A dynamic balance between ARP-1/COUP-TFII, EAR-3/COUP-TFI, and retinoic acid receptor:retinoid X receptor heterodimers regulates Oct-3/4 expression in embryonal carcinoma cells.
The Oct-3/4 transcription factor is a member of the POU family of transcription factors and, as such, probably plays a crucial role in mammalian embryogenesis and differentiation. It is expressed in the earliest stages of embryogenesis and repressed in subsequent stages. Similarly, Oct-3/4 is expressed in embryonal carcinoma (EC) cells and is repressed in retinoic acid (RA)-differentiated EC cells. Previously we have shown that the Oct-3/4 promoter harbors an RA-responsive element, RAREoct, which functions in EC cells as a binding site for positive regulators of transcription and in RA-differentiated EC cells as a binding site for positive regulators of transcription and in RA-differentiated EC cells as a binding site for negative regulators. Our present results demonstrate that in P19 and RA-treated P19 cells, the orphan receptors ARP-1/COUP-TFII and EAR-3/COUP-TFI repress Oct-3/4 promoter activity through the RAREoct site in a dose-dependent manner. While the N-terminal region of the ARP-1/COUP-TFII receptor is dispensable for this repression, the C-terminal domain harbors the silencing region. Interestingly, three different RA receptor:retinoid X receptor (RAR:RXR) heterodimers, RAR alpha:RXR alpha, RAR beta:RXR alpha, and RAR beta:RXR beta, specifically bind and activate Oct-3/4 promoter through the RAREoct site in a ligand-dependent manner. We have shown that antagonism between ARP-1/COUP-TFII or EAR-3/COUP-TFI and the RAR:RXR heterodimers and their intracellular balance modulate Oct-3/4 expression. Oct-3/4 transcriptional repression by the orphan receptors can be overcome by increasing amounts of RAR:RXR heterodimers. Conversely, activation of Oct-3/4 promoter by RAR:RXR heterodimers was completely abolished by EAR-3/COUP-TFI and by ARP-1/COUP-TFII. The orphan receptors bind the RAREoct site with a much higher affinity than the RAR:RXR heterodimers. This high binding affinity provides ARP-1/COUP-TFII and EAR-3/COUP-TFI with the ability to compete with and even displace RAR:RXR from the RAREoct site and subsequently to actively silence the Oct-3/4 promoter. We have shown that RA treatment of EC cells results in up-regulation of ARP-1/COUP-TFII and EAR-3/COUP-TFI expression. Most interestingly, in RA-treated EC cells, the kinetics of Oct-3/4 repression inversely correlates with the kinetics of ARP-1/COUP-TFII and EAR-3/COUP-TFI activation. These findings are in accordance with the suggestion that these orphan receptors participate in controlling a network of transcription factors, among which Oct-3/4 is included, which may establish the pattern of normal gene expression during development.
Ben-Shushan
E
E
Hubert H. Humphrey Center for Experimental Medicine and Cancer Research, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Sharir
H
H
Pikarsky
E
E
Bergman
Y
Y
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D051838:COUP Transcription Factor I; D051837:COUP Transcription Factor II; D051836:COUP Transcription Factors; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050814:Octamer Transcription Factor-3; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D055503:Protein Multimerization; D018168:Receptors, Retinoic Acid / Q000096:biosynthesis / Q000378:metabolism*; D011987:Receptors, Steroid / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D047488:Retinoid X Receptors; D012176:Retinoids / Q000378:metabolism; D013724:Teratoma; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D051838:COUP Transcription Factor I; D051837:COUP Transcription Factor II; D051836:COUP Transcription Factors; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; C497590:Nr2f1 protein, mouse; C497587:Nr2f2 protein, mouse; D050814:Octamer Transcription Factor-3; D009838:Oligodeoxyribonucleotides; C494174:Pou5f1 protein, mouse; D018168:Receptors, Retinoic Acid; D011987:Receptors, Steroid; D011994:Recombinant Proteins; D047488:Retinoid X Receptors; D012176:Retinoids; D014157:Transcription Factors