Binding site cooperativity and dual signal integration in CAP1A1 induction
35
Pascal Schulthess , Albert Braeuning, Alexandra Löffler, Michael Schwarz, and Nils Blüthgen Binding Site Cooperativity and Dual Signal Integration in CYP1A1 Induction 1
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Transcript of Binding site cooperativity and dual signal integration in CAP1A1 induction
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
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