Deletional analysis of the murine IL-12 p35 promoter comparing IFN-gamma and lipopolysaccharide stimulation.
IL-12, pivotal to the development of Th1 cells and formed by association of p35 and p40 subunits, is made by macrophages and the macrophage cell line RAW264.7. In this study, the promoter for p35 was cloned and analyzed. The murine IL-12 p35 gene has promoters upstream from each of the first two exons. The exon 1 and exon 2 promoters, cloned into a reporter vector, were responsive to LPS or IFN-gamma/CD40 ligation in transfected RAW264.7 cells. The exon 2 promoter containing bp -809 to +1 has significant homology to the human p35 promoter. Thus, deletion analysis was performed to determine the regions required for responsiveness to LPS, CD40, and/or IFN-gamma. Base pairs -809 to -740 influenced responsiveness to LPS. In contrast, bp -740to -444 and bp -122 to -100 were required for responses to IFN-gamma, IFN-gamma/LPS, or IFN-gamma/CD40 ligation. Removal of bp -444 to -392 increased the response of the exon 2 promoter to each stimulant. IFN regulatory factor (IRF)-1 is involved in the activity of this promoter at bp -108 to -103 because levels of nuclear IRF-1 correlated with exon 2 promoter activity in response to IFN-gamma and IRF-1 overexpression stimulated and enhanced exon 2 promoter activity. Also, site or deletion mutation of the IRF-1 element at bp -108 to -103 reduced the responsiveness of the promoter and IRF-1 bound to an oligonucleotide containing bp -108 to -103. The data suggest that the response of the p35 promoter to IFN-gamma requires a distinct IRF-1 positive regulatory element at bp -108 to -103.
Kollet
J
J
Department of Oral Biology, University of Nebraska Medical Center, University of Nebraska, Lincoln, NE 68583, USA.
Witek
C
C
Gentry
J D
JD
Liu
X
X
Schwartzbach
S D
SD
Petro
T M
TM
D000818:Animals; D000906:Antibodies / Q000276:immunology; D001483:Base Sequence; D019013:CD40 Antigens / Q000276:immunology; D002460:Cell Line; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000502:physiology; D005091:Exons; D006801:Humans; D050856:Interferon Regulatory Factor-1; D007371:Interferon-gamma / Q000494:pharmacology*; D018664:Interleukin-12 / Q000096:biosynthesis / Q000235:genetics*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000502:physiology; D011401:Promoter Regions, Genetic; D020218:Response Elements; D017384:Sequence Deletion; D013154:Spleen / Q000276:immunology
D000906:Antibodies; D019013:CD40 Antigens; D004268:DNA-Binding Proteins; C494257:IRF1 protein, human; D050856:Interferon Regulatory Factor-1; C494258:Irf1 protein, mouse; D008070:Lipopolysaccharides; D010750:Phosphoproteins; D018664:Interleukin-12; D007371:Interferon-gamma