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

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