Transcript The bHLH-PAS protein ARNT - an activator of ER activity

The bHLH-PAS protein ARNT
- effects on ER signalling
Biological Effects of Dioxin
• Toxic Effects: Wasting, lymph atrophy, endometriosis,
etc
• Teratogenic Effects
• Tumor Promotion
• Enzyme Induction
• Interference with Hormonal signaling (EDC)
The bHLH-PAS proteins
Dimerization
DNA- and
hsp90 binding
Examples:
Ligand- and
hsp90 binding
bHLH PAS Domain
Metabolism of
xenobiotics
Dioxin Receptor, AhR
Partner for AhR and HIF-1a
Sim
CNS growth/differentiation
Regulation of
circadian rhythm
Arnt
Per
A
B
A
B
A
B
A
B
Activation process of the AhR
AhR Nuclear Translocator: ARNT
• Obligatory partner for DR and HIF-1a
• Ligand  DR-ARNT dimers bind to xenobiotic
response elements (XREs) of target genes
• ARNT is constitutively expressed protein
• Nuclear localization
• 3 isoforms identified
ARNT isoforms
•
ARNT-1: ubiquitously expressed
•
ARNT-2: in developing brain, kidney, heart
•
bMAL/ARNT-3: brain, muscle
bHLH
PAS1
PAS2
mARNT-1
791 aa
57%
95%
78%
mARNT-2
712 aa
34%
63%
39%
bMAL/mARNT-3
632 aa
Nuclear receptor superfamily
A/B
AF-1
C
DBD
D
Hinge
E
LBD
AF-2
Ligand regulate receptors like steroid receptors, thyroid
hormaone receptors, retinoid acid receptors and Vitamin D
Orpan receptors
Nuclear receptor signaling
HSP 90
NR Complex
+ligand
NR
+
HSP 90
Complex
transcription
cofactors
HRE
Gene promoter
Nuclear receptor co-factors
Coactivators: SRC family, CBP/p300, PBP, PCAF…
Corepressors: N-CoR, SMRT, DAX-1, SHP…
SRC/p160 Co-activator factors
bHLH PAS
SRC-1
SCR2
(TIF-2)
SRC-3
NR-box
ARNT regulates multiple pathways
p23
hsp90
A
B
DR
A
B
ARNT
p23
hsp90
A
HIF-1a
B
Other nuclear receptors?
Other transcription factors?
ERa and ARNT interact
pSG5-FlagpSG5-Flag-ERa
pCMV-ARNT
+
+
+
+
-
E2
+
+
+
+
Tam
-
IP:anti-Flag
ARNT
WCEs: 10% of input
ARNT
Western: anti-ARNT
ERb and ARNT interact
pSG5pSG5-ERb
pCMV-ARNT
+
+
+
+
-
-
+
+
+
+
E2
Tam
IP:ERb mAb
ARNT
WCEs: 10% input
ARNT
Western: anti-ARNT
ARNT stimulates ERa activity
HeLa/3xERE-TATA-luc
500
450
400
350
300
250
200
150
100
50
0
(-)
E2 10 nM
Tam 1 uM
3xERE pSG5
TATA
ERa
ERa + ERa + ERa + ERa + ERa + ERa +
ARNT ARNT ARNT TIF-2 TIF-2 TIF-2
ARNT
TIF-2
ARNT stimulates ERb activity
HeLa/3xERE-TATA-luc
2000
1800
1600
1400
(-)
E2 10 nM
Tam 1 uM
1200
1000
800
600
400
200
0
3xERE
TATA
luc
ERb
ERb +
ARNT
ERb +
ARNT
ERb +
ARNT
ARNT
ERb +
TIF-2
ERb +
TIF-2
ERb +
TIF-2
TIF-2
ARNT interacts
with the LBD of
ERa/b
The C-Terminal domain of ARNT is required
for co-activation
ARNT is recruited to the pS2 promoter
+ E2
PCR
products
pS2
ERE
-353
-54
TCDD and 3-MC inhibit ERs
transcriptional activity in HC11 cells
120
E2
DPN
PPT
100
% Activity
80
60
40
20
0
-
TCDD 3-MC
TCDD 3-MC
TCDD 3-MC
TCDD 3-MC
3-MC but not TCDD activates ERs
-
E2
3-MC
Antiestrogens inhibit 3-MC induced
transcriptional activity
Hypoxia and TCDD inhibit ER
transcriptional activity in HC11 cells
Activation of the AhR inhibits recruitment
of ARNT to E2 regulated promoters
E2+
NH
E2 TCDD TCDD
Input
NH
E2 TCDD
E2+
TCDD
IgG
Input
a-ERa
IgG
a-ARNT
ARNT
c-Fos
Lactoferrin
ERE
UTR
ERE
Effects of ARNT Si-RNA
SUMMARY
• ARNT interacts with ligand-activated ERa and ERb
• ARNT stimulates both ERa and ERb transcriptional
activity
• ARNT is recruited to the E2 regulated promoters in an
ligand-dependent manner
ARNT regulates multiple cellular pathways
p23
hsp90
A
B
DR
ERa/b
A
B
ARNT
p23
hsp90
A
HIF-1a
B
Other nuclear receptors?
Other transcription factors?
Pauliina Pentinen / Sari Mäkele
University of Turku
Juliette Rambaud / Vincent Laudet
Ecole Normale Supérieure de Lyon
Ingemar Pongratz
Katarina Pettersson
Sara Brunnberg
Elin Swedenborg
Wen Cai
Petra Berg