Transcript Gene Expression

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)
Condition
Response
Pheromone
“smooing”
(mating)
Filamentous growth
(foraging)
Nutrient depletion
Osmotic stress
(high salt concentration)
Glycerol production, cell wall
repair
Cell wall damage
Cell wall repair
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?
Central Dogma


Regulation of expression of genes is crucial
Understanding regulatory processes is a central
problem of biological research
Transcription
Translation
RNA
Protein
Measuring RNA production
Sample 1 >> Sample2
Sample 1 << Sample2
Sample 1  Sample2
Yeast Signaling Pathways
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
Yeast Mating
MATa haploid
Pheromone
 a-factor
 Secreted by MATa
 Sensed by MATα
 α-factor
 Secreted by MATα
 Sensed by MATa
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?


Strong correlation
Results are reproducible
Ste2

Ste2 is a necessary receptor for
pheromone response
Ste12

The whole response appears to
be derived from pathwaydependent activation of Ste12
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
Rst1/2 (Dig1/2)

Rst1/2 KO induces genes
involved in both pheromone
response and filamentous
growth
Pathway interference
Global View
Goals

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


Cells shrink:
damage to cell wall and membrane; increased
protein concentration; problems with pressure for
budding etc.
Increased salt concentration:
changes in protein/protein and protein/DNA
interactions
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
HOG1 pathway
cytoplasm
General
Stress
Factors
Pbs2
Hog1
Smp1
Msn1
Hog1pp
Sko1 Hot1
Msn2/4
?
Msn2/4
Nucleus
Step1: Calibrating the System
Maximal change
Hog1 dependent genes

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)
Hog1 dependent genes
Induced in Hog1Δ
 Repressed by Hog1

Genes of mating and filamentous
growth pathways FUS1, STE2,
TEK1, PGU1,…
Hog1 dependent genes
Repressed in Hog1Δ
 Induced by Hog1
 Either with or without KCl
Hog1 dependent genes
Expression in Hog1Δ changes in
later stages
 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