Download - Cooperativity between aryl hydrocarbon receptor and beta-catenin binding sites in hepatocytes
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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
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Cytochrome P450 (CYP)
‣ oxidate hydrophobic substances hydrophilic substances
➡ easier excretion
‣ essential for metabolizing many drugs & toxins
‣ strong expression in the liver & colon
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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)
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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)
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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
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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
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AhR and ß-catenin converge on CYP1A1 promotor
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Wnt
Dioxin
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AhR and ß-catenin converge on CYP1A1 promotor
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Wnt
Dioxin
Lrp5/6Fzd
b-cat
AhR
Arnt
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AhR and ß-catenin converge on CYP1A1 promotor
5
Wnt
Dioxin
Lrp5/6Fzd
b-cat
AhR
Arnt
TCFCYP1A1
DREDREDRE DRE
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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?
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ABCD
15 human CYP1A1 promotor constructs
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ABCD
15 human CYP1A1 promotor constructs
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ABCD
15 human CYP1A1 promotor constructs
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Luciferase
Dioxin
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Data show more-than-additive effects
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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
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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
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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
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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
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Maximum likelihood fit of thermodynamic model
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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
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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
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Interpretation of model parameters
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strongest binding sites recruit RNA Polymerase
only some cooperations are essential
ABCD
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Interpretation of model parameters
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strongest binding sites recruit RNA Polymerase
only some cooperations are essential
ABCD
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Interpretation of model parameters
10
strongest binding sites recruit RNA Polymerase
only some cooperations are essential
ABCD
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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
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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
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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
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Conclusion
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Conclusion
‣ DRE binding sites
‣ works according to convential paradigm of promotors
‣ 2 strongest binding sites control recruitment of RNAP
‣ cooperativity essential
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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
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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