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.1998 Oct;18(10):6035-43.
doi: 10.1128/MCB.18.10.6035.

The AD1 and AD2 transactivation domains of E2A are essential for the antiapoptotic activity of the chimeric oncoprotein E2A-HLF

Affiliations

The AD1 and AD2 transactivation domains of E2A are essential for the antiapoptotic activity of the chimeric oncoprotein E2A-HLF

T Inukai et al. Mol Cell Biol.1998 Oct.

Abstract

The chimeric oncoprotein E2A-HLF, generated by the t(17;19) chromosomal translocation in pro-B-cell acute lymphoblastic leukemia, incorporates the transactivation domains of E2A and the basic leucine zipper (bZIP) DNA-binding and protein dimerization domain of HLF (hepatic leukemic factor). The ability of E2A-HLF to prolong the survival of interleukin-3 (IL-3)-dependent murine pro-B cells after IL-3 withdrawal suggests that it disrupts signaling pathways normally responsible for cell suicide, allowing the cells to accumulate as transformed lymphoblasts. To determine the structural motifs that contribute to this antiapoptotic effect, we constructed a panel of E2A-HLF mutants and programmed their expression in IL-3-dependent murine pro-B cells (FL5.12 line), using a zinc-inducible vector. Neither the E12 nor the E47 product of the E2A gene nor the wild-type HLF protein was able to protect the cells from apoptosis induced by IL-3 deprivation. Surprisingly, different combinations of disabling mutations within the HLF bZIP domain had little effect on the antiapoptotic property of the chimeric protein, so long as the amino-terminal portion of E2A remained intact. In the context of a bZIP domain defective in DNA binding, mutants retaining either of the two transactivation domains of E2A were able to extend cell survival after growth factor deprivation. Thus, the block of apoptosis imposed by E2A-HLF in pro-B lymphocytes depends critically on the transactivating regions of E2A. Since neither DNA binding nor protein dimerization through the bZIP domain of HLF is required for this effect, we propose mechanisms whereby protein-protein interactions with the amino-terminal region of E2A allow the chimera to act as a transcriptional cofactor to alter the expression of genes regulating the apoptotic machinery in pro-B cells.

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Figures

FIG. 1
FIG. 1
Effects of E2A-HLF protein expression in FL5.12 cells. (A) Immunoblot analysis with an HLF(C) antiserum of five independent E2A-HLF-positive, G418-resistant transfected pools of FL5.12 cells and an empty vector-transfected cell pool, all cultured in the presence (even-numbered lanes) or absence (odd-numbered lanes) of 100 μM ZnSO4. (B) Growth of FL5.12 cells induced to express the E2A-HLF protein, together with growth of control cells transfected with an empty vector. Cells growing exponentially in IL-3-supplemented medium for 16 h in the presence or absence of zinc were adjusted to 5 × 105 cells per ml on day 0 and cultured without IL-3 for 15 days. The growth factor was reintroduced to the cultures on day 15 (arrow). The shaded region indicates the ranges of cell counts for E2A-HLF-transfected pools in the absence of zinc and control cells in the presence or absence of the metal. Each symbol represents a discrete pool of E2A-HLF-transfected, G418-resistant cells; open circles, pool 104; open triangles, pool 112; open squares, pool 116; closed circles, pool 120; and open diamonds, pool 122. (C) Cell cycle phase distribution of a representative pool of transfected cells (pool 116; open squares in panel B) expressing E2A-HLF in the presence (+) or absence (−) of zinc, as determined from DNA histograms obtained by propidium iodide staining and flow cytometry.
FIG. 2
FIG. 2
Antiapoptotic activity of wild-type E2A (E12 and E47) and HLF proteins in FL5.12 cells. (A) Immunoblot analysis with an HLF(C) antiserum (lanes 1 to 6) or E2A antiserum (lanes 7 to 14) of proteins in transfected FL5.12 pools cultured in the presence (even-numbered lanes) or absence (odd-numbered lanes) of zinc. (B) EMSA of DNA-protein complexes formed with an HLF-CS probe in nuclear extracts of transfected FL5.12 clones in the presence of zinc. (C) Comparison of the antiapoptotic activities of normal E2A and HLF proteins with that of the E2A-HLF protein. The numbers of living cells in pools expressing the designated protein are represented by bars in the absence (upper) or presence (lower) of zinc after 4 days of culture without IL-3. The values are the means of results from at least three independently analyzed transfected cell pools; standard deviations are given at the ends of the bars. TAD, transactivation domain; PAR, proline and acidic amino acid-rich region; NLS, nuclear localization signal.
FIG. 3
FIG. 3
Antiapoptotic activities of E2A-HLF and HLF proteins with deletions or amino acid substitutions in the bZIP domain. (A) Immunoblot analysis with an HLF(C) antiserum (lanes 1 to 4) or E2A antiserum (lanes 5 to 12) of proteins in transfected FL5.12 pools cultured in the presence (even-numbered lanes) or absence (odd-numbered lanes) of zinc. (B) EMSA of DNA-protein complexes formed with an HLF-CS probe in nuclear extracts of transfected FL5.12 clones in the presence of zinc. (C) Comparison of the antiapoptotic activity of the protein expressed from each construct with that of the E2A-HLF protein. Diagrams of the E2A-HLF mutant proteins analyzed are shown on the left. Bars indicate the numbers of living cells in each pool that expressed the designated proteins in the absence (upper) or presence (lower) of zinc after 4 days of culture without IL-3. The values are the means of at least three independent pools; standard deviations are given at the ends of the bars. Notation is as for Fig. 2C.
FIG. 4
FIG. 4
Antiapoptotic activities mutations in E2A and E2A-HLF proteins with intact or disabled (BX) HLF DNA-binding domains. (A) Immunoblot analysis with an HLF(C) antiserum of proteins in transfected FL5.12 cell pools cultured in the presence (even-numbered lanes) or absence (odd-numbered lanes) of zinc. (B) EMSA of DNA-protein complexes formed with an HLF-CS probe in nuclear extracts of transfected FL5.12 clones in the presence of zinc. (C) Comparison of the antiapoptotic activities of E2A-HLF proteins with mutations in E2A and intact (left) or disabled (right) HLF DNA-binding domains. Diagrams of the mutant proteins are shown on the left. Bars indicate the numbers of living cells in each pool of transfected cells that expressed the designated proteins in the absence (upper) or presence (lower) of zinc after 4 days of culture without IL-3. The values are means from at least three independent pools; standard deviations are given at the ends of the bars.
FIG. 5
FIG. 5
Subcellular localization of E2A-HLF, HLF, E2A, and representative mutant proteins. FL5.12 cells expressing each construct were immunostained with either IgG-purified anti-HLF(C) serum (A to E) or the anti-E2A serum (F to J). Simultaneous staining with DAPI was performed to permit visualization of cell nuclei (A′ to J′). The FL5.12 cells studied either contained the empty vector (A, A′, F, and F′) or expressed E2A-HLF (B, B′, G, and G′), HLF (C and C′), E2A-HLF/BX (D and D′), Δ1-142 (E and E′), E2A (E12) (H and H′), E2A (E47) (I and I′), or Δ484-574 (J and J′).
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References

    1. Adler S M L, Waterman M L, He X, Rosenfeld M G. Steroid receptor-mediated inhibition of rat prolactin gene expression does not require the receptor DNA-binding domain. Cell. 1988;52:685–695. - PubMed
    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;13:1599–1609. - PMC - PubMed
    1. Askew D S, Ashmun R A, Simmons B C, Cleveland J L. Constitutive c-myc expression in an IL-3-dependent myeloid cell line suppresses cell cycle arrest and accelerates apoptosis. Oncogene. 1991;6:1915–1922. - PubMed
    1. Bain G, Robanus-Maandag E C, Izon D J, Amsen D, Kruisbeek A M, Weintraub B C, Krop I, Schlissel M S, Feeney A J, van Roon M, van der Valk M, te Riele H P J, Berns A, Murre C. E2A proteins are required for proper B-cell development and initiation of immunoglobulin gene rearrangements. Cell. 1994;79:885–892. - PubMed
    1. Bain G, Robanus Maandag E C, te Riele H P, Feeney A J, Sheehy A, Schlissel M, Shinton S A, Hardy R R, Murre C. Both E12 and E47 allow commitment to the B cell lineage. Immunity. 1997;6:145–154. - PubMed

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