1. Combinatorial Control of Toxin Response in C. elegans
Tim Lindblom

2. environment
genome

3. environment
genome
xenobiotics
genes

4. Nuclear Receptors Mediate Toxicogenomic Responses
detoxification
network

5. Nuclear Receptors
cytoplasm
LBD
nucleus
DBD
LBD
DBD

6. ECM
ABC
UGT
SDR
Phase 2
Phase 1
cytoplasm
CYP
GST
nucleus
LBD
DBD

7. Vertebrate Xenosensation
dozens
PXR (Pregnane X Receptor)
CAR (Constitutive Androstane Receptor)
dozens

8. Vertebrate Xenosensation
hyperforin
PXR
CYP2A9 CYP3A4
UGT1A1
MRP2
phenobarbitol
CAR
CYP2C9 CYP2B6
UGT1A1
MRP2 MRP4

9. Nuclear Receptor Crosstalk
PXR or CAR
CYP2B6
UGT1A1
MRP2

10. A Model of Detoxification in C. elegans
ECM
A Model of Detoxification in C. elegans
ABC
UGT
SDR
Phase 2
Phase 1
cytoplasm
CYP
GST
nucleus
LBD
Can C. elegans detoxification inform human studies?
DBD

11. Maximum Parsimony Tree of DBD sequences from Conserved NRs
15 C. elegans NRs
5 of 6 NR subfamilies
no steroid receptors
no clear RXR homolog

12. NHR-8 is a Member of the PXR/VDR Subfamily of Nuclear Receptors

13. GFP
coding
5' (6.8 kb ) 3'
ATG
E  L1 L2 L3 L4 Ad
~ 1.6 kb

14. A deletion in nhr-8 removes the Ligand Binding Domain Sequences
nhr-8(ok186)*
* Produced by The C. elegans Gene Knockout Consortium

15. NHR-8 Contributes to Xenobiotic Resistance
mM Chloroquine
% Survival N2
nhr-8(ok186)
pgp-3(pk18)
nhr-8(ok186); pgp-3(pk18)
mM Colchicine
% Survival

16. NHR-8 ?
PXR & CAR
detox enzymes
& transporters

17. C. elegans’ Detoxification Toolbox
Glutathione S transferases
48 genes
46 mRNAs
1 phenotype
Short Chain Dehydrogenases
68 genes
63 mRNAs
1 phenotype
SDR
GST
CYP
UGT
simplest bioinformatics question - what’s available for detox?
many are likely involved in non-detox
DAF-9 is a CYP that is genetically implicated in controlling the production of a likely dauer hormone
UDP glycosyltransferases
72 genes
71 mRNAs
2 phenotypes
Cytochromes P450
86 genes
84 mRNAs
9 phenotypes
David Nelson, Cytochrome P450 Homepage
drnelson.utmem.edu/CytochromeP450.html
Lindblom, T. & Dodd, A. (2006) Xenobiotic Detoxification in the Nematode Caenorhabditis elegans. Journal of Experimental Zoology 305A, pg

18. Why so many tools? Are they really necessary?
Three Nematode Habitats

19. Brugia malayi is a Causative Agent of Lymphatic Filariasis

20. B. malayi Genome Project

21. Compared to C. elegans, B. malayi has a limited detoxification capacity
* limited EST project and no genetics
C. elegans
Ghedin E, et al (2007) Draft genome of the filarial nematode parasite Brugia malayi. Science 317(5845), pg

22. A Model of Detoxification in C. elegans
ECM
A Model of Detoxification in C. elegans
ABC
UGT
SDR 72 68 53
Phase 2
Phase 1
cytoplasm
CYP
GST 86
nucleus 48
LBD 72
talk briefly about AHR-1 here
NRs are superabundant in the worm genome
also clustered like the DMEs
DBD

23. Affymetrix C. elegans Genechips®
oligonucleotide probe
22,500 transcripts

24. wild type
xenobiotics
nhr-8(ok186)
xenobiotics
wild type
nhr-8(ok186)

25. wild type
xenobiotics
nhr-8(ok186)
xenobiotics
wild type
nhr-8(ok186)
What genes are upregulated
in response to xenobiotics?

26. wild type
xenobiotics
nhr-8(ok186)
xenobiotics
wild type
nhr-8(ok186)
What genes are upregulated
in response to xenobiotics?
Do those genes require NHR-8 for upregulation?

27. wild type
wild type
primaquine
131 genes; 41 best hits
Best hits:
known intestinal expression
various metabolic enzymes
expressed in response to bacterial infection
fat content increased or decreased
no DME’s
nhr-8(ok186)
primaquine
nhr-8(ok186)
42 genes; 5 best hits

28. wild type
wild type
primaquine
Best hits:
intestinal expression
metabolic enzymes
bacterial infection
fat content changes
DMEs: 3 UGTs 2 CYPs 1 SDR 1 GST 1 ABC transporter
299 genes
92 best hits
452 genes
131 best hits
nhr-8(ok186)
primaquine
nhr-8(ok186)

29. Does NHR-8 Regulate Basal Gut Enzyme Expression?
ECM
Does NHR-8 Regulate Basal Gut Enzyme Expression?
ABC
UGT
SDR
Phase 2
Phase 1
cytoplasm
CYP
GST
nucleus
LBD
metabolism?
fat storage?
innate immunity?
DBD

30. Nuclear Receptor Crosstalk
NHR-8
Other Nuclear Receptors? (like the 280+ NRs)

31. RNAi Screening Strategy

32. RNAi Screening Strategy
Wild Type
nhr-8(ok186)

33. RNAi Screening Strategy
Wild Type
nhr-8(ok186)

34. RNAi Screening Strategy
Wild Type
nhr-8(ok186)
934 transcription factors in C. elegans
~300 expressed in the gut

35. Summer 2009 – INBRE Summer Fellowship
RNAi
RNAi
RNAi
Wild Type
nhr-8(ok186)
14 Nuclear Receptors & Primaquine

36. Beau Palmer
Brittany Young
Katee Castleman
Doug Rinehart
Allyn Dodd
Chris Estes
Ryan Thornton
Seth Purcell
Brianne Orr

37. Ann Sluder
Research Triangle Park, NC
Anna Radominska
Little Rock, AR
Michael Lehmann
Fayetteville, AR
Affymetrix Microarray Core
Beaverton, OR
Elodie Ghedin
The Institute for Genomic Research
U. of Pittsburg, PA
NIH; National Center for Research Resources: P20 RR-16460
NIH; National Institute of Environmental Heath Science: 2 R15 ES