GENE SILENCING BY CHICKEN OVALBUMIN UPSTREAM PROMOTER-TRANSCRIPTION FACTOR-I (COUP-TFI) IS MEDIATED BY TRANSCRIPTIONAL COREPRESSORS, NUCLEAR RECEPTOR-COREPRESSOR (N-COR) AND SILENCING MEDIATOR FOR RETINOIC ACID RECEPTOR AND THYROID-HORMONE RECEPTOR (SMRT)

Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) a re orphan receptors that belong to the steroid/thyroid hormone recepto r (TR) superfamily and can repress the transcriptional activity of sev eral target genes; however, the precise mechanism of this repression i s unknown. Transfection of a Gal4 DNA-binding domain fused to the puta tive ligand-binding domain of COUP-TFI (Gal4-COUP-TFI) significantly r epresses the basal transcriptional activity of a reporter gene contain ing Gal4-binding sites. Cotransfection of COUP-TFI can relieve the Gal 4-COUP-TFI repression in a dose-dependent manner. In contrast, COUP-TF I Delta 35, which lacks the repressor domain (the C-terminal 35 amino acids), fails to relieve this repression. This finding suggests that t he repressor domain of COUP-TFI may squelch a limiting amount of corep ressor in HeLa cells. In addition, increasing concentrations of TR bet a also can relieve the COUP-TFI repression in a hormone-sensitive mann er. Similarly, overexpression of increasing concentration of COUP-TFI, but not COUP-TFI Delta 35, can squelch the silencing activity of the unliganded TR beta. Collectively, these results indicate that COUP-TFI and TR beta share a common corepressor(s) for their silencing activit y. To determine which corepressor is involved in the COUP-TF-silencing activity, we used a yeast two-hybrid and in vitro GST pull-down assay s to demonstrate that COUP-TFI can interact with the fragment of N-CoR (nuclear receptor-corepressor) encoding amino acids 921-2453 and the fragments of SMRT (silencing mediator for retinoic acid receptor and T R) encoding amino acids 29-564 and 565-1289, respectively. Interesting ly, the fragment of SMRT encoding amino acids 1192-1495, which strongl y interacts with TR beta, interacts very weakly with COUP-TFI. Further more, overexpression of N-CoR or SMRT potentiates the silencing activi ty of COUP-TFI and can relieve the COUP-TFI-mediated squelching of Gal 4-COUP-TFI activity. Therefore, our studies indicate that N-CoR and SM RT act as corepressors for the COUP-TFI silencing activity.