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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