Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1998 Jun 15;26(12):3034–3043. doi: 10.1093/nar/26.12.3034

Use of RDA analysis of knockout mice to identify myeloid genes regulated in vivo by PU.1 and C/EBPalpha.

A Iwama 1, P Zhang 1, G J Darlington 1, S R McKercher 1, R Maki 1, D G Tenen 1
PMCID: PMC147647  PMID: 9611252

Abstract

PU.1 and C/EBPalpha are transcription factors essential for normal myeloid development. Loss-of-function mutation of PU.1 leads to an absolute block in monocyte/macrophage development and abnormal granulocytic development while that of C/EBPalpha causes a selective block in neutrophilic differentiation. In order to understand these phenotypes, we studied the role of PU.1 and C/EBPalpha in the regulation of myeloid target genes in vivo . Northern blot analysis revealed that mRNAs encoding receptors for M-CSF, G-CSF and GM-CSF, were expressed at low levels in PU.1(-/-) fetal liver compared with wild type. To identify additional myeloid genes regulated by PU.1 and C/EBPalpha, we performed representational difference analysis (RDA), a PCR-based subtractive hybridization using fetal livers from wild type and PU.1 or C/EBPalpha knockout mice. By introducing a new modification of RDA, that of tissue-specific gene suppression, we could selectively identify a set of differentially expressed genes specific to myeloid cells. Differentially expressed genes included both primary and secondary granule protein genes. In addition, eight novel genes were identified that were upregulated in expression during myeloid differentiation. These methods provide a general strategy for elucidating the genes affected in murine knockout models.

Full Text

The Full Text of this article is available as a PDF (313.8 KB).

Selected References

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

  1. Ahne B., Strätling W. H. Characterization of a myeloid-specific enhancer of the chicken lysozyme gene. Major role for an Ets transcription factor-binding site. J Biol Chem. 1994 Jul 8;269(27):17794–17801. [PubMed] [Google Scholar]
  2. Antonson P., Xanthopoulos K. G. Molecular cloning, sequence, and expression patterns of the human gene encoding CCAAT/enhancer binding protein alpha (C/EBP alpha). Biochem Biophys Res Commun. 1995 Oct 4;215(1):106–113. doi: 10.1006/bbrc.1995.2439. [DOI] [PubMed] [Google Scholar]
  3. Berliner N., Hsing A., Graubert T., Sigurdsson F., Zain M., Bruno E., Hoffman R. Granulocyte colony-stimulating factor induction of normal human bone marrow progenitors results in neutrophil-specific gene expression. Blood. 1995 Feb 1;85(3):799–803. [PubMed] [Google Scholar]
  4. Borregaard N., Cowland J. B. Granules of the human neutrophilic polymorphonuclear leukocyte. Blood. 1997 May 15;89(10):3503–3521. [PubMed] [Google Scholar]
  5. Braun B. S., Frieden R., Lessnick S. L., May W. A., Denny C. T. Identification of target genes for the Ewing's sarcoma EWS/FLI fusion protein by representational difference analysis. Mol Cell Biol. 1995 Aug;15(8):4623–4630. doi: 10.1128/mcb.15.8.4623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Chen H. M., Gonzalez D. A., Radomska H. S., Voso M. T., Sun Z., Zhang P., Zhang D. E., Tenen D. G. Two promoters direct expression of the murine Spi-B gene, an Ets family transcription factor. Gene. 1998 Jan 30;207(2):209–218. doi: 10.1016/s0378-1119(97)00629-x. [DOI] [PubMed] [Google Scholar]
  8. Chen H. M., Zhang P., Voso M. T., Hohaus S., Gonzalez D. A., Glass C. K., Zhang D. E., Tenen D. G. Neutrophils and monocytes express high levels of PU.1 (Spi-1) but not Spi-B. Blood. 1995 May 15;85(10):2918–2928. [PubMed] [Google Scholar]
  9. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  10. Costa R. H., Grayson D. R., Xanthopoulos K. G., Darnell J. E., Jr A liver-specific DNA-binding protein recognizes multiple nucleotide sites in regulatory regions of transthyretin, alpha 1-antitrypsin, albumin, and simian virus 40 genes. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3840–3844. doi: 10.1073/pnas.85.11.3840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cross M., Mangelsdorf I., Wedel A., Renkawitz R. Mouse lysozyme M gene: isolation, characterization, and expression studies. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6232–6236. doi: 10.1073/pnas.85.17.6232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Flodby P., Barlow C., Kylefjord H., Ahrlund-Richter L., Xanthopoulos K. G. Increased hepatic cell proliferation and lung abnormalities in mice deficient in CCAAT/enhancer binding protein alpha. J Biol Chem. 1996 Oct 4;271(40):24753–24760. doi: 10.1074/jbc.271.40.24753. [DOI] [PubMed] [Google Scholar]
  14. Ford A. M., Bennett C. A., Healy L. E., Towatari M., Greaves M. F., Enver T. Regulation of the myeloperoxidase enhancer binding proteins Pu1, C-EBP alpha, -beta, and -delta during granulocyte-lineage specification. Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10838–10843. doi: 10.1073/pnas.93.20.10838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fouret P., du Bois R. M., Bernaudin J. F., Takahashi H., Ferrans V. J., Crystal R. G. Expression of the neutrophil elastase gene during human bone marrow cell differentiation. J Exp Med. 1989 Mar 1;169(3):833–845. doi: 10.1084/jem.169.3.833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fukunaga R., Ishizaka-Ikeda E., Seto Y., Nagata S. Expression cloning of a receptor for murine granulocyte colony-stimulating factor. Cell. 1990 Apr 20;61(2):341–350. doi: 10.1016/0092-8674(90)90814-u. [DOI] [PubMed] [Google Scholar]
  17. Goethe R., Loc P. V. The far upstream chicken lysozyme enhancer at -6.1 kilobase, by interacting with NF-M, mediates lipopolysaccharide-induced expression of the chicken lysozyme gene in chicken myelomonocytic cells. J Biol Chem. 1994 Dec 9;269(49):31302–31309. [PubMed] [Google Scholar]
  18. Gruart V., Truong M. J., Plumas J., Zandecki M., Kusnierz J. P., Prin L., Vinatier D., Capron A., Capron M. Decreased expression of eosinophil peroxidase and major basic protein messenger RNAs during eosinophil maturation. Blood. 1992 May 15;79(10):2592–2597. [PubMed] [Google Scholar]
  19. Henkel G. W., McKercher S. R., Yamamoto H., Anderson K. L., Oshima R. G., Maki R. A. PU.1 but not ets-2 is essential for macrophage development from embryonic stem cells. Blood. 1996 Oct 15;88(8):2917–2926. [PubMed] [Google Scholar]
  20. Hohaus S., Petrovick M. S., Voso M. T., Sun Z., Zhang D. E., Tenen D. G. PU.1 (Spi-1) and C/EBP alpha regulate expression of the granulocyte-macrophage colony-stimulating factor receptor alpha gene. Mol Cell Biol. 1995 Oct;15(10):5830–5845. doi: 10.1128/mcb.15.10.5830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hromas R., Orazi A., Neiman R. S., Maki R., Van Beveran C., Moore J., Klemsz M. Hematopoietic lineage- and stage-restricted expression of the ETS oncogene family member PU.1. Blood. 1993 Nov 15;82(10):2998–3004. [PubMed] [Google Scholar]
  22. Hubank M., Schatz D. G. Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucleic Acids Res. 1994 Dec 25;22(25):5640–5648. doi: 10.1093/nar/22.25.5640. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Johnson P. F., Landschulz W. H., Graves B. J., McKnight S. L. Identification of a rat liver nuclear protein that binds to the enhancer core element of three animal viruses. Genes Dev. 1987 Apr;1(2):133–146. doi: 10.1101/gad.1.2.133. [DOI] [PubMed] [Google Scholar]
  24. Khanna-Gupta A., Zibello T., Kolla S., Neufeld E. J., Berliner N. CCAAT displacement protein (CDP/cut) recognizes a silencer element within the lactoferrin gene promoter. Blood. 1997 Oct 1;90(7):2784–2795. [PubMed] [Google Scholar]
  25. 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]
  26. Lerner A., Clayton L. K., Mizoguchi E., Ghendler Y., van Ewijk W., Koyasu S., Bhan A. K., Reinherz E. L. Cross-linking of T-cell receptors on double-positive thymocytes induces a cytokine-mediated stromal activation process linked to cell death. EMBO J. 1996 Nov 1;15(21):5876–5887. [PMC free article] [PubMed] [Google Scholar]
  27. Li F., Rosenberg E., Smith C. I., Notarfrancesco K., Reisher S. R., Shuman H., Feinstein S. I. Correlation of expression of transcription factor C/EBP alpha and surfactant protein genes in lung cells. Am J Physiol. 1995 Aug;269(2 Pt 1):L241–L247. doi: 10.1152/ajplung.1995.269.2.L241. [DOI] [PubMed] [Google Scholar]
  28. Liboi E., Jubinsky P., Andrews N. C., Nathan D. G., Mathey-Prevot B. Enhanced expression of interleukin-3 and granulocyte-macrophage colony-stimulating factor receptor subunits in murine hematopoietic cells stimulated with hematopoietic growth factors. Blood. 1992 Sep 1;80(5):1183–1189. [PubMed] [Google Scholar]
  29. Lieschke G. J., Stanley E., Grail D., Hodgson G., Sinickas V., Gall J. A., Sinclair R. A., Dunn A. R. Mice lacking both macrophage- and granulocyte-macrophage colony-stimulating factor have macrophages and coexistent osteopetrosis and severe lung disease. Blood. 1994 Jul 1;84(1):27–35. [PubMed] [Google Scholar]
  30. Lisitsyn N., Lisitsyn N., Wigler M. Cloning the differences between two complex genomes. Science. 1993 Feb 12;259(5097):946–951. doi: 10.1126/science.8438152. [DOI] [PubMed] [Google Scholar]
  31. Liu F., Wu H. Y., Wesselschmidt R., Kornaga T., Link D. C. Impaired production and increased apoptosis of neutrophils in granulocyte colony-stimulating factor receptor-deficient mice. Immunity. 1996 Nov;5(5):491–501. doi: 10.1016/s1074-7613(00)80504-x. [DOI] [PubMed] [Google Scholar]
  32. McKercher S. R., Torbett B. E., Anderson K. L., Henkel G. W., Vestal D. J., Baribault H., Klemsz M., Feeney A. J., Wu G. E., Paige C. J. Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. EMBO J. 1996 Oct 15;15(20):5647–5658. [PMC free article] [PubMed] [Google Scholar]
  33. Nishinakamura R., Nakayama N., Hirabayashi Y., Inoue T., Aud D., McNeil T., Azuma S., Yoshida S., Toyoda Y., Arai K. Mice deficient for the IL-3/GM-CSF/IL-5 beta c receptor exhibit lung pathology and impaired immune response, while beta IL3 receptor-deficient mice are normal. Immunity. 1995 Mar;2(3):211–222. doi: 10.1016/1074-7613(95)90046-2. [DOI] [PubMed] [Google Scholar]
  34. Oelgeschläger M., Nuchprayoon I., Lüscher B., Friedman A. D. C/EBP, c-Myb, and PU.1 cooperate to regulate the neutrophil elastase promoter. Mol Cell Biol. 1996 Sep;16(9):4717–4725. doi: 10.1128/mcb.16.9.4717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Olson M. C., Scott E. W., Hack A. A., Su G. H., Tenen D. G., Singh H., Simon M. C. PU. 1 is not essential for early myeloid gene expression but is required for terminal myeloid differentiation. Immunity. 1995 Dec;3(6):703–714. doi: 10.1016/1074-7613(95)90060-8. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Rosenberg H. F., Ackerman S. J., Tenen D. G. An alternative method for labeling oligonucleotide probes for screening cDNA libraries. Biotechniques. 1990 Apr;8(4):384–384. [PubMed] [Google Scholar]
  38. Rosmarin A. G., Weil S. C., Rosner G. L., Griffin J. D., Arnaout M. A., Tenen D. G. Differential expression of CD11b/CD18 (Mo1) and myeloperoxidase genes during myeloid differentiation. Blood. 1989 Jan;73(1):131–136. [PubMed] [Google Scholar]
  39. Rothwell V. M., Rohrschneider L. R. Murine c-fms cDNA: cloning, sequence analysis and retroviral expression. Oncogene Res. 1987 Sep-Oct;1(4):311–324. [PubMed] [Google Scholar]
  40. Scott E. W., Fisher R. C., Olson M. C., Kehrli E. W., Simon M. C., Singh H. PU.1 functions in a cell-autonomous manner to control the differentiation of multipotential lymphoid-myeloid progenitors. Immunity. 1997 Apr;6(4):437–447. doi: 10.1016/s1074-7613(00)80287-3. [DOI] [PubMed] [Google Scholar]
  41. Scott E. W., Simon M. C., Anastasi J., Singh H. Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages. Science. 1994 Sep 9;265(5178):1573–1577. doi: 10.1126/science.8079170. [DOI] [PubMed] [Google Scholar]
  42. Scott L. M., Civin C. I., Rorth P., Friedman A. D. A novel temporal expression pattern of three C/EBP family members in differentiating myelomonocytic cells. Blood. 1992 Oct 1;80(7):1725–1735. [PubMed] [Google Scholar]
  43. Shivdasani R. A., Orkin S. H. The transcriptional control of hematopoiesis. Blood. 1996 May 15;87(10):4025–4039. [PubMed] [Google Scholar]
  44. Singh G., Katyal S. L., Brown W. E., Collins D. L., Mason R. J. Pulmonary lysozyme--a secretory protein of type II pneumocytes in the rat. Am Rev Respir Dis. 1988 Nov;138(5):1261–1267. doi: 10.1164/ajrccm/138.5.1261. [DOI] [PubMed] [Google Scholar]
  45. Smas C. M., Sul H. S. Pref-1, a protein containing EGF-like repeats, inhibits adipocyte differentiation. Cell. 1993 May 21;73(4):725–734. doi: 10.1016/0092-8674(93)90252-l. [DOI] [PubMed] [Google Scholar]
  46. Smith L. T., Hohaus S., Gonzalez D. A., Dziennis S. E., Tenen D. G. PU.1 (Spi-1) and C/EBP alpha regulate the granulocyte colony-stimulating factor receptor promoter in myeloid cells. Blood. 1996 Aug 15;88(4):1234–1247. [PubMed] [Google Scholar]
  47. Strobl H., Takimoto M., Majdic O., Fritsch G., Scheinecker C., Höcker P., Knapp W. Myeloperoxidase expression in CD34+ normal human hematopoietic cells. Blood. 1993 Oct 1;82(7):2069–2078. [PubMed] [Google Scholar]
  48. Sturrock A., Franklin K. F., Hoidal J. R. Human proteinase-3 expression is regulated by PU.1 in conjunction with a cytidine-rich element. J Biol Chem. 1996 Dec 13;271(50):32392–32402. doi: 10.1074/jbc.271.50.32392. [DOI] [PubMed] [Google Scholar]
  49. Tenen D. G., Hromas R., Licht J. D., Zhang D. E. Transcription factors, normal myeloid development, and leukemia. Blood. 1997 Jul 15;90(2):489–519. [PubMed] [Google Scholar]
  50. Tobler A., Miller C. W., Johnson K. R., Selsted M. E., Rovera G., Koeffler H. P. Regulation of gene expression of myeloperoxidase during myeloid differentiation. J Cell Physiol. 1988 Aug;136(2):215–225. doi: 10.1002/jcp.1041360203. [DOI] [PubMed] [Google Scholar]
  51. Tondravi M. M., McKercher S. R., Anderson K., Erdmann J. M., Quiroz M., Maki R., Teitelbaum S. L. Osteopetrosis in mice lacking haematopoietic transcription factor PU.1. Nature. 1997 Mar 6;386(6620):81–84. doi: 10.1038/386081a0. [DOI] [PubMed] [Google Scholar]
  52. Tsai S., Bartelmez S., Sitnicka E., Collins S. Lymphohematopoietic progenitors immortalized by a retroviral vector harboring a dominant-negative retinoic acid receptor can recapitulate lymphoid, myeloid, and erythroid development. Genes Dev. 1994 Dec 1;8(23):2831–2841. doi: 10.1101/gad.8.23.2831. [DOI] [PubMed] [Google Scholar]
  53. Voso M. T., Burn T. C., Wulf G., Lim B., Leone G., Tenen D. G. Inhibition of hematopoiesis by competitive binding of transcription factor PU.1. Proc Natl Acad Sci U S A. 1994 Aug 16;91(17):7932–7936. doi: 10.1073/pnas.91.17.7932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wang N. D., Finegold M. J., Bradley A., Ou C. N., Abdelsayed S. V., Wilde M. D., Taylor L. R., Wilson D. R., Darlington G. J. Impaired energy homeostasis in C/EBP alpha knockout mice. Science. 1995 Aug 25;269(5227):1108–1112. doi: 10.1126/science.7652557. [DOI] [PubMed] [Google Scholar]
  55. Yamanaka R., Barlow C., Lekstrom-Himes J., Castilla L. H., Liu P. P., Eckhaus M., Decker T., Wynshaw-Boris A., Xanthopoulos K. G. Impaired granulopoiesis, myelodysplasia, and early lethality in CCAAT/enhancer binding protein epsilon-deficient mice. Proc Natl Acad Sci U S A. 1997 Nov 25;94(24):13187–13192. doi: 10.1073/pnas.94.24.13187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Yamanaka R., Kim G. D., Radomska H. S., Lekstrom-Himes J., Smith L. T., Antonson P., Tenen D. G., Xanthopoulos K. G. CCAAT/enhancer binding protein epsilon is preferentially up-regulated during granulocytic differentiation and its functional versatility is determined by alternative use of promoters and differential splicing. Proc Natl Acad Sci U S A. 1997 Jun 10;94(12):6462–6467. doi: 10.1073/pnas.94.12.6462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Yoshida H., Hayashi S., Kunisada T., Ogawa M., Nishikawa S., Okamura H., Sudo T., Shultz L. D., Nishikawa S. The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature. 1990 May 31;345(6274):442–444. doi: 10.1038/345442a0. [DOI] [PubMed] [Google Scholar]
  58. 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]
  59. Zhang D. E., Zhang P., Wang N. D., Hetherington C. J., Darlington G. J., Tenen D. G. Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAAT enhancer binding protein alpha-deficient mice. Proc Natl Acad Sci U S A. 1997 Jan 21;94(2):569–574. doi: 10.1073/pnas.94.2.569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Zheng W., Flavell R. A. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell. 1997 May 16;89(4):587–596. doi: 10.1016/s0092-8674(00)80240-8. [DOI] [PubMed] [Google Scholar]

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

RESOURCES