Gene%20Expression%20and%20Signaling%20Pathways%20in%20Yeast

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Gene Expression and Signaling Pathways in Yeast
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Classical Genetics

• Genotype
• The genetic makeup of an organism
• Phenotype
• The observed behavior
• Basic Assumption
• Genotype ? phenotype

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Environmental Responses (Yeast)
Condition Response
Pheromone smooing (mating)
Nutrient depletion Filamentous growth (foraging)
Osmotic stress (high salt concentration) Glycerol production, cell wall repair
Cell wall damage Cell wall repair
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Genetic Screens

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

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Central Dogma

• Regulation of expression of genes is crucial
• Understanding regulatory processes is a central
  problem of biological research

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Measuring RNA production
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Yeast Signaling Pathways
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Goals

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

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Yeast Mating
MATa haploid
MATa haploid

• Pheromone
• a-factor
• Secreted by MATa
• Sensed by MATa
• a-factor
• Secreted by MATa
• Sensed by MATa

Diploid
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Genes Involved in Mating Response
MATa a-factor (50nM for 30min) vs MATa

• Each dot average of 4 repeats
• Statistical test finds
• gt 200 genes induced (red)
• gt 200 genes repressed (green)

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Does Dosage Matter?

• Strong correlation
• Results are reproducible

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Ste2

• Ste2 is a necessary receptor for pheromone
  response

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Ste12

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

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Far1
What is the relation between Ste12 and Far1 ??

• Essentially all gene repression requires Far1
• Most of these genes required for G1 cell cycle
  phase

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Rst1/2 (Dig1/2)

• Rst1/2 KO induces genes involved in both
  pheromone response and filamentous growth

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Pathway interference
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Global View
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Global View
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Goals

• Closer look at HOG1 pathway
• Activity of HOG1
• Roles of components upstream of HOG1

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Hyperosmotic Stress

• High salt solution (or other osmolyte) ? water
  drawn out of the cell salt enters the cell
• Cells shrink damage to cell wall and membrane
  increased protein concentration problems with
  pressure for budding etc.
• Increased salt concentrationchanges in
  protein/protein and protein/DNA interactions

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Hyperosmotic Stress Response

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

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HOG1 pathway
cytoplasm
General Stress Factors
Pbs2
Hog1
Msn2/4
Hog1
pp
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Smp1 Msn1
Sko1
Hot1
Msn2/4
Nucleus
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Step1 Calibrating the System
Maximal change
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Hog1 dependent genes

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

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Hog1 dependent genes

• Induced in Hog1?
• Repressed by Hog1
• Genes of mating and filamentous growth pathways
  FUS1, STE2, TEK1, PGU1,

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Hog1 dependent genes

• Repressed in Hog1?
• Induced by Hog1
• Either with or without KCl

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Hog1 dependent genes

• Expression in Hog1? changes in later stages
• Require Hog1 for late stage recovery
• Mostly secondary effects

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Teasing Out Components
Hog1? ? Pbs2 ?
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Teasing Out Components

• Hog1? ? Pbs2 ? ? Ssk1? Ste11?
• Additional inputs to Pbs2?
• Changes in pheromone pathway (Ste11)

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Teasing Out Components

• Ssk1? Ste11? ? Ssk1? Sho1?
• Sho1 leaker than Ste11
• Additional inputs to Ste11?

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Teasing Out Components

• WT ? Ssk1? ? Ste11? ? Sho1?
• Single KO have little effect on response
• Are these receptor redundant?

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Redundancy and dosage