Synthetic lethal analysis of Caenorhabditis elegans posterior embryonic patterning genes identifies conserved genetic interactions

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Synthetic lethal analysis of Caenorhabditis
elegans posteriorembryonic patterning genes
identifies conserved geneticinteractions

• L Ryan Baugh, Joanne C Wen, Andrew A Hill, Donna
  K Slonim, Eugene L Brown Craig P Hunter

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The Hunter Lab at Harvard

• Dr. Craig Hunter
• Examining the mechanism behind systemic RNAi
• Studying the master switch pal-1, involved in
  specifying the fate of the C blastomere

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Background

• Most genes are not essential (i.e. yeast, flies,
  worms)
• 2 possible reasons homology (direct
  compensation) parallel pathways (indirect
  compensation)
• Genes with 1 or more homologs less likely to have
  loss-of-function phenotype
• 2/3 genetic buffering due to homology, implies
  large role for parallel pathways

How do you characterize mechanisms of phenotypic
robustness?
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Background Synthetic Lethality

• Developed by Charlie Boone at U of T
• SGA systematic construction of double mutants
• Cross YFG1 to an array of 5000 ? strains

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Synthetic Lethality

• Identify functional relationships between genes
  pathways
• Shed light on how regulatory networks buffer gene
  function
• Allows for creation of genetic modules
• Helps identify nodes

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The C Blastomere
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pal-1 specifies regulates C lineage

• PAL-1 target genes
• Identified in microarray screen
• Validated using GFP transcriptional reporters
• Many targets are TFs

RNAi of most PAL-1 targets does not result in a
phenotype. Why? Is there overlapping function?
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Goal of paper

• Identify synthetic interactions between pairs of
  PAL-1 targets
• Determine if genetic modules exist that buffer
  loss of proteins in the pal-1 pathway
• Examine the feasibility/reproducibility of double
  RNAi experiments

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Experimental Methods
RNAi Soaked strains of worms in dsRNA
(100-1000bp) -Added minimal T7 promoter to PCR
primers -Amplify DNA for in vitro
transcription -dsRNA reannealed by heating
cooling Attempted assembling matrix with only
RNAi -led to variable, inconsistent
results Examined RNAi-treated progeny for
embryonic lethality -converted lethality to
survival to calculate significance of the
interaction
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Statistical Analysis

1. lethality converted to survival
2. Survival values normalized
3. Calculate significance of interactions using
   students t test (pgt0.05)

Ho Survival of the double disruption (mutation x
RNAi) equals the product of survivals for each
single disruption
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Results
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Which interactions are significant?
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tbx-8 tbx-9 form a module
tbx-9 (RNAi)
tbx-8 (ok656)
Wild-type
tbx-8 (ok656) tbx-9 (RNAi)
tbx-8 (ok656) tbx-9 (RNAi)
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tbx-8 tbx-9 form a module

• Either disruption on their own 1-5 lethality,
  5 of hatchlings display posterior body defects
• Double disruption results in 50 lethality
  severe defects in hatchlings
• tbx-8,9 interactions are conserved in C. briggsae
  display similar expression patterns thus
  module has likely been conserved

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A muscle differentiation module

• Identified a module around hlh-1
• Detected 5 of 6 interactions (plt0.001) between
  hnd-1, hlh-1 and unc-120

• Disruption of any combination of the 3 genes
  results in pat
• Some symmetry, but not interactions are
  symmetrical. Why?

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The hlh-1 module

• hlh-1 is most essential (or most potent) of the 3
  TFs in the module
• hnd-1 is the least essential (or potent)

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Is this module conserved?

• Interactions between bHLH factors in vertebrates
• Relationship between bHLH proteins MADS-box
  regulators (the MEF2 group)

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Criticisms

• No positive controls (i.e. no known interactions
  were used)
• Why choose soaking and not do a comparison to
  feeding injecting?
• Why limit the interactions to lethality? Why not
  search for enhancement of phenotypes (gro, lva,
  lvl etc.)
• Didnt confirm results by doing a dihybrid cross

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Gratuitous Political Cartoons
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The People have spoken!