Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1996 Feb;16(2):685–693. doi: 10.1128/mcb.16.2.685

E2F-1 blocks terminal differentiation and causes proliferation in transgenic megakaryocytes.

C T Guy 1, W Zhou 1, S Kaufman 1, M O Robinson 1
PMCID: PMC231048  PMID: 8552097

Abstract

The transcription factor E2F-1 plays a central role in the cell cycle through its ability to activate genes involved in cell division. E2F-1 activity is regulated by a number of proteins, including the retinoblastoma susceptibility gene product, cyclin-dependent kinases, and their inhibitors, proteins that have been implicated in the control of certain developmental processes. To investigate a potential role of E2F-1 in differentiation, we assayed the ability of megakaryocytes to form platelets in an in vivo transgenic model. E2F-1 expression in megakaryocytes blocked differentiation during maturation, resulting in severe thrombocytopenia. Ultrastructural analysis of megakaryocytes revealed abnormal development characterized by hyperdemarcation of cytoplasmic membranes and reduced numbers of alpha granules. Administration of megakaryocyte growth and development factor or interleukin 6 could not overcome the differentiation block. Additionally, E2F-1 caused massive megakaryocyte accumulation in both normal and ectopic sites, first evident in E15 embryonic liver. Furthermore, significant apoptosis was observed in transgenic megakaryocytes. These data indicate that E2F-1 can prevent terminal differentiation, probably through its cell cycle-stimulatory activity.

Full Text

The Full Text of this article is available as a PDF (2.3 MB).

Selected References

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

  1. An E., Ogata K., Kuriya S., Nomura T. Interleukin-6 and erythropoietin act as direct potentiators and inducers of in vitro cytoplasmic process formation on purified mouse megakaryocytes. Exp Hematol. 1994 Feb;22(2):149–156. [PubMed] [Google Scholar]
  2. Bandara L. R., Buck V. M., Zamanian M., Johnston L. H., La Thangue N. B. Functional synergy between DP-1 and E2F-1 in the cell cycle-regulating transcription factor DRTF1/E2F. EMBO J. 1993 Nov;12(11):4317–4324. doi: 10.1002/j.1460-2075.1993.tb06116.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bartley T. D., Bogenberger J., Hunt P., Li Y. S., Lu H. S., Martin F., Chang M. S., Samal B., Nichol J. L., Swift S. Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl. Cell. 1994 Jul 1;77(7):1117–1124. doi: 10.1016/0092-8674(94)90450-2. [DOI] [PubMed] [Google Scholar]
  4. Beijersbergen R. L., Kerkhoven R. M., Zhu L., Carlée L., Voorhoeve P. M., Bernards R. E2F-4, a new member of the E2F gene family, has oncogenic activity and associates with p107 in vivo. Genes Dev. 1994 Nov 15;8(22):2680–2690. doi: 10.1101/gad.8.22.2680. [DOI] [PubMed] [Google Scholar]
  5. Clarke A. R., Maandag E. R., van Roon M., van der Lugt N. M., van der Valk M., Hooper M. L., Berns A., te Riele H. Requirement for a functional Rb-1 gene in murine development. Nature. 1992 Sep 24;359(6393):328–330. doi: 10.1038/359328a0. [DOI] [PubMed] [Google Scholar]
  6. Dimri G. P., Hara E., Campisi J. Regulation of two E2F-related genes in presenescent and senescent human fibroblasts. J Biol Chem. 1994 Jun 10;269(23):16180–16186. [PubMed] [Google Scholar]
  7. Dou Q. P., Zhao S., Levin A. H., Wang J., Helin K., Pardee A. B. G1/S-regulated E2F-containing protein complexes bind to the mouse thymidine kinase gene promoter. J Biol Chem. 1994 Jan 14;269(2):1306–1313. [PubMed] [Google Scholar]
  8. Fisher D. E. Apoptosis in cancer therapy: crossing the threshold. Cell. 1994 Aug 26;78(4):539–542. doi: 10.1016/0092-8674(94)90518-5. [DOI] [PubMed] [Google Scholar]
  9. Flemington E. K., Speck S. H., Kaelin W. G., Jr E2F-1-mediated transactivation is inhibited by complex formation with the retinoblastoma susceptibility gene product. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):6914–6918. doi: 10.1073/pnas.90.15.6914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ginsberg D., Vairo G., Chittenden T., Xiao Z. X., Xu G., Wydner K. L., DeCaprio J. A., Lawrence J. B., Livingston D. M. E2F-4, a new member of the E2F transcription factor family, interacts with p107. Genes Dev. 1994 Nov 15;8(22):2665–2679. doi: 10.1101/gad.8.22.2665. [DOI] [PubMed] [Google Scholar]
  11. Girling R., Partridge J. F., Bandara L. R., Burden N., Totty N. F., Hsuan J. J., La Thangue N. B. A new component of the transcription factor DRTF1/E2F. Nature. 1993 Mar 4;362(6415):83–87. doi: 10.1038/362083a0. [DOI] [PubMed] [Google Scholar]
  12. Gu W., Schneider J. W., Condorelli G., Kaushal S., Mahdavi V., Nadal-Ginard B. Interaction of myogenic factors and the retinoblastoma protein mediates muscle cell commitment and differentiation. Cell. 1993 Feb 12;72(3):309–324. doi: 10.1016/0092-8674(93)90110-c. [DOI] [PubMed] [Google Scholar]
  13. Halevy O., Novitch B. G., Spicer D. B., Skapek S. X., Rhee J., Hannon G. J., Beach D., Lassar A. B. Correlation of terminal cell cycle arrest of skeletal muscle with induction of p21 by MyoD. Science. 1995 Feb 17;267(5200):1018–1021. doi: 10.1126/science.7863327. [DOI] [PubMed] [Google Scholar]
  14. Helin K., Harlow E., Fattaey A. Inhibition of E2F-1 transactivation by direct binding of the retinoblastoma protein. Mol Cell Biol. 1993 Oct;13(10):6501–6508. doi: 10.1128/mcb.13.10.6501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Helin K., Wu C. L., Fattaey A. R., Lees J. A., Dynlacht B. D., Ngwu C., Harlow E. Heterodimerization of the transcription factors E2F-1 and DP-1 leads to cooperative trans-activation. Genes Dev. 1993 Oct;7(10):1850–1861. doi: 10.1101/gad.7.10.1850. [DOI] [PubMed] [Google Scholar]
  16. Hoffman R. Regulation of megakaryocytopoiesis. Blood. 1989 Sep;74(4):1196–1212. [PubMed] [Google Scholar]
  17. Hsiao K. M., McMahon S. L., Farnham P. J. Multiple DNA elements are required for the growth regulation of the mouse E2F1 promoter. Genes Dev. 1994 Jul 1;8(13):1526–1537. doi: 10.1101/gad.8.13.1526. [DOI] [PubMed] [Google Scholar]
  18. Ishibashi T., Kimura H., Shikama Y., Uchida T., Kariyone S., Hirano T., Kishimoto T., Takatsuki F., Akiyama Y. Interleukin-6 is a potent thrombopoietic factor in vivo in mice. Blood. 1989 Sep;74(4):1241–1244. [PubMed] [Google Scholar]
  19. Ivey-Hoyle M., Conroy R., Huber H. E., Goodhart P. J., Oliff A., Heimbrook D. C. Cloning and characterization of E2F-2, a novel protein with the biochemical properties of transcription factor E2F. Mol Cell Biol. 1993 Dec;13(12):7802–7812. doi: 10.1128/mcb.13.12.7802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jacks T., Fazeli A., Schmitt E. M., Bronson R. T., Goodell M. A., Weinberg R. A. Effects of an Rb mutation in the mouse. Nature. 1992 Sep 24;359(6393):295–300. doi: 10.1038/359295a0. [DOI] [PubMed] [Google Scholar]
  21. Johnson D. G., Cress W. D., Jakoi L., Nevins J. R. Oncogenic capacity of the E2F1 gene. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12823–12827. doi: 10.1073/pnas.91.26.12823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Johnson D. G., Ohtani K., Nevins J. R. Autoregulatory control of E2F1 expression in response to positive and negative regulators of cell cycle progression. Genes Dev. 1994 Jul 1;8(13):1514–1525. doi: 10.1101/gad.8.13.1514. [DOI] [PubMed] [Google Scholar]
  23. Johnson D. G., Schwarz J. K., Cress W. D., Nevins J. R. Expression of transcription factor E2F1 induces quiescent cells to enter S phase. Nature. 1993 Sep 23;365(6444):349–352. doi: 10.1038/365349a0. [DOI] [PubMed] [Google Scholar]
  24. Kaelin W. G., Jr, Krek W., Sellers W. R., DeCaprio J. A., Ajchenbaum F., Fuchs C. S., Chittenden T., Li Y., Farnham P. J., Blanar M. A. Expression cloning of a cDNA encoding a retinoblastoma-binding protein with E2F-like properties. Cell. 1992 Jul 24;70(2):351–364. doi: 10.1016/0092-8674(92)90108-o. [DOI] [PubMed] [Google Scholar]
  25. Krek W., Livingston D. M., Shirodkar S. Binding to DNA and the retinoblastoma gene product promoted by complex formation of different E2F family members. Science. 1993 Dec 3;262(5139):1557–1560. doi: 10.1126/science.8248803. [DOI] [PubMed] [Google Scholar]
  26. La Thangue N. B. DRTF1/E2F: an expanding family of heterodimeric transcription factors implicated in cell-cycle control. Trends Biochem Sci. 1994 Mar;19(3):108–114. doi: 10.1016/0968-0004(94)90202-x. [DOI] [PubMed] [Google Scholar]
  27. Lam E. W., Watson R. J. An E2F-binding site mediates cell-cycle regulated repression of mouse B-myb transcription. EMBO J. 1993 Jul;12(7):2705–2713. doi: 10.1002/j.1460-2075.1993.tb05932.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lee E. Y., Chang C. Y., Hu N., Wang Y. C., Lai C. C., Herrup K., Lee W. H., Bradley A. Mice deficient for Rb are nonviable and show defects in neurogenesis and haematopoiesis. Nature. 1992 Sep 24;359(6393):288–294. doi: 10.1038/359288a0. [DOI] [PubMed] [Google Scholar]
  29. Lees J. A., Saito M., Vidal M., Valentine M., Look T., Harlow E., Dyson N., Helin K. The retinoblastoma protein binds to a family of E2F transcription factors. Mol Cell Biol. 1993 Dec;13(12):7813–7825. doi: 10.1128/mcb.13.12.7813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lok S., Kaushansky K., Holly R. D., Kuijper J. L., Lofton-Day C. E., Oort P. J., Grant F. J., Heipel M. D., Burkhead S. K., Kramer J. M. Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo. Nature. 1994 Jun 16;369(6481):565–568. doi: 10.1038/369565a0. [DOI] [PubMed] [Google Scholar]
  31. Mudrak I., Ogris E., Rotheneder H., Wintersberger E. Coordinated trans activation of DNA synthesis- and precursor-producing enzymes by polyomavirus large T antigen through interaction with the retinoblastoma protein. Mol Cell Biol. 1994 Mar;14(3):1886–1892. doi: 10.1128/mcb.14.3.1886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mudryj M., Hiebert S. W., Nevins J. R. A role for the adenovirus inducible E2F transcription factor in a proliferation dependent signal transduction pathway. EMBO J. 1990 Jul;9(7):2179–2184. doi: 10.1002/j.1460-2075.1990.tb07387.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Neuman E., Flemington E. K., Sellers W. R., Kaelin W. G., Jr Transcription of the E2F-1 gene is rendered cell cycle dependent by E2F DNA-binding sites within its promoter. Mol Cell Biol. 1994 Oct;14(10):6607–6615. doi: 10.1128/mcb.14.10.6607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nevins J. R. E2F: a link between the Rb tumor suppressor protein and viral oncoproteins. Science. 1992 Oct 16;258(5081):424–429. doi: 10.1126/science.1411535. [DOI] [PubMed] [Google Scholar]
  35. Novak E. K., Reddington M., Zhen L., Stenberg P. E., Jackson C. W., McGarry M. P., Swank R. T. Inherited thrombocytopenia caused by reduced platelet production in mice with the gunmetal pigment gene mutation. Blood. 1995 Apr 1;85(7):1781–1789. [PubMed] [Google Scholar]
  36. Odell T. T., Jr, Jackson C. W. Polyploidy and maturation of rat megakaryocytes. Blood. 1968 Jul;32(1):102–110. [PubMed] [Google Scholar]
  37. Parker S. B., Eichele G., Zhang P., Rawls A., Sands A. T., Bradley A., Olson E. N., Harper J. W., Elledge S. J. p53-independent expression of p21Cip1 in muscle and other terminally differentiating cells. Science. 1995 Feb 17;267(5200):1024–1027. doi: 10.1126/science.7863329. [DOI] [PubMed] [Google Scholar]
  38. Qin X. Q., Livingston D. M., Kaelin W. G., Jr, Adams P. D. Deregulated transcription factor E2F-1 expression leads to S-phase entry and p53-mediated apoptosis. Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):10918–10922. doi: 10.1073/pnas.91.23.10918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ravid K., Beeler D. L., Rabin M. S., Ruley H. E., Rosenberg R. D. Selective targeting of gene products with the megakaryocyte platelet factor 4 promoter. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1521–1525. doi: 10.1073/pnas.88.4.1521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Robinson M. O., Simon M. I. Determining transcript number using the polymerase chain reaction: Pgk-2, mP2, and PGK-2 transgene mRNA levels during spermatogenesis. Nucleic Acids Res. 1991 Apr 11;19(7):1557–1562. doi: 10.1093/nar/19.7.1557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Robinson M. O., Zhou W., Hokom M., Danilenko D. M., Hsu R. Y., Atherton R. E., Xu W., Mu S., Saris C. J., Swift S. The tsA58 simian virus 40 large tumor antigen disrupts megakaryocyte differentiation in transgenic mice. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12798–12802. doi: 10.1073/pnas.91.26.12798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sala A., Nicolaides N. C., Engelhard A., Bellon T., Lawe D. C., Arnold A., Graña X., Giordano A., Calabretta B. Correlation between E2F-1 requirement in the S phase and E2F-1 transactivation of cell cycle-related genes in human cells. Cancer Res. 1994 Mar 15;54(6):1402–1406. [PubMed] [Google Scholar]
  43. Schulze A., Zerfass K., Spitkovsky D., Henglein B., Jansen-Dürr P. Activation of the E2F transcription factor by cyclin D1 is blocked by p16INK4, the product of the putative tumor suppressor gene MTS1. Oncogene. 1994 Dec;9(12):3475–3482. [PubMed] [Google Scholar]
  44. Schwarz J. K., Bassing C. H., Kovesdi I., Datto M. B., Blazing M., George S., Wang X. F., Nevins J. R. Expression of the E2F1 transcription factor overcomes type beta transforming growth factor-mediated growth suppression. Proc Natl Acad Sci U S A. 1995 Jan 17;92(2):483–487. doi: 10.1073/pnas.92.2.483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Shan B., Lee W. H. Deregulated expression of E2F-1 induces S-phase entry and leads to apoptosis. Mol Cell Biol. 1994 Dec;14(12):8166–8173. doi: 10.1128/mcb.14.12.8166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Shan B., Zhu X., Chen P. L., Durfee T., Yang Y., Sharp D., Lee W. H. Molecular cloning of cellular genes encoding retinoblastoma-associated proteins: identification of a gene with properties of the transcription factor E2F. Mol Cell Biol. 1992 Dec;12(12):5620–5631. doi: 10.1128/mcb.12.12.5620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Shivdasani R. A., Rosenblatt M. F., Zucker-Franklin D., Jackson C. W., Hunt P., Saris C. J., Orkin S. H. Transcription factor NF-E2 is required for platelet formation independent of the actions of thrombopoietin/MGDF in megakaryocyte development. Cell. 1995 Jun 2;81(5):695–704. doi: 10.1016/0092-8674(95)90531-6. [DOI] [PubMed] [Google Scholar]
  48. Singh P., Wong S. H., Hong W. Overexpression of E2F-1 in rat embryo fibroblasts leads to neoplastic transformation. EMBO J. 1994 Jul 15;13(14):3329–3338. doi: 10.1002/j.1460-2075.1994.tb06635.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Skapek S. X., Rhee J., Spicer D. B., Lassar A. B. Inhibition of myogenic differentiation in proliferating myoblasts by cyclin D1-dependent kinase. Science. 1995 Feb 17;267(5200):1022–1024. doi: 10.1126/science.7863328. [DOI] [PubMed] [Google Scholar]
  50. Slansky J. E., Li Y., Kaelin W. G., Farnham P. J. A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter. Mol Cell Biol. 1993 Mar;13(3):1610–1618. doi: 10.1128/mcb.13.3.1610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Surh C. D., Sprent J. T-cell apoptosis detected in situ during positive and negative selection in the thymus. Nature. 1994 Nov 3;372(6501):100–103. doi: 10.1038/372100a0. [DOI] [PubMed] [Google Scholar]
  52. Szekely L., Jiang W. Q., Bulic-Jakus F., Rosen A., Ringertz N., Klein G., Wiman K. G. Cell type and differentiation dependent heterogeneity in retinoblastoma protein expression in SCID mouse fetuses. Cell Growth Differ. 1992 Mar;3(3):149–156. [PubMed] [Google Scholar]
  53. Wiman K. G. The retinoblastoma gene: role in cell cycle control and cell differentiation. FASEB J. 1993 Jul;7(10):841–845. doi: 10.1096/fasebj.7.10.8393817. [DOI] [PubMed] [Google Scholar]
  54. Wu X., Levine A. J. p53 and E2F-1 cooperate to mediate apoptosis. Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3602–3606. doi: 10.1073/pnas.91.9.3602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Xu G., Livingston D. M., Krek W. Multiple members of the E2F transcription factor family are the products of oncogenes. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1357–1361. doi: 10.1073/pnas.92.5.1357. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES