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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