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

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