Abstract
The Oct-3/4 gene product, which belongs to the POU family of transcription factors, is a good candidate for regulating initial differentiation decisions. It is expressed in the earliest stages of embryogenesis and repressed in subsequent stages. Retinoic acid (RA)-induced differentiation of embryonal carcinoma (EC) cells is accompanied by decreased expression of the Oct-3/4 gene. Previous findings show that sequences in the Oct-3/4 enhancer region (designated RARE1) are targets for RA-mediated repression (H. Okazawa, K. Okamoto, F. Ishino, T. Ishino-Kaneko, S. Takeda, Y. Toyoda, M. Muramatsu, and H. Hamada, EMBO J. 10:2997-3005, 1991). Our present results demonstrate conclusively that the TATA-less Oct-3/4 promoter is also a target for RA-induced repression. We identified a novel cis element in the Oct-3/4 promoter harbors a putative Sp1 binding site and a RA-responsive element (designated RAREoct), which are juxtaposed to one another. Protein binding to the Sp1 site is independent of protein binding to the RAREoct sequence. Unlike the RARE1 situated in the Oct-3/4 enhancer which does not contain a typical RAR recognition site, the RAREoct identified in this study consists of three directly repeated motifs that exhibit extensive homology to RARE sequences located in RA-responsive genes. Moreover, the RAREoct shows different DNA-binding characteristics and DNase I footprint patterns with nuclear proteins isolated from undifferentiated versus RA-differentiated EC cells. This suggests that the RAREoct element binds different nuclear proteins in RA-treated and untreated EC cells which most probably belong to the RA receptor, retinoid X receptor, or orphan receptor families of transcription factors. Using site-directed mutagenesis, we show that the RAREoct contributes to the transcriptional activation of Oct-3/4 promoter in P19 cells and, most interestingly, mediates the RA-induced repression in RA-differentiated EC cells. Thus, the RAREoct element could be one of the points of integration of several signalling pathways influencing Oct-3/4 expression. In accordance with the suggestion that suppression of Oct-3/4 expression is a crucial step during embryogenesis, the Oct-3/4 upstream region contains multiple targets for RA-induced repression, probably to ensure accurate and prompt repression of Oct-3/4 expression. It is possible that these repressors are differentially used at specific stages of development in response to various signals.
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- Baniahmad A., Köhne A. C., Renkawitz R. A transferable silencing domain is present in the thyroid hormone receptor, in the v-erbA oncogene product and in the retinoic acid receptor. EMBO J. 1992 Mar;11(3):1015–1023. doi: 10.1002/j.1460-2075.1992.tb05140.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ben-Shushan E., Pikarsky E., Klar A., Bergman Y. Extinction of Oct-3/4 gene expression in embryonal carcinoma x fibroblast somatic cell hybrids is accompanied by changes in the methylation status, chromatin structure, and transcriptional activity of the Oct-3/4 upstream region. Mol Cell Biol. 1993 Feb;13(2):891–901. doi: 10.1128/mcb.13.2.891. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
- Cooney A. J., Tsai S. Y., O'Malley B. W., Tsai M. J. Chicken ovalbumin upstream promoter transcription factor (COUP-TF) dimers bind to different GGTCA response elements, allowing COUP-TF to repress hormonal induction of the vitamin D3, thyroid hormone, and retinoic acid receptors. Mol Cell Biol. 1992 Sep;12(9):4153–4163. doi: 10.1128/mcb.12.9.4153. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Durand B., Saunders M., Leroy P., Leid M., Chambon P. All-trans and 9-cis retinoic acid induction of CRABPII transcription is mediated by RAR-RXR heterodimers bound to DR1 and DR2 repeated motifs. Cell. 1992 Oct 2;71(1):73–85. doi: 10.1016/0092-8674(92)90267-g. [DOI] [PubMed] [Google Scholar]
- Fridovich-Keil J. L., Gudas J. M., Bryan I. B., Pardee A. B. Improved expression vectors for eukaryotic promoter/enhancer studies. Biotechniques. 1991 Nov;11(5):572–579. [PubMed] [Google Scholar]
- Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Garner M. M., Revzin A. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 1981 Jul 10;9(13):3047–3060. doi: 10.1093/nar/9.13.3047. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gorman C. M., Merlino G. T., Willingham M. C., Pastan I., Howard B. H. The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6777–6781. doi: 10.1073/pnas.79.22.6777. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Heyman R. A., Mangelsdorf D. J., Dyck J. A., Stein R. B., Eichele G., Evans R. M., Thaller C. 9-cis retinoic acid is a high affinity ligand for the retinoid X receptor. Cell. 1992 Jan 24;68(2):397–406. doi: 10.1016/0092-8674(92)90479-v. [DOI] [PubMed] [Google Scholar]
- Husmann M., Lehmann J., Hoffmann B., Hermann T., Tzukerman M., Pfahl M. Antagonism between retinoic acid receptors. Mol Cell Biol. 1991 Aug;11(8):4097–4103. doi: 10.1128/mcb.11.8.4097. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Janson L., Pettersson U. Cooperative interactions between transcription factors Sp1 and OTF-1. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4732–4736. doi: 10.1073/pnas.87.12.4732. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kastner P., Krust A., Mendelsohn C., Garnier J. M., Zelent A., Leroy P., Staub A., Chambon P. Murine isoforms of retinoic acid receptor gamma with specific patterns of expression. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2700–2704. doi: 10.1073/pnas.87.7.2700. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kliewer S. A., Umesono K., Mangelsdorf D. J., Evans R. M. Retinoid X receptor interacts with nuclear receptors in retinoic acid, thyroid hormone and vitamin D3 signalling. Nature. 1992 Jan 30;355(6359):446–449. doi: 10.1038/355446a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ladias J. A., Karathanasis S. K. Regulation of the apolipoprotein AI gene by ARP-1, a novel member of the steroid receptor superfamily. Science. 1991 Feb 1;251(4993):561–565. doi: 10.1126/science.1899293. [DOI] [PubMed] [Google Scholar]
- Lehmann J. M., Zhang X. K., Pfahl M. RAR gamma 2 expression is regulated through a retinoic acid response element embedded in Sp1 sites. Mol Cell Biol. 1992 Jul;12(7):2976–2985. doi: 10.1128/mcb.12.7.2976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Leid M., Kastner P., Lyons R., Nakshatri H., Saunders M., Zacharewski T., Chen J. Y., Staub A., Garnier J. M., Mader S. Purification, cloning, and RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently. Cell. 1992 Jan 24;68(2):377–395. doi: 10.1016/0092-8674(92)90478-u. [DOI] [PubMed] [Google Scholar]
- Leroy P., Krust A., Zelent A., Mendelsohn C., Garnier J. M., Kastner P., Dierich A., Chambon P. Multiple isoforms of the mouse retinoic acid receptor alpha are generated by alternative splicing and differential induction by retinoic acid. EMBO J. 1991 Jan;10(1):59–69. doi: 10.1002/j.1460-2075.1991.tb07921.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Levin A. A., Sturzenbecker L. J., Kazmer S., Bosakowski T., Huselton C., Allenby G., Speck J., Kratzeisen C., Rosenberger M., Lovey A. 9-cis retinoic acid stereoisomer binds and activates the nuclear receptor RXR alpha. Nature. 1992 Jan 23;355(6358):359–361. doi: 10.1038/355359a0. [DOI] [PubMed] [Google Scholar]
- Lipkin S. M., Nelson C. A., Glass C. K., Rosenfeld M. G. A negative retinoic acid response element in the rat oxytocin promoter restricts transcriptional stimulation by heterologous transactivation domains. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1209–1213. doi: 10.1073/pnas.89.4.1209. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lucas P. C., O'Brien R. M., Mitchell J. A., Davis C. M., Imai E., Forman B. M., Samuels H. H., Granner D. K. A retinoic acid response element is part of a pleiotropic domain in the phosphoenolpyruvate carboxykinase gene. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2184–2188. doi: 10.1073/pnas.88.6.2184. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mangelsdorf D. J., Borgmeyer U., Heyman R. A., Zhou J. Y., Ong E. S., Oro A. E., Kakizuka A., Evans R. M. Characterization of three RXR genes that mediate the action of 9-cis retinoic acid. Genes Dev. 1992 Mar;6(3):329–344. doi: 10.1101/gad.6.3.329. [DOI] [PubMed] [Google Scholar]
- Mangelsdorf D. J., Umesono K., Kliewer S. A., Borgmeyer U., Ong E. S., Evans R. M. A direct repeat in the cellular retinol-binding protein type II gene confers differential regulation by RXR and RAR. Cell. 1991 Aug 9;66(3):555–561. doi: 10.1016/0092-8674(81)90018-0. [DOI] [PubMed] [Google Scholar]
- Marks M. S., Hallenbeck P. L., Nagata T., Segars J. H., Appella E., Nikodem V. M., Ozato K. H-2RIIBP (RXR beta) heterodimerization provides a mechanism for combinatorial diversity in the regulation of retinoic acid and thyroid hormone responsive genes. EMBO J. 1992 Apr;11(4):1419–1435. doi: 10.1002/j.1460-2075.1992.tb05187.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
- McBurney M. W., Rogers B. J. Isolation of male embryonal carcinoma cells and their chromosome replication patterns. Dev Biol. 1982 Feb;89(2):503–508. doi: 10.1016/0012-1606(82)90338-4. [DOI] [PubMed] [Google Scholar]
- Mendelsohn C., Ruberte E., Chambon P. Retinoid receptors in vertebrate limb development. Dev Biol. 1992 Jul;152(1):50–61. doi: 10.1016/0012-1606(92)90155-a. [DOI] [PubMed] [Google Scholar]
- Mietus-Snyder M., Sladek F. M., Ginsburg G. S., Kuo C. F., Ladias J. A., Darnell J. E., Jr, Karathanasis S. K. Antagonism between apolipoprotein AI regulatory protein 1, Ear3/COUP-TF, and hepatocyte nuclear factor 4 modulates apolipoprotein CIII gene expression in liver and intestinal cells. Mol Cell Biol. 1992 Apr;12(4):1708–1718. doi: 10.1128/mcb.12.4.1708. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nagata T., Segars J. H., Levi B. Z., Ozato K. Retinoic acid-dependent transactivation of major histocompatibility complex class I promoters by the nuclear hormone receptor H-2RIIBP in undifferentiated embryonal carcinoma cells. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):937–941. doi: 10.1073/pnas.89.3.937. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nagpal S., Saunders M., Kastner P., Durand B., Nakshatri H., Chambon P. Promoter context- and response element-dependent specificity of the transcriptional activation and modulating functions of retinoic acid receptors. Cell. 1992 Sep 18;70(6):1007–1019. doi: 10.1016/0092-8674(92)90250-g. [DOI] [PubMed] [Google Scholar]
- Nicholson R. C., Mader S., Nagpal S., Leid M., Rochette-Egly C., Chambon P. Negative regulation of the rat stromelysin gene promoter by retinoic acid is mediated by an AP1 binding site. EMBO J. 1990 Dec;9(13):4443–4454. doi: 10.1002/j.1460-2075.1990.tb07895.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Okamoto K., Okazawa H., Okuda A., Sakai M., Muramatsu M., Hamada H. A novel octamer binding transcription factor is differentially expressed in mouse embryonic cells. Cell. 1990 Feb 9;60(3):461–472. doi: 10.1016/0092-8674(90)90597-8. [DOI] [PubMed] [Google Scholar]
- Okazawa H., Okamoto K., Ishino F., Ishino-Kaneko T., Takeda S., Toyoda Y., Muramatsu M., Hamada H. The oct3 gene, a gene for an embryonic transcription factor, is controlled by a retinoic acid repressible enhancer. EMBO J. 1991 Oct;10(10):2997–3005. doi: 10.1002/j.1460-2075.1991.tb07850.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Reue K., Leff T., Breslow J. L. Human apolipoprotein CIII gene expression is regulated by positive and negative cis-acting elements and tissue-specific protein factors. J Biol Chem. 1988 May 15;263(14):6857–6864. [PubMed] [Google Scholar]
- Robidoux S., Gosselin P., Harvey M., Leclerc S., Guérin S. L. Transcription of the mouse secretory protease inhibitor p12 gene is activated by the developmentally regulated positive transcription factor Sp1. Mol Cell Biol. 1992 Sep;12(9):3796–3806. doi: 10.1128/mcb.12.9.3796. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rosenfeld M. G. POU-domain transcription factors: pou-er-ful developmental regulators. Genes Dev. 1991 Jun;5(6):897–907. doi: 10.1101/gad.5.6.897. [DOI] [PubMed] [Google Scholar]
- Rosner M. H., Vigano M. A., Ozato K., Timmons P. M., Poirier F., Rigby P. W., Staudt L. M. A POU-domain transcription factor in early stem cells and germ cells of the mammalian embryo. Nature. 1990 Jun 21;345(6277):686–692. doi: 10.1038/345686a0. [DOI] [PubMed] [Google Scholar]
- Rottman J. N., Widom R. L., Nadal-Ginard B., Mahdavi V., Karathanasis S. K. A retinoic acid-responsive element in the apolipoprotein AI gene distinguishes between two different retinoic acid response pathways. Mol Cell Biol. 1991 Jul;11(7):3814–3820. doi: 10.1128/mcb.11.7.3814. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ruvkun G., Finney M. Regulation of transcription and cell identity by POU domain proteins. Cell. 1991 Feb 8;64(3):475–478. doi: 10.1016/0092-8674(91)90227-p. [DOI] [PubMed] [Google Scholar]
- Saffer J. D., Jackson S. P., Annarella M. B. Developmental expression of Sp1 in the mouse. Mol Cell Biol. 1991 Apr;11(4):2189–2199. doi: 10.1128/mcb.11.4.2189. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schöler H. R., Balling R., Hatzopoulos A. K., Suzuki N., Gruss P. Octamer binding proteins confer transcriptional activity in early mouse embryogenesis. EMBO J. 1989 Sep;8(9):2551–2557. doi: 10.1002/j.1460-2075.1989.tb08393.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schöler H. R., Dressler G. R., Balling R., Rohdewohld H., Gruss P. Oct-4: a germline-specific transcription factor mapping to the mouse t-complex. EMBO J. 1990 Jul;9(7):2185–2195. doi: 10.1002/j.1460-2075.1990.tb07388.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schöler H. R., Ruppert S., Suzuki N., Chowdhury K., Gruss P. New type of POU domain in germ line-specific protein Oct-4. Nature. 1990 Mar 29;344(6265):435–439. doi: 10.1038/344435a0. [DOI] [PubMed] [Google Scholar]
- Schüle R., Rangarajan P., Yang N., Kliewer S., Ransone L. J., Bolado J., Verma I. M., Evans R. M. Retinoic acid is a negative regulator of AP-1-responsive genes. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6092–6096. doi: 10.1073/pnas.88.14.6092. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sladek F. M., Zhong W. M., Lai E., Darnell J. E., Jr Liver-enriched transcription factor HNF-4 is a novel member of the steroid hormone receptor superfamily. Genes Dev. 1990 Dec;4(12B):2353–2365. doi: 10.1101/gad.4.12b.2353. [DOI] [PubMed] [Google Scholar]
- Smith W. C., Nakshatri H., Leroy P., Rees J., Chambon P. A retinoic acid response element is present in the mouse cellular retinol binding protein I (mCRBPI) promoter. EMBO J. 1991 Aug;10(8):2223–2230. doi: 10.1002/j.1460-2075.1991.tb07758.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sucov H. M., Murakami K. K., Evans R. M. Characterization of an autoregulated response element in the mouse retinoic acid receptor type beta gene. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5392–5396. doi: 10.1073/pnas.87.14.5392. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tabin C. J. Retinoids, homeoboxes, and growth factors: toward molecular models for limb development. Cell. 1991 Jul 26;66(2):199–217. doi: 10.1016/0092-8674(91)90612-3. [DOI] [PubMed] [Google Scholar]
- Tran P., Zhang X. K., Salbert G., Hermann T., Lehmann J. M., Pfahl M. COUP orphan receptors are negative regulators of retinoic acid response pathways. Mol Cell Biol. 1992 Oct;12(10):4666–4676. doi: 10.1128/mcb.12.10.4666. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vasios G. W., Gold J. D., Petkovich M., Chambon P., Gudas L. J. A retinoic acid-responsive element is present in the 5' flanking region of the laminin B1 gene. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9099–9103. doi: 10.1073/pnas.86.23.9099. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang L. H., Tsai S. Y., Cook R. G., Beattie W. G., Tsai M. J., O'Malley B. W. COUP transcription factor is a member of the steroid receptor superfamily. Nature. 1989 Jul 13;340(6229):163–166. doi: 10.1038/340163a0. [DOI] [PubMed] [Google Scholar]
- Widom R. L., Rhee M., Karathanasis S. K. Repression by ARP-1 sensitizes apolipoprotein AI gene responsiveness to RXR alpha and retinoic acid. Mol Cell Biol. 1992 Aug;12(8):3380–3389. doi: 10.1128/mcb.12.8.3380. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wigler M., Pellicer A., Silverstein S., Axel R., Urlaub G., Chasin L. DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1373–1376. doi: 10.1073/pnas.76.3.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yu V. C., Delsert C., Andersen B., Holloway J. M., Devary O. V., När A. M., Kim S. Y., Boutin J. M., Glass C. K., Rosenfeld M. G. RXR beta: a coregulator that enhances binding of retinoic acid, thyroid hormone, and vitamin D receptors to their cognate response elements. Cell. 1991 Dec 20;67(6):1251–1266. doi: 10.1016/0092-8674(91)90301-e. [DOI] [PubMed] [Google Scholar]
- Zelent A., Mendelsohn C., Kastner P., Krust A., Garnier J. M., Ruffenach F., Leroy P., Chambon P. Differentially expressed isoforms of the mouse retinoic acid receptor beta generated by usage of two promoters and alternative splicing. EMBO J. 1991 Jan;10(1):71–81. doi: 10.1002/j.1460-2075.1991.tb07922.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhang X. K., Hoffmann B., Tran P. B., Graupner G., Pfahl M. Retinoid X receptor is an auxiliary protein for thyroid hormone and retinoic acid receptors. Nature. 1992 Jan 30;355(6359):441–446. doi: 10.1038/355441a0. [DOI] [PubMed] [Google Scholar]
- de Thé H., Vivanco-Ruiz M. M., Tiollais P., Stunnenberg H., Dejean A. Identification of a retinoic acid responsive element in the retinoic acid receptor beta gene. Nature. 1990 Jan 11;343(6254):177–180. doi: 10.1038/343177a0. [DOI] [PubMed] [Google Scholar]