Abstract
The E2A-HLF fusion gene, formed by the t(17;19)(q22;p13) translocation in childhood acute pro-B-cell leukemia, encodes a hybrid protein that contains the paired trans-activation domains of E2A (E12/E47) linked to the basic region/leucine zipper DNA-binding and dimerization domain of hepatic leukemia factor (HLF). To assess the transforming potential of this novel gene, we introduced it into NIH 3T3 murine fibroblasts by using an expression vector that also contained the neomycin resistance gene. Cells selected for resistance to the neomycin analog G418 formed aberrant colonies in monolayer cultures, marked by increased cell density and altered morphology. Transfected cells also grew readily in soft agar, producing colonies whose sizes correlated with E2A-HLF expression levels. Subclones expanded from colonies with high levels of the protein reproducibly formed tumors in nude mice and grew to higher plateau-phase cell densities in reduced-serum conditions than did parental NIH 3T3 cells. By contrast, NIH 3T3 cells expressing mutant E2A-HLF proteins that lacked either of the bipartite E2A trans-activation domains or the HLF leucine zipper domain failed to show oncogenic properties, including anchorage-independent cell growth. Thus, both of the E2A trans-activation motifs and the HLF leucine zipper dimerization domain are essential for the transforming potential of the chimeric E2A-HLF protein, suggesting a model in which aberrant regulation of the expression pattern of downstream target genes contributes to leukemogenesis.
Full Text
The Full Text of this article is available as a PDF (1.6 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Aronheim A., Shiran R., Rosen A., Walker M. D. The E2A gene product contains two separable and functionally distinct transcription activation domains. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):8063–8067. doi: 10.1073/pnas.90.17.8063. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bram R. J., Hung D. T., Martin P. K., Schreiber S. L., Crabtree G. R. Identification of the immunophilins capable of mediating inhibition of signal transduction by cyclosporin A and FK506: roles of calcineurin binding and cellular location. Mol Cell Biol. 1993 Aug;13(8):4760–4769. doi: 10.1128/mcb.13.8.4760. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Drolet D. W., Scully K. M., Simmons D. M., Wegner M., Chu K. T., Swanson L. W., Rosenfeld M. G. TEF, a transcription factor expressed specifically in the anterior pituitary during embryogenesis, defines a new class of leucine zipper proteins. Genes Dev. 1991 Oct;5(10):1739–1753. doi: 10.1101/gad.5.10.1739. [DOI] [PubMed] [Google Scholar]
- Guadagno T. M., Ohtsubo M., Roberts J. M., Assoian R. K. A link between cyclin A expression and adhesion-dependent cell cycle progression. Science. 1993 Dec 3;262(5139):1572–1575. doi: 10.1126/science.8248807. [DOI] [PubMed] [Google Scholar]
- Han E. K., Guadagno T. M., Dalton S. L., Assoian R. K. A cell cycle and mutational analysis of anchorage-independent growth: cell adhesion and TGF-beta 1 control G1/S transit specifically. J Cell Biol. 1993 Jul;122(2):461–471. doi: 10.1083/jcb.122.2.461. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Henthorn P., Kiledjian M., Kadesch T. Two distinct transcription factors that bind the immunoglobulin enhancer microE5/kappa 2 motif. Science. 1990 Jan 26;247(4941):467–470. doi: 10.1126/science.2105528. [DOI] [PubMed] [Google Scholar]
- Hunger S. P., Brown R., Cleary M. L. DNA-binding and transcriptional regulatory properties of hepatic leukemia factor (HLF) and the t(17;19) acute lymphoblastic leukemia chimera E2A-HLF. Mol Cell Biol. 1994 Sep;14(9):5986–5996. doi: 10.1128/mcb.14.9.5986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hunger S. P., Ohyashiki K., Toyama K., Cleary M. L. Hlf, a novel hepatic bZIP protein, shows altered DNA-binding properties following fusion to E2A in t(17;19) acute lymphoblastic leukemia. Genes Dev. 1992 Sep;6(9):1608–1620. doi: 10.1101/gad.6.9.1608. [DOI] [PubMed] [Google Scholar]
- Inaba T., Roberts W. M., Shapiro L. H., Jolly K. W., Raimondi S. C., Smith S. D., Look A. T. Fusion of the leucine zipper gene HLF to the E2A gene in human acute B-lineage leukemia. Science. 1992 Jul 24;257(5069):531–534. doi: 10.1126/science.1386162. [DOI] [PubMed] [Google Scholar]
- Inaba T., Shapiro L. H., Funabiki T., Sinclair A. E., Jones B. G., Ashmun R. A., Look A. T. DNA-binding specificity and trans-activating potential of the leukemia-associated E2A-hepatic leukemia factor fusion protein. Mol Cell Biol. 1994 May;14(5):3403–3413. doi: 10.1128/mcb.14.5.3403. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Khatib Z. A., Inaba T., Valentine M., Look A. T. Chromosomal localization and cDNA cloning of the human DBP and TEF genes. Genomics. 1994 Sep 15;23(2):344–351. doi: 10.1006/geno.1994.1510. [DOI] [PubMed] [Google Scholar]
- Lassar A. B., Davis R. L., Wright W. E., Kadesch T., Murre C., Voronova A., Baltimore D., Weintraub H. Functional activity of myogenic HLH proteins requires hetero-oligomerization with E12/E47-like proteins in vivo. Cell. 1991 Jul 26;66(2):305–315. doi: 10.1016/0092-8674(91)90620-e. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Mueller C. R., Maire P., Schibler U. DBP, a liver-enriched transcriptional activator, is expressed late in ontogeny and its tissue specificity is determined posttranscriptionally. Cell. 1990 Apr 20;61(2):279–291. doi: 10.1016/0092-8674(90)90808-r. [DOI] [PubMed] [Google Scholar]
- 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]
- Quong M. W., Massari M. E., Zwart R., Murre C. A new transcriptional-activation motif restricted to a class of helix-loop-helix proteins is functionally conserved in both yeast and mammalian cells. Mol Cell Biol. 1993 Feb;13(2):792–800. doi: 10.1128/mcb.13.2.792. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rabbitts T. H. Translocations, master genes, and differences between the origins of acute and chronic leukemias. Cell. 1991 Nov 15;67(4):641–644. doi: 10.1016/0092-8674(91)90057-6. [DOI] [PubMed] [Google Scholar]
- Roberts C. W., Shutter J. R., Korsmeyer S. J. Hox11 controls the genesis of the spleen. Nature. 1994 Apr 21;368(6473):747–749. doi: 10.1038/368747a0. [DOI] [PubMed] [Google Scholar]
- Roussel M. F., Sherr C. J. Mouse NIH 3T3 cells expressing human colony-stimulating factor 1 (CSF-1) receptors overgrow in serum-free medium containing human CSF-1 as their only growth factor. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7924–7927. doi: 10.1073/pnas.86.20.7924. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shapiro L. H., Ashmun R. A., Roberts W. M., Look A. T. Separate promoters control transcription of the human aminopeptidase N gene in myeloid and intestinal epithelial cells. J Biol Chem. 1991 Jun 25;266(18):11999–12007. [PubMed] [Google Scholar]
- 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]
- Vinson C. R., Sigler P. B., McKnight S. L. Scissors-grip model for DNA recognition by a family of leucine zipper proteins. Science. 1989 Nov 17;246(4932):911–916. doi: 10.1126/science.2683088. [DOI] [PubMed] [Google Scholar]