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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)

Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030

Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) are orphan receptors that belong to the steroid/thyroid hormone receptor (TR) superfamily and can repress the transcriptional activity of several target genes; however, the precise mechanism of this repression is unknown. Transfection of a Gal4 DNA-binding domain fused to the putative ligand-binding domain of COUP-TFI (Gal4-COUP-TFI) significantly represses the basal transcriptional activity of a reporter gene containing Gal4-binding sites. Cotransfection of COUP-TFI can relieve the Gal4-COUP-TFI repression in a dose-dependent manner. In contrast, COUP-TFI35, which lacks the repressor domain (the C-terminal 35 amino acids), fails to relieve this repression. This finding suggests that the repressor domain of COUP-TFI may squelch a limiting amount of corepressor in HeLa cells. In addition, increasing concentrations of TRß also can relieve the COUP-TFI repression in a hormone-sensitive manner. Similarly, overexpression of increasing concentration of COUP-TFI, but not COUP-TFI35, can squelch the silencing activity of the unliganded TRß. Collectively, these results indicate that COUP-TFI and TRß share a common corepressor(s) for their silencing activity. To determine which corepressor is involved in the COUP-TF-silencing activity, we used a yeast two-hybrid and in vitro GST pull-down assays 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 TR) encoding amino acids 29–564 and 565-1289, respectively. Interestingly, the fragment of SMRT encoding amino acids 1192–1495, which strongly interacts with TRß, interacts very weakly with COUP-TFI. Furthermore, overexpression of N-CoR or SMRT potentiates the silencing activity of COUP-TFI and can relieve the COUP-TFI-mediated squelching of Gal4-COUP-TFI activity. Therefore, our studies indicate that N-CoR and SMRT act as corepressors for the COUP-TFI silencing activity.

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