Engineering a simpler pheromone response pathway

1. Engineering a simpler pheromone response pathway Alex Mallet Endy Lab MIT

2. Regulation of pheromone pathway (Dohlman and Thorner, 2001)

3. 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

4. Complexity of genomic organization Chr I (100kb bins) Chr II Chr III Chr IV Chr V Chr VI 16 genes involved in signal transduction pathway from Ste2 to Ste12 are scattered across 10 chromosomes Chr VII Chr VIII Chr IX Chr X Chr XI Chr XII Target promoters identified by Zeitlinger et al are scattered across all 16 chromosomes Chr XIII Chr XIV Chr XV Chr XVI

5. 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

6. 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”)

7. 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 Motivation

8. 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)

9. 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) 5’ sequence, with binding sites for Ste12, Mcm1, Dig1, Fkh1 Ste2 coding sequence Ste2 3’ sequence Custom UAS for single TF e.g. TetR Custom core promoter Ste2 coding sequence Custom 3’ sequence

10. Next steps • Do some actual lab work  • Planning to start with Ste2

11. Acknowledgements Molecular Sciences Kirsten Benjamin Richard Yu MIT Endy Lab Natalie Kuldell Harvard Fred Winston U. of Washington Stan Fields Funding: MIT CSBi PhD Program

12. Questions, comments ?

13. Backup

14. 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