Characterization of a negative retinoic acid response element in the murine Oct4 promoter.

Abstract

Expression of Oct4 in embryonic stem cells is controlled by a distal upstream stem cell-specific enhancer that is deactivated during retinoic acid (RA)-induced differentiation by an indirect mechanism not involving binding of RA receptors (H. Okazawa, K. Okamoto, F. Ishino, T. Ishino-Kaneko, S. Takeda, Y. Toyoda, M. Muramatsu, and H. Hamada, EMBO J. 10:2997EMBO J. 10: -3005, 1991). Here we report that in RA-treated P19 embryonal carcinoma cells the Oct4 promoter is also subject to negative regulation by RA. The minimal Oct4 promoter sequence mediating repression consists of a promoter-proximal sequence containing a GC-rich SP1 consensus-like sequence and several hormone response element half-sites that can be arranged into direct repeats with different spacing. The GC box binds a nuclear factor that is invariably present in undifferentiated and RA-treated differentiated P19 cells. By contrast, the hormone response element-containing sequence binds factors that are induced following RA treatment. Mutational analysis and competition experiments show that the functional entity binding the RA-induced factor is a direct repeat sequence with a spacing of one nucleotide, previously shown to be a binding site for COUP transcription factors (COUP-TFs). Cotransfected orphan receptors COUP-TF1, ARP-1, and EAR-2 were able to repress the activity of Oct4 promoter-driven reporters in P19 EC cells, albeit with different efficiencies. Furthermore, the negative transcriptional effect of COUP-TFs is dominant over the activating effect of the Oct4 embryonic stem cell-specific enhancer. These results show that negative regulation of Oct4 expression during RA-induced differentiation of embryonic stem cells is controlled by two different mechanisms, including deactivation of the embryonic stem cell-specific enhancer and promoter silencing by orphan nuclear hormone receptors.Among the gene families that play an important role during both vertebrate and invertebrate development is the POU family of transcriptional regulators. Their conserved DNAbinding motif, first recognized among the Pit-1/GHF-1, Octl and Oct2, and Unc83 proteins (27), is involved in binding to the octamer motif (18,95,96), present in the enhancer/ promoter of a variety of eukaryotic genes (77, 85). The Oct family presently consists of a score of members, including Octl (87), Oct2 (56,78), Oct4 (59,68,82), and Oct6 (53,55,88), that, with the exception of Octl, have been implicated in tissue-and stage-specific transcriptional regulation in line with their restricted expression patterns during early embryogenesis and in adult tissues (67,72,79). Expression of Oct4 during early mouse development is restricted to pluripotent stem cells as found in the inner cell mass of blastocyst stage embryos, the pluripotent primitive ectoderm, primordial germ cells, oocytes, and spermatids (68,80,81). In line with its expression in the inner cell mass and primitive neuroectoderm of early mouse embryos, the Oct4 gene is expressed at high levels in both embryonic stem (ES) cells and em...

Rex-1, a Gene Encoding a Transcription Factor Expressed in the Early Embryo, Is Regulated via Oct-3/4 and Oct-6 Binding to an Octamer Site and a Novel Protein, Rox-1, Binding to an Adjacent Site

“…Mutations either in the Octa site or in the Rox-1 binding sequences compromise the ability of Oct-3/4 to activate the Rex-1 promoter. Oct-3/4 protein possesses the ability to enhance expression of several additional genes, including the platelet-derived growth factor ␣ receptor (PDGF␣R [25]) and fibroblast growth factor (FGF-4) genes (10,31,54,75). Interestingly, nucleotides juxtaposed to the octamer motif were found to play a key role in positively (10 g) and a ␤-Gal ⌬DB-containing reference plasmid (1 g) in the absence (Ϫ) or presence of 10 g of wild-type Oct-6, N-terminus-deleted Oct-6 (N157 or N229), C-terminus-deleted Oct-6 (N2C52), and DNA-binding-domain-deleted Oct-6 (N229C52).…”

“…Oct-3/4 gene expression is regulated by RA through its enhancer and promoter elements (3,38,41,45,54,66,74). Whereas no RA-responsive elements (RARE) were identified in the enhancer region, the Oct-3/4 promoter contains a RARE motif (designated RAREoct) which contributes to transcriptional activation in EC cells and mediates the RAinduced repression in RA-differentiated EC cells (45,54).…”

“…Whereas no RA-responsive elements (RARE) were identified in the enhancer region, the Oct-3/4 promoter contains a RARE motif (designated RAREoct) which contributes to transcriptional activation in EC cells and mediates the RAinduced repression in RA-differentiated EC cells (45,54). Inhibition of Oct-3/4 expression was shown to occur through the binding of ARP-I/COUP-TFII and EAR-3/COUP-TFI orphan receptors to the RAREoct site.…”

“…Inhibition of Oct-3/4 expression was shown to occur through the binding of ARP-I/COUP-TFII and EAR-3/COUP-TFI orphan receptors to the RAREoct site. These orphan receptors bind to the RAREoct site with a high affinity and actively silence the promoter activity (3,54,66).…”

Non-cell autonomous induction of apoptosis and loss of posterior structures by activation domain-specific interactions of Oct-1 in the Xenopus embryo

“…0.75 fmole RNA corresponds to respectively 703 pg antisense RNA, 703 pg NPC RNA, 418 pg NP RNA, 510 pg PC RNA, 215 pg P RNA, 405 pg C RNA. For promoter analysis, 50 pg pXSna-Luc and 50 pg b-actin-CAT (Schoorlemmer et al, 1994), and 0.75 fmole Oct-1 RNA was co-injected. In most experiments, injections were made in the animal half of the embryo during the first cell cycle of the embryo.…”