. Gene Expression and Signaling Pathways in Yeast

Classical Genetics Genotype: The genetic makeup of an organism Phenotype: The observed “behavior” Basic Assumption: Genotype  phenotype

Environmental Responses (Yeast) ConditionResponse Pheromone“smooing” (mating) Nutrient depletionFilamentous growth (foraging) Osmotic stress (high salt concentration) Glycerol production, cell wall repair Cell wall damageCell wall repair

Genetic Screens u Search for genes whose removal (KO) disrupt desired response u Classical genetic screens identified many genes that are involved in yeast response to environmental cues. Can we understand these processes better?

Central Dogma Transcription RNA Translation Protein u Regulation of expression of genes is crucial u Understanding regulatory processes is a central problem of biological research

Measuring RNA production Sample 1 >> Sample2 Sample 1 << Sample2 Sample 1  Sample2

Yeast Signaling Pathways

Goals u Comprehensive map of genes involved in different responses u Understanding the function of different components in the pathway Meta-goal: u Utility of gene expression for studying signaling pathways

Yeast Mating Pheromone u a-factor  Secreted by MATa  Sensed by MATα u α-factor  Secreted by MATα  Sensed by MATa MATa haploid MATα haploid Diploid

Genes Involved in Mating Response MATa +α-factor (50nM for 30min) vs MATa Each dot – average of 4 repeats Statistical test finds > 200 genes induced (red) > 200 genes repressed (green)

Does Dosage Matter? u Strong correlation u Results are reproducible

Ste2 u Ste2 is a necessary receptor for pheromone response

Ste12 u The whole response appears to be derived from pathway- dependent activation of Ste12

Far1 u Essentially all gene repression requires Far1 u Most of these genes required for G1 cell cycle phase What is the relation between Ste12 and Far1 ??

Rst1/2 (Dig1/2) u Rst1/2 KO induces genes involved in both pheromone response and filamentous growth

Pathway interference

Global View

Goals u Closer look at HOG1 pathway  Activity of HOG1  Roles of components upstream of HOG1

Hyperosmotic Stress High salt solution (or other osmolyte)  water drawn out of the cell; salt enters the cell u Cells shrink: damage to cell wall and membrane; increased protein concentration; problems with pressure for budding etc. u Increased salt concentration: changes in protein/protein and protein/DNA interactions

Hyperosmotic Stress Response u Production of an osmolyte:  Glycerol is synthesized to balance osmolarity inside/outside  Change in metabolism to accommodate this u Removal of salt:  Upregulation/downregulation of appropriate transporters for NaCl, KCl and other ions u Dealing with stress:  Expression of chaperones and other general stress response genes  Shutting down non-essential stuff

HOG1 pathway Pbs2 Hog1 pp cytoplasm Nucleus Smp1 Msn1 Sko1Hot1 Msn2/4 ? General Stress Factors

Step1: Calibrating the System Maximal change

Hog1 dependent genes u 579 Hog1 dependent genes  2-fold response to KCl  3-fold response to Hog1Δ  Both in at least two time points (Note differences from previous paper)

Induced in Hog1Δ u Repressed by Hog1 u Genes of mating and filamentous growth pathways FUS1, STE2, TEK1, PGU1,… Hog1 dependent genes

Repressed in Hog1Δ u Induced by Hog1 u Either with or without KCl Hog1 dependent genes

u Expression in Hog1Δ changes in later stages u Require Hog1 for late stage recovery Mostly secondary effects

Teasing Out Components Hog1Δ  = Pbs2 Δ

Teasing Out Components Hog1Δ  = Pbs2 Δ  Ssk1Δ + Ste11Δ Additional inputs to Pbs2? Changes in pheromone pathway (Ste11)

Teasing Out Components Ssk1Δ + Ste11Δ  Ssk1Δ + Sho1Δ Sho1 “leaker” than Ste11 Additional inputs to Ste11?

Teasing Out Components WT  Ssk1Δ  Ste11Δ  Sho1Δ Single KO have little effect on response Are these receptor redundant?

Redundancy and dosage