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 tran scription factors and, as such, probably plays a crucial role in mamma lian embryogenesis and differentiation. It is expressed in the earlies t stages of embryogenesis and repressed in subsequent stages. Similarl y, Oct-3/4 is expressed in embryonal carcinoma (EC) cells and is repre ssed in retinoic acid (RA)-differentiated EC cells. previously we have shown,vn that the Oct-3/4 promoter harbors an RA-responsive element, RAREoct, which functions in EC cells as a binding site for positive re gulators of transcription and in RA-differentiated EC cells as a bindi ng 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 RAREo ct 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-te rminal domain harbors the silencing region. Interestingly, three diffe rent RA receptor:retinoid X receptor (RAR:RXR) heterodimers, RAR alpha :RXR alpha, RAR beta:RXR alpha, and RAR beta:RXR beta, specifically bi nd and activate Oct-3/4 promoter through the RAREoct site in a ligand- dependent manner. We have shown that antagonism between ARP-1/COUP-TFI I or EAR-3/COUP-TFI and the RAR:RXR heterodimers and their intracellul ar balance modulate Oct-3/4 expression. Oct-3/4 transcriptional repres sion by the orphan receptors can be overcome by increasing amounts of RAR:RXR heterodimers. Conversely, activation of Oct-3/4 promoter by RA R:RXR heterodimers was completely abolished by EAR-3/COUP-TFI and by A RP-1/COUP-TFII. The orphan receptors bind the RAREoct site with a much higher affinity than the RAR:RXR heterodimers. This high binding affi nity provides ARP-1/COUP-TFII and EAR-3/COUP-TFI with the ability to c ompete with and even displace RAR:RXR from the RAREoct site and subseq uently 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 kine tics of ARP-1/COUP-TFII and EAR-3ICOUP-TFI activation. These findings are in accordance with the suggestion that these orphan receptors part icipate in controlling a network of transcription factors, among which Oct-3/4 is included, which may establish the pattern of normal gene e xpression during development.