W. Zhang et al., MOLECULAR-CLONING AND CHARACTERIZATION OF NF-IL3A, A TRANSCRIPTIONAL ACTIVATOR OF THE HUMAN INTERLEUKIN-3 PROMOTER, Molecular and cellular biology, 15(11), 1995, pp. 6055-6063
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.