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

bHLHPAS DomainBABABABA

Dioxin Receptor, AhRArntSim

Per

Metabolism of xenobiotics

CNS growth/differentiation

Regulation of circadian rhythm

Partner for AhR and HIF-1

Examples:

DimerizationDNA- and

hsp90 binding

Ligand- andhsp90 binding

Ligand BindingDimerizationTCDDARNT

ARNT

hsp90

DNA Binding, TransactivationNUCLEUSCYTOPLASM

hsp90

Activation process of the AhR

AhR Nuclear Translocator: ARNT

• Obligatory partner for DR and HIF-1• 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

bHLH PAS1 PAS2

mARNT-1791 aa

mARNT-2712 aa

95% 78%

• ARNT-1: ubiquitously expressed

• ARNT-2: in developing brain, kidney, heart

• bMAL/ARNT-3: brain, muscle

bMAL/mARNT-3632 aa

63% 39%

57%

34%

A/B C D EAF-1 DBD Hinge LBD AF-2

Ligand regulate receptors like steroid receptors, thyroid

hormaone receptors, retinoid acid receptors and Vitamin D

Orpan receptors

Nuclear receptor superfamily

Nuclear receptor signaling

NRHSP 90 Complex

+ligand

NR+

HSP 90 Complex

HREcofactors

transcription

Gene promoter

Coactivators: SRC family, CBP/p300, PBP, PCAF…

Corepressors: N-CoR, SMRT, DAX-1, SHP…

Nuclear receptor co-factors

SRC-1

SCR2(TIF-2)

SRC-3

bHLH PAS

NR-box

SRC/p160 Co-activator factors

A B

p23

hsp90XAP-2

A B

A B

p23

hsp90

DR

HIF-1Other nuclear receptors?Other transcription factors?

ARNT

ARNT regulates multiple pathways

E2 Tam- -

pCMV-ARNT

pSG5-Flag-ER

pSG5-Flag- - - - +

+ + + -

+ + + +

IP:anti-Flag ARNT

ARNTWCEs: 10% of input

ER and ARNT interact

Western: anti-ARNT

E2 Tam

pCMV-ARNT

pSG5-ER

pSG5- + - - -

- + + +

+ + + +

IP:ER mAb ARNT

WCEs: 10% input

ER and ARNT interact

Western: anti-ARNT

ARNT

- -

ARNT stimulates ER activity

ARNT TIF-2

HeLa/3xERE-TATA-luc

050

100150200250

300350400450500

3xERETATA

pSG5ERa

ERa +ARNT

ERa +ARNT

ERa +ARNT

ERa +TIF-2

ERa +TIF-2

ERa +TIF-2

(-)E2 10 nMTam 1 uM

ARNT stimulates ER activity

ARNT TIF-2

HeLa/3xERE-TATA-luc

0

200

400

600

800

1000

1200

1400

1600

1800

2000

3xERETATAluc

ERb ERb +ARNT

ERb +ARNT

ERb +ARNT

ERb +TIF-2

ERb +TIF-2

ERb +TIF-2

(-)E2 10 nMTam 1 uM

200400600

ER-LBDARNTARNTER-LBD

Tam2E-ERER−ΔABARNTARNT10002000300040002E- 50010001500

ERER−ΔABARNTARNT2E-ARNT interacts

with the LBD of ER/

The C-Terminal domain of ARNT is required for co-activation

ER250500750

-ARNTARNT 603ARNT 407

C E2-5001000150020002500

ERβ-ARNTARNT 603ARNT 407

D E2-ERβ250-ARNTΔbHLHΔPASERα

500-ARNTΔbHLHΔPAS500750A E2-

100015002000B E2-

EREpS2-353

-54

+ E2

ARNT is recruited to the pS2 promoter

PCR products

TCDD and 3-MC inhibit ERs transcriptional activity in HC11 cells

0

20

40

60

80

100

120

- TCDD 3-MC TCDD 3-MC TCDD 3-MC TCDD 3-MC

% Activity

E2 DPN PPT

3-MC but not TCDD activates ERs

0

20

40

60

80

100

120

140

160

180

ERa

ERb

E2- 3-MC

Antiestrogens inhibit 3-MC induced transcriptional activity

0

20

40

60

80

100

120

140

160

180

- E2 3MC 3MC+ICI 3MC+4OHT

ERa

ERb

HC11-ERE

0

20

40

60

80

100

120

(-) E2 10 nM Tam 1 uM ICI 100 nM

% Activity

DMSO

TCDD

Hypoxia

Hypoxia+TCDD

Hypoxia and TCDD inhibit ER transcriptional activity in HC11 cells

E2+

TCDDE2NH

IgG

-ER

TCDD

Input

Activation of the AhR inhibits recruitment of ARNT to E2 regulated promoters

ARNT

IgG

-ARNT

Input

E2+TCDD

E2NH TCDD

c-Fos Lactoferrin

ERE

UTR

ERE

Effects of ARNT Si-RNA

0

10

20

30

40

50

60

70

scrambled siRNA 24h

Fold ind

(-)

E2

E2+TCDD

SUMMARY

• ARNT interacts with ligand-activated ER and ER

• ARNT stimulates both ER and ER transcriptional activity

• ARNT is recruited to the E2 regulated promoters in an ligand-dependent manner

A B

p23

hsp90XAP-2

A B

A B

p23

hsp90

DR

HIF-1

ER/

Other nuclear receptors?Other transcription factors?

ARNT

ARNT regulates multiple cellular pathways

Ingemar Pongratz

Katarina Pettersson

Wen Cai

Elin Swedenborg

Petra Berg

Pauliina Pentinen / Sari MäkeleUniversity of Turku

Sara Brunnberg

Juliette Rambaud / Vincent LaudetEcole Normale Supérieure de Lyon