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

Binding Site Cooperativity and Dual Signal Integration in CYP1A1 Induction

1

CYP1A expression in liver

2Braeuning, A. & Schwarz, M. Biol. Chem. 391, 139–148 (2010)

rela

tive

mR

NA

exp

ress

ion [a

. u.]

0

3

6

9

100

150

200

250

300

TCDD – +

CYP1A1primary mousehepatocytes

n=5

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

CYP1A expression in liver

WT

CYP1A

P

C

2Braeuning, A. & Schwarz, M. Biol. Chem. 391, 139–148 (2010)

rela

tive

mR

NA

exp

ress

ion [a

. u.]

0

3

6

9

100

150

200

250

300

TCDD – +

CYP1A1primary mousehepatocytes

n=5

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

CYP1A expression in liver

WT

CYP1A

P

C

ß-ca

t k.o

.

P

C

2Braeuning, A. & Schwarz, M. Biol. Chem. 391, 139–148 (2010)

rela

tive

mR

NA

exp

ress

ion [a

. u.]

0

3

6

9

100

150

200

250

300

TCDD – +

CYP1A1primary mousehepatocytes

n=5

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

Wnt

TCDD

AhR

Arnt

Lrp5/6Fzd

b-cat

AhR and ß-catenin converge on CYP1A1 promoter

3

TCFCYP1A1

DREDREDRE DRE RNAP

Wnt

TCDD

AhR

Arnt

Lrp5/6Fzd

b-cat

AhR and ß-catenin converge on CYP1A1 promoter

3

TCFCYP1A1

DREDREDRE DRE RNAP

TCFCYP1A1

DREDREDRE DRE RNAP

Wnt

TCDD

AhR

Arnt

Lrp5/6Fzd

b-cat

AhR and ß-catenin converge on CYP1A1 promoter

3

TCFCYP1A1

DREDREDRE DRE RNAP

TCFCYP1A1

DREDREDRE DRE RNAP

How do DREs cooperate?

Wnt

TCDD

AhR

Arnt

Lrp5/6Fzd

b-cat

AhR and ß-catenin converge on CYP1A1 promoter

3

TCFCYP1A1

DREDREDRE DRE RNAP

TCFCYP1A1

DREDREDRE DRE RNAP

How are 2 signals integrated?

How do DREs cooperate?

Experimental setup

4

Experimental setup

4

Experimental setup

4

TCDD

Luciferase

1D data shows cooperativity of binding sites

5

ABCD T

relat

ive lu

cifer

ase

activ

ity [R

LU]

1

2

3

TCDD [nM]100 101 102

AAA

AA

AAA AA

A

A AA

BBBBBBBB B B B B B B

Active Sites

1D data shows cooperativity of binding sites

5

ABCD T

relat

ive lu

cifer

ase

activ

ity [R

LU]

1

2

3

TCDD [nM]100 101 102

AAA

AA

AAA AA

A

A AA

BBBBBBBB B B B B B B

Active SitesABAB

AB

ABABAB

AB

AB

ABAB

AB AB

AB

AB

Modeling approach

6

promoter logic

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

Modeling approach

6

promoter logic

XYKPKXKY

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

Modeling approach

6

promoter logic

XYKPKXKY

EX

EY

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

Modeling approach

6

promoter logic

XYKPKXKY

EX

EY

EXY

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

Interpretation of resulting model parameters

7

strongest binding sites recruit RNA Polymerase

B site most important cooperation partner

ABCD

Interpretation of resulting model parameters

7

strongest binding sites recruit RNA Polymerase

B site most important cooperation partner

ABCD

Interpretation of resulting model parameters

7

strongest binding sites recruit RNA Polymerase

B site most important cooperation partner

ABCD

Interpretation of resulting model parameters

7

strongest binding sites recruit RNA Polymerase

B site most important cooperation partner

ABCDHow are 2 signals integrated?

CYP1A1 expression modulated by ß-catenin & TCDD

8Braeuning, A., Köhle, C., Buchmann, A. & Schwarz, M. Toxicol. Sci. 122, 16–25 (2011).

TCDD –

ß-catenin +

–

–

+

–

+

+

*

*175

125

75

1

0

55.1c

CYP1A1

rela

tive m

RN

A e

xpre

ssio

n [a

. u.]

CYP1A1 expression modulated by ß-catenin & TCDD

8Braeuning, A., Köhle, C., Buchmann, A. & Schwarz, M. Toxicol. Sci. 122, 16–25 (2011).

AhR expression not modulated by ß-catenin & TCDD

TCDD –

ß-catenin +

–

–

+

–

+

+

*

*175

125

75

1

0

55.1c

CYP1A1

rela

tive m

RN

A e

xpre

ssio

n [a

. u.]

0

0.4

0.6

1AhR

Experimental setup

9

ß-catenin

TCDD

Luciferase

Experimental setup

9

ß-catenin

TCDD

Luciferase

2D data shows interplay of ß-catenin & TCDD

10

ATBCD (WT)

ßòJH[LUPU

�HJ[P]P[`�B�

D 100

93

67

53

29

28

;*++�BU4D100 101 1020

ABCD

;*++�BU4D100 101 1020

AT5

4

3

2

1

SVN�YLS��S\J��HJ[P]P[`�B93<D

;*++�BU4D100 101 1020

DATA

ABCD T

2D data shows interplay of ß-catenin & TCDD

10

PL model unable to describe ß-catenin integration

ATBCD (WT)

ßòJH[LUPU

�HJ[P]P[`�B�

D 100

93

67

53

29

28

;*++�BU4D100 101 1020

ABCD

;*++�BU4D100 101 1020

AT5

4

3

2

1

SVN�YLS��S\J��HJ[P]P[`�B93<D

;*++�BU4D100 101 1020

DATA

ABCD T

ßòJH[LUPU

�HJ[P]P[`�B�

D 100

93

67

53

29

28

;*++�BU4D100 101 1020

;*++�BU4D100 101 1020

;*++�BU4D100 101 1020

SVN�YLS��S\J��HJ[P]P[`�B93<D

5

4

3

2

1MO

DEL

2D data shows interplay of ß-catenin & TCDD

10

PL model unable to describe ß-catenin integration

ATBCD (WT)

ßòJH[LUPU

�HJ[P]P[`�B�

D 100

93

67

53

29

28

;*++�BU4D100 101 1020

ABCD

;*++�BU4D100 101 1020

AT5

4

3

2

1

SVN�YLS��S\J��HJ[P]P[`�B93<D

;*++�BU4D100 101 1020

DATA

ABCD T

ßòJH[LUPU

�HJ[P]P[`�B�

D 100

93

67

53

29

28

;*++�BU4D100 101 1020

;*++�BU4D100 101 1020

;*++�BU4D100 101 1020

SVN�YLS��S\J��HJ[P]P[`�B93<D

5

4

3

2

1MO

DEL

Modeling approach – adding TF formation

11

XYKPKXKY

EX

EY

EXY

promoter logic

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

Modeling approach – adding TF formation

11

XYKPKXKY

EX

EY

EXYAhR Arnt

TCDD ß-catenin

promoter logictranscription factor formation +

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

PL + TFF model capture dual signal integration

12

ATBCD (WT)

ßòJH[LUPU

�HJ[P]P[`�B�

D 100

93

67

53

29

28

;*++�BU4D100 101 1020

ABCD

;*++�BU4D100 101 1020

AT5

4

3

2

1

SVN�YLS��S\J��HJ[P]P[`�B93<D

;*++�BU4D100 101 1020

DATA

ßòJH[LUPU

�HJ[P]P[`�B�

D 100

93

67

53

29

28

;*++�BU4D100 101 1020

;*++�BU4D100 101 1020

;*++�BU4D100 101 1020

SVN�YLS��S\J��HJ[P]P[`�B93<D

5

4

3

2

1MO

DEL

ABCD T

Conclusions

13

‣ Cooperativity of CYP1A1 promoter binding sites‣ 2 strongest binding sites control recruitment of RNAP

‣ second DRE is most important cooperation partner

‣ Dual signal integration on CYP1A1 promoter‣ simple promoter logic not sufficient to capture effect of

ß-catenin on CYP1A1 expression‣ additional transcription factor formation necessary‣ combined models capture dual signal integration

Conclusions

13

‣ Cooperativity of CYP1A1 promoter binding sites‣ 2 strongest binding sites control recruitment of RNAP

‣ second DRE is most important cooperation partner

‣ Dual signal integration on CYP1A1 promoter‣ simple promoter logic not sufficient to capture effect of

ß-catenin on CYP1A1 expression‣ additional transcription factor formation necessary‣ combined models capture dual signal integration

Conclusions

13

‣ Cooperativity of CYP1A1 promoter binding sites‣ 2 strongest binding sites control recruitment of RNAP

‣ second DRE is most important cooperation partner

‣ Dual signal integration on CYP1A1 promoter‣ simple promoter logic not sufficient to capture effect of

ß-catenin on CYP1A1 expression‣ additional transcription factor formation necessary‣ combined models capture dual signal integration

Acknowledgements

‣ Nils Blüthgen‣ Pawel Durek‣ Raphaela

Fritsche‣ Manuela

Benary‣ Anja Sieber

‣ Nadine Schmidt‣ Kajetan Bentele‣ Bertram Klinger‣ Johannes Meisig‣ Jörn Schmiedel‣ Franziska Witzel

Systems Biology of Molecular Networks Group @ Charité

‣ Michael Schwarz‣ Albert Braeuning‣ Alexandra Löffler

Department of Toxicology @ Uni Tübingen

Funding

14