1. The bHLH-PAS protein ARNT - effects on ER signalling

2. 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 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

4. Activation process of the AhR

5. 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

6. ARNT isoforms bHLHPAS1PAS2 mARNT-1 791 aa mARNT-2 712 aa 95%78% ARNT-1: ubiquitously expressed ARNT-2: in developing brain, kidney, heart bMAL/ARNT-3: brain, muscle bMAL/mARNT-3 632 aa 63% 39% 57% 34%

7. 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

8. Nuclear receptor signaling

9. Coactivators: SRC family, CBP/p300, PBP, PCAF… Corepressors: N-CoR, SMRT, DAX-1, SHP… Nuclear receptor co-factors

10. SRC-1 SCR2 (TIF-2) SRC-3 bHLH PAS NR-box SRC/p160 Co-activator factors

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

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

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

14. ARNT stimulates ER  activity ARNTTIF-2

15. ARNT stimulates ER  activity ARNTTIF-2

16. ARNT interacts with the LBD of ER  / 

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

18. ERE pS2 -353 -54 + E2 ARNT is recruited to the pS2 promoter PCR products

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

20. 3-MC but not TCDD activates ERs E2E2 - 3-MC

21. Antiestrogens inhibit 3-MC induced transcriptional activity

22. Hypoxia and TCDD inhibit ER transcriptional activity in HC11 cells

23. 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

24. Effects of ARNT Si-RNA

25. 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

26. 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

27. 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