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Combinatorial Control of Toxin Response in C. elegans
Tim Lindblom
environment genome
environment genome
xenobiotics genes
Nuclear Receptors MediateToxicogenomic Responses
detoxificationnetwork
Nuclear Receptors
LBD
DB
D
nucleus
cytoplasm
LBD DBD
LBD
DB
D
nucleus
cytoplasm
SDR
CYP
UGT
GST
ABC
Phase 2 Phase 1
ECM
Vertebrate Xenosensation
dozens
PXR (Pregnane X Receptor)CAR (Constitutive Androstane
Receptor)
dozens
Vertebrate Xenosensation
CYP2A9 CYP3A4UGT1A1MRP2
PXR
hyperforin
CYP2C9 CYP2B6UGT1A1
MRP2 MRP4
CAR
phenobarbitol
Nuclear Receptor Crosstalk
CYP2B6UGT1A1MRP2
PXR or CAR
LBD
DB
D
nucleus
cytoplasm
SDR
CYP
UGT
GST
ABC
Phase 2 Phase 1
ECM
A Model of Detoxification in C. elegans
Can C. elegans detoxification inform human studies?
Maximum ParsimonyTree of DBD sequences
from Conserved NRs
• 15 C. elegans NRs
• 5 of 6 NR subfamilies
• no steroid receptors
• no clear RXR homolog
NHR-8 is a Member of the PXR/VDR Subfamily of Nuclear Receptors
GFP coding5' (6.8 kb) 3'
ATG
E L1 L2 L3 L4 Ad
~ 1.6 kb
nhr-8(ok186)*
nhr-8
A deletion in nhr-8 removes theLigand Binding Domain Sequences
* Produced by The C. elegans Gene Knockout Consortium
mM Colchicine
% S
urvi
val
mM Chloroquine
% S
urvi
val
N2nhr-8(ok186)pgp-3(pk18)nhr-8(ok186); pgp-3(pk18)
NHR-8 Contributes to Xenobiotic Resistance
PXR & CAR
NHR-8
detox enzymes & transporters
?
C. elegans’ Detoxification Toolbox
Short Chain Dehydrogenases 68 genes 63 mRNAs 1 phenotype
Cytochromes P450 86 genes 84 mRNAs 9 phenotypes
UDP glycosyltransferases 72 genes 71 mRNAs 2 phenotypes
Glutathione S transferases 48 genes 46 mRNAs 1 phenotype
David Nelson, Cytochrome P450 Homepagedrnelson.utmem.edu/CytochromeP450.html Lindblom, T. & Dodd, A. (2006) Xenobiotic Detoxification in the
Nematode Caenorhabditis elegans. Journal of Experimental Zoology 305A, pg. 720-730.
SDR
CYP UGT
GST
Why so many tools?Are they really necessary?
Three Nematode Habitats
Brugia malayi is a Causative Agent of Lymphatic Filariasis
B. malayi Genome Project
Compared to C. elegans, B. malayi has a limited detoxification capacity
B. malayi
C. elegans
* limited EST project and no genetics
Ghedin E, et al (2007) Draft genome of the filarial nematode parasite Brugia malayi. Science 317(5845), pg. 1756-60.
68
48 86
72
53
LBD
DB
D
nucleus
cytoplasm
SDR
CYP
UGT
GST
ABC
Phase 2 Phase 1
ECM
A Model of Detoxification in C. elegans
72
Affymetrix C. elegans Genechips®
oligonucleotide probe22,500 transcripts
wild typewild type
xenobioticsnhr-8(ok186)
nhr-8(ok186)xenobiotics
wild typewild type
xenobioticsnhr-8(ok186)
nhr-8(ok186)xenobiotics
What genes are upregulatedin response to xenobiotics?
wild typewild type
xenobioticsnhr-8(ok186)
nhr-8(ok186)xenobiotics
What genes are upregulatedin response to xenobiotics?
Do those genes require NHR-8 for upregulation?
wild type
nhr-8(ok186)
wild typeprimaquine
nhr-8(ok186)primaquine
131 genes; 41 best hits
42 genes; 5 best hits
Best hits: • known intestinal expression• various metabolic enzymes• expressed in response to bacterial infection• fat content increased or decreased• no DME’s
wild type
nhr-8(ok186)
wild typeprimaquine
nhr-8(ok186)primaquine
452 genes131 best hits
299 genes92 best hits
Best hits: • intestinal expression• metabolic enzymes• bacterial infection• fat content changes• DMEs:
3 UGTs2 CYPs1 SDR1 GST1 ABC transporter
LBD
DB
D
nucleus
cytoplasm
SDR
CYP
UGT
GST
ABC
Phase 2 Phase 1
ECM
Does NHR-8 Regulate Basal Gut Enzyme Expression?
metabolism?fat storage?innate immunity?
Nuclear Receptor Crosstalk
NHR-8
Other Nuclear Receptors? (like the 280+ NRs)
RNAi Screening Strategy
RNAi Screening Strategy
Wild Type
nhr-8(ok186)
RNAi Screening Strategy
Wild Type
nhr-8(ok186)
RNAi Screening Strategy
Wild Type
nhr-8(ok186)
RNAi RNAi RNAi
934 transcription factors in C. elegans~300 expressed in the gut
Summer 2009 – INBRE Summer Fellowship
Wild Type
nhr-8(ok186)
RNAi RNAi RNAi
14 Nuclear Receptors & Primaquine
Beau PalmerBrittany YoungKatee CastlemanDoug RinehartAllyn DoddChris EstesRyan ThorntonSeth PurcellBrianne Orr
Anna RadominskaLittle Rock, AR
NIH; National Center for Research Resources: P20 RR-16460 NIH; National Institute of Environmental Heath Science: 2 R15 ES013128-02
Ann SluderResearch Triangle Park, NC
Affymetrix Microarray CoreBeaverton, OR
Elodie GhedinThe Institute for Genomic ResearchU. of Pittsburg, PA
Michael LehmannFayetteville, AR