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Functional characterization of the promoter region of the human EVI1 gene in acute myeloid leukemia: RUNX1 and ELK1 directly regulate its transcription

Oncogene volume 32, pages 2069–2078 (2013)Cite this article

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Abstract

The EVI1 gene (3q26) codes for a transcription factor with important roles in normal hematopoiesis and leukemogenesis. High expression of EVI1 is a negative prognostic indicator of survival in acute myeloid leukemia (AML) irrespective of the presence of 3q26 rearrangements. However, the only known mechanisms that lead to EVI1 overexpression are 3q aberrations, and the MLL-ENL oncoprotein, which activates the transcription of EVI1 in hematopoietic stem cells. Our aim was to characterize the functional promoter region of EVI1, and to identify transcription factors involved in the regulation of this gene. Generation of seven truncated constructs and luciferase reporter assays allowed us to determine a 318-bp region as the minimal promoter region of EVI1. Site-directed mutagenesis and chromatin immunoprecipitation (ChIP) assays identified RUNX1 and ELK1 as putative transcription factors of EVI1. Furthermore, knockdown of RUNX1 and ELK1 led to EVI1 downregulation, and their overexpression to upregulation of EVI1. Interestingly, in a series of patient samples with AML at diagnosis, we found a significant positive correlation between EVI1 and RUNX1 at protein level. Moreover, we identified one of the roles of RUNX1 in the activation of EVI1 during megakaryocytic differentiation. EVI1 knockdown significantly inhibited the expression of megakaryocytic markers after treating K562 cells with TPA, as happens when knocking down RUNX1. In conclusion, we define the minimal promoter region of EVI1 and demonstrate that RUNX1 and ELK1, two proteins with essential functions in hematopoiesis, regulate EVI1 in AML. Furthermore, our results show that one of the mechanisms by which RUNX1 regulates the transcription of EVI1 is by acetylation of the histone H3 on its promoter region. This study opens new directions to further understand the mechanisms of EVI1 overexpressing leukemias.

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References

  1. Goyama S, Yamamoto G, Shimabe M, Sato T, Ichikawa M, Ogawa S et al. Evi-1 is a critical regulator for hematopoietic stem cells and transformed leukemic cells. Cell Stem Cell 2008; 3: 207–220.

    Article  CAS  Google Scholar 

  2. Yuasa H, Oike Y, Iwama A, Nishikata I, Sugiyama D, Perkins A et al. Oncogenic transcription factor Evi1 regulates hematopoietic stem cell proliferation through GATA-2 expression. EMBO J 2005; 24: 1976–1987.

    Article  CAS  Google Scholar 

  3. Shimizu S, Nagasawa T, Katoh O, Komatsu N, Yokota J, Morishita K . EVI1 is expressed in megakaryocyte cell lineage and enforced expression of EVI1 in UT-7/GM cells induces megakaryocyte differentiation. Biochem Biophys Res Commun 2002; 292: 609–616.

    Article  CAS  Google Scholar 

  4. Dohner H, Estey EH, Amadori S, Appelbaum FR, Buchner T, Burnett AK et al. Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood 2010; 115: 453–474.

    Article  Google Scholar 

  5. Lugthart S, van Drunen E, van Norden Y, van Hoven A, Erpelinck CA, Valk PJ et al. High EVI1 levels predict adverse outcome in acute myeloid leukemia: prevalence of EVI1 overexpression and chromosome 3q26 abnormalities underestimated. Blood 2008; 111: 4329–4337.

    Article  CAS  Google Scholar 

  6. Haas K, Kundi M, Sperr WR, Esterbauer H, Ludwig WD, Ratei R et al. Expression and prognostic significance of different mRNA 5'-end variants of the oncogene EVI1 in 266 patients with de novo AML: EVI1 and MDS1/EVI1 overexpression both predict short remission duration. Genes Chromosomes Cancer 2008; 47: 288–298.

    Article  CAS  Google Scholar 

  7. Groschel S, Lugthart S, Schlenk RF, Valk PJ, Eiwen K, Goudswaard C et al. High EVI1 expression predicts outcome in younger adult patients with acute myeloid leukemia and is associated with distinct cytogenetic abnormalities. J Clin Oncol 2010; 28: 2101–2107.

    Article  Google Scholar 

  8. Vazquez I, Maicas M, Cervera J, Agirre X, Marin-Bejar O, Marcotegui N et al. Down-regulation of EVI1 is associated with epigenetic alterations and good prognosis in patients with acute myeloid leukemia. Haematologica 96: 1448–1456.

    Article  CAS  Google Scholar 

  9. Wieser R . The oncogene and developmental regulator EVI1: expression, biochemical properties, and biological functions. Gene 2007; 396: 346–357.

    Article  CAS  Google Scholar 

  10. Nucifora G, Laricchia-Robbio L, Senyuk V . EVI1 and hematopoietic disorders: history and perspectives. Gene 2006; 368: 1–11.

    Article  CAS  Google Scholar 

  11. Laricchia-Robbio L, Fazzina R, Li D, Rinaldi CR, Sinha KK, Chakraborty S et al. Point mutations in two EVI1 Zn fingers abolish EVI1-GATA1 interaction and allow erythroid differentiation of murine bone marrow cells. Mol Cell Biol 2006; 26: 7658–7666.

    Article  CAS  Google Scholar 

  12. Laricchia-Robbio L, Premanand K, Rinaldi CR, Nucifora G . EVI1 Impairs myelopoiesis by deregulation of PU.1 function. Cancer Res 2009; 69: 1633–1642.

    Article  CAS  Google Scholar 

  13. Senyuk V, Sinha KK, Li D, Rinaldi CR, Yanamandra S, Nucifora G . Repression of RUNX1 activity by EVI1: a new role of EVI1 in leukemogenesis. Cancer Res 2007; 67: 5658–5666.

    Article  CAS  Google Scholar 

  14. Vazquez I, Maicas M, Cervera J, Agirre X, Marin-Bejar O, Marcotegui N et al. Down-regulation of EVI1 is associated with epigenetic alterations and good prognosis in patients with acute myeloid leukemia. Haematologica 2011; 96: 1448–1456.

    Article  CAS  Google Scholar 

  15. Arai S, Yoshimi A, Shimabe M, Ichikawa M, Nakagawa M, Imai Y et al. Evi-1 is a transcriptional target of mixed-lineage leukemia oncoproteins in hematopoietic stem cells. Blood 2010; 117: 6304–6314.

    Article  Google Scholar 

  16. Fujiwara T, O’Geen H, Keles S, Blahnik K, Linnemann AK, Kang YA et al. Discovering hematopoietic mechanisms through genome-wide analysis of GATA factor chromatin occupancy. Mol Cell 2009; 36: 667–681.

    Article  CAS  Google Scholar 

  17. Oakford PC, James SR, Qadi A, West AC, Ray SN, Bert AG et al. Transcriptional and epigenetic regulation of the GM-CSF promoter by RUNX1. Leuk Res 2010; 34: 1203–1213.

    Article  CAS  Google Scholar 

  18. Mikhail FM, Sinha KK, Saunthararajah Y, Nucifora G . Normal and transforming functions of RUNX1: a perspective. J Cell Physiol 2006; 207: 582–593.

    Article  CAS  Google Scholar 

  19. Gaidzik VI, Bullinger L, Schlenk RF, Zimmermann AS, Rock J, Paschka P et al. RUNX1 mutations in acute myeloid leukemia: results from a comprehensive genetic and clinical analysis from the AML study group. J Clin Oncol 2011; 29: 1364–1372.

    Article  Google Scholar 

  20. Elagib KE, Racke FK, Mogass M, Khetawat R, Delehanty LL, Goldfarb AN . RUNX1 and GATA-1 coexpression and cooperation in megakaryocytic differentiation. Blood 2003; 101: 4333–4341.

    Article  CAS  Google Scholar 

  21. Pencovich N, Jaschek R, Tanay A, Groner Y . Dynamic combinatorial interactions of RUNX1 and cooperating partners regulates megakaryocytic differentiation in cell line models. Blood 2011; 117: e1–14.

    Article  CAS  Google Scholar 

  22. Bingemann SC, Konrad TA, Wieser R . Zinc finger transcription factor ecotropic viral integration site 1 is induced by all-trans retinoic acid (ATRA) and acts as a dual modulator of the ATRA response. FEBS J 2009; 276: 6810–6822.

    Article  CAS  Google Scholar 

  23. Aytekin M, Vinatzer U, Musteanu M, Raynaud S, Wieser R . Regulation of the expression of the oncogene EVI1 through the use of alternative mRNA 5'-ends. Gene 2005; 356: 160–168.

    Article  CAS  Google Scholar 

  24. Goldfarb AN . Megakaryocytic programming by a transcriptional regulatory loop: a circle connecting RUNX1, GATA-1, and P-TEFb. J Cell Biochem 2009; 107: 377–382.

    Article  CAS  Google Scholar 

  25. Terui K, Takahashi Y, Kitazawa J, Toki T, Yokoyama M, Ito E . Expression of transcription factors during megakaryocytic differentiation of CD34+ cells from human cord blood induced by thrombopoietin. Tohoku J Exp Med 2000; 192: 259–273.

    Article  CAS  Google Scholar 

  26. Osato M . Point mutations in the RUNX1/AML1 gene: another actor in RUNX leukemia. Oncogene 2004; 23: 4284–4296.

    Article  CAS  Google Scholar 

  27. Zhang L, Li Z, Yan J, Pradhan P, Corpora T, Cheney MD et al. Mutagenesis of the Runt domain defines two energetic hot spots for heterodimerization with the core binding factor beta subunit. J Biol Chem 2003; 278: 33097–33104.

    Article  CAS  Google Scholar 

  28. Langabeer SE, Gale RE, Rollinson SJ, Morgan GJ, Linch DC . Mutations of the AML1 gene in acute myeloid leukemia of FAB types M0 and M7. Genes Chromosomes Cancer 2002; 34: 24–32.

    Article  CAS  Google Scholar 

  29. Osato M, Asou N, Abdalla E, Hoshino K, Yamasaki H, Okubo T et al. Biallelic and heterozygous point mutations in the runt domain of the AML1/PEBP2alphaB gene associated with myeloblastic leukemias. Blood 1999; 93: 1817–1824.

    CAS  PubMed  Google Scholar 

  30. Yordy JS, Muise-Helmericks RC . Signal transduction and the Ets family of transcription factors. Oncogene 2000; 19: 6503–6513.

    Article  CAS  Google Scholar 

  31. Wasylyk B, Hahn SL, Giovane A . The Ets family of transcription factors. Eur J Biochem 1993; 211: 7–18.

    Article  CAS  Google Scholar 

  32. Yasar D, Karadogan I, Alanoglu G, Akkaya B, Luleci G, Salim O et al. Array comparative genomic hybridization analysis of adult acute leukemia patients. Cancer Genet Cytogenet 2010; 197: 122–129.

    Article  CAS  Google Scholar 

  33. Cruzalegui FH, Cano E, Treisman R . ERK activation induces phosphorylation of Elk-1 at multiple S/T-P motifs to high stoichiometry. Oncogene 1999; 18: 7948–7957.

    Article  CAS  Google Scholar 

  34. Garcia J, de Gunzburg J, Eychene A, Gisselbrecht S, Porteu F . Thrombopoietin-mediated sustained activation of extracellular signal-regulated kinase in UT7-Mpl cells requires both Ras-Raf-1- and Rap1-B-Raf-dependent pathways. Mol Cell Biol 2001; 21: 2659–2670.

    Article  CAS  Google Scholar 

  35. Biggs JR, Peterson LF, Zhang Y, Kraft AS, Zhang DE . AML1/RUNX1 phosphorylation by cyclin-dependent kinases regulates the degradation of AML1/RUNX1 by the anaphase-promoting complex. Mol Cell Biol 2006; 26: 7420–7429.

    Article  CAS  Google Scholar 

  36. Fernandez-Alvarez A, Soledad Alvarez M, Cucarella C, Casado M . Characterization of the human insulin-induced gene 2 (INSIG2) promoter: the role of Ets-binding motifs. J Biol Chem 2010; 285: 11765–11774.

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Dr Hipskind (Hannover Medical School, Germany) for the plasmid pCDNA3-ELK1, Dr Zhang (Department of Molecular and Experimental Medicine, La Jolla, CA, USA) for the plasmid pFlagCMV2–AML1B (Addgene plasmid 12504), and Dr Arenas and Dr Guruceaga (Department of Proteomics, Genomics and Bioinformatics, CIMA) for technical support. This work was supported by Ministerio Educación y Ciencia (AP2007-03879), Ministerio Ciencia e Innovación (PI081687), Departamento Salud del Gobierno de Navarra (14/2008), ISCIII-RTICC (RD06/0020/0078) and Fundación para la Investigación Médica Aplicada y UTE (Spain).

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Authors and Affiliations

  1. Division of Oncology, Laboratory of Genetics, CIMA, University of Navarra, Pamplona, Spain

    M Maicas, I Vázquez, C Vicente, M A García-Sánchez, N Marcotegui, L Urquiza & M D Odero

  2. Department of Genetics, University of Navarra, Pamplona, Spain

    M Maicas, M J Calasanz & M D Odero

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  1. M Maicas
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Correspondence to M D Odero.

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Maicas, M., Vázquez, I., Vicente, C. et al. Functional characterization of the promoter region of the human EVI1 gene in acute myeloid leukemia: RUNX1 and ELK1 directly regulate its transcription. Oncogene 32, 2069–2078 (2013). https://doi.org/10.1038/onc.2012.222

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