Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 Sep;17(9):5127–5135. doi: 10.1128/mcb.17.9.5127

Core binding factor cannot synergistically activate the myeloperoxidase proximal enhancer in immature myeloid cells without c-Myb.

M Britos-Bray 1, A D Friedman 1
PMCID: PMC232363  PMID: 9271390

Abstract

The myeloperoxidase (MPO) gene is transcribed specifically in immature myeloid cells and is regulated in part by a 414-bp proximal enhancer. Mutation of a core binding factor (CBF)-binding site at -288 decreased enhancer activity 30-fold in 32D cl3 myeloid cells cultured in granulocyte colony-stimulating factor (G-CSF). A novel functional analysis, linking the CBF-binding site to an enhancer deletion series, located at -147 an evolutionarily conserved c-Myb-binding site which was required for optimal enhancer activity and synergy with CBF in 32D cells. These sites cooperated in isolation and independent of a precise spacing. Deletional analysis carried out in the absence of the c-Myb-binding site at -147 located at -301 a second c-Myb-binding site which also synergized with CBF to activate the enhancer. A GA-rich region at -162 contributed to cooperation with CBF when the adjacent c-Myb-binding site was intact. Mutation of both c-Myb-binding sites in the context of the entire enhancer greatly impaired activation by endogenous CBF in 32D cells. Similarly, activation by c-Myb was impaired in constructs lacking the CBF-binding site. CBF and c-Myb were required for induction of MPO proximal enhancer activity when 32D cells differentiated in response to G-CSF. A fusion protein containing the Gal4 DNA-binding domain and the AML-1B activation domain, amino acids 216 to 480, activated transcription alone and cooperatively with c-Myb in nonmyeloid CV-1 cells. Determining how CBF and c-Myb synergize in myeloid cells might contribute to our understanding of leukemogenesis by the AML1-ETO, AML1-MDS1, CBFbeta-SMMHC, and v-Myb oncoproteins.

Full Text

The Full Text of this article is available as a PDF (428.1 KB).

Selected References

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

  1. Bae S. C., Ogawa E., Maruyama M., Oka H., Satake M., Shigesada K., Jenkins N. A., Gilbert D. J., Copeland N. G., Ito Y. PEBP2 alpha B/mouse AML1 consists of multiple isoforms that possess differential transactivation potentials. Mol Cell Biol. 1994 May;14(5):3242–3252. doi: 10.1128/mcb.14.5.3242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bruhn L., Munnerlyn A., Grosschedl R. ALY, a context-dependent coactivator of LEF-1 and AML-1, is required for TCRalpha enhancer function. Genes Dev. 1997 Mar 1;11(5):640–653. doi: 10.1101/gad.11.5.640. [DOI] [PubMed] [Google Scholar]
  3. Citro G., Perrotti D., Cucco C., D'Agnano I., Sacchi A., Zupi G., Calabretta B. Inhibition of leukemia cell proliferation by receptor-mediated uptake of c-myb antisense oligodeoxynucleotides. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):7031–7035. doi: 10.1073/pnas.89.15.7031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dai P., Akimaru H., Tanaka Y., Hou D. X., Yasukawa T., Kanei-Ishii C., Takahashi T., Ishii S. CBP as a transcriptional coactivator of c-Myb. Genes Dev. 1996 Mar 1;10(5):528–540. doi: 10.1101/gad.10.5.528. [DOI] [PubMed] [Google Scholar]
  5. Dudek H., Tantravahi R. V., Rao V. N., Reddy E. S., Reddy E. P. Myb and Ets proteins cooperate in transcriptional activation of the mim-1 promoter. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1291–1295. doi: 10.1073/pnas.89.4.1291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Friedman A. D., Britos-Bray M., Suzow J. The murine myeloperoxidase gene contains a bipartite distal enhancer, including a novel region regulated by PEBP2/CBF. Leuk Res. 1996 Oct;20(10):809–815. doi: 10.1016/s0145-2126(96)00035-5. [DOI] [PubMed] [Google Scholar]
  8. Friedman A. D., Krieder B. L., Venturelli D., Rovera G. Transcriptional regulation of two myeloid-specific genes, myeloperoxidase and lactoferrin, during differentiation of the murine cell line 32D C13. Blood. 1991 Nov 1;78(9):2426–2432. [PubMed] [Google Scholar]
  9. Friedman A. D., Landschulz W. H., McKnight S. L. CCAAT/enhancer binding protein activates the promoter of the serum albumin gene in cultured hepatoma cells. Genes Dev. 1989 Sep;3(9):1314–1322. doi: 10.1101/gad.3.9.1314. [DOI] [PubMed] [Google Scholar]
  10. Friedman A. D. Regulation of immature myeloid cell differentiation by PEBP2/CBF, Myb, C/EBP and Ets family members. Curr Top Microbiol Immunol. 1996;211:149–157. doi: 10.1007/978-3-642-85232-9_15. [DOI] [PubMed] [Google Scholar]
  11. Gewirtz A. M., Anfossi G., Venturelli D., Valpreda S., Sims R., Calabretta B. G1/S transition in normal human T-lymphocytes requires the nuclear protein encoded by c-myb. Science. 1989 Jul 14;245(4914):180–183. doi: 10.1126/science.2665077. [DOI] [PubMed] [Google Scholar]
  12. Hernandez-Munain C., Krangel M. S. Regulation of the T-cell receptor delta enhancer by functional cooperation between c-Myb and core-binding factors. Mol Cell Biol. 1994 Jan;14(1):473–483. doi: 10.1128/mcb.14.1.473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hernandez-Munain C., Krangel M. S. c-Myb and core-binding factor/PEBP2 display functional synergy but bind independently to adjacent sites in the T-cell receptor delta enhancer. Mol Cell Biol. 1995 Jun;15(6):3090–3099. doi: 10.1128/mcb.15.6.3090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hu M., Krause D., Greaves M., Sharkis S., Dexter M., Heyworth C., Enver T. Multilineage gene expression precedes commitment in the hemopoietic system. Genes Dev. 1997 Mar 15;11(6):774–785. doi: 10.1101/gad.11.6.774. [DOI] [PubMed] [Google Scholar]
  15. Levanon D., Negreanu V., Bernstein Y., Bar-Am I., Avivi L., Groner Y. AML1, AML2, and AML3, the human members of the runt domain gene-family: cDNA structure, expression, and chromosomal localization. Genomics. 1994 Sep 15;23(2):425–432. doi: 10.1006/geno.1994.1519. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Meyers S., Downing J. R., Hiebert S. W. Identification of AML-1 and the (8;21) translocation protein (AML-1/ETO) as sequence-specific DNA-binding proteins: the runt homology domain is required for DNA binding and protein-protein interactions. Mol Cell Biol. 1993 Oct;13(10):6336–6345. doi: 10.1128/mcb.13.10.6336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Meyers S., Lenny N., Hiebert S. W. The t(8;21) fusion protein interferes with AML-1B-dependent transcriptional activation. Mol Cell Biol. 1995 Apr;15(4):1974–1982. doi: 10.1128/mcb.15.4.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Miyoshi H., Shimizu K., Kozu T., Maseki N., Kaneko Y., Ohki M. t(8;21) breakpoints on chromosome 21 in acute myeloid leukemia are clustered within a limited region of a single gene, AML1. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10431–10434. doi: 10.1073/pnas.88.23.10431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mucenski M. L., McLain K., Kier A. B., Swerdlow S. H., Schreiner C. M., Miller T. A., Pietryga D. W., Scott W. J., Jr, Potter S. S. A functional c-myb gene is required for normal murine fetal hepatic hematopoiesis. Cell. 1991 May 17;65(4):677–689. doi: 10.1016/0092-8674(91)90099-k. [DOI] [PubMed] [Google Scholar]
  21. Ness S. A., 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. doi: 10.1101/gad.7.5.749. [DOI] [PubMed] [Google Scholar]
  22. Nuchprayoon I., Meyers S., Scott L. M., Suzow J., Hiebert S., Friedman A. D. 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. doi: 10.1128/mcb.14.8.5558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nuchprayoon I., Simkevich C. P., Luo M., Friedman A. D., Rosmarin A. G. GABP cooperates with c-Myb and C/EBP to activate the neutrophil elastase promoter. Blood. 1997 Jun 15;89(12):4546–4554. [PubMed] [Google Scholar]
  24. Oelgeschläger M., Krieg J., Lüscher-Firzlaff J. M., Lüscher B. Casein kinase II phosphorylation site mutations in c-Myb affect DNA binding and transcriptional cooperativity with NF-M. Mol Cell Biol. 1995 Nov;15(11):5966–5974. doi: 10.1128/mcb.15.11.5966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Ogawa E., Inuzuka M., Maruyama M., Satake M., Naito-Fujimoto M., Ito Y., Shigesada K. Molecular cloning and characterization of PEBP2 beta, the heterodimeric partner of a novel Drosophila runt-related DNA binding protein PEBP2 alpha. Virology. 1993 May;194(1):314–331. doi: 10.1006/viro.1993.1262. [DOI] [PubMed] [Google Scholar]
  27. Ogawa E., Maruyama M., Kagoshima H., Inuzuka M., Lu J., Satake M., Shigesada K., Ito Y. PEBP2/PEA2 represents a family of transcription factors homologous to the products of the Drosophila runt gene and the human AML1 gene. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6859–6863. doi: 10.1073/pnas.90.14.6859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Okuda T., van Deursen J., Hiebert S. W., Grosveld G., Downing J. R. AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis. Cell. 1996 Jan 26;84(2):321–330. doi: 10.1016/s0092-8674(00)80986-1. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Patel G., Kreider B., Rovera G., Reddy E. P. v-myb blocks granulocyte colony-stimulating factor-induced myeloid cell differentiation but not proliferation. Mol Cell Biol. 1993 Apr;13(4):2269–2276. doi: 10.1128/mcb.13.4.2269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sadowski I., Ptashne M. A vector for expressing GAL4(1-147) fusions in mammalian cells. Nucleic Acids Res. 1989 Sep 25;17(18):7539–7539. doi: 10.1093/nar/17.18.7539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Shapiro L. H. Myb and Ets proteins cooperate to transactivate an early myeloid gene. J Biol Chem. 1995 Apr 14;270(15):8763–8771. doi: 10.1074/jbc.270.15.8763. [DOI] [PubMed] [Google Scholar]
  35. Smith M. In vitro mutagenesis. Annu Rev Genet. 1985;19:423–462. doi: 10.1146/annurev.ge.19.120185.002231. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Suzow J., Friedman A. D. The murine myeloperoxidase promoter contains several functional elements, one of which binds a cell type-restricted transcription factor, myeloid nuclear factor 1 (MyNF1). Mol Cell Biol. 1993 Apr;13(4):2141–2151. doi: 10.1128/mcb.13.4.2141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Valtieri M., Tweardy D. J., Caracciolo D., Johnson K., Mavilio F., Altmann S., Santoli D., Rovera G. Cytokine-dependent granulocytic differentiation. Regulation of proliferative and differentiative responses in a murine progenitor cell line. J Immunol. 1987 Jun 1;138(11):3829–3835. [PubMed] [Google Scholar]
  39. Wang Q., Stacy T., Binder M., Marin-Padilla M., Sharpe A. H., Speck N. A. Disruption of the Cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis. Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3444–3449. doi: 10.1073/pnas.93.8.3444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wang Q., Stacy T., Miller J. D., Lewis A. F., Gu T. L., Huang X., Bushweller J. H., Bories J. C., Alt F. W., Ryan G. The CBFbeta subunit is essential for CBFalpha2 (AML1) function in vivo. Cell. 1996 Nov 15;87(4):697–708. doi: 10.1016/s0092-8674(00)81389-6. [DOI] [PubMed] [Google Scholar]
  41. Wang S., Wang Q., Crute B. E., Melnikova I. N., Keller S. R., Speck N. A. Cloning and characterization of subunits of the T-cell receptor and murine leukemia virus enhancer core-binding factor. Mol Cell Biol. 1993 Jun;13(6):3324–3339. doi: 10.1128/mcb.13.6.3324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Zaiman A. L., Lenz J. Transcriptional activation of a retrovirus enhancer by CBF (AML1) requires a second factor: evidence for cooperativity with c-Myb. J Virol. 1996 Aug;70(8):5618–5629. doi: 10.1128/jvi.70.8.5618-5629.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zhang D. E., Hetherington C. J., Meyers S., Rhoades K. L., Larson C. J., Chen H. M., Hiebert S. W., Tenen D. G. CCAAT enhancer-binding protein (C/EBP) and AML1 (CBF alpha2) synergistically activate the macrophage colony-stimulating factor receptor promoter. Mol Cell Biol. 1996 Mar;16(3):1231–1240. doi: 10.1128/mcb.16.3.1231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. Zhu J., Bennett C. A., MacGregor A. D., Greaves M. F., Goodwin G. H., Ford A. M. A myeloid-lineage-specific enhancer upstream of the mouse myeloperoxidase (MPO) gene. Leukemia. 1994 May;8(5):717–723. [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES