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 human interleukin-3 (IL-3) gene, we screened a lambda 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 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.
Zhang
W
W
Laboratory of Molecular Aspects of Hematopoiesis, Sloan-Kettering Institute, New York, New York, USA.
Zhang
J
J
Kornuc
M
M
Kwan
K
K
Frank
R
R
Nimer
S D
SD
D000595:Amino Acid Sequence; D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors; D003001:Cloning, Molecular; D016384:Consensus Sequence; D018983:DNA Footprinting; D018076:DNA, Complementary / Q000235:genetics; D051790:G-Box Binding Factors; D015870:Gene Expression; D006801:Humans; D007377:Interleukin-3 / Q000235:genetics*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics*
D050976:Basic-Leucine Zipper Transcription Factors; D018076:DNA, Complementary; D051790:G-Box Binding Factors; D007377:Interleukin-3; C497440:NFIL3 protein, human; D009687:Nuclear Proteins; D012333:RNA, Messenger; D015534:Trans-Activators