Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 Jan;17(1):81–88. doi: 10.1128/mcb.17.1.81

The Hox cooperativity motif of the chimeric oncoprotein E2a-Pbx1 is necessary and sufficient for oncogenesis.

C P Chang 1, I de Vivo 1, M L Cleary 1
PMCID: PMC231732  PMID: 8972188

Abstract

E2a-Pbx1 chimeric oncoproteins result from fusion of the E2A and PBX1 genes at the sites of t(1;19) chromosomal translocations in a subset acute lymphoblastic leukemias. Experimentally, E2a-Pbx1 transforms a variety of cell types, including fibroblasts, myeloid progenitors, and lymphoblasts. Structure-function studies have shown that contributions from both E2a and Pbx1 are necessary for oncogenesis, but the Pbx1 homeodomain is dispensable and the required portion of Pbx1 has not been delineated. In this study, we used deletional and site-directed mutagenesis to identify portions of Pbx1 necessary for oncogenic and transcriptional activities of E2a-Pbx1. These studies defined a motif (named the Hox cooperativity motif [HCM]) carboxy terminal to the Pbx homeodomain that is required for cooperative DNA binding, cellular transcriptional activity, and the oncogenic potential of E2a-Pbx1. The HCM is highly conserved throughout the Pbx/exd subfamily of divergent homeodomain proteins and functions in DNA-binding assays as a potential contact site for Hox dimerization. E2a-Pbx1 proteins with interstitial deletion or single-point mutations in the HCM could neither activate transcription in cellular assays nor transform NIH 3T3 cells. An E2a-Pbx1 mutant containing 50 amino acids of Pbx1b spanning the HCM but lacking the homeodomain was capable of inducing fibroblast transformation. Thus, the HCM is a necessary and sufficient contribution of Pbx1 for oncogenesis induced by E2a-Pbx1 and accounts for its homeodomain-independent transforming properties. Since subtle alterations of the Pbx HCM result in complete abrogation of transforming activity whereas the homeodomain is entirely dispensable, we conclude that interactions mediated by the HCM are more important for transformation by E2a-Pbx1 than interactions with cognate Pbx DNA sites.

Full Text

The Full Text of this article is available as a PDF (584.5 KB).

Selected References

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

  1. Ananthan J., Baler R., Morrissey D., Zuo J., Lan Y., Weir M., Voellmy R. Synergistic activation of transcription is mediated by the N-terminal domain of Drosophila fushi tarazu homeoprotein and can occur without DNA binding by the protein. Mol Cell Biol. 1993 Mar;13(3):1599–1609. doi: 10.1128/mcb.13.3.1599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Borrow J., Shearman A. M., Stanton V. P., Jr, Becher R., Collins T., Williams A. J., Dubé I., Katz F., Kwong Y. L., Morris C. The t(7;11)(p15;p15) translocation in acute myeloid leukaemia fuses the genes for nucleoporin NUP98 and class I homeoprotein HOXA9. Nat Genet. 1996 Feb;12(2):159–167. doi: 10.1038/ng0296-159. [DOI] [PubMed] [Google Scholar]
  3. Bürglin T. R., Ruvkun G. New motif in PBX genes. Nat Genet. 1992 Aug;1(5):319–320. doi: 10.1038/ng0892-319. [DOI] [PubMed] [Google Scholar]
  4. Chan S. K., Jaffe L., Capovilla M., Botas J., Mann R. S. The DNA binding specificity of Ultrabithorax is modulated by cooperative interactions with extradenticle, another homeoprotein. Cell. 1994 Aug 26;78(4):603–615. doi: 10.1016/0092-8674(94)90525-8. [DOI] [PubMed] [Google Scholar]
  5. Chang C. P., Brocchieri L., Shen W. F., Largman C., Cleary M. L. Pbx modulation of Hox homeodomain amino-terminal arms establishes different DNA-binding specificities across the Hox locus. Mol Cell Biol. 1996 Apr;16(4):1734–1745. doi: 10.1128/mcb.16.4.1734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chang C. P., Shen W. F., Rozenfeld S., Lawrence H. J., Largman C., Cleary M. L. Pbx proteins display hexapeptide-dependent cooperative DNA binding with a subset of Hox proteins. Genes Dev. 1995 Mar 15;9(6):663–674. doi: 10.1101/gad.9.6.663. [DOI] [PubMed] [Google Scholar]
  7. Corsetti M. T., Levi G., Lancia F., Sanseverino L., Ferrini S., Boncinelli E., Corte G. Nucleolar localisation of three Hox homeoproteins. J Cell Sci. 1995 Jan;108(Pt 1):187–193. doi: 10.1242/jcs.108.1.187. [DOI] [PubMed] [Google Scholar]
  8. Dedera D. A., Waller E. K., LeBrun D. P., Sen-Majumdar A., Stevens M. E., Barsh G. S., Cleary M. L. Chimeric homeobox gene E2A-PBX1 induces proliferation, apoptosis, and malignant lymphomas in transgenic mice. Cell. 1993 Sep 10;74(5):833–843. doi: 10.1016/0092-8674(93)90463-z. [DOI] [PubMed] [Google Scholar]
  9. Fibi M., Zink B., Kessel M., Colberg-Poley A. M., Labeit S., Lehrach H., Gruss P. Coding sequence and expression of the homeobox gene Hox 1.3. Development. 1988 Feb;102(2):349–359. doi: 10.1242/dev.102.2.349. [DOI] [PubMed] [Google Scholar]
  10. Jacobs Y., Vierra C., Nelson C. E2A expression, nuclear localization, and in vivo formation of DNA- and non-DNA-binding species during B-cell development. Mol Cell Biol. 1993 Dec;13(12):7321–7333. doi: 10.1128/mcb.13.12.7321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Johnson F. B., Parker E., Krasnow M. A. Extradenticle protein is a selective cofactor for the Drosophila homeotics: role of the homeodomain and YPWM amino acid motif in the interaction. Proc Natl Acad Sci U S A. 1995 Jan 31;92(3):739–743. doi: 10.1073/pnas.92.3.739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kamps M. P., Baltimore D. E2A-Pbx1, the t(1;19) translocation protein of human pre-B-cell acute lymphocytic leukemia, causes acute myeloid leukemia in mice. Mol Cell Biol. 1993 Jan;13(1):351–357. doi: 10.1128/mcb.13.1.351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kamps M. P., Look A. T., Baltimore D. The human t(1;19) translocation in pre-B ALL produces multiple nuclear E2A-Pbx1 fusion proteins with differing transforming potentials. Genes Dev. 1991 Mar;5(3):358–368. doi: 10.1101/gad.5.3.358. [DOI] [PubMed] [Google Scholar]
  14. Kamps M. P., Murre C., Sun X. H., Baltimore D. A new homeobox gene contributes the DNA binding domain of the t(1;19) translocation protein in pre-B ALL. Cell. 1990 Feb 23;60(4):547–555. doi: 10.1016/0092-8674(90)90658-2. [DOI] [PubMed] [Google Scholar]
  15. Kamps M. P., Wright D. D., Lu Q. DNA-binding by oncoprotein E2a-Pbx1 is important for blocking differentiation but dispensable for fibroblast transformation. Oncogene. 1996 Jan 4;12(1):19–30. [PubMed] [Google Scholar]
  16. Kessel M., Schulze F., Fibi M., Gruss P. Primary structure and nuclear localization of a murine homeodomain protein. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5306–5310. doi: 10.1073/pnas.84.15.5306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kiledjian M., Kadesch T. Analysis of the human liver/bone/kidney alkaline phosphatase promoter in vivo and in vitro. Nucleic Acids Res. 1990 Feb 25;18(4):957–961. doi: 10.1093/nar/18.4.957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Knoepfler P. S., Kamps M. P. The pentapeptide motif of Hox proteins is required for cooperative DNA binding with Pbx1, physically contacts Pbx1, and enhances DNA binding by Pbx1. Mol Cell Biol. 1995 Oct;15(10):5811–5819. doi: 10.1128/mcb.15.10.5811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. LeBrun D. P., Cleary M. L. Fusion with E2A alters the transcriptional properties of the homeodomain protein PBX1 in t(1;19) leukemias. Oncogene. 1994 Jun;9(6):1641–1647. [PubMed] [Google Scholar]
  20. Lu Q., Kamps M. P. Structural determinants within Pbx1 that mediate cooperative DNA binding with pentapeptide-containing Hox proteins: proposal for a model of a Pbx1-Hox-DNA complex. Mol Cell Biol. 1996 Apr;16(4):1632–1640. doi: 10.1128/mcb.16.4.1632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lu Q., Knoepfler P. S., Scheele J., Wright D. D., Kamps M. P. Both Pbx1 and E2A-Pbx1 bind the DNA motif ATCAATCAA cooperatively with the products of multiple murine Hox genes, some of which are themselves oncogenes. Mol Cell Biol. 1995 Jul;15(7):3786–3795. doi: 10.1128/mcb.15.7.3786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lu Q., Wright D. D., Kamps M. P. Fusion with E2A converts the Pbx1 homeodomain protein into a constitutive transcriptional activator in human leukemias carrying the t(1;19) translocation. Mol Cell Biol. 1994 Jun;14(6):3938–3948. doi: 10.1128/mcb.14.6.3938. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Monica K., Galili N., Nourse J., Saltman D., Cleary M. L. PBX2 and PBX3, new homeobox genes with extensive homology to the human proto-oncogene PBX1. Mol Cell Biol. 1991 Dec;11(12):6149–6157. doi: 10.1128/mcb.11.12.6149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Monica K., LeBrun D. P., Dedera D. A., Brown R., Cleary M. L. Transformation properties of the E2a-Pbx1 chimeric oncoprotein: fusion with E2a is essential, but the Pbx1 homeodomain is dispensable. Mol Cell Biol. 1994 Dec;14(12):8304–8314. doi: 10.1128/mcb.14.12.8304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nakamura T., Largaespada D. A., Lee M. P., Johnson L. A., Ohyashiki K., Toyama K., Chen S. J., Willman C. L., Chen I. M., Feinberg A. P. Fusion of the nucleoporin gene NUP98 to HOXA9 by the chromosome translocation t(7;11)(p15;p15) in human myeloid leukaemia. Nat Genet. 1996 Feb;12(2):154–158. doi: 10.1038/ng0296-154. [DOI] [PubMed] [Google Scholar]
  26. Neuteboom S. T., Peltenburg L. T., van Dijk M. A., Murre C. The hexapeptide LFPWMR in Hoxb-8 is required for cooperative DNA binding with Pbx1 and Pbx2 proteins. Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9166–9170. doi: 10.1073/pnas.92.20.9166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nourse J., Mellentin J. D., Galili N., Wilkinson J., Stanbridge E., Smith S. D., Cleary M. L. Chromosomal translocation t(1;19) results in synthesis of a homeobox fusion mRNA that codes for a potential chimeric transcription factor. Cell. 1990 Feb 23;60(4):535–545. doi: 10.1016/0092-8674(90)90657-z. [DOI] [PubMed] [Google Scholar]
  28. Numata S., Kato K., Horibe K. New E2A/PBX1 fusion transcript in a patient with t(1;19)(q23;p13) acute lymphoblastic leukemia. Leukemia. 1993 Sep;7(9):1441–1444. [PubMed] [Google Scholar]
  29. Peers B., Sharma S., Johnson T., Kamps M., Montminy M. The pancreatic islet factor STF-1 binds cooperatively with Pbx to a regulatory element in the somatostatin promoter: importance of the FPWMK motif and of the homeodomain. Mol Cell Biol. 1995 Dec;15(12):7091–7097. doi: 10.1128/mcb.15.12.7091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Peifer M., Wieschaus E. Mutations in the Drosophila gene extradenticle affect the way specific homeo domain proteins regulate segmental identity. Genes Dev. 1990 Jul;4(7):1209–1223. doi: 10.1101/gad.4.7.1209. [DOI] [PubMed] [Google Scholar]
  31. Phelan M. L., Rambaldi I., Featherstone M. S. Cooperative interactions between HOX and PBX proteins mediated by a conserved peptide motif. Mol Cell Biol. 1995 Aug;15(8):3989–3997. doi: 10.1128/mcb.15.8.3989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pöpperl H., Bienz M., Studer M., Chan S. K., Aparicio S., Brenner S., Mann R. S., Krumlauf R. Segmental expression of Hoxb-1 is controlled by a highly conserved autoregulatory loop dependent upon exd/pbx. Cell. 1995 Jun 30;81(7):1031–1042. doi: 10.1016/s0092-8674(05)80008-x. [DOI] [PubMed] [Google Scholar]
  33. Rauskolb C., Peifer M., Wieschaus E. extradenticle, a regulator of homeotic gene activity, is a homolog of the homeobox-containing human proto-oncogene pbx1. Cell. 1993 Sep 24;74(6):1101–1112. doi: 10.1016/0092-8674(93)90731-5. [DOI] [PubMed] [Google Scholar]
  34. Schulze F., Chowdhury K., Zimmer A., Drescher U., Gruss P. The murine homeo box gene product, Hox 1.1 protein, is growth-controlled and associated with chromatin. Differentiation. 1987;36(2):130–137. doi: 10.1111/j.1432-0436.1987.tb00187.x. [DOI] [PubMed] [Google Scholar]
  35. Shen W. F., Chang C. P., Rozenfeld S., Sauvageau G., Humphries R. K., Lu M., Lawrence H. J., Cleary M. L., Largman C. Hox homeodomain proteins exhibit selective complex stabilities with Pbx and DNA. Nucleic Acids Res. 1996 Mar 1;24(5):898–906. doi: 10.1093/nar/24.5.898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Van Dijk M. A., Voorhoeve P. M., Murre C. Pbx1 is converted into a transcriptional activator upon acquiring the N-terminal region of E2A in pre-B-cell acute lymphoblastoid leukemia. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6061–6065. doi: 10.1073/pnas.90.13.6061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. van Dijk M. A., Murre C. extradenticle raises the DNA binding specificity of homeotic selector gene products. Cell. 1994 Aug 26;78(4):617–624. doi: 10.1016/0092-8674(94)90526-6. [DOI] [PubMed] [Google Scholar]

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

RESOURCES