Variability in gene expression underlies incomplete
penetrance in Caenorhabditis elegans
Arjun Raj1*, Scott Rifkin2*, Erik...
Why are individuals different?




                            Genetic variation
Why are individuals different?




                            Genetic variation




     Environmental variation
What about random variation?
Random fluctuations in gene expression lead to
cell-to-cell variations in mRNA and protein number




Elowitz et al, 2002 a...
Random fluctuations in gene expression lead to
cell-to-cell variations in mRNA and protein number




Elowitz et al, 2002  ...
Random fluctuations in gene expression lead to
cell-to-cell variations in mRNA and protein number




Maamar and Raj et al,...
Noisy gene expression can be useful for microbes




                          Competent
                            cells...
What about multicellular organisms?
Gene expression in mammalian cells occurs in
bursts
                                  Raj et al., PLoS Biology, 2006
     ...
What about multicellular organisms?

                  Gene expression can
                    be very noisy...




      ...
What about multicellular organisms?

       Gene expression can     So you might expect a
         be very noisy...     lo...
What about multicellular organisms?

       Gene expression can      But the results often
         be very noisy...      ...
Wild-type C. elegans embryos develop robustly
Wild-type C. elegans embryos develop robustly
Incompletely penetrant mutations reveal alternate
cell fates


                                    Birefringent gut granul...
Does variability in gene expression underlie the
incomplete penetrance of mutant phenotypes?
The C. elegans intestine is composed of
            E cell descendants

                                         P0


    ...
Intestinal fate is specified by a gene cascade

                          Cell lineage                                     ...
Intestinal fate is specified by a gene cascade

                          Cell lineage                                     ...
Wild-type embryos all express elt-2

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

                                        elt-2
                    ...
Wild-type (N2)




DAPI (nuclei)                 end-3




                med-1,2




                          elt-2
 en...
Wild type expression is very regular


                                Wild-type (N2)
                      600


        ...
Mutant expression patterns are highly variable


                     Wild-type (N2)                                Mutant...
Mutant contain premature stop codons in skn-1


                                                 Wild-type (N2)           ...
elt-2 expresses in a bimodal fashion


                                                 Wild-type (N2)                    ...
med-1,2 and end-3 expression is low


                                                 Wild-type (N2)                     ...
med-1,2 and end-3 expression is low


                                                 Wild-type (N2)                     ...
end-1 expression is highly variable


                                                 Wild-type (N2)                     ...
Could end-1 be a “controller” for elt-2 expression?

                                 Wild-type (N2)
                elt-2...
Are end-1 and elt-2 correlated?


                                                 Wild-type (N2)                        M...
A lower threshold for elt-2 expression results in
                          lower penetrance of the mutant phenotype

    ...
A lower threshold for elt-2 expression results in
                          lower penetrance of the mutant phenotype

    ...
Knocking out end-1 has virtually no effect


                                             Wild-type (N2)                  ...
Knocking out end-3 affects both end-1 and elt-2


                                             Wild-type (N2)             ...
Transcriptional bursts lead to high variability




                                     Vargas et al., PNAS, 2005
       ...
Bursts may be caused by chromatin remodeling
BB38-13   ARI        28 January 2009     0:45




                   OFF

   ...
Knocking down hda-1 rescues the mutant
 phenotype by reducing variability in end-1


                                     ...
Knocking down hda-1 rescues the mutant
            phenotype by reducing variability in end-1

600                  Wild-t...
Variability in gene expression may underlie
incomplete penetrance of human disease alleles
Variability in gene expression may underlie
incomplete penetrance of human disease alleles

       Genetic variation

    ...
Thanks
  Scott Rifkin   Alexander van Oudenaarden            Sanjay Tyagi
    UCSD                     MIT                ...
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Variability in gene expression underlies incomplete penetrance

  1. 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. 2. Why are individuals different? Genetic variation
  3. 3. Why are individuals different? Genetic variation Environmental variation
  4. 4. What about random variation?
  5. 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. 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. 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. 8. Noisy gene expression can be useful for microbes Competent cells Maamar and Raj et al., Science, 2007
  9. 9. What about multicellular organisms?
  10. 10. Gene expression in mammalian cells occurs in bursts Raj et al., PLoS Biology, 2006 Chubb et al., Curr Biol, 2006
  11. 11. What about multicellular organisms? Gene expression can be very noisy... Raj et al, 2006
  12. 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. 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. 14. Wild-type C. elegans embryos develop robustly
  15. 15. Wild-type C. elegans embryos develop robustly
  16. 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. 17. Does variability in gene expression underlie the incomplete penetrance of mutant phenotypes?
  18. 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. 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. 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. 21. Wild-type embryos all express elt-2 elt-2 Nuclei
  22. 22. Mutations to skn-1 result in variable elt-2 expression elt-2 Nuclei
  23. 23. Wild-type (N2) DAPI (nuclei) end-3 med-1,2 elt-2 end-1
  24. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 37. Transcriptional bursts lead to high variability Vargas et al., PNAS, 2005 Raj et al., PLoS Biology, 2006
  38. 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. 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. 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. 41. Variability in gene expression may underlie incomplete penetrance of human disease alleles
  42. 42. Variability in gene expression may underlie incomplete penetrance of human disease alleles Genetic variation Random variation Environmental variation
  43. 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

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