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GENE EXPRESSION PATTERNS AS A TOOL FOR BIO-ANALYSIS

B. Hock, M. Alberti, U. Kausch, J. Budczies, N. Theilacker and R. Leibiger

Technische Universität München

• Scope of bio-analysis

• Environmental analysis at the transcriptome level

• qPCR and microarrays as tools

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• Analysis of biomolecules (e.g., proteins und nucleic acids),c.f. Lottspeich, F., Zorbas, H. (1998): Bioanalytik. Spektrum Akademischer Verlag, Heidelberg, Berlin.

• Analysis with biomolecules Major applications of bioanalytical methods in medicine, pharmacology, food analysis and environmental monitoring.Use of biomolecules, whole cells and organisms.

Definition of bio-analysis

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Scope of bio-analysis• Biomolecular recognition

Basic principle: Specific binding of target moleculesof biological or non-biological origin by biomolecules.

• Examples for relevant biomoleculesEnzymes, antibodies, receptors, nucleic acids, and complex structures (ribosomes, organelles, cells).

• Biological responses are registered- at the level of the binding event (e.g., immunoassays, receptor assays)- at the level of signal transduction (e.g., reporter gene systems, gene expression analysis)- at the level of metabolism, development and reproduction (e.g., vitellogenin as an endpoint for endocrine disruption)

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• Chemicals that interrupt the endocrine system

• comprise exogenous natural or anthropogenic

agents

• produce adverse effects not only at the level of the

individual, but also of the population and the

community

Endocrine disruptors

Wildlife species suffering endocrine disruption*

• ‘Feminisation’ of some species of fish-eating birds

(e.g. bald eagles, herons)

• ‘Feminisation’ of alligators in Florida

• ‘Feminisation’ of some species of top predator fish

(e.g. swordfish)

• Intersexuality in polar bears

• ‘Feminisation’ of the Florida panther

• ‘Feminisation’ of fish in Europe

• ‘Masculinisation’ of fish in U.S.A.

* list provided by John Sumpter

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Biomolecular recognition of estrogensfollowed by signal transduction

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Vitellogenin as a biomarker

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Test organism zebrafish

Environmental genomics

Exposure units for zebrafish

(1 - 500 ng/L 17ß-estradiol)

♀

♂

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Exposure units for zebrafish

1

23

4

55

6677

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1: Fresh water; 2: Overflow; 3: Heating tank (400L); 4: Pump; 5: Chemical pump6: Exposure tanks (20L); 7: Overflow; 8: Activated charcoal filter

Flow-through: 21 mL/min

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ö

ß -

-

productsubstrate

17ß-estradiol-BSA

estrogen receptor (ER)

anti-ER-antibody (biotinylated)

streptavidin

POD-biotin

ELRA:Enzyme-Linked Receptor Assay in microwell format for high-troughput screening of environmental samples

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Monitoring of 17ß-estradiol in tanks by ELRA

100 200 300 400 500

100

200

300

400

500

Mea

sure

d co

ncen

trat

ion

[ng/

L]

Calculated dose [ng/L]

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Quantification of gene expression(vtg 1, ef1a) by qPCR

• Exposure of zebrafish for 11 days(0; 1; 10; 100; 200; 300; 400; 500 ng/L 17ß-estradiol), two replicates

• Preparation of mRNA from liver and gonad tissue

• cDNA synthesis

• Quantitative PCR using specific primers

• Examination of threshold level

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LightCycler®

15Vitellogenin Elongation factor 1α Negative

controls

LightCycler®

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0 1 10 100 200 300 400 5000,00

0,05

0,10

0,15

0,20

0,25

0,30

0,35

Rel

ativ

e E

xpre

ssio

n R

atio

17ß-estradiol [ng/L]

Gene expression of vitellogenin 1

in male liver tissue

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DNA microarrays

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cy3

cy5

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Zebrafish 14k Microarray

X axis = intensity of control,Y axis = intensity of exposed

2 x 7k-Arrays = 14,000gene spots

Vitellogenin

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Bioinformatic tools are required to detect regulated genes in an organism.

Distinction between significant effects due to treatment and natural variability within a population is crucial

Normalization on-chip normalization (non-linear)inter-chip normalization (linear)

Detection of differentially expressed genesp-value thresholdingsignificance assessment by sample permutations

Data evaluation

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Data normalization

Raw data (mean signal minus mean background)

Normalized data (on- and inter-chip normalization)

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MA Plot

M: fold change (between exposed and control fish)

A: average expression value(in exposed and control fish)

M = log2 (xexposed / xcontrol)A = ½ log2 (xexposed * xcontrol)

Mean expression values over 10 arrays

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Exposure to 17β-estradiol (male liver tissue from 10 exposed and 10 control fish)

• Exposure for 11 days with 500 ng/L 17β-estradiol

• Selection of (potentially) differential genes by thresholding

p-values from 1-sample t-test

• 186 probes identified (116 up-regulated, 70 down-regulated)

• Signifance assessment by sample permutations

• Number of selected genes is significantly higher than

expected from the null distribution (p=0.008)

• Estimated sensitivity 89%

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Up-regulated genes Fold

change Name Function

128.9 vitellogenin 1; vg1 egg yolk protein, lipid transporter activity

31.0 homeo box a3a; hoxa3a homeobox gene, embryonic development

15.9 vitellogenin 3; vg3 egg yolk protein, lipid transporter activity

9.8 vitellogenin 3; vg3 egg yolk protein, lipid transporter activity

6.8 nothepsin; nots liver-specific aspartic protease, similar to mammalian cathepsin E and D, function: post-translational processing of vitellogenin in liver prior to secrection into the blood stream

6.7 estrogen receptor; er nuclear receptor, ligand-inducible transcription factor

6.1 rev-erb beta 2 nuclear receptor, "orphan receptor", DNA-dependent regulation of transcription

5.9 activin A receptor, type IB; tarama

membrane-bound, kinase activity, receptor activity

5.7 decapentaplegic and vg-related 1; dvr1

regulation of cell cycle and cell proliferation, growth factor activity

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Up-regulated genes (continued) Fold

change Name Function

3.4 mesoderm posterior b; mespb

anterior/posterior pattern formation, somitogenesis, transcription factor activity

1.9 Kallmann syndrome 1b sequence; kal1b

prior pubertal development; congenital, isolated, idiopathic hypogonadotropic hypogonadism (IHH) and anosmia

1.5 one-eyed pinhead; oep somitogenesis, determination of left/right symmetry,germ cell migration, induction of positive chemotaxis, mesoderm development, notochord development

1.4 translocon-associated protein beta signal sequence receptor beta

embryonic development

1.4 steroidogenic acute regulatory protein; star

steroid biosynthesis, cholesterol and lipid binding, cholesterol transporter activity

1.4 \bg738177 gi:14087866 fp05c04.y1 zebrafish gridded kidney danio rerio cdna clone

embryonic axis specification

1.3 T-cell acute lymphocytic leukemia 1, tal-1

transcription factor activity

1.3 LIM domain only 2; lmo2 erythrocyte differentiation

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Fold change Name Function

-2.7 pleiotrophin 1; plei1effects on neural crest and floorplate development, growth factor activity

-2.4 carboxypeptidase A proteolysis and peptidolysis

-2.1 GATA-binding protein 2; zg2 DNA-dependent, regulation of transcription

-1.9 ephrin B2a; efnb2a neurogenesis, synaptic target recognition

-1.8 caspase 8; casp8 regulation of apoptosis, proteolysis and peptidolysis

-1.8 POU domain gene 50; pou50 DNA-dependent, development: transverse and longitudinal subdivisions of the embryoniczebrafish forebrain, regulation of transcription

-1.7 leukocyte cell derived chemotaxin 1; peroxisome proliferator activated

embryonic development: cartilage morphogenesis

-1.5 receptor alpha; ppara DNA-dependent, regulation of transcription

-1.4 transcription factor 7-like 1a; tcf3 embryonic development, determination of anterior/posterior axis, regulation of transcription

-1.4 HIV-1 Tat interactive protein 2; tip30 immune-type receptor gene

-1.4 runt-related transcription factor 1; runxa

hematopoietic development, DNA-dependent, ATP- and DNA-binding, regulation of transcription

-1.3 transcription factor AP2 alpha 2neural plate border and neural crest cells during somitogenesis

-1.3 claudin c; cldnc structual molecule activity

Down-regulated genes

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Conclusions and outlook

• A set of biomarkers is available to examine estrogen exposure

• Potential use of biomarker arrays to discriminate fordifferent classes of endocrine disruptors

• Strong influence of estrogen on developmental events in male fish

• Elucidation of signal transduction pathways and networksfeasible on the basis of up- and down-regulated genes

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Acknowledgements• Dr. Martin Seifert• Steffi Haindl• Martin Alberti • Ulf Kausch• Dr. Jan Budczies• Robert Leibiger• Nora Theilacker

• European Union for supporting the EDEN project

• METROPOLIS hock@wzw.tum.de