Pascal Schulthess, Albert Braeuning, Alexandra Löffler, Michael Schwarz, and Nils Blüthgen

Cooperativity between Aryl Hydrocarbon Receptor and ß-catenin Binding Sites in Hepatocytes

1

Cytochrome P450 (CYP)

‣ oxidate hydrophobic substances hydrophilic substances

➡ easier excretion

‣ essential for metabolizing many drugs & toxins

‣ strong expression in the liver & colon

2

�

CYP1A1 expression gradient in liver lobules

normal liver ß-catenin k.o.

CYP1

A1

P

C

P

C

3Braeuning, A. & Schwarz, M. Biol. Chem. 391, 139–148 (2010)

CYP1A1 expression gradient in liver lobules

normal liver ß-catenin k.o.

CYP1

A1

P

C

P

C

3Braeuning, A. & Schwarz, M. Biol. Chem. 391, 139–148 (2010)

CYP1A1 expression induced by Wnt & Dioxin

4Braeuning, A., Köhle, C., Buchmann, A. & Schwarz, M. Toxicol. Sci. 122, 16–25 (2011).

*rela

tive m

RN

A e

xpre

ssio

n

0

3

6

9

100

150

200

250

300

Dioxin –Wnt3a –

–+

CYP1A1n=5primary mouse

hepatocytes

CYP1A1 expression induced by Wnt & Dioxin

4Braeuning, A., Köhle, C., Buchmann, A. & Schwarz, M. Toxicol. Sci. 122, 16–25 (2011).

*

++

+–

*rela

tive m

RN

A e

xpre

ssio

n

0

3

6

9

100

150

200

250

300

Dioxin –Wnt3a –

–+

CYP1A1n=5primary mouse

hepatocytes

AhR and ß-catenin converge on CYP1A1 promotor

5

Wnt

Dioxin

AhR and ß-catenin converge on CYP1A1 promotor

5

Wnt

Dioxin

Lrp5/6Fzd

b-cat

AhR

Arnt

AhR and ß-catenin converge on CYP1A1 promotor

5

Wnt

Dioxin

Lrp5/6Fzd

b-cat

AhR

Arnt

TCFCYP1A1

DREDREDRE DRE

AhR and ß-catenin converge on CYP1A1 promotor

5

Wnt

Dioxin

Lrp5/6Fzd

b-cat

AhR

Arnt

TCFCYP1A1

DREDREDRE DRE

How do the binding sites cooperate?

ABCD

15 human CYP1A1 promotor constructs

6

ABCD

15 human CYP1A1 promotor constructs

6

ABCD

15 human CYP1A1 promotor constructs

6

Luciferase

Dioxin

Data show more-than-additive effects

7

ABACBCADBDCD

ABCABDACDBCD

ABCD

rela

tive lu

cife

rase

activ

ity [M

]

1

2

3

4

5

6

7

8

Dioxin [nM]100 101 102

1 Activator 2 Activators 3 Activators

WT

rela

tive lu

cife

rase

activity [M

]1

2

3

4

5

6

7

8

Dioxin [nM]100 101 102

Dioxin [nM]100 101 102

rela

tive lu

cife

rase

activ

ity [M

]

1

2

3

4

5

6

7

8

ABCD

Parameters of thermodynamic model

8Bintu, L. et al. Curr. Opin. Genet. Dev. 15, 116–124 (2005).

Fold change model expressed with mass action kinetics

ABKPKAKB

EAB EA

EB

Parameters of thermodynamic model

8Bintu, L. et al. Curr. Opin. Genet. Dev. 15, 116–124 (2005).

Fold change model expressed with mass action kinetics

ABKPKAKB

EAB EA

EB

Parameters of thermodynamic model

8Bintu, L. et al. Curr. Opin. Genet. Dev. 15, 116–124 (2005).

Fold change model expressed with mass action kinetics

ABKPKAKB

EAB EA

EB

Maximum likelihood fit of thermodynamic model

9

ABACBCADBDCD

ABCABDACDBCD

ABCD

rela

tive lu

cife

rase

activ

ity [M

]

1

2

3

4

5

6

7

8

Dioxin [nM]100 101 102

1 Activator 2 Activators 3 Activators

WT

rela

tive lu

cife

rase

activity [M

]1

2

3

4

5

6

7

8

Dioxin [nM]100 101 102

Dioxin [nM]100 101 102

rela

tive lu

cife

rase

activ

ity [M

]

1

2

3

4

5

6

7

8

ABCD

Maximum likelihood fit of thermodynamic model

9

ABACBCADBDCD

ABCABDACDBCD

ABCD

rela

tive lu

cife

rase

activ

ity [M

]

1

2

3

4

5

6

7

8

Dioxin [nM]100 101 102

1 Activator 2 Activators 3 Activators

WT

rela

tive lu

cife

rase

activity [M

]1

2

3

4

5

6

7

8

Dioxin [nM]100 101 102

Dioxin [nM]100 101 102

rela

tive lu

cife

rase

activ

ity [M

]

1

2

3

4

5

6

7

8

ABCD

Prediction of thermodynamic model

Interpretation of model parameters

10

strongest binding sites recruit RNA Polymerase

only some cooperations are essential

ABCD

Interpretation of model parameters

10

strongest binding sites recruit RNA Polymerase

only some cooperations are essential

ABCD

Interpretation of model parameters

10

strongest binding sites recruit RNA Polymerase

only some cooperations are essential

ABCD

TCF binding site interactions not trivial

11Dioxin [nM]100 101 102

rel.

luc a

ct.

[M

]

1

3

5

7 –T+T

rel.

luc a

ct.

1.2

0

2.4 *

–T +T

Experiments

high

low

basal transcription basal transcription

Thermodynamic model

–T +T –T +T

TCF binding site interactions not trivial

11Dioxin [nM]100 101 102

rel.

luc a

ct.

[M

]

1

3

5

7 –T+T

rel.

luc a

ct.

1.2

0

2.4 *

–T +T

Experiments

high

low

basal transcription basal transcription

Thermodynamic model

–T +T –T +T

TCF binding site interactions not trivial

11Dioxin [nM]100 101 102

rel.

luc a

ct.

[M

]

1

3

5

7 –T+T

rel.

luc a

ct.

1.2

0

2.4 *

–T +T

Experiments

high

low

basal transcription basal transcription

Thermodynamic model

–T +T –T +T

Conclusion

12

Conclusion

‣ DRE binding sites

‣ works according to convential paradigm of promotors

‣ 2 strongest binding sites control recruitment of RNAP

‣ cooperativity essential

12

Conclusion

‣ DRE binding sites

‣ works according to convential paradigm of promotors

‣ 2 strongest binding sites control recruitment of RNAP

‣ cooperativity essential

‣ TCF binding site

‣ contradicts convential paradigm of promotors

‣ needs active regulation

‣ Mechanism?

‣ needs further experiments

12

Acknowledgements

‣ Nils Blüthgen‣ Pawel Durek‣ Raphaela

Fritsche‣ Anja Sieber‣ Nadine Schmidt‣ Kajetan Bentele

‣ Bertram Klinger‣ Johannes Meisig‣ Jörn Schmiedel‣ Franziska Witzel‣ Martina

Klünemann

Systems Biology of Molecular Networks Group @ Charité

‣ Michael Schwarz‣ Albert Braeuning‣ Alexandra Löffler

Department of Toxicology @ Uni Tübingen

Funding

13