synthetic lethal analysis of caenorhabditis elegans posterior embryonic patterning genes identifies...
TRANSCRIPT
Synthetic lethal analysis of Caenorhabditis elegans
posteriorembryonic patterning genes identifies conserved genetic
interactionsL Ryan Baugh, Joanne C Wen, Andrew A Hill, Donna K Slonim, Eugene L Brown & Craig P
Hunter*
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
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?
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
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
The C Blastomere
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?
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
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
Statistical Analysis
1. % lethality converted to % survival
2. Survival values normalized
3. Calculate significance of interactions using students t test (p>0.05)Ho: Survival of the double disruption (mutation x RNAi)
equals the product of survivals for each single disruption
Results
Which interactions are significant?
interaction
interaction
tbx-8 & tbx-9 form a module
Wild-type tbx-9 (RNAi) tbx-8 (ok656)
tbx-8 (ok656); tbx-9 (RNAi) tbx-8 (ok656); tbx-9 (RNAi)
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
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?
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
Is this module conserved?•Interactions between bHLH factors in vertebrates
•Relationship between bHLH proteins & MADS-box regulators (the MEF2 group)
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
Gratuitous Political Cartoons
The People have spoken!Plebiscite results
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1 2
pe
rce
nti
le
Property
Food/beer?