Abstract
The erythropoietin (Epo) gene is regulated by hypoxia-inducible cis-acting elements in the promoter and in a 3' enhancer, both of which contain consensus hexanucleotide hormone receptor response elements which are important for function. A group of 11 orphan nuclear receptors, transcribed and translated in vitro, were screened by the electrophoretic mobility shift assay. Of these, hepatic nuclear factor 4 (HNF-4), TR2-11, ROR alpha 1, and EAR3/COUP-TF1 bound specifically to the response elements in the Epo promoter and enhancer and, except for ROR alpha 1, formed DNA-protein complexes that had mobilities similar to those observed in nuclear extracts of the Epo-producing cell line Hep3B. Moreover, both anti-HNF-4 and anti-COUP antibodies were able to supershift complexes in Hep3B nuclear extracts. Like Epo, HNF-4 is expressed in kidney, liver, and Hep3B cells but not in HeLa cells. Transfection of a plasmid expressing HNF-4 into HeLa cells enabled an eightfold increase in the hypoxic induction of a luciferase reporter construct which contains the minimal Epo enhancer and Epo promoter, provided that the nuclear hormone receptor consensus DNA elements in both the promoter and the enhancer were intact. The augmentation by HNF-4 in HeLa cells could be abrogated by cotransfection with HNF-4 delta C, which retains the DNA binding domain of HNF-4 but lacks the C-terminal activation domain. Moreover, the hypoxia-induced expression of the endogenous Epo gene was significantly inhibited in Hep3B cells stably transfected with HNF-4 delta C. On the other hand, cotransfection of EAR3/COUP-TF1 and the Epo reporter either with HNF-4 into HeLa cells or alone into Hep3B cells suppressed the hypoxia induction of the Epo reporter. These electrophoretic mobility shift assay and functional experiments indicate that HNF-4 plays a critical positive role in the tissue-specific and hypoxia-inducible expression of the Epo gene, whereas the COUP family has a negative modulatory role.
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- Baes M., Gulick T., Choi H. S., Martinoli M. G., Simha D., Moore D. D. A new orphan member of the nuclear hormone receptor superfamily that interacts with a subset of retinoic acid response elements. Mol Cell Biol. 1994 Mar;14(3):1544–1552. doi: 10.1128/mcb.14.3.1544. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Beck I., Ramirez S., Weinmann R., Caro J. Enhancer element at the 3'-flanking region controls transcriptional response to hypoxia in the human erythropoietin gene. J Biol Chem. 1991 Aug 25;266(24):15563–15566. [PubMed] [Google Scholar]
- Beck I., Weinmann R., Caro J. Characterization of hypoxia-responsive enhancer in the human erythropoietin gene shows presence of hypoxia-inducible 120-Kd nuclear DNA-binding protein in erythropoietin-producing and nonproducing cells. Blood. 1993 Aug 1;82(3):704–711. [PubMed] [Google Scholar]
- Blanchard K. L., Acquaviva A. M., Galson D. L., Bunn H. F. Hypoxic induction of the human erythropoietin gene: cooperation between the promoter and enhancer, each of which contains steroid receptor response elements. Mol Cell Biol. 1992 Dec;12(12):5373–5385. doi: 10.1128/mcb.12.12.5373. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chang C., Kokontis J., Acakpo-Satchivi L., Liao S., Takeda H., Chang Y. Molecular cloning of new human TR2 receptors: a class of steroid receptor with multiple ligand-binding domains. Biochem Biophys Res Commun. 1989 Dec 15;165(2):735–741. doi: 10.1016/s0006-291x(89)80028-2. [DOI] [PubMed] [Google Scholar]
- Chang C., Kokontis J. Identification of a new member of the steroid receptor super-family by cloning and sequence analysis. Biochem Biophys Res Commun. 1988 Sep 15;155(2):971–977. doi: 10.1016/s0006-291x(88)80591-6. [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]
- Evans R. M. The steroid and thyroid hormone receptor superfamily. Science. 1988 May 13;240(4854):889–895. doi: 10.1126/science.3283939. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Giguère V., Tini M., Flock G., Ong E., Evans R. M., Otulakowski G. Isoform-specific amino-terminal domains dictate DNA-binding properties of ROR alpha, a novel family of orphan hormone nuclear receptors. Genes Dev. 1994 Mar 1;8(5):538–553. doi: 10.1101/gad.8.5.538. [DOI] [PubMed] [Google Scholar]
- Glass C. K., DiRenzo J., Kurokawa R., Han Z. H. Regulation of gene expression by retinoic acid receptors. DNA Cell Biol. 1991 Nov;10(9):623–638. doi: 10.1089/dna.1991.10.623. [DOI] [PubMed] [Google Scholar]
- Goldberg M. A., Gaut C. C., Bunn H. F. Erythropoietin mRNA levels are governed by both the rate of gene transcription and posttranscriptional events. Blood. 1991 Jan 15;77(2):271–277. [PubMed] [Google Scholar]
- Goldberg M. A., Glass G. A., Cunningham J. M., Bunn H. F. The regulated expression of erythropoietin by two human hepatoma cell lines. Proc Natl Acad Sci U S A. 1987 Nov;84(22):7972–7976. doi: 10.1073/pnas.84.22.7972. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Imagawa S., Goldberg M. A., Doweiko J., Bunn H. F. Regulatory elements of the erythropoietin gene. Blood. 1991 Jan 15;77(2):278–285. [PubMed] [Google Scholar]
- Jacobs K., Shoemaker C., Rudersdorf R., Neill S. D., Kaufman R. J., Mufson A., Seehra J., Jones S. S., Hewick R., Fritsch E. F. Isolation and characterization of genomic and cDNA clones of human erythropoietin. 1985 Feb 28-Mar 6Nature. 313(6005):806–810. doi: 10.1038/313806a0. [DOI] [PubMed] [Google Scholar]
- Jelkmann W. Erythropoietin: structure, control of production, and function. Physiol Rev. 1992 Apr;72(2):449–489. doi: 10.1152/physrev.1992.72.2.449. [DOI] [PubMed] [Google Scholar]
- Kimura A., Nishiyori A., Murakami T., Tsukamoto T., Hata S., Osumi T., Okamura R., Mori M., Takiguchi M. Chicken ovalbumin upstream promoter-transcription factor (COUP-TF) represses transcription from the promoter of the gene for ornithine transcarbamylase in a manner antagonistic to hepatocyte nuclear factor-4 (HNF-4). J Biol Chem. 1993 May 25;268(15):11125–11133. [PubMed] [Google Scholar]
- Kliewer S. A., Umesono K., Heyman R. A., Mangelsdorf D. J., Dyck J. A., Evans R. M. Retinoid X receptor-COUP-TF interactions modulate retinoic acid signaling. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1448–1452. doi: 10.1073/pnas.89.4.1448. [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]
- Kliewer S. A., Umesono K., Noonan D. J., Heyman R. A., Evans R. M. Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors. Nature. 1992 Aug 27;358(6389):771–774. doi: 10.1038/358771a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
- Ladias J. A., Hadzopoulou-Cladaras M., Kardassis D., Cardot P., Cheng J., Zannis V., Cladaras C. Transcriptional regulation of human apolipoprotein genes ApoB, ApoCIII, and ApoAII by members of the steroid hormone receptor superfamily HNF-4, ARP-1, EAR-2, and EAR-3. J Biol Chem. 1992 Aug 5;267(22):15849–15860. [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]
- Lin F. K., Suggs S., Lin C. H., Browne J. K., Smalling R., Egrie J. C., Chen K. K., Fox G. M., Martin F., Stabinsky Z. Cloning and expression of the human erythropoietin gene. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7580–7584. doi: 10.1073/pnas.82.22.7580. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Madan A., Curtin P. T. A 24-base-pair sequence 3' to the human erythropoietin gene contains a hypoxia-responsive transcriptional enhancer. Proc Natl Acad Sci U S A. 1993 May 1;90(9):3928–3932. doi: 10.1073/pnas.90.9.3928. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Maxwell P. H., Pugh C. W., Ratcliffe P. J. Inducible operation of the erythropoietin 3' enhancer in multiple cell lines: evidence for a widespread oxygen-sensing mechanism. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2423–2427. doi: 10.1073/pnas.90.6.2423. [DOI] [PMC free article] [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]
- Miyajima N., Kadowaki Y., Fukushige S., Shimizu S., Semba K., Yamanashi Y., Matsubara K., Toyoshima K., Yamamoto T. Identification of two novel members of erbA superfamily by molecular cloning: the gene products of the two are highly related to each other. Nucleic Acids Res. 1988 Dec 9;16(23):11057–11074. doi: 10.1093/nar/16.23.11057. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Miyata K. S., Zhang B., Marcus S. L., Capone J. P., Rachubinski R. A. Chicken ovalbumin upstream promoter transcription factor (COUP-TF) binds to a peroxisome proliferator-responsive element and antagonizes peroxisome proliferator-mediated signaling. J Biol Chem. 1993 Sep 15;268(26):19169–19172. [PubMed] [Google Scholar]
- Nakshatri H., Chambon P. The directly repeated RG(G/T)TCA motifs of the rat and mouse cellular retinol-binding protein II genes are promiscuous binding sites for RAR, RXR, HNF-4, and ARP-1 homo- and heterodimers. J Biol Chem. 1994 Jan 14;269(2):890–902. [PubMed] [Google Scholar]
- Nordeen S. K. Luciferase reporter gene vectors for analysis of promoters and enhancers. Biotechniques. 1988 May;6(5):454–458. [PubMed] [Google Scholar]
- Porter D. L., Goldberg M. A. Regulation of erythropoietin production. Exp Hematol. 1993 Mar;21(3):399–404. [PubMed] [Google Scholar]
- Pugh C. W., Tan C. C., Jones R. W., Ratcliffe P. J. Functional analysis of an oxygen-regulated transcriptional enhancer lying 3' to the mouse erythropoietin gene. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10553–10557. doi: 10.1073/pnas.88.23.10553. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schuster S. J., Badiavas E. V., Costa-Giomi P., Weinmann R., Erslev A. J., Caro J. Stimulation of erythropoietin gene transcription during hypoxia and cobalt exposure. Blood. 1989 Jan;73(1):13–16. [PubMed] [Google Scholar]
- Semenza G. L., Dureza R. C., Traystman M. D., Gearhart J. D., Antonarakis S. E. Human erythropoietin gene expression in transgenic mice: multiple transcription initiation sites and cis-acting regulatory elements. Mol Cell Biol. 1990 Mar;10(3):930–938. doi: 10.1128/mcb.10.3.930. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Semenza G. L., Koury S. T., Nejfelt M. K., Gearhart J. D., Antonarakis S. E. Cell-type-specific and hypoxia-inducible expression of the human erythropoietin gene in transgenic mice. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8725–8729. doi: 10.1073/pnas.88.19.8725. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Semenza G. L., Nejfelt M. K., Chi S. M., Antonarakis S. E. Hypoxia-inducible nuclear factors bind to an enhancer element located 3' to the human erythropoietin gene. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5680–5684. doi: 10.1073/pnas.88.13.5680. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Semenza G. L., Traystman M. D., Gearhart J. D., Antonarakis S. E. Polycythemia in transgenic mice expressing the human erythropoietin gene. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2301–2305. doi: 10.1073/pnas.86.7.2301. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Semenza G. L., Wang G. L. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol Cell Biol. 1992 Dec;12(12):5447–5454. doi: 10.1128/mcb.12.12.5447. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shapiro D. J., Sharp P. A., Wahli W. W., Keller M. J. A high-efficiency HeLa cell nuclear transcription extract. DNA. 1988 Jan-Feb;7(1):47–55. doi: 10.1089/dna.1988.7.47. [DOI] [PubMed] [Google Scholar]
- Sladek F. M. Orphan receptor HNF-4 and liver-specific gene expression. Receptor. 1993 Fall;3(3):223–232. [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]
- Thomas H. E., Stunnenberg H. G., Stewart A. F. Heterodimerization of the Drosophila ecdysone receptor with retinoid X receptor and ultraspiracle. Nature. 1993 Apr 1;362(6419):471–475. doi: 10.1038/362471a0. [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]
- Umesono K., Murakami K. K., Thompson C. C., Evans R. M. Direct repeats as selective response elements for the thyroid hormone, retinoic acid, and vitamin D3 receptors. Cell. 1991 Jun 28;65(7):1255–1266. doi: 10.1016/0092-8674(91)90020-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wahli W., Martinez E. Superfamily of steroid nuclear receptors: positive and negative regulators of gene expression. FASEB J. 1991 Jun;5(9):2243–2249. doi: 10.1096/fasebj.5.9.1860615. [DOI] [PubMed] [Google Scholar]
- Wang G. L., Semenza G. L. Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia. J Biol Chem. 1993 Oct 15;268(29):21513–21518. [PubMed] [Google Scholar]
- Wang G. L., Semenza G. L. General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia. Proc Natl Acad Sci U S A. 1993 May 1;90(9):4304–4308. doi: 10.1073/pnas.90.9.4304. [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]
- de Wet J. R., Wood K. V., DeLuca M., Helinski D. R., Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987 Feb;7(2):725–737. doi: 10.1128/mcb.7.2.725. [DOI] [PMC free article] [PubMed] [Google Scholar]