Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1998 Feb 15;330(Pt 1):335–343. doi: 10.1042/bj3300335

Functional analysis of the endothelial cell-specific Tie2/Tek promoter identifies unique protein-binding elements.

B M Fadel 1, S C Boutet 1, T Quertermous 1
PMCID: PMC1219145  PMID: 9461528

Abstract

To investigate the molecular basis of endothelial cell-specific gene expression, we have examined the DNA sequences and the cognate DNA-binding proteins that mediate transcription of the murine tie2/tek gene. Reporter transfection experiments conformed with earlier findings in transgenic mice, indicating that the upstream promoter of Tie2/Tek is capable of activating transcription in an endothelial cell-specific fashion. These experiments have also allowed the identification of a single upstream inhibitory region (region I) and two positive regulatory regions (regions U and A) in the proximal promoter. Electrophoretic mobility-shift assays have allowed further characterization of three novel DNA-binding sequences associated with these regions and have provided preliminary characterization of the protein factors binding to these elements. Two of the elements (U and A) confer increased transcription on a heterologous promoter, with element U functioning in an endothelial-cell-selective manner. By employing embryonic endothelial-like yolk sac cells in parallel with adult-derived endothelial cells, we have identified differences in functional activity and protein binding that may reflect mechanisms for specifying developmental regulation of tie2/tek expression. Further study of the DNA and protein elements characterized in these experiments is likely to provide new insight into the molecular basis of developmental- and cell-specific gene expression in the endothelium.

Full Text

The Full Text of this article is available as a PDF (476.1 KB).

Selected References

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

  1. Aird W. C., Jahroudi N., Weiler-Guettler H., Rayburn H. B., Rosenberg R. D. Human von Willebrand factor gene sequences target expression to a subpopulation of endothelial cells in transgenic mice. Proc Natl Acad Sci U S A. 1995 May 9;92(10):4567–4571. doi: 10.1073/pnas.92.10.4567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andersen B., Pearse R. V., 2nd, Schlegel P. N., Cichon Z., Schonemann M. D., Bardin C. W., Rosenfeld M. G. Sperm 1: a POU-domain gene transiently expressed immediately before meiosis I in the male germ cell. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11084–11088. doi: 10.1073/pnas.90.23.11084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Andersen B., Schonemann M. D., Flynn S. E., Pearse R. V., 2nd, Singh H., Rosenfeld M. G. Skn-1a and Skn-1i: two functionally distinct Oct-2-related factors expressed in epidermis. Science. 1993 Apr 2;260(5104):78–82. doi: 10.1126/science.7682011. [DOI] [PubMed] [Google Scholar]
  4. Cao Z., Umek R. M., McKnight S. L. Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. Genes Dev. 1991 Sep;5(9):1538–1552. doi: 10.1101/gad.5.9.1538. [DOI] [PubMed] [Google Scholar]
  5. Coffin J. D., Poole T. J. Embryonic vascular development: immunohistochemical identification of the origin and subsequent morphogenesis of the major vessel primordia in quail embryos. Development. 1988 Apr;102(4):735–748. doi: 10.1242/dev.102.4.735. [DOI] [PubMed] [Google Scholar]
  6. Coffin J. D., Poole T. J. Endothelial cell origin and migration in embryonic heart and cranial blood vessel development. Anat Rec. 1991 Nov;231(3):383–395. doi: 10.1002/ar.1092310312. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Dumont D. J., Gradwohl G., Fong G. H., Puri M. C., Gertsenstein M., Auerbach A., Breitman M. L. Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, tek, reveal a critical role in vasculogenesis of the embryo. Genes Dev. 1994 Aug 15;8(16):1897–1909. doi: 10.1101/gad.8.16.1897. [DOI] [PubMed] [Google Scholar]
  9. Ellis J., Tan-Un K. C., Harper A., Michalovich D., Yannoutsos N., Philipsen S., Grosveld F. A dominant chromatin-opening activity in 5' hypersensitive site 3 of the human beta-globin locus control region. EMBO J. 1996 Feb 1;15(3):562–568. [PMC free article] [PubMed] [Google Scholar]
  10. Fong G. H., Rossant J., Gertsenstein M., Breitman M. L. Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature. 1995 Jul 6;376(6535):66–70. doi: 10.1038/376066a0. [DOI] [PubMed] [Google Scholar]
  11. Freytag S. O., Paielli D. L., Gilbert J. D. Ectopic expression of the CCAAT/enhancer-binding protein alpha promotes the adipogenic program in a variety of mouse fibroblastic cells. Genes Dev. 1994 Jul 15;8(14):1654–1663. doi: 10.1101/gad.8.14.1654. [DOI] [PubMed] [Google Scholar]
  12. Harats D., Kurihara H., Belloni P., Oakley H., Ziober A., Ackley D., Cain G., Kurihara Y., Lawn R., Sigal E. Targeting gene expression to the vascular wall in transgenic mice using the murine preproendothelin-1 promoter. J Clin Invest. 1995 Mar;95(3):1335–1344. doi: 10.1172/JCI117784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hariharan N., Perry R. P. Functional dissection of a mouse ribosomal protein promoter: significance of the polypyrimidine initiator and an element in the TATA-box region. Proc Natl Acad Sci U S A. 1990 Feb;87(4):1526–1530. doi: 10.1073/pnas.87.4.1526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Herr W., Cleary M. A. The POU domain: versatility in transcriptional regulation by a flexible two-in-one DNA-binding domain. Genes Dev. 1995 Jul 15;9(14):1679–1693. doi: 10.1101/gad.9.14.1679. [DOI] [PubMed] [Google Scholar]
  15. Kadonaga J. T., Tjian R. Affinity purification of sequence-specific DNA binding proteins. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5889–5893. doi: 10.1073/pnas.83.16.5889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kingston R. E., Bunker C. A., Imbalzano A. N. Repression and activation by multiprotein complexes that alter chromatin structure. Genes Dev. 1996 Apr 15;10(8):905–920. doi: 10.1101/gad.10.8.905. [DOI] [PubMed] [Google Scholar]
  17. Korhonen J., Lahtinen I., Halmekytö M., Alhonen L., Jänne J., Dumont D., Alitalo K. Endothelial-specific gene expression directed by the tie gene promoter in vivo. Blood. 1995 Sep 1;86(5):1828–1835. [PubMed] [Google Scholar]
  18. Lee M. E., Bloch K. D., Clifford J. A., Quertermous T. Functional analysis of the endothelin-1 gene promoter. Evidence for an endothelial cell-specific cis-acting sequence. J Biol Chem. 1990 Jun 25;265(18):10446–10450. [PubMed] [Google Scholar]
  19. Lee M. E., Temizer D. H., Clifford J. A., Quertermous T. Cloning of the GATA-binding protein that regulates endothelin-1 gene expression in endothelial cells. J Biol Chem. 1991 Aug 25;266(24):16188–16192. [PubMed] [Google Scholar]
  20. Morishita K., Johnson D. E., Williams L. T. A novel promoter for vascular endothelial growth factor receptor (flt-1) that confers endothelial-specific gene expression. J Biol Chem. 1995 Nov 17;270(46):27948–27953. doi: 10.1074/jbc.270.46.27948. [DOI] [PubMed] [Google Scholar]
  21. Noden D. M. Embryonic origins and assembly of blood vessels. Am Rev Respir Dis. 1989 Oct;140(4):1097–1103. doi: 10.1164/ajrccm/140.4.1097. [DOI] [PubMed] [Google Scholar]
  22. Noden D. M. Origins and assembly of avian embryonic blood vessels. Ann N Y Acad Sci. 1990;588:236–249. doi: 10.1111/j.1749-6632.1990.tb13214.x. [DOI] [PubMed] [Google Scholar]
  23. Nothias J. Y., Majumder S., Kaneko K. J., DePamphilis M. L. Regulation of gene expression at the beginning of mammalian development. J Biol Chem. 1995 Sep 22;270(38):22077–22080. doi: 10.1074/jbc.270.38.22077. [DOI] [PubMed] [Google Scholar]
  24. Osada S., Yamamoto H., Nishihara T., Imagawa M. DNA binding specificity of the CCAAT/enhancer-binding protein transcription factor family. J Biol Chem. 1996 Feb 16;271(7):3891–3896. doi: 10.1074/jbc.271.7.3891. [DOI] [PubMed] [Google Scholar]
  25. Pardanaud L., Altmann C., Kitos P., Dieterlen-Lievre F., Buck C. A. Vasculogenesis in the early quail blastodisc as studied with a monoclonal antibody recognizing endothelial cells. Development. 1987 Jun;100(2):339–349. doi: 10.1242/dev.100.2.339. [DOI] [PubMed] [Google Scholar]
  26. Pardanaud L., Yassine F., Dieterlen-Lievre F. Relationship between vasculogenesis, angiogenesis and haemopoiesis during avian ontogeny. Development. 1989 Mar;105(3):473–485. doi: 10.1242/dev.105.3.473. [DOI] [PubMed] [Google Scholar]
  27. Patterson C., Perrella M. A., Hsieh C. M., Yoshizumi M., Lee M. E., Haber E. Cloning and functional analysis of the promoter for KDR/flk-1, a receptor for vascular endothelial growth factor. J Biol Chem. 1995 Sep 29;270(39):23111–23118. doi: 10.1074/jbc.270.39.23111. [DOI] [PubMed] [Google Scholar]
  28. Quertermous E. E., Hidai H., Blanar M. A., Quertermous T. Cloning and characterization of a basic helix-loop-helix protein expressed in early mesoderm and the developing somites. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7066–7070. doi: 10.1073/pnas.91.15.7066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sato T. N., Tozawa Y., Deutsch U., Wolburg-Buchholz K., Fujiwara Y., Gendron-Maguire M., Gridley T., Wolburg H., Risau W., Qin Y. Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Nature. 1995 Jul 6;376(6535):70–74. doi: 10.1038/376070a0. [DOI] [PubMed] [Google Scholar]
  30. Schlaeger T. M., Qin Y., Fujiwara Y., Magram J., Sato T. N. Vascular endothelial cell lineage-specific promoter in transgenic mice. Development. 1995 Apr;121(4):1089–1098. doi: 10.1242/dev.121.4.1089. [DOI] [PubMed] [Google Scholar]
  31. Schreiber E., Harshman K., Kemler I., Malipiero U., Schaffner W., Fontana A. Astrocytes and glioblastoma cells express novel octamer-DNA binding proteins distinct from the ubiquitous Oct-1 and B cell type Oct-2 proteins. Nucleic Acids Res. 1990 Sep 25;18(18):5495–5503. doi: 10.1093/nar/18.18.5495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Shalaby F., Rossant J., Yamaguchi T. P., Gertsenstein M., Wu X. F., Breitman M. L., Schuh A. C. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature. 1995 Jul 6;376(6535):62–66. doi: 10.1038/376062a0. [DOI] [PubMed] [Google Scholar]
  33. Tapscott S. J., Lassar A. B., Weintraub H. A novel myoblast enhancer element mediates MyoD transcription. Mol Cell Biol. 1992 Nov;12(11):4994–5003. doi: 10.1128/mcb.12.11.4994. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES