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 organizationChr I

Chr II

Chr III

Chr IV

Chr V

Chr VI

Chr VII

Chr VIII

Chr IX

Chr X

Chr XI

Chr XII

Chr XIII

Chr XIV

Chr XV

Chr XVI

16 genes involved in signal transduction pathway from Ste2 to Ste12 are scattered across 10 chromosomes

Target promoters identified by Zeitlinger et al are scattered across all 16 chromosomes

(100kb bins)

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, separately-

inducible/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

• Re-engineered Ste2

– 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)

Custom UAS for single TF e.g. TetR

Custom core promoter

Ste2 coding sequence

Custom 3’ sequence

5’ sequence, with binding sites for Ste12, Mcm1, Dig1, Fkh1

Ste2 coding sequence

Ste2 3’ sequence

Next steps

• Do some actual lab work • Planning to start with Ste2

Acknowledgements

MIT

Endy Lab

Natalie Kuldell

Harvard

Fred Winston

Molecular Sciences

Kirsten Benjamin

Richard Yu

Funding: MIT CSBi PhD Program

U. of Washington

Stan Fields

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