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Combinatorial Control of Toxin Response in C. elegans Tim Lindblom environment genome environment genome xenobiotics genes Nuclear Receptors Mediate Toxicogenomic Responses detoxification network Nuclear Receptors cytoplasm nucleus LBD LBD DBD ECM UGT SDR Phase 2 Phase 1 GST cytoplasm nucleus CYP LBD DBD dozens Vertebrate Xenosensation PXR (Pregnane X CAR (Constitutive Receptor) Androstane Receptor) dozens Vertebrate Xenosensation hyperforin PXR CYP2A9 CYP3A4 UGT1A1 MRP2 phenobarbitol CAR CYP2C9 CYP2B6 UGT1A1 MRP2 MRP4 Nuclear Receptor Crosstalk PXR or CAR CYP2B6 UGT1A1 MRP2 ECM A Model of Detoxification in C. elegans UGT SDR Phase 2 Phase 1 GST cytoplasm nucleus CYP LBD DBD Can C. elegans detoxification inform human studies? 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 NHR-8 is a Member of the PXR/VDR Subfamily of Nuclear Receptors ATG GFPcoding 3' 5' (6.8kb) E L1 L2 L3 L4 Ad ~ 1.6 kb A deletion in nhr-8 removes the Ligand Binding Domain Sequences nhr-8 nhr-8(ok186)* * Produced by The C. elegans Gene Knockout Consortium % Survival NHR-8 Contributes to Xenobiotic Resistance % Survival mM Chloroquine mM Colchicine N2 nhr-8(ok186) pgp-3(pk18) nhr-8(ok186); pgp-3(pk18) NHR-8 ? PXR & CAR detox enzymes & transporters C. elegans’ Detoxification Toolbox Short Chain Dehydrogenases 68 genes 63 mRNAs 1 phenotype SDR CYP Cytochromes P450 86 genes 84 mRNAs 9 phenotypes David Nelson, Cytochrome P450 Homepage drnelson.utmem.edu/CytochromeP450.html Glutathione S transferases 48 genes 46 mRNAs 1 phenotype GST UGT UDP glycosyltransferases 72 genes 71 mRNAs 2 phenotypes Lindblom, T. & Dodd, A. (2006) Xenobiotic Detoxification in the Nematode Caenorhabditis elegans. Journal of Experimental Zoology 305A, pg. 720-730. 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 * 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. 1756-60. ECM A Model of Detoxification in C. elegans 72 UGT 53 Phase 2 48 GST 68 SDR Phase 1 cytoplasm nucleus 86 CYP 72 LBD DBD Affymetrix C. elegans Genechips® oligonucleotide probe 22,500 transcripts wild type wild type xenobiotics nhr-8(ok186) nhr-8(ok186) xenobiotics wild type wild type xenobiotics What genes are upregulated in response to xenobiotics? nhr-8(ok186) nhr-8(ok186) xenobiotics wild type wild type xenobiotics What genes are upregulated in response to xenobiotics? nhr-8(ok186) nhr-8(ok186) xenobiotics Do those genes require NHR-8 for upregulation? 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) 42 genes; 5 best hits nhr-8(ok186) primaquine wild type 299 genes 92 best hits nhr-8(ok186) 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 452 genes 131 best hits nhr-8(ok186) primaquine ECM Does NHR-8 Regulate Basal Gut Enzyme Expression? UGT SDR Phase 2 Phase 1 GST cytoplasm nucleus CYP LBD DBD 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 RNAi RNAi RNAi Wild Type nhr-8(ok186) 934 transcription factors in C. elegans ~300 expressed in the gut Summer 2009 – INBRE Summer Fellowship RNAi RNAi RNAi Wild Type nhr-8(ok186) 14 Nuclear Receptors & Primaquine Beau Palmer Brittany Young Katee Castleman Doug Rinehart Allyn Dodd Chris Estes Ryan Thornton Seth Purcell Brianne Orr Ann Sluder Research Triangle Park, NC Affymetrix Microarray Core Beaverton, OR Michael Lehmann Fayetteville, AR Anna Radominska Little Rock, AR 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 ES013128-02
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