Identification of COUP-TF as a transcriptional repressor of the c-mos proto-oncogene

The c-mos proto-oncogene is specifically expressed in the male and female g erm cells of the mouse and other vertebrates. We previously identified a 15 -base pair sequence element (B2) as the binding site of a candidate repress or of c-mos transcription in somatic cells. In the present study, we used t he yeast one-hybrid system to isolate HeLa cell cDNAs encoding proteins tha t specifically bound to the c-mos B2 element. Nucleotide sequencing identif ied several of the clones isolated in this screen as the orphan nuclear rec eptors COUP-TFI and COUP-TFII. A COUP-TF-binding site was then identified w ithin the B2 sequence. Complexes formed between purified COUP-TFs and the c -mos B2 probe comigrated in electrophoretic mobility shift assays with thos e formed using whole nuclear extracts of NIH 3T3 or HeLa cells. Moreover, t he complexes formed with NIH 3T3 nuclear extracts and B2 probe were supersh ifted with antibody against COUP-TF, identifying COUP-TF as the candidate r epressor previously detected in these somatic cell extracts. Substitution o f a consensus COUP-TF-binding site for the c-mos negative regulatory elemen t suppressed expression from the c-mos promoter in transfected somatic cell s, demonstrating the functional activity of COUP-TF as a repressor of c-mos transcription.