Transcript Slide 1

Transcription factors regulating expression of AQY1 and
screens for germination mutants
Cecilia Geijer
Dept. of Cell and Molecular Biology
Göteborg University
March 5 2007
Life cycle of budding yeast S. cerevisiae
Sporulation in S. cerevisiae – the process
•
Sporulation encompasses two overlapping
processes: meiosis and spore formation.
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Cells go through meiosis which leads to the
generation of four haploid nuclei
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Plasma membranes for four daugther cells are
constructed within the mother cell cytoplasm,
and these surrounds the four haploid nuclei.
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Spore wall synthesis occurs.
Aquaporins
• Aquaporins mediate the
transport of water across
biological membranes. Some
also transport other substrates
such as glycerol, urea and
nitrogen.
• The genome of S cerevisiae
contains four aquaporin
genes.
Yeast aquaporin 1 (Aqy1) is involved in sporulation
Northern Blot analysis of AQY1 in strain SK1.
Low basal level of expression of AQY1 in
vegetative cells. Upregulation of expression ~8
h after transfer to sporulation medium (1% KAc)
(The Saccharomyces cerevisiae aquaporin Aqy1 is
involved in sporulation, Sidoux-Walter et al, PNAS 2004)
AQY1/AQY1-GFP
diploids – expression in
two out of four spores
suggests role in later
stages of sporulation
Workpackage 1 Deliverable 9
Putative transcription factor binding sites for the AQY1
promoter
Consensus sites identified:
• Msn2/4 • Gcr1
• Fkh1/2 • Mcm1
• War1
• Ash1
• Nrg1
• Mot3
• Xbp1
• Tec1
No sporulation-specific
transcription factor hits!
www.yeastract.com
Workpackage 1 Deliverable 9
AQY1prom-lacZ fusion construct
Reporter gene lacZ system
AQY1prom
lacZ
Yeast lacZ reporter plasmid to search for transcription factors regulating
expression of AQY1
• X-gal overlay assay
• β-galactosidase activity assay ONPG
Workpackage 1 Deliverable 9
X-gal overlay assay
BY4741 KO collection used to search for transcription factors
wild type
tec1Δ
mot3Δ
war1Δ
X-gal overlay assay works to identify
transcription factors regulating the AQY1
expression in vegetative cells
fkh2Δ_1
fkh2Δ_2
Preliminary data:
fkh2Δ strain seem to have lower
expression of AQY1 than wild type
Workpackage 1 Deliverable 9
Sporulation plates with X-gal overlay assay
SK1a/α
ε1278ba/α
Spores treated with chloroform and overlayed with agarose X-gal are not
turning blue – this assay probably not suitable for spores.
Workpackage 1 Deliverable 9
Conclusions - transcription factor search
• Stationary phase cells, grown on YPD plates for 36h, express AQY1.
Search for transcription factors regulating the expression of AQY1
revealed Fkh2 as a potential candidate.
• X-gal overlay assay is not suitable for spores, instead βgalactosidase activity assay using ONPG.
• Transcription factors regulating late sporulation induced genes are
not yet identified. Finding regulators of AQY1 will point to
transcription factors that possibly regulates other late sporulation
genes as well.
Workpackage 1 Deliverable 9
Germination – search for mutants
What gene products are
essential for the germination
process in Saccharomyces
cerevisiae?
Involvement of actin and polarisome in morphological
change during spore germination of Saccharomyces
cerevisiae
Workpackage 2 Deliverable 14
Keiko Kono, Rino Matsunaga, Aiko Hirata, Genjiro Suzuki,
Mitsuhiro Abe, Yoshikazu Ohya
YEAST 2005
Workpackage 4 Deliverable 23
Screen for Germination mutants using
heterozygous mutants
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Heterozygous diploids – wild type and mutant mated to create
diploid with one functional allele of gene of interest. Deletion of
gene by insertion of KanMX cassette at gene locus.
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Heterozygous diploids – all cells can go through sporulation
with one functional copy of the gene as diploid. Hoping to find
true germination mutants avoiding sporulation mutants.
Workpackage 2 Deliverable 14
Workpackage 4 Deliverable 23
Germination mutants – heterozygous diploids
Sporulation of heterozygous diploids with mutated
genes of interest.
Tetrad dissection of spores
Germination on geneticin plates selecting for haploid
cells carrying deletion cassette with geneticin
resistance marker
Workpackage 2 Deliverable 14
Workpackage 4 Deliverable 23
Germination mutants – heterozygous diploids
Problem:
Proteins inherited from the mother diploid are enough to allow mutants to
germinate. Also true for kanMX – spores without the kanMX cassette
germinates and can divide to the 50-cells stage on geneticin plates.
Potential:
Preliminary data indicates that rho1Δ haploids derived from heterozygous
dipolids can not germinate.
Rho1 - GTP-binding protein of the rho subfamily of Ras-like proteins,
involved in establishment of cell polarity; regulates protein kinase C
(Pkc1) and the cell wall synthesizing enzyme 1,3-beta-glucan synthase.
Workpackage 2 Deliverable 14
Workpackage 4 Deliverable 23
Synthetic Genetic Array (SGA) Analysis
MATa (-met) deletion collection carrying reporter MFA1pr-HIS3, only
expressed in haploid a cells
Mate with deletion collection of alpha cells (-lys)
Homozygous diploids, each diploid with both alleles of one particular
gene deleted. Select for diploids on selective plates containing no
Metionine and no Lysine
Sporulate diploids
Germinate spores on selective plates containing no Histidine. Only
haploid a cells with the MFA1pr-HIS3 cassette will grow on plates.
Screen for germination mutants, ie empty spots on SGA plate
Synthetic Genetic Array (SGA) Analysis
Will find:
Sporulation mutants, aneuploid strains,
true germination mutants (?)
Will not find:
Essential genes
Workpackage 2 Deliverable 14
Workpackage 4 Deliverable 23
Conclusions – germination mutants
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Screening for germination mutants using heterozygous diploids is
time consuming and results hard to interpret since proteins from
the mother diploid take mutants through germination.
•
Screening for germination mutants using SGA Analysis has
potential, but also drawbacks – not only germination mutants will
be picked up but also sporulation mutants. Which is which?
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Another possibility is to use mutants with temperature sensitive
alleles for essential genes. Will potentially identify mutants that
can germinate but not grow vegetatively.
Workpackage 2 Deliverable 14
Workpackage 4 Deliverable 23
Control Mechanisms of Dormancy and
Germination of The Baker’s Yeast S. cerevisiae
Spore
Ivan Pirkov
Dept. Of Cell and Molecular Biology
Göteborg University
Present Focus
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The baker’s yeast Saccharomyces cerevisiae produces a dormant stage,
the spore
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The present aim of the project is to investigate how the yeast spore is
reactivated from its dormant stage
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We are currently studying the global gene expression changes upon
induction of germination using microarray analysis
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Expectations - To identify pathways and specific genes that are
associated with spore germination
Microarray on Germinating Yeast Spores (WP2)
The experimental outline
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Diploid cells were sporulated in 1% KAc solution
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The spores were left resting at 4C in 0.5% TritonX-100 solution for at least 14 days
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Spores were then transferred to rich nutrient growth medium containing 2% glucose (YPD) OR only in 2%
glucose solution
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Samples for total RNA extraction were taken in a logarithmic time-fashion, 0, 4, 8, 16 min… etc. up to 128
min
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Resting spores were used as reference sample
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The experiment was done in three independent replicates
Spore Germination
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Is most efficient when a readily fermentable carbon source is present – e.g. glucose,
fructose, galactose
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Presence of just a carbon source is sufficient for germination initiation
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Metabolism of the carbon source is necessary for germination, mere presence is not enough
Percent of spores that have
germinated after 24 hrs
sensing of S. cerevisiae Y55 spores
Herman and Rine (1997),Glucose
EMBO J, 16:6171-6181
100
90
80
70
60
50
40
30
20
10
0
1000
100
10
1
mM Glucose
0,1
0,01
0,001
Spore Germination
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RNA synthesis increases within minutes upon addition of glucose and nutrients
Brengues et al (2002), JBC, 277:40505-40512
Microarray on Germinating Yeast Spores (WP2)
Number of genes significantly up/down-regulated (2-fold) relative to resting spores
experiment)
(2% Glucose
300
No. of genes up/down
200
100
0
0
4
8
16
32
48
100
200
300
400
Time samples [min]
64
96
128
Up regulated
Down regulated
Microarray on Germinating Yeast Spores (WP2)
Clustering of significantly up/down-regulated genes (2-fold) relative resting spores
experiment)
1
9
0
8
7
6 5
4
3
2
1
(2%Glucose
Microarray on Germinating Yeast Spores (WP2)
Clustering result (GO Annotations) of significantly up/down-regulated (2-fold) relative to resting
spores (2%Glucose experiment)
Groups of up-regulated genes
(Cluster: 2, 5, 7, 8 and 9)
• Amino acid biosynthesis and degradation
(2) (7) (9)
• Ribosome biogenesis (2) (5) (7)
• Stress response (2)
• Glucose transport and signaling (3) (5)
• Protein folding and stabilization (8)
• Ion transport (2) (8) (9)
Groups of down-regulated genes
(Cluster: 1, 3, 4, 6 and 10)
• Stress response (1)
• Meiosis (1)
• Glycolysis and gluconeogenesis (3)
• Fatty acid oxidation (3)
• TCA-cycle (4)
• Glyoxylate cycle (4)
• Oxidative phosphorylation and
transport (4)
electron
• Biosynthesis of Glycogen and Trehalose (4)
(10)
Microarray on Germinating Yeast Spores (WP2)
Commitment-step synchrony of S. cerevisiae Y55
No. of genes significantly up/down-regulated (2-fold) relative resting spores
spores in YPD
experiment)
(YPD
No. of genes up/down
600
400
200
0
0
200
400
600
800
Percent of spores commited for germination
100
90
80
70
60
50
4
8
16
32
48
64
96
128
40
30
20
10
0
0
15
30
45
60
75
90
105 120 135 150 165 180 195
Time samples
[min]
Time [min]
Up regulated
Down regulated
Microarray on Germinating Yeast Spores (WP2)
Clustering of significantly up/down-regulated genes (2-fold) relative resting spores
(YPD experiment)
9
8
7
6
5
4
3
2
1
Microarray on Germinating Yeast Spores (WP2)
Clustering result (GO Annotations) of significantly up/down-regulated (2-fold) relative resting spores
(YPD experiment)
Groups of up-regulated genes
(Cluster: 3, 4, 6, 7 and 9)
Groups of down-regulated genes
(Cluster: 1, 2, 5 and 8)
• Ribosome biogenesis (3) (4) (7)
• Stress response (1) (2)
•Transcription and Translation (3) (4) (6)
(7)
• Meiosis (1)
• Nucleotide metabolism (3)
• Pheromone response – mating type
determination (4)
• Glycolysis and gluconeogenesis (1) (2)
• Fatty acid oxidation and transport (2)
• TCA-cycle (2)
• Glucose transport and signaling (6)
•Peroxisome and vacuole (2)
• Protein folding and stabilization (8)
• Glyoxylate cycle (2)
•Ion transport (6) (9)
• Oxidative phosphorylation and e--transport (2) (8)
•RAS protein signal tranduction (2) (8)
• Metabolism of Glycogen and Trehalose (2) (8)
• Amino acid biosynthesis (5)
• Redox homeostasis (5)
Microarray on Germinating Yeast Spores (WP2)
•
Potential problems
– Different mRNA to total RNA ratios between reference and
time-samples
– Global shifts in mRNA population during germination
Future Perspectives
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Analyze the microarray results in more detail to look for transcriptional patterns
 Publication of the results (WP2)
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Identifying genes that are expressed in resting spores and not in growing cells
and vice versa  Done, data need to be analyzed (WP1, WP2)
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Work with Cecilia to construct the homozygote deletion strain and to screen for
mutants that are unable to germinate (WP2)
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Continue with the Long-term dormancy experiment (WP1, WP2)
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Extract and analyze proteins from resting spores of different age (WP1)
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Analyze contents of resting spores  DTU (WP1)
CMB - Cell and Molecular Biology - Group Stefan Hohmann
Avi Ericsson, 2007
Total number of up/down regulated
genes in the YPD experiment
CMB - Cell and Molecular Biology - Group Stefan Hohmann
Avi Ericsson, 2007
Control experiment
• Hybridize 32 and 46 min against each
other
• Hybridize 96 and 128 min against each
other
• And then compare the ”real” and
”expected” values of up/down regulation
CMB - Cell and Molecular Biology - Group Stefan Hohmann
Avi Ericsson, 2007
Control experiment schematically
Resting spores
Time point X
CMB - Cell and Molecular Biology - Group Stefan Hohmann
Time point Y
Avi Ericsson, 2007
CMB - Cell and Molecular Biology - Group Stefan Hohmann
Avi Ericsson, 2007
CMB - Cell and Molecular Biology - Group Stefan Hohmann
Avi Ericsson, 2007
Conclusions and future work
• The correlation is not too bad! Which
might indicate that we probably do not
have a problem.
• To be sure - we have to measure the
mRNA concentrations in our samples.
• When we are sure – publish!
CMB - Cell and Molecular Biology - Group Stefan Hohmann
Avi Ericsson, 2007
Acknowledgements
to
The Hohmann lab
Microarray on Germinating Yeast Spores (WP2)
Test – mRNA to total RNA ratios
Total RNA from sample X
Normalization of the data
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•
•
Avi will explain the normalization method
No external control mRNA was used
Potential problems
–
–
•
•
Different mRNA to total RNA ratios between reference and
time-samples
Global shifts in mRNA population during germination
Total RNA from sample X
Oligo dT + Random
primers, Label with
Cy5
Only oligo dT,
Label with Cy5
Cy5 labeled cDNA from only
mRNA
How to solve these problems?
Suggestions?
Cy 5 labeled cDNA from total
RNA
Cy5 signal in cDNA from mRNA (M) and total
RNA (T)
0M
48M
128M
0T
48T
0x
5x
25x
Ratio mRNA vs. total RNA
0min
48min
128min
??
??
??
128T