1. Variability in gene expression underlies incomplete
penetrance in Caenorhabditis elegans
Arjun Raj1*, Scott Rifkin2*, Erik Andersen3, Alexander van Oudenaarden4
1 University of Pennsylvania, Department of Bioengineering
2 University of California, San Diego
3 Princeton University
4 Massachusetts Institute of Technology
* Equal contributions

2. Why are individuals different?
Genetic variation

3. Why are individuals different?
Genetic variation
Environmental variation

4. What about random variation?

5. Random fluctuations in gene expression lead to
cell-to-cell variations in mRNA and protein number
Elowitz et al, 2002 al, 2007
Maamar and Raj et Raser and O’Shea, 2004
A.R., unpublished observations Raj et al, 2006
Raj and van Oudenaarden, Cell, 2008

6. Random fluctuations in gene expression lead to
cell-to-cell variations in mRNA and protein number
Elowitz et al, 2002 Raser and O’Shea, 2004 Raj et al, 2006
Maamar and Raj et al, 2007 A.R., unpublished observations Raj et al, 2006

7. Random fluctuations in gene expression lead to
cell-to-cell variations in mRNA and protein number
Maamar and Raj et al, 2007 A.R., unpublished observations Raj et al, 2006
Raj et al. Nature Methods, 2008
www.singlemoleculefish.com
Femino et al. Science, 1998

8. Noisy gene expression can be useful for microbes
Competent
cells
Maamar and Raj et al., Science, 2007

9. What about multicellular organisms?

10. Gene expression in mammalian cells occurs in
bursts
Raj et al., PLoS Biology, 2006
Chubb et al., Curr Biol, 2006

11. What about multicellular organisms?
Gene expression can
be very noisy...
Raj et al, 2006

12. What about multicellular organisms?
Gene expression can So you might expect a
be very noisy... lot of random variation...
Raj et al, 2006

13. What about multicellular organisms?
Gene expression can But the results often
be very noisy... come out the same.
Raj et al, 2006 Dianne Arbus, Identical Twins, 1967

14. Wild-type C. elegans embryos develop robustly

15. Wild-type C. elegans embryos develop robustly

16. Incompletely penetrant mutations reveal alternate
cell fates
Birefringent gut granules
• Many mutants are incompletely
penetrant, meaning that not all
mutant organisms display the
mutant phenotype.

17. Does variability in gene expression underlie the
incomplete penetrance of mutant phenotypes?

18. The C. elegans intestine is composed of
E cell descendants
P0
AB P1
EMS
ABa ABp
P2
MS E
C
P3
D
P4
Neural Pharynx Epidermal Blast Cell Muscle Intestine Germline
(Figure adapted from Baugh et al., Development 2003)

19. Intestinal fate is specified by a gene cascade
Cell lineage Gene Regulatory Network
P0 skn-1
AB P1
ABa ABp
EMS
P2 med-1,2
MS E
C
end-3 end-1
P3
D
OR
P4
elt-2
Neural Pharynx Epidermal Blast Cell Muscle Intestine Germline Intestine-specific
genes
(Figure adapted from Baugh et al., Development 2003)
Morris Maduro
Joel Rothman
Jim McGhee
Bruce Bowerman

20. Intestinal fate is specified by a gene cascade
Cell lineage Gene Regulatory Network
P0 skn-1
AB P1
ABa ABp
EMS
P2 med-1,2
MS E
C
end-3 end-1
P3
D
OR
P4
elt-2
Neural Pharynx Epidermal Blast Cell Muscle Intestine Germline Intestine-specific
genes
(Figure adapted from Baugh et al., Development 2003)
Morris Maduro
Joel Rothman
Jim McGhee
Bruce Bowerman

21. Wild-type embryos all express elt-2
elt-2
Nuclei

22. Mutations to skn-1 result in variable elt-2 expression
elt-2
Nuclei

23. Wild-type (N2)
DAPI (nuclei) end-3
med-1,2
elt-2
end-1

24. Wild type expression is very regular
Wild-type (N2)
600
med-1,2
0
600
end-3
0
600
end-1
0
600
elt-2
0
0 50 100 150 200
Number of nuclei

25. Mutant expression patterns are highly variable
Wild-type (N2) Mutant (zu135)
600 600
med-1,2
0 0
600 600
end-3
0 0
600 600
end-1
0 0
600 600
elt-2
0 0
0 50 100 150 200 0 50 100 150 200
Number of nuclei Number of nuclei

26. Mutant contain premature stop codons in skn-1
Wild-type (N2) Mutant (zu135)
600
med-1,2
skn-1 (mutant)
0
med-1,2
end-3 end-1 600
end-3
OR
elt-2 0
600
end-1
Intestine-specific
genes 0
600
elt-2
0
0 50 100 150 200 0 50 100 150 200
Number of nuclei Number of nuclei

27. elt-2 expresses in a bimodal fashion
Wild-type (N2) Mutant (zu135)
600
med-1,2
skn-1 (mutant)
0
med-1,2
end-3 end-1 600
end-3
OR
elt-2 0
(bimodal)
600
end-1
Intestine-specific
genes 0
600
elt-2
20%
0
0 50 100 150 200 0 50 100 150 200
Number of nuclei Number of nuclei

28. med-1,2 and end-3 expression is low
Wild-type (N2) Mutant (zu135)
600
med-1,2
skn-1 (mutant)
0
med-1,2
end-3 end-1 600
end-3
OR
elt-2 0
(bimodal)
600
end-1
Intestine-specific
genes 0
600
elt-2
20%
0
0 50 100 150 200 0 50 100 150 200
Number of nuclei Number of nuclei

29. med-1,2 and end-3 expression is low
Wild-type (N2) Mutant (zu135)
600
med-1,2
skn-1 (mutant)
0
med-1,2
end-3 end-1 600
end-3
OR
elt-2 0
(bimodal)
600
end-1
Intestine-specific
genes 0
600
elt-2
20%
0
0 50 100 150 200 0 50 100 150 200
Number of nuclei Number of nuclei

30. end-1 expression is highly variable
Wild-type (N2) Mutant (zu135)
600
med-1,2
skn-1 (mutant)
0
med-1,2
end-3 end-1 600
(variable)
end-3
OR
elt-2 0
(bimodal)
600
end-1
Intestine-specific
genes 0
600
elt-2
20%
0
0 50 100 150 200 0 50 100 150 200
Number of nuclei Number of nuclei

31. Could end-1 be a “controller” for elt-2 expression?
Wild-type (N2)
elt-2 OFF elt-2 ON
Mutant phenotype Normal phenotype
Threshold
skn-1 mutants
elt-2 OFF elt-2 ON
Mutant phenotype Normal phenotype
end-1 expression level

32. Are end-1 and elt-2 correlated?
Wild-type (N2) Mutant (zu135)
600
med-1,2
skn-1 (mutant)
0
med-1,2
end-3 end-1 600
(variable)
end-3
OR
elt-2 0
(bimodal)
600
end-1
Intestine-specific
genes 0
600
elt-2
20%
0
0 50 100 150 200 0 50 100 150 200
Number of nuclei Number of nuclei

33. A lower threshold for elt-2 expression results in
lower penetrance of the mutant phenotype
zu129 zu135 zu67
Lower penetrance Higher penetrance Higher penetrance
elt-2 transcript number
400 400 400
200 200 200
0 0 0
0 200 400 0 200 400 0 200 400
end-1 transcript number
WT (N2)
skn-1 mutant

34. A lower threshold for elt-2 expression results in
lower penetrance of the mutant phenotype
zu129 zu135 zu67
Lower penetrance Higher penetrance Higher penetrance
elt-2 transcript number
400 400 400
Threshold
Threshold
Threshold
200 200 200
0 0 0
0 200 400 0 200 400 0 200 400
end-1 transcript number
WT (N2)
skn-1 mutant

35. Knocking out end-1 has virtually no effect
Wild-type (N2) end-1 mutant
600
med-1,2
skn-1
0
med-1,2
end-3 end-1 600
end-3
OR
elt-2 0
600
end-1
Intestine-specific
genes 0
600
elt-2
0
0 50 100 150 200 0 50 100 150 200
Number of nuclei Number of nuclei

36. Knocking out end-3 affects both end-1 and elt-2
Wild-type (N2) end-3 mutant
600
med-1,2
skn-1
0
med-1,2
end-3 end-1 600
end-3
OR
elt-2 0
600
end-1
Intestine-specific
genes 0
600
elt-2
0
0 50 100 150 200 0 50 100 150 200
Number of nuclei Number of nuclei

37. Transcriptional bursts lead to high variability
Vargas et al., PNAS, 2005
Raj et al., PLoS Biology, 2006

38. Bursts may be caused by chromatin remodeling
BB38-13 ARI 28 January 2009 0:45
OFF
γ λ
δ
ON
High burst frequency Low burst frequency
λ>δ λ<δ
Fast transcription Slow transcription Fast transcription Slow transcription
large small Raj etlarge PLoS
al., Biology, small
2006
γ>δ Burst Burst
approximation approximation
Fast valid valid
inactivation
f cells

39. Knocking down hda-1 rescues the mutant
phenotype by reducing variability in end-1
skn-1 mutant skn-1 hda-1 mutant
600
med-1,2
skn-1 (mutant)
hda-1
(RNAi) 0
med-1,2
end-3 end-1 600
(less variable)
end-3
OR
elt-2 0
(rescued)
600
end-1
Intestine-specific
genes 0
600
elt-2
0
0 50 100 150 200 0 50 100 150 200
Number of nuclei Number of nuclei

40. Knocking down hda-1 rescues the mutant
phenotype by reducing variability in end-1
600 Wild-type (N2) skn-1 mutant skn-1 hda-1 mutant
end-1
0
0 50 100 150 200 0 50 100 150 200 0 50 100 150 200
Number of nuclei Number of nuclei Number of nuclei
18 17 8
0 250 500 0 250 500 0 250 500
Number of end-1 transcripts Number of end-1 transcripts Number of end-1 transcripts
Coefficient of variation: 0.20 Coefficient of variation: 0.69 Coefficient of variation: 0.44

41. Variability in gene expression may underlie
incomplete penetrance of human disease alleles

42. Variability in gene expression may underlie
incomplete penetrance of human disease alleles
Genetic variation
Random variation
Environmental variation

43. Thanks
Scott Rifkin Alexander van Oudenaarden Sanjay Tyagi
UCSD MIT PHRI
Erik Anderson www.singlemoleculefish.com
Bob Horvitz
MIT
Funding
National Science Foundation
Burroughs-Wellcome Fund