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