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.1997 Mar 18;94(6):2609-14.
doi: 10.1073/pnas.94.6.2609.

Pivotal role for the NFIL3/E4BP4 transcription factor in interleukin 3-mediated survival of pro-B lymphocytes

Affiliations

Pivotal role for the NFIL3/E4BP4 transcription factor in interleukin 3-mediated survival of pro-B lymphocytes

S Ikushima et al. Proc Natl Acad Sci U S A..

Abstract

The E2A-HLF (hepatic leukemia factor) oncoprotein, generated in pro-B lymphocytes by fusion of the trans-activation domain of E2A to the basic region/leucine zipper (bZIP) domain of HLF, functions as an anti-apoptotic transcription factor in leukemic cell transformation. When introduced into interleukin 3 (IL-3)-dependent mouse pro-B lymphocytes, E2A-HLF prevents apoptosis induced by growth factor deprivation, suggesting that IL-3 mediates cell survival through activation of a transcription factor whose activity can be constitutively replaced by the chimeric oncoprotein. We considered four bZIP transcription factors as candidates for this putative IL-3-regulated factor, each of which binds avidly to the DNA consensus sequence recognized by E2A-HLF and is related to the Caenorhabditis elegans CES-2 (cell death specification protein) neuron-specific mediator of cell death. The expression and binding activity of the Nfil3 protein (also called E4bp4), but not of Hlf, Dbp, or Tef, was found to be regulated by IL-3 in mouse pro-B cell lines (Baf-3 and FL5.12). Northern blot analysis showed that Nfil3/E4bp4 is regulated as a "delayed-early" IL-3-responsive gene, requiring de novo protein synthesis. In the absence of IL-3, enforced expression of the human NFIL3/E4BP4 cDNA promoted the survival but not the growth of IL-3-dependent pro-B cells. Our results implicate NFIL3/E4BP4 (nuclear factor regulated by IL-3/adenovirus E4 promoter binding protein) in a distinct growth factor-regulated signaling pathway that is responsible for the survival of early B-cell progenitors, and whose alteration by E2A-HLF leads to childhood B lineage leukemia.

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Figures

Figure 1
Figure 1
Nfil3/E4bp4 binding activity is regulated by IL-3. (A) Nuclear extracts prepared from IL-3-dependent mouse pro-B cells (Baf-3 or FL5.12) were studied by electrophoretic mobility shift assay with a32P-labeled oligonucleotide probe containing the HLF consensus binding sequence (GTTACGTAAC; lanes 1–10). Lanes 2–5 and 7–10 show results at the indicated intervals after withdrawal of IL-3 from the medium; lanes 1 and 6 depict findings for stable culture in IL-3. Brackets indicate Nfil3/E4bp4-DNA complexes (lanes 1–5), and solid arrowheads indicate the supershifted complex that was evident after incubation with antiserum specific for Nfil3/E4bp4 (lanes 6–10). As a control for extract integrity, the mobility shift assay was also performed with an SP1 transcription factor consensus sequence probe on extracts from cells growing in IL-3 (lane 11) and after its withdrawal (lanes 12 and 13). (B) Same as inA, except that extracts were analyzed from cells deprived of ligand for 8 hr (lanes 1, 6, and 11) and after the indicated intervals following IL-3 addition (lanes 2–5, 7–10, and 12). (C) Western blot analysis showing levels of the ≈56- to 58-kDa Nfil3/E4bp4 protein in Baf-3 and FL5.12 cells deprived of IL-3 for 8 hr (lane 1) and at 1-hr intervals following reinstatement of the cytokines. Nfil3/E4bp4 protein levels were 4.1- and 4.5-fold higher, respectively, in Baf-3 and FL5.12 cells, 4 hr after IL-3 restimulation (lane 5) than in cells deprived of IL-3 for 8 hr (lane 1). Nfil3/E4bp4 levels in cells growing continuously in IL-3 were 2.5- (Baf-3 cells) and 1.8-fold (FL5.12 cells) higher than in IL-3-deprived cells (lane 1).
Figure 2
Figure 2
Nfil3/E4bp4 expression is regulated by IL-3. (A) Northern blot analysis of total RNA (20 μg per lane) prepared from Baf-3 (Upper) or FL5.12 (Lower) pro-B lymphocytes were grown in IL-3 (lane 1) and then deprived of the ligand for the indicated intervals (lanes 2–5). Cells starved of ligand for 8 hr (lane 7) were restimulated with 10 ng/ml of murine IL-3 and incubated for the indicated times (lanes 8–11). Blots were hybridized with anNfil3/E4bp4 probe and stripped and rehybridized with the GAPDH control probe. (B) Induction ofNfil3/E4bp4 by IL-3 requires protein synthesis. Asynchronously growing FL5.12 cells were deprived of IL-3 for 16 hr (lane 1) and stimulated with either IL-3 alone for the indicated times (lanes 2–6), or with cycloheximide (CHX, 20 μg/ml) alone (lanes 7–11), or were pretreated with CHX for 15 min and incubated in both IL-3 and CHX for the indicated times (lanes 12–16). Northern blots were sequentially hybridized with probes forNfil3/E4bp4,c-fos, andGAPDH.
Figure 3
Figure 3
Transfected pro-B cell clones conditionally overexpressing NFIL3/E4BP4. (A) Antibody-perturbed electrophoretic mobility shift assay of FL5.12 clones expressing NFIL3/E4BP4 from the zinc-regulated pMT-CB6+ expression plasmid. Solid arrowheads indicate NFIL3/E4BP4–DNA complexes from each of four independent clones grown in the presence (even lanes) or absence (odd lanes) of zinc, in the presence of IL-3 for 24 hr (+IL-3,Upper) or 16 hr after its removal (−IL-3,Lower). (B) A time course of NFIL3/E4BP4 binding activity in FL5.12 cells bearing the empty zinc-regulated vector (Upper) or the vector containing NFIL3/E4BP4 (clone 9;Lower). The mobility assay was performed with extracts from cells cultured in the presence (lanes 6–10) or absence (lanes 1–5) of zinc, and in the presence of IL-3 (lanes 1 and 6), or at the indicated intervals after its removal. Solid arrowheads indicate NFIL3/E4BP4–DNA complexes that supershifted with E4BP4 antiserum.
Figure 4
Figure 4
NFIL3/E4BP4 suppresses apoptosis of IL-3-deprived murine pro-B cells. (A) Numbers of viable cells at serial times after IL-3 withdrawal in the absence of zinc for FL5.12 clones transfected with zinc-regulatable NFIL3/E4BP4 plasmid or the empty vector (means of triplicate counts, with bars showing standard deviations). (B) Viable cell counts of the same clones after IL-3 withdrawal and culture in the presence of zinc to induce NFIL3/E4BP4 expression. Representative data from seven independent experiments are presented; comparable results were obtained with nine independently derived pools of cells transfected with the NFIL3/E4BP4 plasmid (data not shown).
Figure 5
Figure 5
Model of NFIL3/E4BP4 function in cell survival pathways and a proposed mechanism of action of the E2A-HLF oncoprotein in the transformation of pro-B lymphocytes. Expression of theNFIL3/E4BP4 gene is tightly regulated by IL-3 through an undefined signal transduction pathway emanating from the ligand-activated IL-3 receptor. The NFIL3/E4BP4 transcription factor binds to a consensus sequence in the promoter of downstream target genes, leading to trans-activation of critical survival signals. Because HLF and NFIL3/E4BP4 can recognize the same DNA binding site, the constitutively expressed E2A-HLF oncoprotein can replace the function of the normal IL-3-regulated transcription factor, accounting for its anti-apoptotic role in leukemogenesis. An additional prediction of the model is that the consensus binding site (TTACGTAA) could serve as a versatile regulatory switch, allowing other, still-to-be-identified mammalian CES-2 homologues to contribute to programmed cell death by acting as trans-repressors of this pathway.
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