Transcript Synthetic lethal analysis of Caenorhabditis elegans posterior

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*
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-offunction 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
RNAi of most PAL-1 targets does not result in•Identified
a
in
phenotype. Why? Is there overlapping function?
microarray screen
•Validated using
GFP
transcriptional
reporters
•Many targets are
TF’s
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-9 (RNAi)
tbx-8 (ok656)
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)
wildtype
pat
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
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