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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)

Nutrient depletion Filamentous growth (foraging)

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

Transcription

RNA

Translation

Protein

Regulation of expression of genes is crucial Understanding regulatory processes is a central

problem of biological research

Measuring RNA production

Sample 1 >> Sample2

Sample 1 << Sample2Sample 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

Pheromone a-factor

Secreted by MATa Sensed by MATα

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?

Strong correlation Results are reproducible

Ste2

Ste2 is a necessary receptor for pheromone response

Ste12

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

Far1

Essentially all gene repression requires Far1

Most of these genes required for G1 cell cycle phase

What is the relation between Ste12 and Far1 ??

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

Pbs2

Hog1

Hog1pp

cytoplasm

Nucleus

Smp1Msn1

Sko1 Hot1 Msn2/4

?

Msn2/4

GeneralStressFactors

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)

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

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