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) 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-TFI delta35, 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 TRbeta also can relieve the COUP-TFI repression in a hormone-sensitive manner. Similarly, overexpression of increasing concentration of COUP-TFI, but not COUP-TFI delta35, can squelch the silencing activity of the unliganded TRbeta. Collectively, these results indicate that COUP-TFI and TRbeta 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 TRbeta, 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.
Shibata
H
H
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Nawaz
Z
Z
Tsai
S Y
SY
O'Malley
B W
BW
Tsai
M J
MJ
D000818:Animals; D051838:COUP Transcription Factor I; D002645:Chickens; D004268:DNA-Binding Proteins / Q000235:genetics*; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics*; D056971:Nuclear Receptor Co-Repressor 1; D056985:Nuclear Receptor Co-Repressor 2; D011401:Promoter Regions, Genetic; D011965:Receptors, Glucocorticoid / Q000235:genetics*; D011988:Receptors, Thyroid Hormone / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic
D051838:COUP Transcription Factor I; D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; C539127:NCOR1 protein, human; C539130:NCOR2 protein, human; C497589:NR2F1 protein, human; D009687:Nuclear Proteins; D056971:Nuclear Receptor Co-Repressor 1; D056985:Nuclear Receptor Co-Repressor 2; D011965:Receptors, Glucocorticoid; D011988:Receptors, Thyroid Hormone; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors