Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1996 Dec 1;24(23):4768–4774. doi: 10.1093/nar/24.23.4768

The role of the near upstream sequence in hypoxia-induced expression of the erythropoietin gene.

M Gupta 1, E Goldwasser 1
PMCID: PMC146316  PMID: 8972864

Abstract

Transcription of the erythropoietin (epo) gene is regulated in response to tissue hypoxia. In this study we show that constructs containing 117 bp of the epo promoter sequence cloned upstream of a luciferase reporter, respond to hypoxia when transfected into the human hepatoma cell line, Hep3B. The sequence -61 to -45 (EP17) relative to the transcription start of the murine epo gene imparted an approximately 4-fold induction of reporter gene expression due to hypoxia. Internal deletion of EP17 resulted in loss of induction by hypoxia without altering basal expression of the 117 bp epo promoter reporter construct. Mutagenesis studies showed that the bases at positions -53, -59, from -49 to -51 and from -55 to -57 are essential for hypoxic induction. The EP17 sequence is required for the 3' enhancer element of the epo gene to be maximally functional. Gel shift and UV cross-linking experiments showed the presence in Hep3B nuclear extracts, of two protein factors with approximate molecular weights of 52 kDa and 25 kDa that bind to EP17. Introduction of specific mutations in the EP17 region that abolish induction by hypoxia, also eliminated the binding of one or both of these factors. These experiments demonstrate a role for the proximal region of the epo promoter in hypoxic induction of the epo gene.

Full Text

The Full Text of this article is available as a PDF (195.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. 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]
  2. Beru N., McDonald J., Lacombe C., Goldwasser E. Expression of the erythropoietin gene. Mol Cell Biol. 1986 Jul;6(7):2571–2575. doi: 10.1128/mcb.6.7.2571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beru N., Smith D., Goldwasser E. Evidence suggesting negative regulation of the erythropoietin gene by ribonucleoprotein. J Biol Chem. 1990 Aug 25;265(24):14100–14104. [PubMed] [Google Scholar]
  4. 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]
  5. D'Andrea A. D., Lodish H. F., Wong G. G. Expression cloning of the murine erythropoietin receptor. Cell. 1989 Apr 21;57(2):277–285. doi: 10.1016/0092-8674(89)90965-3. [DOI] [PubMed] [Google Scholar]
  6. DesJardins E., Hay N. Repeated CT elements bound by zinc finger proteins control the absolute and relative activities of the two principal human c-myc promoters. Mol Cell Biol. 1993 Sep;13(9):5710–5724. doi: 10.1128/mcb.13.9.5710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Florini J. R., Magri K. A., Ewton D. Z., James P. L., Grindstaff K., Rotwein P. S. "Spontaneous" differentiation of skeletal myoblasts is dependent upon autocrine secretion of insulin-like growth factor-II. J Biol Chem. 1991 Aug 25;266(24):15917–15923. [PubMed] [Google Scholar]
  8. Galson D. L., Tsuchiya T., Tendler D. S., Huang L. E., Ren Y., Ogura T., Bunn H. F. The orphan receptor hepatic nuclear factor 4 functions as a transcriptional activator for tissue-specific and hypoxia-specific erythropoietin gene expression and is antagonized by EAR3/COUP-TF1. Mol Cell Biol. 1995 Apr;15(4):2135–2144. doi: 10.1128/mcb.15.4.2135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Goldberg M. A., Dunning S. P., Bunn H. F. Regulation of the erythropoietin gene: evidence that the oxygen sensor is a heme protein. Science. 1988 Dec 9;242(4884):1412–1415. doi: 10.1126/science.2849206. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Grueneberg D. A., Natesan S., Alexandre C., Gilman M. Z. Human and Drosophila homeodomain proteins that enhance the DNA-binding activity of serum response factor. Science. 1992 Aug 21;257(5073):1089–1095. doi: 10.1126/science.257.5073.1089. [DOI] [PubMed] [Google Scholar]
  12. Gumucio D. L., Rood K. L., Blanchard-McQuate K. L., Gray T. A., Saulino A., Collins F. S. Interaction of Sp1 with the human gamma globin promoter: binding and transactivation of normal and mutant promoters. Blood. 1991 Oct 1;78(7):1853–1863. [PubMed] [Google Scholar]
  13. Gupta M. P., Gupta M., Zak R. An E-box/M-CAT hybrid motif and cognate binding protein(s) regulate the basal muscle-specific and cAMP-inducible expression of the rat cardiac alpha-myosin heavy chain gene. J Biol Chem. 1994 Nov 25;269(47):29677–29687. [PubMed] [Google Scholar]
  14. Hagen G., Müller S., Beato M., Suske G. Cloning by recognition site screening of two novel GT box binding proteins: a family of Sp1 related genes. Nucleic Acids Res. 1992 Nov 11;20(21):5519–5525. doi: 10.1093/nar/20.21.5519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Imagawa S., Izumi T., Miura Y. Positive and negative regulation of the erythropoietin gene. J Biol Chem. 1994 Mar 25;269(12):9038–9044. [PubMed] [Google Scholar]
  16. JACOBSON L. O., GOLDWASSER E., FRIED W., PLZAK L. Role of the kidney in erythropoiesis. Nature. 1957 Mar 23;179(4560):633–634. doi: 10.1038/179633a0. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Jones N. Transcriptional regulation by dimerization: two sides to an incestuous relationship. Cell. 1990 Apr 6;61(1):9–11. doi: 10.1016/0092-8674(90)90207-u. [DOI] [PubMed] [Google Scholar]
  19. Lobanenkov V. V., Nicolas R. H., Adler V. V., Paterson H., Klenova E. M., Polotskaja A. V., Goodwin G. H. A novel sequence-specific DNA binding protein which interacts with three regularly spaced direct repeats of the CCCTC-motif in the 5'-flanking sequence of the chicken c-myc gene. Oncogene. 1990 Dec;5(12):1743–1753. [PubMed] [Google Scholar]
  20. 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]
  21. Mayeux P., Billat C., Jacquot R. The erythropoietin receptor of rat erythroid progenitor lens. Characterization and affinity cross-linkage. J Biol Chem. 1987 Oct 15;262(29):13985–13990. [PubMed] [Google Scholar]
  22. Ptashne M. How eukaryotic transcriptional activators work. Nature. 1988 Oct 20;335(6192):683–689. doi: 10.1038/335683a0. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Rosenthal N. Identification of regulatory elements of cloned genes with functional assays. Methods Enzymol. 1987;152:704–720. doi: 10.1016/0076-6879(87)52075-4. [DOI] [PubMed] [Google Scholar]
  25. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. 1977. Biotechnology. 1992;24:104–108. [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Simmons D. M., Voss J. W., Ingraham H. A., Holloway J. M., Broide R. S., Rosenfeld M. G., Swanson L. W. Pituitary cell phenotypes involve cell-specific Pit-1 mRNA translation and synergistic interactions with other classes of transcription factors. Genes Dev. 1990 May;4(5):695–711. doi: 10.1101/gad.4.5.695. [DOI] [PubMed] [Google Scholar]
  29. Wang G. L., Jiang B. H., Rue E. A., Semenza G. L. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5510–5514. doi: 10.1073/pnas.92.12.5510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. 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]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES