaeA protein from
A. nidulans is only soluble as a MBP
fusion protein, which has proved to be uninformative for
in vitro activity assays. A partial
proteolysis study was performed to identify soluble domains that could be
amenable to in vitro analysis.
Soluble truncation mutants were identified and have proved useful for
in vitro methyltransferase activity
assays. Validation of the truncated LaeA proteins was carried out through
successful in vivo complementation of
a ∆laeA mutant. These truncation
mutants are functionally equivalent to the full-length protein by restoring
sterigmatocystin (ST) biosynthesis to wild type levels.
Using the truncated LaeA protein, we have confirmed binding of S-adenosyl-L-methionine
(methyl group donor) and have identified a methyltransferase activity. Each
candidate protein substrate’s site of methylation is being mapped by trypsin
digestion coupled with LC/MS. The
in vivo role of LaeA methylation will
be evaluated with point mutants for effects on ST biosynthesis.
Our findings confirm LaeA has methyltransferase activity and provide the
first functional insights into the mechanism of LaeA regulation of secondary
metabolism.