Molecular Cloning and Characterization of NF-IL3A, a Transcriptional Activator of the Human Interleukin-3 Promoter

Abstract

To isolate transcription factors important in the regulation of the human interleukin-3 (IL-3) gene, we screened a gt11 cDNA library, constructed from phytohemagglutinin-stimulated human T-cell RNA, with a probe containing regulatory sequences in the upstream region of the IL-3 gene (located from bp ؊165 to ؊128 and referred to as the DNase I footprint A region). We isolated a 0.96-kb cDNA that encoded a basic amino acid domain and a leucine zipper domain and used the ''rapid amplification and cloning of 3 ends'' technique to isolate the 3 half of the cDNA clone, generating a 1.9-kb full-length cDNA clone. Using in vitro-translated protein, which we call NF-IL3A, we defined the IL-3 promoter sequences bound by NF-IL3A in DNase I footprinting assays as TAATTACGTCTG and, using gel shift assays, defined ATTACG as the minimal sequence Interleukin-3 (IL-3) is a multilineage hematopoietic growth factor which stimulates the proliferation of hematopoietic progenitor cells and enhances the functional activity of mature effector cells (27,36). The expression of IL-3 is restricted to activated, but not resting, T cells, natural killer (NK) cells, and mast cell lines (9,27,29). In order to examine the regulatory mechanisms controlling the expression of IL-3, several investigators, including ourselves, have identified regulatory elements in the 5Ј-flanking region of the IL-3 promoter (3,10,13,19,21,22,33,35). These regulatory elements include a consensus AP-1 binding site, which can bind c-Fos/c-Jun heterodimers (13), several consensus binding sites for ets proteins (13), a CD28 response element (12, 35), and two regions identified by DNase I footprinting experiments as the A region and the B region (35). The A region contains regulatory sequences important in the inducible expression of 33,35), and methylation interference experiments have identified several distinct regions within this A region that are involved in DNAprotein interactions (10, 37). Electrophoretic mobility shift assays (EMSAs) combined with UV cross-linking have identified several proteins of 56 to 65 kDa that are capable of binding to the IL-3 A region (37).To identify and characterize IL-3 A region-binding proteins, we screened a phytohemagglutinin (PHA)-stimulated human T-cell cDNA gt11 expression library with a 32 P-labeled, multimerized, double-stranded IL-3 A region synthetic oligonucleotide probe. Approximately one million recombinant clones were screened, and one clone that generated a ␤-galactosidase fusion product that bound to the IL-3 A region but not to unrelated control DNA was identified. DNA sequence analysis of this clone demonstrated the presence of a potential DNAbinding basic-amino-acid region and a leucine zipper repeat. The 3Ј portion of the cDNA clone was not present, so rapid amplification and cloning of 3Ј ends (3Ј RACE) was performed to generate a full-length cDNA clone.A 1.9-kb full-length cDNA that contains the entire coding region for a 58-kDa protein that we call NF-IL3A was generated. In vitro-translated NF-IL3A binds specifi...

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

“…Although NFIL3͞E4BP4 has been shown to trans-activate the IL-3 promoter in T cells (20), its effects on the survival of FL5.12 pro-B cells do not appear to be mediated through an autocrine mechanism, as IL-3 mRNA was not detectable in clones overexpressing NFIL3͞E4BP4, and conditioned media from these cells did not promote the growth or survival of wild-type cells (data not shown). In addition, murine lymphoblasts with enforced expression of NFIL3͞E4BP4 accumulated in G 0 ͞G 1 phase in the absence of IL-3, and the administration of zinc in the presence of IL-3 did not effect cell growth rate (data not shown).…”

“…Functional studies indicate a positive regulatory role for E2A-HLF in gene expression, mediated through two separate domains (AD1 and AD2) in the E2A fusion element that act as potent trans-activators of gene expression in several contexts (12,13,(29)(30)(31). By contrast, NFIL3͞E4BP4 can function as either a trans-repressor or trans-activator of transcription in different cell types and promoter contexts (18)(19)(20). The C. elegans cell death specification protein CES-2 is highly related to HLF and NFIL3͞E4BP4 in its bZIP domain, and it binds to the same DNA consensus sequence (11).…”

“…These include members of the mammalian proline-and acidic amino acid-rich (PAR) subfamily of bZIP proteins: HLF (8,9,(12)(13)(14), DBP (albumin gene promoter D-box binding protein) (15,16), and TEF (thyrotroph embryonic factor) (17). We also analyzed a related bZIP protein called E4BP4, isolated by its ability to recognize the proximal activating transcription factor binding site of the adenovirus E4 promoter (18,19), and later independently identified as NFIL3, a protein expressed in T cells and capable of binding to a similar sequence motif in the 5Ј flanking region of the human IL-3 promoter (20). Here we report that murine Nfil3͞E4bp4 is regulated by IL-3 and that enforced expression of its human counterpart in IL-3-deprived cells prevents apoptosis.…”

Extensive genetic variation in somatic human tissues

“…shows an increase in signal of a ∼21.2-kb region on chromosome 9 of subject 6 in liver tissue DNA when hybridized against pancreas reference tissue DNA, suggesting a duplication of a region encompassing the NFIL3 gene in liver tissue. Expression of NFIL3 was previously reported in human liver tissue (22). Fig.…”

Virally mediated MafB transduction induces the monocyte commitment of human CD34+ hematopoietic stem/progenitor cells

“…Similarly, MafB transduced CD34 þ cells exhibited an increased expression of genes coding for typical macrophage granule proteins (Cathepsins, RNAses) and metalloproteinases (MMPs), 18,19 Analysis of cell cycle regulators revealed an increased mRNA expression of the GADD45B growth arrest/proapoptotic gene 21 and a clear downregulation of universally recognized proliferation markers as the c-myc proto-oncogene, 22 the CDC25A phosphatase 23 and the TFDP2 transcription factor 24 (Figure 7).…”