Microarray Profiling of Genes Differentially Expressed during Erythroid Differentiation of Murine Erythroleukemia Cells

  • Heo, Hyen Seok (Department of Life Science, College of Natural Sciences, Hanyang University) ;
  • Kim, Ju Hyun (Department of Life Science, College of Natural Sciences, Hanyang University) ;
  • Lee, Young Jin (Department of Life Science, College of Natural Sciences, Hanyang University) ;
  • Kim, Sung-Hyun (Department of Life Science, College of Natural Sciences, Hanyang University) ;
  • Cho, Yoon Shin (Department of Life Science, College of Natural Sciences, Hanyang University) ;
  • Kim, Chul Geun (Department of Life Science, College of Natural Sciences, Hanyang University)
  • Received : 2005.02.01
  • Accepted : 2005.05.17
  • Published : 2005.08.31


Murine erythroleukemia (MEL) cells are widely used to study erythroid differentiation thanks to their ability to terminally differentiate in vitro in response to chemical induction. At the molecular level, not much is known of their terminal differentiation apart from activation of adult-type globin gene expression. We examined changes in gene expression during the terminal differentiation of these cells using microarray-based technology. We identified 180 genes whose expression changed significantly during differentiation. The microarray data were analyzed by hierarchical and k-means clustering and confirmed by semi-quantitative RT-PCR. We identified several genes including H1f0, Bnip3, Mgl2, ST7L, and Cbll1 that could be useful markers for erythropoiesis. These genetic markers should be a valuable resource both as potential regulators in functional studies of erythroid differentiation, and as straightforward cell type markers.


Supported by : Ministry of Health and Welfare of Korea


  1. Chen, Z. X., Banks, J., Rifkind, R. A., and Marks, P. A. (1982) Inducer-mediated commitment of murine erythroleukemia cells to differentiation: a multistep process. Proc. Natl. Acad. Sci. USA 79, 471-475
  2. Little, J. A., Dempsey, N. J., Tuchman, M., and Ginder, G. D. (1995) Metabolic persistence of fetal hemoglobin. Blood 85, 1712-1718
  3. Osborne, H. B. and Chabanas, A. (1984) Kinetics of histone H10 accumulation and commitment to differentiation in murine erythroleukemia cells. Exp. Cell. Res. 152, 449-458 https://doi.org/10.1016/0014-4827(84)90646-3
  4. Pettersson, M., Bessonova, M., Gu, H. F., Groop, L. C., and Jonsson, J. I. (2000) Characterization, chromosomal localization, and expression during hematopoietic differentiation of the gene encoding Arl6ip, ADP-ribosylation-like factor-6 interacting protein (ARL6). Genomics 68, 351-354 https://doi.org/10.1006/geno.2000.6278
  5. Ruff, P., Speicher, D. W., and Husain-Chishti, A. (1991) Molecular identification of a major palmitoylated erythrocyte membrane protein containing the src homology 3 motif. Proc. Natl. Acad. Sci. USA 88, 6595-6569
  6. Tabilio, A., Pelicci, P. G., Vinci, G., Mannoni, P., Civin, C. I., et al. (1983) Myeloid and megakaryocytic properties of K-562 cell lines. Cancer Res. 43, 4569-4574
  7. Zlatanova, J. and Doenecke, D. (1994) Histone H1 zero: a major player in cell differentiation? FASEB J. 8, 1260-1268
  8. Orkin, S. H. (1996) Development of the hematopoietic system. Curr. Opin. Genet. Dev. 6, 597-602 https://doi.org/10.1016/S0959-437X(96)80089-X
  9. Trimborn, T., Gribnau, J., Grosveld, F., and Fraser, P. (1999) Mechanisms of developmental control of transcription in the murine alpha- and beta-globin loci. Genes Dev. 13, 112-124 https://doi.org/10.1101/gad.13.1.112
  10. Tusher, V. G., Tibshirani, R., and Chu, G. (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci. USA 98, 5116-5121
  11. Taylor, G. A. M., Jeffers, D. A., Largaespada, N. A., Jenkins, N. G., Copeland, G. F., et al. (1996) Identification of a novel GTPase, the inducibly expressed GTPase, that accumulates in response to interferon-gamma. J. Biol. Chem. 271, 20399- 20405 https://doi.org/10.1074/jbc.271.34.20399
  12. Testa, U. (2004) Apoptotic mechanisms in the control of erythropoiesis. Leukemia 18, 1176-1199 https://doi.org/10.1038/sj.leu.2403383
  13. Benezra, R., Davis, R. L., Lockshon, D., Turner, D. L., and Weintraub, H. (1990b) The protein Id: a negative regulator of helix-loop-helix DNA binding proteins. Cell 61, 49-59 https://doi.org/10.1016/0092-8674(90)90214-Y
  14. Migliaccio, A. R. and Migliaccio, G. (1998) The making of an erythroid cell. Molecular control of hematopoiesis. Biotherapy 10, 251-268 https://doi.org/10.1007/BF02678546
  15. Benezra, R., Davis, R. L., Lassar, A., Tapscott, S., Thayer, M., et al. (1990a) Id: a negative regulator of helix-loop-helix DNA binding proteins. Control of terminal myogenic differentiation. Ann. N. Y. Acad. Sci. 599, 1-11 https://doi.org/10.1111/j.1749-6632.1990.tb42359.x
  16. Cheng, G. H. and Skoultchi, A. I. (1989) Rapid induction of polyadenylated H1 histone mRNAs in mouse erythroleukemia cells is regulated by c-myc. Mol. Cell. Biol. 9, 2332- 2340
  17. Fujita, Y., Krause, G., Scheffner, M., Zechner, D., Leddy, H. E., et al. (2002) Hakai, a c-Cbl-like protein, ubiquitinates and induces endocytosis of the E-cadherin complex. Nat. Cell. Biol. 4, 222-231 https://doi.org/10.1038/ncb758
  18. Keppel, F., Allet, B., and Eisen, H. (1977) Appearance of a chromatin protein during the erythroid differentiation of Friend virus-transformed cells. Proc. Natl. Acad. Sci. USA 74, 653-656
  19. Vannucchi, A. M., Linari, S., Cellai, C., Koury, M. J., and Paoletti, F. (1997) Constitutive and inducible expression of megakaryocyte-specific genes in Friend erythroleukaemia cells. Br. J. Haematol. 99, 500-508 https://doi.org/10.1046/j.1365-2141.1997.4273230.x
  20. Choi, I., Muta, K., Wickrema, A., Krantz, S. B., Nishimura, J., et al. (2000) Interferon gamma delays apoptosis of mature erythroid progenitor cells in the absence of erythropoietin. Blood 95, 3742-3749
  21. Palis, J., Robertson, S., Kennedy, M., Wall, C., and Keller, G. (1999) Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse. Development 126, 5073-5084
  22. Verdel, A., Seigneurin-Berny, D., Faure, A. K., Eddahbi, M., Khochbin, S., et al. (2003) HDAC6-induced premature chromatin compaction in mouse oocytes and fertilised eggs. Zygote 11, 323-328 https://doi.org/10.1017/S0967199403002387
  23. Haslauer, M., Baltensperger, K., and Porzig, H. (1999) Erythropoietin- and stem cell factor-induced DNA synthesis in normal human erythroid progenitor cells requires activation of protein kinase C-alpha and is strongly inhibited by thrombin. Blood 94, 114-126
  24. Perry, C. and Soreq, H. (2002) Transcriptional regulation of erythropoiesis. Fine tuning of combinatorial multi-domain elements. Eur. J. Biochem. 269, 3607-3618 https://doi.org/10.1046/j.1432-1033.2002.02999.x
  25. Yang, Y. H., Dudoit, S., Luu, P., Lin, D. M., Peng, V., et al. (2002) Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 30, e15 https://doi.org/10.1093/nar/30.4.e15
  26. Marks, P. A., Sheffery, M., Ramsay, R., Ikeda, K., and Rifkind, R. A. (1987) Induction of transformed cells to terminal differentiation. Ann. N. Y. Acad. Sci. 511, 246-255 https://doi.org/10.1111/j.1749-6632.1987.tb36252.x
  27. Palis, J. and Segel, G. B. (1998) Developmental biology of erythropoiesis. Blood Rev. 12, 106-114 https://doi.org/10.1016/S0268-960X(98)90022-4
  28. Yasuda, M., Theodorakis, P., Subramanian, T., and Chinnadurai, G. (1998) Adenovirus E1B-19K/BCL-2 interacting protein BNIP3 contains a BH3 domain and a mitochondrial targeting sequence. J. Biol. Chem. 273, 12415-12421 https://doi.org/10.1074/jbc.273.20.12415
  29. Koh, P. O., Noh, H. S., Kim, Y. S., Cheon, E. W., Kim, H. J., et al. (2003) Cellular localization of pitutary adenylate cyclaseactivating polypeptide in the rat testis. Mol. Cells 15, 271- 276
  30. Marks, P. A., Chen, Z., Banks, J., and Rifkind, R. A. (1983) Erythroleukemia cells: variants inducible for hemoglobin synthesis without commitment to terminal cell division. Proc. Natl. Acad. Sci. USA 80, 2281-2284
  31. Fujiwara, Y., Browne, C. P., Cunniff, K., Goff, S. C., and Orkin, S. H. (1996) Arrested development of embryonic red cell precursors in mouse embryos lacking transcription factor GATA-1. Proc. Natl. Acad. Sci. USA 93, 12355-12358
  32. Ray, R., Chen, G., Vande, V. C., Cizeau, J., Park, J. H., et al. (2000) BNIP3 heterodimerizes with Bcl-2/Bcl-X(L) and induces cell death independent of a Bcl-2 homology 3 (BH3) domain at both mitochondrial and nonmitochondrial sites. J. Biol. Chem. 275, 1439-1448 https://doi.org/10.1074/jbc.275.2.1439
  33. Sherlock, G. (2000) Analysis of large-scale gene expression data. Curr. Opin. Immunol. 12, 201-205 https://doi.org/10.1016/S0952-7915(99)00074-6
  34. Dame, C. and Juul, S. E. (2000) The switch from fetal to adult erythropoiesis. Clin. Perinatol. 27, 507-526 https://doi.org/10.1016/S0095-5108(05)70036-1
  35. Dudoit, S., Yang, Y. H., Callow, M. J., and Speed, T. P. (2002) Statistical methods for identifying differentially expressed genes in replicated cDNA microarray experiments. Statistica. Sinica. 12, 111-139
  36. Tsiftsoglou, A. S. and Robinson, S. H. (1985) Differentiation of leukemic cell lines: a review focusing on murine erythroleukemia and human HL-60 cells. Int. J. Cell. Cloning 3, 349- 366 https://doi.org/10.1002/stem.5530030602
  37. Itoh, T., Itoh, A., and Pleasure, D. (2003) Bcl-2-related protein family gene expression during oligodendroglial differentiation. J. Neurochem. 85, 1500-1512 https://doi.org/10.1046/j.1471-4159.2003.01795.x
  38. Rutledge, R. G., Neelin, J. M., and Seligy, V. L. (1984) Uncoupled synthesis of H1o-like histone H1s during late erythropoiesis in Xenopus laevis. Eur. J. Biochem. 144, 191-198 https://doi.org/10.1111/j.1432-1033.1984.tb08448.x
  39. Green, A. R., Lints, T., Visvader, J., Harvey, R., and Begley, C. G. (1992) SCL is coexpressed with GATA-1 in hemopoietic cells but is also expressed in developing brain. Oncogene 7, 653-660
  40. Koutzamani, E., Loborg, H., Sarg, B., Lindner, H. H., and Rundquist, I. (2002) Linker histone subtype composition and affinity for chromatin in situ in nucleated mature erythrocytes. J. Biol. Chem. 277, 44688-44694 https://doi.org/10.1074/jbc.M203533200
  41. Pevny, L., Simon, M. C., Robertson, E., Klein, W. H., Tsai, S. F., et al. (1991) Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1. Nature 349, 257-260 https://doi.org/10.1038/349257a0
  42. Rekhtman, N., Radparvar, F., Evans, T., and Skoultchi, A. I. (1999) Direct interaction of hematopoietic transcription factors PU.1 and GATA-1: functional antagonism in erythroid cells. Genes Dev. 13, 1398-1411 https://doi.org/10.1101/gad.13.11.1398
  43. Eisen, M. B., Spellman, P. T., Brown, P. O., and Botstein, D. (1998) Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. USA 95, 14863-14868
  44. Leonard, M., Brice, M., Engel, J. D., and Papayannopoulou, T. (1993) Dynamics of GATA transcription factor expression during erythroid differentiation. Blood 82, 1071-1079
  45. Lister, J., Forrester, W. C., and Baron, M. H. (1995) Inhibition of an erythroid differentiation switch by the helix-loop-helix protein Id1. J. Biol. Chem. 270, 17939-17946 https://doi.org/10.1074/jbc.270.30.17939
  46. Dzierzak, E. and Medvinsky, A. (1995) Mouse embryonic hematopoiesis. Trends. Genet. 11, 359-366 https://doi.org/10.1016/S0168-9525(00)89107-6
  47. Manduchi, E., Scearce, L. M., Brestelli, J. E., Grant, G. R., Kaestner, K. H., et al. (2002) Comparison of different labeling methods for two-channel high-density microarray experiments. Physiol. Genomics 10, 169-179
  48. Shoji, W., Yamamoto, T., and Obinata, M. (1994) The helixloop- helix protein Id inhibits differentiation of murine erythroleukemia cells. J. Biol. Chem. 269, 5078-5084
  49. Leder, A. and Leder, P. (1975) Butyric acid, a potent inducer of erythroid differentiation in cultured erythroleukemic cells. Cell 5, 319-322 https://doi.org/10.1016/0092-8674(75)90107-5
  50. Scott, E. W., Simon, M. C., Anastasi, J., and Singh, H. (1994) Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages. Science 265, 1573-1577 https://doi.org/10.1126/science.8079170
  51. Starck, J., Cohet, N., Gonnet, C., Sarrazin, S., Doubeikovskaia, Z., et al. (2003) Functional cross-antagonism between transcription factors FLI-1 and EKLF. Mol. Cell. Biol. 23, 1390- 1402 https://doi.org/10.1128/MCB.23.4.1390-1402.2003
  52. Wade, P. A. (2001) Transcriptional control at regulatory check-points by histone deacetylases: molecular connections between cancer and chromatin. Hum. Mol. Genet. 10, 693-698 https://doi.org/10.1093/hmg/10.7.693