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 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