The bHLH-PAS protein ARNT - effects on ER signalling

1
The bHLH-PAS protein ARNT - effects on ER
signalling
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Biological Effects of Dioxin

• Toxic Effects Wasting, lymph atrophy,
  endometriosis, etc
• Teratogenic Effects
• Tumor Promotion
• Enzyme Induction
• Interference with Hormonal signaling (EDC)

3
The bHLH-PAS proteins
Dimerization DNA- and hsp90 binding
Ligand- and hsp90 binding
Examples
Metabolism of xenobiotics
Partner for AhR and HIF-1a
CNS growth/differentiation
Regulation of circadian rhythm
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Activation process of the AhR
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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

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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
39
34
63
bMAL/mARNT-3 632 aa
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Nuclear receptor superfamily
Nuclear receptor superfamily
Nuclear receptor superfamily
A/B C D
E
A/B C D
E
A/B C D
E
AF-1 DBD Hinge
LBD AF-2
AF-1 DBD Hinge
LBD AF-2
AF-1 DBD Hinge
LBD AF-2
Ligand regulate receptors like steroid
receptors, thyroid hormaone receptors, retinoid
acid receptors and Vitamin D
Ligand regulate receptors like steroid
receptors, thyroid hormaone receptors, retinoid
acid receptors and Vitamin D
Ligand regulate receptors like steroid
receptors, thyroid hormaone receptors, retinoid
acid receptors and Vitamin D
Orpan receptors
Orpan receptors
Orpan receptors
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Nuclear receptor signaling
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Nuclear receptor co-factors
Coactivators SRC family, CBP/p300, PBP, PCAF

Corepressors N-CoR, SMRT, DAX-1, SHP
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SRC/p160 Co-activator factors
bHLH PAS
SRC-1
SCR2 (TIF-2)
SRC-3
NR-box
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ARNT regulates multiple pathways
DR
A
B
ARNT
p23
hsp90
Other nuclear receptors? Other transcription
factors?
A
B
HIF-1a
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ERa and ARNT interact
- - -
-

pSG5-Flag-
pSG5-Flag-ERa
pCMV-ARNT
-
-
E2
Tam
IPanti-Flag
ARNT
ARNT
WCEs 10 of input
Western anti-ARNT
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ER? and ARNT interact
- - -
-

pSG5-
pSG5-ERb
pCMV-ARNT
-
-
E2
Tam
IPERb mAb
ARNT
ARNT
WCEs 10 input
Western anti-ARNT
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ARNT stimulates ER? activity
ARNT
TIF-2
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ARNT stimulates ER? activity
ARNT
TIF-2
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ARNT interacts with the LBD of ERa/b
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The C-Terminal domain of ARNT is required for
co-activation
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ARNT is recruited to the pS2 promoter
E2
PCR products
-54
-353
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TCDD and 3-MC inhibit ERs transcriptional
activity in HC11 cells
E2
DPN
PPT
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3-MC but not TCDD activates ERs
-
E2
3-MC
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Antiestrogens inhibit 3-MC induced
transcriptional activity
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Hypoxia and TCDD inhibit ER transcriptional
activity in HC11 cells
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Activation of the AhR inhibits recruitment of
ARNT to E2 regulated promoters
E2 TCDD
E2 TCDD
E2
NH
TCDD
Input
E2
NH
TCDD
Input
IgG
IgG
a-ERa
a-ARNT
ARNT
c-Fos
Lactoferrin
ERE
ERE
UTR
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Effects of ARNT Si-RNA
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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

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ARNT regulates multiple cellular pathways
DR
ERa/b
A
B
ARNT
p23
hsp90
Other nuclear receptors? Other transcription
factors?
A
B
HIF-1a
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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