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 LBD nucleus DBD LBD DBD

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

Vertebrate Xenosensation dozens PXR (Pregnane X Receptor) CAR (Constitutive 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

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

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

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

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

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

NHR-8 ? PXR & CAR detox enzymes & transporters

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. 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 * 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.

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

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

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

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

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?

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

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)

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

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) 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 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 ES013128-02