1. Synthetic lethal analysis of Caenorhabditis elegans posterior embryonic patterning genes identifies conserved genetic interactions L Ryan Baugh, Joanne C Wen, Andrew A Hill, Donna K Slonim, Eugene L Brown & Craig P Hunter*

2. 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

3. 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?

4. Background: Synthetic Lethality Developed by Charlie Boone at U of T SGA= systematic construction of double mutants Cross YFG1 to an array of ~ 5000 Δ strains

5. 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

6. The C Blastomere

7. pal-1 specifies & regulates C lineage PAL-1 target genes Identified in microarray screen Validated using GFP transcriptional reporters Many targets are TF’s RNAi of most PAL-1 targets does not result in a phenotype. Why? Is there overlapping function?

8. 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

9. 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

10. Statistical Analysis 1.% lethality converted to % survival 2.Survival values normalized 3.Calculate significance of interactions using students t test (p>0.05) H o : Survival of the double disruption (mutation x RNAi) equals the product of survivals for each single disruption

11. Results

12. Which interactions are significant? interaction

13. tbx-8 & tbx-9 form a module Wild-type tbx-9 (RNAi)tbx-8 (ok656) tbx-8 (ok656); tbx-9 (RNAi)

14. 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

15. A muscle differentiation module Identified a module around hlh-1 Detected 5 of 6 interactions (p<0.001) 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?

16. The hlh-1 module hlh-1 is most essential (or most potent) of the 3 TF’s in the module hnd-1 is the least essential (or potent) patwildtype

17. Is this module conserved? Interactions between bHLH factors in vertebrates Relationship between bHLH proteins & MADS-box regulators (the MEF2 group)

18. 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.) Didn’t confirm results by doing a dihybrid cross

19. Gratuitous Political Cartoons

20. The People have spoken!