The bHLH-PAS protein ARNT - effects on ER signalling

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 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

4. Activation process of the AhR

5. AhRNuclear 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

6. 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% 39% 63% bMAL/mARNT-3 632 aa

7. 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

8. Nuclear receptor signaling

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

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

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

12. ERa and ARNT interact pSG5-Flag- pSG5-Flag-ERa pCMV-ARNT - - E2 Tam IP:anti-Flag ARNT WCEs: 10% of input ARNT Western: anti-ARNT

13. ER and ARNT interact pSG5- pSG5-ERb pCMV-ARNT - - E2 Tam IP:ERb mAb ARNT ARNT WCEs: 10% input Western: anti-ARNT

14. ARNT stimulates ER activity ARNT TIF-2

15. ARNT stimulates ER activity ARNT TIF-2

16. ARNT interacts with the LBD of ERa/b

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

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

19. TCDD and 3-MC inhibit ERs transcriptional activity in HC11 cells PPT E2 DPN

20. 3-MC but not TCDD activates ERs - E2 3-MC

21. Antiestrogens inhibit 3-MC induced transcriptional activity

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

23. Activation of the AhR inhibits recruitment of ARNT to E2 regulated promoters E2+ TCDD E2+ TCDD NH E2 TCDD Input NH E2 TCDD Input IgG IgG a-ERa a-ARNT ARNT c-Fos Lactoferrin ERE ERE UTR

24. Effects of ARNT Si-RNA

25. 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

26. p23 hsp90 XAP-2 A B ARNT regulates multiple cellular pathways DR ERa/b A B ARNT p23 hsp90 Other nuclear receptors? Other transcription factors? A B HIF-1a

27. 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