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. 1994 Aug 16;91(17):7932–7936. doi: 10.1073/pnas.91.17.7932

Inhibition of hematopoiesis by competitive binding of transcription factor PU.1.

M T Voso 1, T C Burn 1, G Wulf 1, B Lim 1, G Leone 1, D G Tenen 1
PMCID: PMC44518  PMID: 7520173

Abstract

Transcription factors have been shown to play a role as "master switch" factors in the programming of hematopoietic cell commitment and differentiation. PU.1 is a hematopoietic-specific member of the Ets family of transcription factors. In human bone marrow CD34-enriched progenitor cells, PU.1 expression was upregulated during the early phases of granulocytic/monocytic differentiation, preceding expression of its target genes encoding CD11b and the macrophage-colony-stimulating factor receptor, whereas PU.1 was expressed at stable levels throughout erythroid differentiation. To study PU.1 function, we synthesized double-stranded phosphorothioate oligonucleotides containing a characterized PU.1 site and demonstrated their ability to specifically compete for PU.1 DNA binding. When added to CD34+ cells in vitro, wild-type PU.1-binding oligonucleotides significantly blocked hematopoietic colony formation, whereas mutated PU.1 oligonucleotides which no longer bind PU.1 had no specific inhibitory effect. These results demonstrate that PU.1 is developmentally upregulated during normal human myelopoiesis and that the function of PU.1 is critical for the development of in vitro hematopoiesis.

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Selected References

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

  1. Andrews R. G., Bryant E. M., Bartelmez S. H., Muirhead D. Y., Knitter G. H., Bensinger W., Strong D. M., Bernstein I. D. CD34+ marrow cells, devoid of T and B lymphocytes, reconstitute stable lymphopoiesis and myelopoiesis in lethally irradiated allogeneic baboons. Blood. 1992 Oct 1;80(7):1693–1701. [PubMed] [Google Scholar]
  2. Andrews R. G., Singer J. W., Bernstein I. D. Monoclonal antibody 12-8 recognizes a 115-kd molecule present on both unipotent and multipotent hematopoietic colony-forming cells and their precursors. Blood. 1986 Mar;67(3):842–845. [PubMed] [Google Scholar]
  3. Berenson R. J., Andrews R. G., Bensinger W. I., Kalamasz D., Knitter G., Buckner C. D., Bernstein I. D. Antigen CD34+ marrow cells engraft lethally irradiated baboons. J Clin Invest. 1988 Mar;81(3):951–955. doi: 10.1172/JCI113409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bielinska A., Shivdasani R. A., Zhang L. Q., Nabel G. J. Regulation of gene expression with double-stranded phosphorothioate oligonucleotides. Science. 1990 Nov 16;250(4983):997–1000. doi: 10.1126/science.2237444. [DOI] [PubMed] [Google Scholar]
  5. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  6. Clusel C., Ugarte E., Enjolras N., Vasseur M., Blumenfeld M. Ex vivo regulation of specific gene expression by nanomolar concentration of double-stranded dumbbell oligonucleotides. Nucleic Acids Res. 1993 Jul 25;21(15):3405–3411. doi: 10.1093/nar/21.15.3405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Eck S. L., Perkins N. D., Carr D. P., Nabel G. J. Inhibition of phorbol ester-induced cellular adhesion by competitive binding of NF-kappa B in vivo. Mol Cell Biol. 1993 Oct;13(10):6530–6536. doi: 10.1128/mcb.13.10.6530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Eisenbeis C. F., Singh H., Storb U. PU.1 is a component of a multiprotein complex which binds an essential site in the murine immunoglobulin lambda 2-4 enhancer. Mol Cell Biol. 1993 Oct;13(10):6452–6461. doi: 10.1128/mcb.13.10.6452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fairbairn L. J., Cowling G. J., Reipert B. M., Dexter T. M. Suppression of apoptosis allows differentiation and development of a multipotent hemopoietic cell line in the absence of added growth factors. Cell. 1993 Sep 10;74(5):823–832. doi: 10.1016/0092-8674(93)90462-y. [DOI] [PubMed] [Google Scholar]
  10. Gabbianelli M., Sargiacomo M., Pelosi E., Testa U., Isacchi G., Peschle C. "Pure" human hematopoietic progenitors: permissive action of basic fibroblast growth factor. Science. 1990 Sep 28;249(4976):1561–1564. doi: 10.1126/science.2218497. [DOI] [PubMed] [Google Scholar]
  11. Galson D. L., Hensold J. O., Bishop T. R., Schalling M., D'Andrea A. D., Jones C., Auron P. E., Housman D. E. Mouse beta-globin DNA-binding protein B1 is identical to a proto-oncogene, the transcription factor Spi-1/PU.1, and is restricted in expression to hematopoietic cells and the testis. Mol Cell Biol. 1993 May;13(5):2929–2941. doi: 10.1128/mcb.13.5.2929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Keller G., Kennedy M., Papayannopoulou T., Wiles M. V. Hematopoietic commitment during embryonic stem cell differentiation in culture. Mol Cell Biol. 1993 Jan;13(1):473–486. doi: 10.1128/mcb.13.1.473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Klemsz M. J., Maki R. A., Papayannopoulou T., Moore J., Hromas R. Characterization of the ets oncogene family member, fli-1. J Biol Chem. 1993 Mar 15;268(8):5769–5773. [PubMed] [Google Scholar]
  14. Klemsz M. J., McKercher S. R., Celada A., Van Beveren C., Maki R. A. The macrophage and B cell-specific transcription factor PU.1 is related to the ets oncogene. Cell. 1990 Apr 6;61(1):113–124. doi: 10.1016/0092-8674(90)90219-5. [DOI] [PubMed] [Google Scholar]
  15. Lautenberger J. A., Burdett L. A., Gunnell M. A., Qi S., Watson D. K., O'Brien S. J., Papas T. S. Genomic dispersal of the ets gene family during metazoan evolution. Oncogene. 1992 Sep;7(9):1713–1719. [PubMed] [Google Scholar]
  16. Moreau-Gachelin F., Tavitian A., Tambourin P. Spi-1 is a putative oncogene in virally induced murine erythroleukaemias. Nature. 1988 Jan 21;331(6153):277–280. doi: 10.1038/331277a0. [DOI] [PubMed] [Google Scholar]
  17. Mouthon M. A., Bernard O., Mitjavila M. T., Romeo P. H., Vainchenker W., Mathieu-Mahul D. Expression of tal-1 and GATA-binding proteins during human hematopoiesis. Blood. 1993 Feb 1;81(3):647–655. [PubMed] [Google Scholar]
  18. Pahl H. L., Scheibe R. J., Zhang D. E., Chen H. M., Galson D. L., Maki R. A., Tenen D. G. The proto-oncogene PU.1 regulates expression of the myeloid-specific CD11b promoter. J Biol Chem. 1993 Mar 5;268(7):5014–5020. [PubMed] [Google Scholar]
  19. Pevny L., Simon M. C., Robertson E., Klein W. H., Tsai S. F., D'Agati V., Orkin S. H., Costantini F. Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1. Nature. 1991 Jan 17;349(6306):257–260. doi: 10.1038/349257a0. [DOI] [PubMed] [Google Scholar]
  20. Pongubala J. M., Nagulapalli S., Klemsz M. J., McKercher S. R., Maki R. A., Atchison M. L. PU.1 recruits a second nuclear factor to a site important for immunoglobulin kappa 3' enhancer activity. Mol Cell Biol. 1992 Jan;12(1):368–378. doi: 10.1128/mcb.12.1.368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ray D., Bosselut R., Ghysdael J., Mattei M. G., Tavitian A., Moreau-Gachelin F. Characterization of Spi-B, a transcription factor related to the putative oncoprotein Spi-1/PU.1. Mol Cell Biol. 1992 Oct;12(10):4297–4304. doi: 10.1128/mcb.12.10.4297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schuetze S., Stenberg P. E., Kabat D. The Ets-related transcription factor PU.1 immortalizes erythroblasts. Mol Cell Biol. 1993 Sep;13(9):5670–5678. doi: 10.1128/mcb.13.9.5670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Shin M. K., Koshland M. E. Ets-related protein PU.1 regulates expression of the immunoglobulin J-chain gene through a novel Ets-binding element. Genes Dev. 1993 Oct;7(10):2006–2015. doi: 10.1101/gad.7.10.2006. [DOI] [PubMed] [Google Scholar]
  24. Simon M. C., Pevny L., Wiles M. V., Keller G., Costantini F., Orkin S. H. Rescue of erythroid development in gene targeted GATA-1- mouse embryonic stem cells. Nat Genet. 1992 May;1(2):92–98. doi: 10.1038/ng0592-92. [DOI] [PubMed] [Google Scholar]
  25. Sposi N. M., Zon L. I., Carè A., Valtieri M., Testa U., Gabbianelli M., Mariani G., Bottero L., Mather C., Orkin S. H. Cell cycle-dependent initiation and lineage-dependent abrogation of GATA-1 expression in pure differentiating hematopoietic progenitors. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6353–6357. doi: 10.1073/pnas.89.14.6353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Strauss L. C., Rowley S. D., La Russa V. F., Sharkis S. J., Stuart R. K., Civin C. I. Antigenic analysis of hematopoiesis. V. Characterization of My-10 antigen expression by normal lymphohematopoietic progenitor cells. Exp Hematol. 1986 Oct;14(9):878–886. [PubMed] [Google Scholar]
  27. Visvader J. E., Elefanty A. G., Strasser A., Adams J. M. GATA-1 but not SCL induces megakaryocytic differentiation in an early myeloid line. EMBO J. 1992 Dec;11(12):4557–4564. doi: 10.1002/j.1460-2075.1992.tb05557.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wulf G. M., Adra C. N., Lim B. Inhibition of hematopoietic development from embryonic stem cells by antisense vav RNA. EMBO J. 1993 Dec 15;12(13):5065–5074. doi: 10.1002/j.1460-2075.1993.tb06200.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Zhang D. E., Hetherington C. J., Chen H. M., Tenen D. G. The macrophage transcription factor PU.1 directs tissue-specific expression of the macrophage colony-stimulating factor receptor. Mol Cell Biol. 1994 Jan;14(1):373–381. doi: 10.1128/mcb.14.1.373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Zon L. I., Youssoufian H., Mather C., Lodish H. F., Orkin S. H. Activation of the erythropoietin receptor promoter by transcription factor GATA-1. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10638–10641. doi: 10.1073/pnas.88.23.10638. [DOI] [PMC free article] [PubMed] [Google Scholar]

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