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 Metabolism of xenobiotics CNS growth/differentiation Regulation of circadian rhythm Partner for AhR and HIF-1  Examples: Dimerization DNA- and hsp90 binding Ligand- and hsp90 binding

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 bHLHPAS1PAS2 mARNT aa mARNT aa 95%78% ARNT-1: ubiquitously expressed ARNT-2: in developing brain, kidney, heart bMAL/ARNT-3: brain, muscle bMAL/mARNT aa 63% 39% 57% 34%

Nuclear receptor superfamily A/B C D E AF-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 A/B C D E AF-1 DBD Hinge LBD AF-2 Ligand regulate receptors like steroid receptors, thyroid hormaone receptors, retinoid acid receptors and Vitamin D Orpan receptors A/B C D E AF-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

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

AB p23 hsp90 XAP-2 AB AB p23 hsp90 DR HIF-1  Other nuclear receptors? Other transcription factors? ARNT ARNT regulates multiple pathways

E2Tam -- pCMV-ARNT pSG5-Flag-ER  pSG5-Flag IP:anti-FlagARNT WCEs: 10% of input ER  and ARNT interact Western: anti-ARNT

E2Tam pCMV-ARNT pSG5-ER  pSG IP:ER  mAb ARNT WCEs: 10% input ER  and ARNT interact Western: anti-ARNT ARNT --

ARNT stimulates ER  activity ARNTTIF-2

ARNT stimulates ER  activity ARNTTIF-2

ARNT interacts with the LBD of ER  / 

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

ERE pS E2 ARNT is recruited to the pS2 promoter PCR products

TCDD and 3-MC inhibit ERs transcriptional activity in HC11 cells E2E2 DPN PPT

3-MC but not TCDD activates ERs E2E2 - 3-MC

Antiestrogens inhibit 3-MC induced transcriptional activity

Hypoxia and TCDD inhibit ER transcriptional activity in HC11 cells

E2+ TCDD E2NH IgG  -ER  TCDD Input Activation of the AhR inhibits recruitment of ARNT to E 2 regulated promoters ARNT IgG  -ARNT Input E2+ TCDD E2NHTCDD c-FosLactoferrin ERE UTR ERE

Effects of ARNT Si-RNA

SUMMARY ARNT interacts with ligand-activated ER  and ER  ARNT stimulates both ER  and ER  transcriptional activity ARNT is recruited to the E 2 regulated promoters in an ligand-dependent manner

AB p23 hsp90 XAP-2 AB AB 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 Mäkele University of Turku Sara Brunnberg Juliette Rambaud / Vincent Laudet Ecole Normale Supérieure de Lyon