Engineering a simpler pheromone response pathway

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Engineering a simpler pheromone response pathway

• Alex Mallet
• Endy Lab
• MIT

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Regulation of pheromone pathway (Dohlman and
Thorner, 2001)
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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 arent
• Some mating genes are cell-cycle regulated eg
  Fus1, Sst2
• Positive and negative feedback loops (eg Ste2,
  Sst2)
• Feedforward loops e.g. Ste12 -gt Kar4 Ste12, Kar4
  -gt Kar3
• Multiple types of regulation for single gene eg
  Sst2, Fus1

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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
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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

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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)

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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

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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)

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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
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Next steps

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

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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
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Questions, comments ?
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Backup
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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