• Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
    Be the first to comment
    Be the first to like this
No Downloads

Views

Total Views
5,483
On Slideshare
0
From Embeds
0
Number of Embeds
4

Actions

Shares
Downloads
0
Comments
0
Likes
0

Embeds 0

No embeds

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
    No notes for slide

Transcript

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variations in mRNA and protein number Elowitz et al, 2002 al, 2007 Maamar and Raj et Raser and O’Shea, 2004 A.R., unpublished observations Raj et al, 2006 Raj and van Oudenaarden, Cell, 2008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in mRNA and protein number Elowitz et al, 2002 Raser and O’Shea, 2004 Raj et al, 2006 Maamar and Raj et al, 2007 A.R., unpublished observations Raj et al, 2006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in gene expression lead to cell-to-cell variations in mRNA and protein number Maamar and Raj et al, 2007 A.R., unpublished observations Raj et al, 2006 Raj et al.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