MOLECULAR-CLONING AND CHARACTERIZATION OF NF-IL3A, A TRANSCRIPTIONAL ACTIVATOR OF THE HUMAN INTERLEUKIN-3 PROMOTER

To isolate transcription factors important in the regulation of the hu man interleukin-3 (IL-3) gene we screened a lambda gtll 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 amp lification and cloning of 3' ends'' technique to isolate the 3' half o f 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 p romoter sequences bound by NF-IL3A in DNase I footprinting assays as T AATTACGTCTG 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 stimul ated T-cell lines in similar amounts, although the level of NF-IL3A mR NA increases after T-cell activation in several mature T-cell lines. T he NF-IL3A protein is nearly identical to a recently identified transc riptional 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 acetyltr ansferase gene plasmids that contain the A region; this effect require s the presence of an intact NF-IL3A binding site. One or more copies o f the A region also confer NF-IL3A responsiveness on a heterologous pr omoter in T cells. NF-IL3A appears to play an important role in the ex pression of IL-3 by T cells.