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Chicken ovalbumin upstream promoter-transcription factor (COUP-TF) is an orphan nuclear receptor that can bind DNA as a homodimer or a heterodimer with retinoid X receptor or thyroid hormone receptor. COUP-TF exhibits multiple tissue-specific functions and these include silencing of other nuclear receptors and transcription factors. Ligand-dependent activation of COUP-TF has hitherto not been reported. Studies in this laboratory have demonstrated that different structural classes of triaryl methanes containing two bis-indole substituents activate several orphan receptors and a subclass of 1,1-bis(3’-indolyl)-1-(heteroaromatic)methanes were identified as COUP-TF1 activators. In MCF-7 breast cancer cells transfected with chimeric Gal4-COUP-TF1-FL (full length) expression and a pGal4 construct containing 5 tandem Gal4 response elements linked to a luciferase receptor gene, 10-20uM 1,1-bis(3’-indolyl)-1-(4-pyridyl)methane (DIM-C-Pyr-4) induced transactivation. The effect of several other analogs containing heteroaromatic rings including the 3- and 2-pyridyl isomers were also investigated and exhibited minimal activation of Gal4-COUP-TF1. We also determined the effects of DIM-C-Pyr-4 on activation of chimeric COUP-TF1-ΔC (aa 1-211) and COUP-TF1-ΔN (aa 151-423) and in which regions of the ligand binding domain E and the A/B domain were deleted respectively. DIM-C-Pyr-4 induced transactivation > 15 fold in cells transfected with COUP-TF1-ΔC (aa 1-211) whereas decreased (>3 fold) but significant transactivation was observed in cells transfected with COUP-TF1-ΔN (aa 151-423). Cotreatment with phosphatidylinositol 3-kinase (PI3K) inhibitor blocked the DIM-C-Pyr-4-induced transcriptional activation of COUP-TF1-ΔC (aa 1-211) by 70%. We also investigated ligand-dependent interaction of Gal4-coactivator (SRC-1, SRC-2, SRC-3, TRAP220, CARM1 and PGC-1) and VP16-COUP-TF1 (full-length) in a mammalian two-hybrid assay in LNCaP cells and SRC-1 and SRC-2 were the major COUP-TF1 interacting coactivators. This represents the first report of COUP-TF1 activation and current studies are investigating the mechanisms of this response and the effects on gene expression.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]