Molecular cloning and characterization of NF-IL3A, a transcriptional activator of the human interleukin-3 promoter

Wei Zhang, Jin Zhang, Masayo Kornuc, Karen Kwan, Richard Frank, Stephen D. Nimer

Research output: Contribution to journalArticle

98 Scopus citations

Abstract

To isolate transcription factors important in the regulation of the human interleukin-3 (IL-3) gene, we screened a λgt11 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 1 footprinting assays as TAATTACGTCTG and, using gel shift assays, defined ATTACG as the minimal sequence required for binding of NF-IL3A in vitro. Proteins that bind to the NF-IL3A binding site are found in both unstimulated and stimulated T-cell lines in similar amounts, although the level of NF-IL3A mRNA increases after T-cell activation in several mature T-cell lines. The NF-IL3A protein is nearly identical to a recently identified transcriptional repressor protein, E4BP4, and NF-IL3A binds specifically to regulatory sequences in both the adenovirus E4 promoter and the human gamma interferon promoter. Cotransfection experiments demonstrate that introduction of an expression vector containing the NF-IL3A cDNA into resting T cells transactivates IL-3 promoter-chloramphenicol acetyltransferase gene plasmids that contain the A region; this effect requires the presence of an intact NF-IL3A binding site. One or more copies of the A region also confer NF-IL3A responsiveness on a heterologous promoter in T cells. NF-IL3A appears to play an important role in the expression of IL-3 by T cells.

Original languageEnglish (US)
Pages (from-to)6055-6063
Number of pages9
JournalMolecular and cellular biology
Volume15
Issue number11
DOIs
StatePublished - Nov 1995

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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