transcription factors regulating expression of aqy1 and screens for germination mutants cecilia...

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.

• Cells go through meiosis which leads to the generation of four haploid nuclei

• Plasma membranes for four daugther cells are constructed within the mother cell cytoplasm, and these surrounds the four haploid nuclei.

• 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.

http://nobelprize.org/chemistry/laureates/2003/public.html

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

www.yeastract.com

• Msn2/4

• Fkh1/2

• War1

• Nrg1

• Xbp1

• Gcr1

• Mcm1

• Ash1

• Mot3

• Tec1

Consensus sites identified:

No sporulation-specific transcription factor hits!


AQY1prom-lacZ fusion construct

Reporter gene lacZ system

Yeast lacZ reporter plasmid to search for transcription factors regulating expression of AQY1

• X-gal overlay assay

• β-galactosidase activity assay ONPG

AQY1prom lacZ


X-gal overlay assay

BY4741 KO collection used to search for transcription factors

Preliminary data:

fkh2Δ strain seem to have lower expression of AQY1 than wild type

wild type

tec1Δ

mot3Δ

war1Δ

fkh2Δ_1

fkh2Δ_2

X-gal overlay assay works to identify transcription factors regulating the AQY1 expression in vegetative cells


Sporulation plates with X-gal overlay assay

Spores treated with chloroform and overlayed with agarose X-gal are not turning blue – this assay probably not suitable for spores.

SK1a/α

ε1278ba/α


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 β-galactosidase 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.


Germination – search for mutants

Involvement of actin and polarisome in morphological change during spore germination of Saccharomyces cerevisiaeKeiko Kono, Rino Matsunaga, Aiko Hirata, Genjiro Suzuki, Mitsuhiro Abe, Yoshikazu Ohya YEAST 2005

What gene products are essential for the germination process in Saccharomyces cerevisiae?



Screen for Germination mutants using heterozygous mutants

• 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.

• 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.





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

Germination mutants – heterozygous diploids

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.



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

Will find:

Sporulation mutants, aneuploid strains, true germination mutants (?)

Will not find:

Essential genes

Synthetic Genetic Array (SGA) Analysis



Conclusions – germination mutants

• 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?

• Another possibility is to use mutants with temperature sensitive alleles for essential genes. Will potentially identify mutants that can germinate but not grow vegetatively.



Control Mechanisms of Dormancy and Control Mechanisms of Dormancy and Germination of The Baker’s Yeast Germination of The Baker’s Yeast S. cerevisiaeS. cerevisiae

SporeSpore

Ivan PirkovIvan Pirkov

Dept. Of Cell and Molecular BiologyDept. Of Cell and Molecular BiologyGöteborg UniversityGöteborg University

Present FocusPresent Focus

• The baker’s yeast Saccharomyces cerevisiae produces a dormant stage, the spore

• The present aim of the project is to investigate how the yeast spore is reactivated from its dormant stage

• We are currently studying the global gene expression changes upon induction of germination using microarray analysis

• Expectations - To identify pathways and specific genes that are associated with spore germination

Microarray on Germinating Yeast Spores Microarray on Germinating Yeast Spores (WP2)(WP2)

The experimental outline

• Diploid cells were sporulated in 1% KAc solution

• The spores were left resting at 4C in 0.5% TritonX-100 solution for at least 14 days

• Spores were then transferred to rich nutrient growth medium containing 2% glucose (YPD) OR only in 2%

glucose solution

• Samples for total RNA extraction were taken in a logarithmic time-fashion, 0, 4, 8, 16 min… etc. up to 128

min

• Resting spores were used as reference sample

• The experiment was done in three independent replicates

Spore GerminationSpore Germination

• Is most efficient when a readily fermentable carbon source is present – e.g. glucose,

fructose, galactose– Presence of just a carbon source is sufficient for germination initiation

– Metabolism of the carbon source is necessary for germination, mere presence is not enough

Herman and Rine (1997), EMBO J, 16:6171-6181Glucose sensing of S. cerevisiae Y55 spores

0

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0,0010,010,11101001000

mM Glucose

Perc

en

t o

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ore

s t

hat

have

germ

inate

d a

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24 h

rs

• RNA synthesis increases within minutes upon addition of glucose and nutrients

Brengues et al (2002), JBC, 277:40505-40512

Spore GerminationSpore Germination

Number of genes significantly up/down-regulated (2-fold) relative to resting spores (2% Glucose experiment)

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0

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0 4 8 16 32 48 64 96 128

Time samples [min]

No

. of

gen

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p/d

ow

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Up regulated

Down regulated


Clustering of significantly up/down-regulated genes (2-fold) relative resting spores (2%Glucose experiment)

12345678910


Clustering result (GO Annotations) of significantly up/down-regulated (2-fold) relative to resting spores (2%Glucose experiment)

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 electron transport (4)

• Biosynthesis of Glycogen and Trehalose (4) (10)

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)


800

600

400

200

0

200

400

600

0 4 8 16 32 48 64 96 128

Time samples [min]

No

. of

gen

es u

p/d

ow

n

Up regulatedDown regulated

No. of genes significantly up/down-regulated (2-fold) relative resting spores (YPD experiment)


Commitment-step synchrony of S. cerevisiae Y55 spores in YPD

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ore

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om

mit

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r g

erm

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Clustering of significantly up/down-regulated genes (2-fold) relative resting spores (YPD experiment)

16 5 4 3 29 8 7


Clustering result (GO Annotations) of significantly up/down-regulated (2-fold) relative resting spores (YPD experiment)

Groups of down-regulated genes(Cluster: 1, 2, 5 and 8)

• Stress response (1) (2)

• Meiosis (1)

• Glycolysis and gluconeogenesis (1) (2)

• Fatty acid oxidation and transport (2)

• TCA-cycle (2)

•Peroxisome and vacuole (2)

• Glyoxylate cycle (2)

• 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)

Groups of up-regulated genes(Cluster: 3, 4, 6, 7 and 9)

• Ribosome biogenesis (3) (4) (7)

•Transcription and Translation (3) (4) (6) (7)

• Nucleotide metabolism (3)

• Pheromone response – mating type determination (4)

• Glucose transport and signaling (6)

• Protein folding and stabilization (8)

•Ion transport (6) (9)


• Potential problems

– Different mRNA to total RNA ratios between reference and time-samples

– Global shifts in mRNA population during germination


Future PerspectivesFuture Perspectives

• Analyze the microarray results in more detail to look for transcriptional patterns Publication of the results (WP2)

• Identifying genes that are expressed in resting spores and not in growing cells and vice versa Done, data need to be analyzed (WP1, WP2)

• Work with Cecilia to construct the homozygote deletion strain and to screen for mutants that are unable to germinate (WP2)

• Continue with the Long-term dormancy experiment (WP1, WP2)

• Extract and analyze proteins from resting spores of different age (WP1)

• Analyze contents of resting spores DTU (WP1)

Avi Ericsson, 2007CMB - 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


Avi Ericsson, 2007

Control experiment schematically

Resting spores

Time point X Time point Y


Avi Ericsson, 2007CMB - 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!


Acknowledgementsto

The Hohmann lab

Normalization of the data

• 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

• How to solve these problems?• Suggestions?

48T 128T128M48M0M 0T

0x

5x

25x

Cy5 signal in cDNA from mRNA (M) and total RNA (T)

Total RNA from sample X Total RNA from sample X

Only oligo dT, Label with Cy5

Oligo dT + Random primers, Label with

Cy5

Cy5 labeled cDNA from only mRNA

Cy 5 labeled cDNA from total RNA

Test – mRNA to total RNA ratios

0min 48min 128min

Ratio mRNA vs. total RNA

?? ?? ??


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