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

Abstract

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.

MicroRNA-302 Increases Reprogramming Efficiency via Repression of NR2F2

“…We found that Lv‐shNR2F2 increased pOCT4‐Luc reporter activity (Fig. 3B), confirming previous studies showing that NR2F2 silences OCT4 by directly binding to its promoter [35]. We next transfected the promoter reporter (p302‐Luc) into hESCs along with a lentivirus expressing NR2F2 (Lv‐NR2F2) or Lv‐GFP (control).…”

MicroRNA-302 Increases Reprogramming Efficiency via Repression of NR2F2

“…We found that Lv‐shNR2F2 increased pOCT4‐Luc reporter activity (Fig. 3B), confirming previous studies showing that NR2F2 silences OCT4 by directly binding to its promoter [35]. We next transfected the promoter reporter (p302‐Luc) into hESCs along with a lentivirus expressing NR2F2 (Lv‐NR2F2) or Lv‐GFP (control).…”

Role of COUP‐TFI during retinoic acid‐induced differentiation of P19 cells to endodermal cells

“…Consistent with the findings of Ben‐Shushan et al (1995), COUP‐TFI and COUP‐TFII mRNA levels inP19 cells were found to be highly elevated upon RA treatment with the maximal increase after 72 h. In addition, only variant 1 of COUP‐TFII was found to display this RA‐dependent increase in mRNA levels. Furthermore, the increase in COUP‐TFI and COUP‐TFII variant 1 mRNA levels is not a direct response to RA but rather requires the RA‐dependent increase in PBX expression.…”

Expression of Mirlet7 Family MicroRNAs in Response to Retinoic Acid-Induced Spermatogonial Differentiation in Mice1

“…We found that both RARA and RARG interact with the Pou5f1 promoter in untreated P19 cells, but not in RA-treated P19 cells. This result is consistent with the previous observation that in P19 cells, the Pou5f1 promoter was occupied by RAR/RXR heterodimers before RA treatment, but this occupancy was lost following the treatment [41,42], demonstrating the specificity of our findings.…”

MicroRNA-302 Increases Reprogramming Efficiency via Repression of NR2F2

“…We found that Lv‐shNR2F2 increased pOCT4‐Luc reporter activity (Fig. 3B), confirming previous studies showing that NR2F2 silences OCT4 by directly binding to its promoter [35]. We next transfected the promoter reporter (p302‐Luc) into hESCs along with a lentivirus expressing NR2F2 (Lv‐NR2F2) or Lv‐GFP (control).…”

Role of COUP‐TFI during retinoic acid‐induced differentiation of P19 cells to endodermal cells

“…Consistent with the findings of Ben‐Shushan et al (1995), COUP‐TFI and COUP‐TFII mRNA levels inP19 cells were found to be highly elevated upon RA treatment with the maximal increase after 72 h. In addition, only variant 1 of COUP‐TFII was found to display this RA‐dependent increase in mRNA levels. Furthermore, the increase in COUP‐TFI and COUP‐TFII variant 1 mRNA levels is not a direct response to RA but rather requires the RA‐dependent increase in PBX expression.…”

Expression of Mirlet7 Family MicroRNAs in Response to Retinoic Acid-Induced Spermatogonial Differentiation in Mice1

“…We found that both RARA and RARG interact with the Pou5f1 promoter in untreated P19 cells, but not in RA-treated P19 cells. This result is consistent with the previous observation that in P19 cells, the Pou5f1 promoter was occupied by RAR/RXR heterodimers before RA treatment, but this occupancy was lost following the treatment [41,42], demonstrating the specificity of our findings.…”

MicroRNA-302 Increases Reprogramming Efficiency via Repression of NR2F2

“…We found that Lv‐shNR2F2 increased pOCT4‐Luc reporter activity (Fig. 3B), confirming previous studies showing that NR2F2 silences OCT4 by directly binding to its promoter [35]. We next transfected the promoter reporter (p302‐Luc) into hESCs along with a lentivirus expressing NR2F2 (Lv‐NR2F2) or Lv‐GFP (control).…”

Role of COUP‐TFI during retinoic acid‐induced differentiation of P19 cells to endodermal cells

“…Consistent with the findings of Ben‐Shushan et al (1995), COUP‐TFI and COUP‐TFII mRNA levels inP19 cells were found to be highly elevated upon RA treatment with the maximal increase after 72 h. In addition, only variant 1 of COUP‐TFII was found to display this RA‐dependent increase in mRNA levels. Furthermore, the increase in COUP‐TFI and COUP‐TFII variant 1 mRNA levels is not a direct response to RA but rather requires the RA‐dependent increase in PBX expression.…”

Expression of Mirlet7 Family MicroRNAs in Response to Retinoic Acid-Induced Spermatogonial Differentiation in Mice1

“…We found that both RARA and RARG interact with the Pou5f1 promoter in untreated P19 cells, but not in RA-treated P19 cells. This result is consistent with the previous observation that in P19 cells, the Pou5f1 promoter was occupied by RAR/RXR heterodimers before RA treatment, but this occupancy was lost following the treatment [41,42], demonstrating the specificity of our findings.…”