Transcript Jan06_Alpha_Project_Retreat

Engineering a simpler
pheromone response pathway
Alex Mallet
Endy Lab
MIT
Regulation of pheromone pathway (Dohlman and Thorner, 2001)
Transcriptional complexity
• Transcriptional response
– ~ 200 genes upregulated, ~200 genes downregulated after
exposure to pheromone (Roberts et al, 2000)
• Transcriptional regulation
– Ste12 binds to ~115 promoters on exposure to pheromone
(Zeitlinger et al, 2003)
– Some mating genes are induced by pheromone, others
aren’t
– Some mating genes are cell-cycle regulated eg Fus1, Sst2
– Positive and negative feedback loops (eg Ste2, Sst2)
– Feedforward loops e.g. Ste12 -> Kar4; Ste12, Kar4 ->
Kar3
– Multiple types of regulation for single gene eg Sst2, Fus1
Complexity of genomic
organization
Chr I
Chr II
(100kb bins)
Chr III
Chr IV
Chr V
Chr VI
Chr VII
Chr VIII
Chr IX
16 genes involved in signal
transduction pathway from
Ste2 to Ste12 are scattered
across 10 chromosomes
Chr X
Chr XI
Chr XII
Chr XIII
Chr XIV
Chr XV
Chr XVI
Target promoters
identified by
Zeitlinger et al are
scattered across all
16 chromosomes
Complexity is undesirable
• Difficult to understand, even
qualitatively
– E.g. which bits of regulation are essential ?
• Difficult to model accurately
• Difficult to manipulate experimentally
– Hard to manipulate many genes at once
– Hard to control multiple genes
simultaneously
Proposed project
• Re-engineer pheromone pathway for simpler
transcriptional characteristics and experimental
manipulation
•
•
•
Custom (simpler) transcriptional control
– Get rid of cell-cycle regulation
– Get rid of feedback loops
– Express genes from custom constitutive or inducible/repressible
promoters
Simpler response
– Remove genes known to be involved in, but not essential to, mating
Easier to manipulate
– Put all genes involved on single plasmid/YAC
– Subdivide pathway: divide genes into independent, separatelyinducible/repressible subsystems (eg “ligand manufacture and export
subsystem”, “MAPK cascade subsystem”)
Motivation
• Simpler pathway is easier to model and manipulate
• Engineered GPCR-MAPK cascade signal
transduction system can be reused
• Getting rid of (some of) the regulation will tell us how
essential these levels of regulation are
• Validation of existing state of knowledge about
genes involved in yeast mating response
• Engineering lessons in:
– Building a large pathway
– Designing independent subsystems and getting
them to interoperate successfully in yeast
– Designing a debuggable pathway
Some proposed subsystems and
changes
• Receptor subsystem: Ste2
– Remove cell-cycle, Ste12, Mcm1 regulation
• Pheromone manufacture and export: Mfa1,
Mfa2, Ste6, Bar1
– Remove cell-cycle, Ste12 regulation
• G-proteins: Gpa1, Ste4, Ste18
– Remove Ste12 regulation of Gpa1
– Remove Sst2 phosphatase regulation of Gpa1 (knock
out Sst2 or remove Ste12 control of Sst2)
Example: re-engineering Ste2
• Wild-type Ste2
5’ sequence, with binding sites
for Ste12, Mcm1, Dig1, Fkh1
Ste2 coding
sequence
Ste2 3’
sequence
• Re-engineered Ste2
Custom UAS for
single TF e.g. TetR
Custom core
promoter
Ste2 coding
sequence
Custom 3’
sequence
– Custom 3’ sequence: CYC1 terminator
– Custom core promoter: CYC1 core promoter and 5’ UTR, with
single TATA box
– Custom UAS: contains binding motif for single TF (currently
based on database of ~100 motifs, will expand using
TRANSFAC)
Next steps
• Do some actual lab work 
• Planning to start with Ste2
Acknowledgements
MIT
Molecular Sciences
Endy Lab
Kirsten Benjamin
Natalie Kuldell
Richard Yu
Harvard
U. of Washington
Fred Winston
Stan Fields
Funding: MIT CSBi PhD Program
Questions, comments ?
Backup
Pheromone response regulation
• Pheromone response is subject to many layers
of regulation
–
–
–
–
–
–
Phosphorylation/Dephosphorylation
Transcriptional regulation
Protein stability
Receptor endocytosis
Protein localization
Ligand export and degradation
• My focus is on the transcriptional characteristics
of the pathway