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Mol Cell Biol. 1995 October; 15(10): 5830–5845.
PMCID: PMC230835

PU.1 (Spi-1) and C/EBP alpha regulate expression of the granulocyte-macrophage colony-stimulating factor receptor alpha gene.

Abstract

Growth factor receptors play an important role in hematopoiesis. In order to further understand the mechanisms directing the expression of these key regulators of hematopoiesis, we initiated a study investigating the transcription factors activating the expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha gene. Here, we demonstrate that the human GM-CSF receptor alpha promoter directs reporter gene activity in a tissue-specific fashion in myelomonocytic cells, which correlates with its expression pattern as analyzed by reverse transcription PCR. The GM-CSF receptor alpha promoter contains an important functional site between positions -53 and -41 as identified by deletion analysis of reporter constructs. We show that the myeloid and B cell transcription factor PU.1 binds specifically to this site. Furthermore, we demonstrate that a CCAAT site located upstream of the PU.1 site between positions -70 and -54 is involved in positive-negative regulation of the GM-CSF receptor alpha promoter activity. C/EBP alpha is the major CCAAT/enhancer-binding protein (C/EBP) form binding to this site in nuclear extracts of U937 cells. Point mutations of either the PU.1 site or the C/EBP site that abolish the binding of the respective factors result in a significant decrease of GM-CSF receptor alpha promoter activity in myelomonocytic cells only. Furthermore, we demonstrate that in myeloid and B cell extracts, PU.1 forms a novel, specific, more slowly migrating complex (PU-SF) when binding the GM-CSF receptor alpha promoter PU.1 site. This is the first demonstration of a specific interaction with PU.1 on a myeloid PU.1 binding site. The novel complex is distinct from that described previously as binding to B cell enhancer sites and can be formed by addition of PU.1 to extracts from certain nonmyeloid cell types which do not express PU.1, including T cells and epithelial cells, but not from erythroid cells. Furthermore, we demonstrate that the PU-SF complex binds to PU.1 sites found on a number of myeloid promoters, and its formation requires an intact PU.1 site adjacent to a single-stranded region. Expression of PU.1 in nonmyeloid cells can activate the GM-CSF receptor alpha promoter. Deletion of the amino-terminal region of PU.1 results in a failure to form the PU-SF complex and in a concomitant loss of transactivation, suggesting that formation of the PU-SF complex is of functional importance for the activity of the GM-CSF receptor alpha promoter. Finally, we demonstrate that C/EBP alpha can also active the GM-CSF receptor alpha promoter in nonmyeloid cells. These results suggest that PU.1 and C/EBP alpha direct the cell-type-specific expression of GM-CSF receptor alpha, further establish the role of PU.1 as a key regulator of hematopoiesis, and point to C/EBP alpha as an additional important factor in this process.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Ahne B, Strätling WH. 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]
  • Arcari P, Martinelli R, Salvatore F. The complete sequence of a full length cDNA for human liver glyceraldehyde-3-phosphate dehydrogenase: evidence for multiple mRNA species. Nucleic Acids Res. 1984 Dec 11;12(23):9179–9189. [PMC free article] [PubMed]
  • Bretz JD, Williams SC, Baer M, Johnson PF, Schwartz RC. C/EBP-related protein 2 confers lipopolysaccharide-inducible expression of interleukin 6 and monocyte chemoattractant protein 1 to a lymphoblastic cell line. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7306–7310. [PubMed]
  • Burk O, Mink S, Ringwald M, Klempnauer KH. Synergistic activation of the chicken mim-1 gene by v-myb and C/EBP transcription factors. EMBO J. 1993 May;12(5):2027–2038. [PubMed]
  • Cao Z, Umek RM, McKnight SL. Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. Genes Dev. 1991 Sep;5(9):1538–1552. [PubMed]
  • Chen HM, Zhang P, Voso MT, Hohaus S, Gonzalez DA, Glass CK, Zhang DE, Tenen DG. Neutrophils and monocytes express high levels of PU.1 (Spi-1) but not Spi-B. Blood. 1995 May 15;85(10):2918–2928. [PubMed]
  • Dignam JD, Lebovitz RM, Roeder RG. 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. [PMC free article] [PubMed]
  • DiPersio J, Billing P, Kaufman S, Eghtesady P, Williams RE, Gasson JC. Characterization of the human granulocyte-macrophage colony-stimulating factor receptor. J Biol Chem. 1988 Feb 5;263(4):1834–1841. [PubMed]
  • Duncan R, Bazar L, Michelotti G, Tomonaga T, Krutzsch H, Avigan M, Levens D. A sequence-specific, single-strand binding protein activates the far upstream element of c-myc and defines a new DNA-binding motif. Genes Dev. 1994 Feb 15;8(4):465–480. [PubMed]
  • Eichbaum QG, Iyer R, Raveh DP, Mathieu C, Ezekowitz RA. Restriction of interferon gamma responsiveness and basal expression of the myeloid human Fc gamma R1b gene is mediated by a functional PU.1 site and a transcription initiator consensus. J Exp Med. 1994 Jun 1;179(6):1985–1996. [PMC free article] [PubMed]
  • Eisenbeis CF, 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. [PMC free article] [PubMed]
  • Emanuel PD, Bates LJ, Zhu SW, Castleberry RP, Gualtieri RJ, Zuckerman KS. The role of monocyte-derived hemopoietic growth factors in the regulation of myeloproliferation in juvenile chronic myelogenous leukemia. Exp Hematol. 1991 Nov;19(10):1017–1024. [PubMed]
  • Feinman R, Qiu WQ, Pearse RN, Nikolajczyk BS, Sen R, Sheffery M, Ravetch JV. PU.1 and an HLH family member contribute to the myeloid-specific transcription of the Fc gamma RIIIA promoter. EMBO J. 1994 Aug 15;13(16):3852–3860. [PubMed]
  • Friedman AD, Landschulz WH, McKnight SL. CCAAT/enhancer binding protein activates the promoter of the serum albumin gene in cultured hepatoma cells. Genes Dev. 1989 Sep;3(9):1314–1322. [PubMed]
  • Gearing DP, King JA, Gough NM, Nicola NA. Expression cloning of a receptor for human granulocyte-macrophage colony-stimulating factor. EMBO J. 1989 Dec 1;8(12):3667–3676. [PubMed]
  • Goebl MK. The PU.1 transcription factor is the product of the putative oncogene Spi-1. Cell. 1990 Jun 29;61(7):1165–1166. [PubMed]
  • Goodbourn S, Maniatis T. Overlapping positive and negative regulatory domains of the human beta-interferon gene. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1447–1451. [PubMed]
  • Haas JG, Ströbel M, Leutz A, Wendelgass P, Müller C, Sterneck E, Riethmüller G, Ziegler-Heitbrock HW. Constitutive monocyte-restricted activity of NF-M, a nuclear factor that binds to a C/EBP motif. J Immunol. 1992 Jul 1;149(1):237–243. [PubMed]
  • Hagemeier C, Bannister AJ, Cook A, Kouzarides T. The activation domain of transcription factor PU.1 binds the retinoblastoma (RB) protein and the transcription factor TFIID in vitro: RB shows sequence similarity to TFIID and TFIIB. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1580–1584. [PubMed]
  • Johnson PF, McKnight SL. Eukaryotic transcriptional regulatory proteins. Annu Rev Biochem. 1989;58:799–839. [PubMed]
  • Jubinsky PT, Laurie AS, Nathan DG, Yetz-Aldepe J, Sieff CA. Expression and function of the human granulocyte-macrophage colony-stimulating factor receptor alpha subunit. Blood. 1994 Dec 15;84(12):4174–4185. [PubMed]
  • Katz S, Kowenz-Leutz E, Müller C, Meese K, Ness SA, Leutz A. The NF-M transcription factor is related to C/EBP beta and plays a role in signal transduction, differentiation and leukemogenesis of avian myelomonocytic cells. EMBO J. 1993 Apr;12(4):1321–1332. [PubMed]
  • Klemsz MJ, Maki RA, 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]
  • Klemsz MJ, McKercher SR, Celada A, Van Beveren C, Maki RA. The macrophage and B cell-specific transcription factor PU.1 is related to the ets oncogene. Cell. 1990 Apr 6;61(1):113–124. [PubMed]
  • Kominato Y, Galson D, Waterman WR, Webb AC, Auron PE. Monocyte expression of the human prointerleukin 1 beta gene (IL1B) is dependent on promoter sequences which bind the hematopoietic transcription factor Spi-1/PU.1. Mol Cell Biol. 1995 Jan;15(1):58–68. [PMC free article] [PubMed]
  • Kuipers OP, Boot HJ, de Vos WM. Improved site-directed mutagenesis method using PCR. Nucleic Acids Res. 1991 Aug 25;19(16):4558–4558. [PMC free article] [PubMed]
  • Lehming N, Thanos D, Brickman JM, Ma J, Maniatis T, Ptashne M. An HMG-like protein that can switch a transcriptional activator to a repressor. Nature. 1994 Sep 8;371(6493):175–179. [PubMed]
  • Lin FT, Lane MD. CCAAT/enhancer binding protein alpha is sufficient to initiate the 3T3-L1 adipocyte differentiation program. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8757–8761. [PubMed]
  • Maury W. Monocyte maturation controls expression of equine infectious anemia virus. J Virol. 1994 Oct;68(10):6270–6279. [PMC free article] [PubMed]
  • Maxam AM, Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. [PubMed]
  • Michelotti EF, Tomonaga T, Krutzsch H, Levens D. Cellular nucleic acid binding protein regulates the CT element of the human c-myc protooncogene. J Biol Chem. 1995 Apr 21;270(16):9494–9499. [PubMed]
  • Miyajima A, Mui AL, Ogorochi T, Sakamaki K. Receptors for granulocyte-macrophage colony-stimulating factor, interleukin-3, and interleukin-5. Blood. 1993 Oct 1;82(7):1960–1974. [PubMed]
  • 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. [PubMed]
  • Moulton KS, Semple K, Wu H, Glass CK. Cell-specific expression of the macrophage scavenger receptor gene is dependent on PU.1 and a composite AP-1/ets motif. Mol Cell Biol. 1994 Jul;14(7):4408–4418. [PMC free article] [PubMed]
  • Nakagawa Y, Kosugi H, Miyajima A, Arai K, Yokota T. Structure of the gene encoding the alpha subunit of the human granulocyte-macrophage colony stimulating factor receptor. Implications for the evolution of the cytokine receptor superfamily. J Biol Chem. 1994 Apr 8;269(14):10905–10912. [PubMed]
  • Natsuka S, Akira S, Nishio Y, Hashimoto S, Sugita T, Isshiki H, Kishimoto T. Macrophage differentiation-specific expression of NF-IL6, a transcription factor for interleukin-6. Blood. 1992 Jan 15;79(2):460–466. [PubMed]
  • Ness SA, Kowenz-Leutz E, Casini T, Graf T, Leutz A. Myb and NF-M: combinatorial activators of myeloid genes in heterologous cell types. Genes Dev. 1993 May;7(5):749–759. [PubMed]
  • Nordeen SK. Luciferase reporter gene vectors for analysis of promoters and enhancers. Biotechniques. 1988 May;6(5):454–458. [PubMed]
  • Nuchprayoon I, Meyers S, Scott LM, Suzow J, Hiebert S, Friedman AD. PEBP2/CBF, the murine homolog of the human myeloid AML1 and PEBP2 beta/CBF beta proto-oncoproteins, regulates the murine myeloperoxidase and neutrophil elastase genes in immature myeloid cells. Mol Cell Biol. 1994 Aug;14(8):5558–5568. [PMC free article] [PubMed]
  • Pahl HL, Burn TC, Tenen DG. Optimization of transient transfection into human myeloid cell lines using a luciferase reporter gene. Exp Hematol. 1991 Nov;19(10):1038–1041. [PubMed]
  • Pahl HL, Scheibe RJ, Zhang DE, Chen HM, Galson DL, Maki RA, Tenen DG. The proto-oncogene PU.1 regulates expression of the myeloid-specific CD11b promoter. J Biol Chem. 1993 Mar 5;268(7):5014–5020. [PubMed]
  • Park LS, Friend D, Gillis S, Urdal DL. Characterization of the cell surface receptor for human granulocyte/macrophage colony-stimulating factor. J Exp Med. 1986 Jul 1;164(1):251–262. [PMC free article] [PubMed]
  • Park LS, Waldron PE, Friend D, Sassenfeld HM, Price V, Anderson D, Cosman D, Andrews RG, Bernstein ID, Urdal DL. Interleukin-3, GM-CSF, and G-CSF receptor expression on cell lines and primary leukemia cells: receptor heterogeneity and relationship to growth factor responsiveness. Blood. 1989 Jul;74(1):56–65. [PubMed]
  • Paul R, Schuetze S, Kozak SL, Kozak CA, Kabat D. The Sfpi-1 proviral integration site of Friend erythroleukemia encodes the ets-related transcription factor Pu.1. J Virol. 1991 Jan;65(1):464–467. [PMC free article] [PubMed]
  • Perez C, Coeffier E, Moreau-Gachelin F, Wietzerbin J, Benech PD. Involvement of the transcription factor PU.1/Spi-1 in myeloid cell-restricted expression of an interferon-inducible gene encoding the human high-affinity Fc gamma receptor. Mol Cell Biol. 1994 Aug;14(8):5023–5031. [PMC free article] [PubMed]
  • Pongubala JM, Nagulapalli S, Klemsz MJ, McKercher SR, Maki RA, Atchison ML. 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. [PMC free article] [PubMed]
  • Pongubala JM, Van Beveren C, Nagulapalli S, Klemsz MJ, McKercher SR, Maki RA, Atchison ML. Effect of PU.1 phosphorylation on interaction with NF-EM5 and transcriptional activation. Science. 1993 Mar 12;259(5101):1622–1625. [PubMed]
  • Pope RM, Leutz A, Ness SA. C/EBP beta regulation of the tumor necrosis factor alpha gene. J Clin Invest. 1994 Oct;94(4):1449–1455. [PMC free article] [PubMed]
  • Raines MA, Liu L, Quan SG, Joe V, DiPersio JF, Golde DW. Identification and molecular cloning of a soluble human granulocyte-macrophage colony-stimulating factor receptor. Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):8203–8207. [PubMed]
  • Rapoport AP, Abboud CN, DiPersio JF. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF): receptor biology, signal transduction, and neutrophil activation. Blood Rev. 1992 Mar;6(1):43–57. [PubMed]
  • Ray D, Bosselut R, Ghysdael J, Mattei MG, 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. [PMC free article] [PubMed]
  • Rosmarin AG, Caprio D, Levy R, Simkevich C. CD18 (beta 2 leukocyte integrin) promoter requires PU.1 transcription factor for myeloid activity. Proc Natl Acad Sci U S A. 1995 Jan 31;92(3):801–805. [PubMed]
  • Schuetze S, Paul R, Gliniak BC, Kabat D. Role of the PU.1 transcription factor in controlling differentiation of Friend erythroleukemia cells. Mol Cell Biol. 1992 Jul;12(7):2967–2975. [PMC free article] [PubMed]
  • Schuetze S, Stenberg PE, Kabat D. The Ets-related transcription factor PU.1 immortalizes erythroblasts. Mol Cell Biol. 1993 Sep;13(9):5670–5678. [PMC free article] [PubMed]
  • Scott EW, Simon MC, 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. [PubMed]
  • Scott LM, Civin CI, Rorth P, Friedman AD. A novel temporal expression pattern of three C/EBP family members in differentiating myelomonocytic cells. Blood. 1992 Oct 1;80(7):1725–1735. [PubMed]
  • Shibuya T, Mak TW. Isolation and induction of erythroleukemic cell lines with properties of erythroid progenitor burst-forming cell (BFU-E) and erythroid precursor cell (CFU-E). Proc Natl Acad Sci U S A. 1983 Jun;80(12):3721–3725. [PubMed]
  • Shin MK, Koshland ME. 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. [PubMed]
  • Shuman JD, Vinson CR, McKnight SL. Evidence of changes in protease sensitivity and subunit exchange rate on DNA binding by C/EBP. Science. 1990 Aug 17;249(4970):771–774. [PubMed]
  • Skalnik DG, Strauss EC, Orkin SH. CCAAT displacement protein as a repressor of the myelomonocytic-specific gp91-phox gene promoter. J Biol Chem. 1991 Sep 5;266(25):16736–16744. [PubMed]
  • Staudt LM, Singh H, Sen R, Wirth T, Sharp PA, Baltimore D. A lymphoid-specific protein binding to the octamer motif of immunoglobulin genes. Nature. 1986 Oct 16;323(6089):640–643. [PubMed]
  • Sterneck E, Müller C, Katz S, Leutz A. Autocrine growth induced by kinase type oncogenes in myeloid cells requires AP-1 and NF-M, a myeloid specific, C/EBP-like factor. EMBO J. 1992 Jan;11(1):115–126. [PubMed]
  • Sun S, Stoflet ES, Cogan JG, Strauch AR, Getz MJ. Negative regulation of the vascular smooth muscle alpha-actin gene in fibroblasts and myoblasts: disruption of enhancer function by sequence-specific single-stranded-DNA-binding proteins. Mol Cell Biol. 1995 May;15(5):2429–2436. [PMC free article] [PubMed]
  • Voso MT, Burn TC, Wulf G, Lim B, Leone G, Tenen DG. 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. [PubMed]
  • Wang ZY, Lin XH, Nobyuoshi M, Qui QQ, Deuel TF. Binding of single-stranded oligonucleotides to a non-B-form DNA structure results in loss of promoter activity of the platelet-derived growth factor A-chain gene. J Biol Chem. 1992 Jul 5;267(19):13669–13674. [PubMed]
  • Wognum AW, Westerman Y, Visser TP, Wagemaker G. Distribution of receptors for granulocyte-macrophage colony-stimulating factor on immature CD34+ bone marrow cells, differentiating monomyeloid progenitors, and mature blood cell subsets. Blood. 1994 Aug 1;84(3):764–774. [PubMed]
  • Zhang DE, Fujioka K, Hetherington CJ, Shapiro LH, Chen HM, Look AT, Tenen DG. Identification of a region which directs the monocytic activity of the colony-stimulating factor 1 (macrophage colony-stimulating factor) receptor promoter and binds PEBP2/CBF (AML1). Mol Cell Biol. 1994 Dec;14(12):8085–8095. [PMC free article] [PubMed]
  • Zhang DE, Hetherington CJ, Chen HM, Tenen DG. 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. [PMC free article] [PubMed]

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