A novel cis-acting element required for lipopolysaccharide-induced transcription of the murine interleukin-1 beta gene.
Regulatory elements important for transcription of the murine interleukin-1 beta (IL-1 beta) gene lie within a DNase I-hypersensitive region located > 2,000 bp upstream from the transcription start site. We have identified within this region a novel positive regulatory element that is required for activation of an IL-1 beta promoter-chloramphenicol acetyltransferase (CAT) fusion gene in the murine macrophage line RAW264.7. Electrophoretic mobility shift analysis of the 3' portion (-2315 to -2106) of the hypersensitive region revealed at least two nuclear factor binding sites, one of which is located between positions -2285 and -2256. Competitive inhibition studies localized the binding site to a 15-bp sequence between -2285 and -2271. Nuclear factor binding was lost by mutation of the 6-bp sequence from -2280 to -2275. The specific retarded complex formed with RAW264.7 nuclear extract was not detected under similar conditions with nuclear extracts from RLM-11, a murine T-cell line which does not express IL-1 beta RNA. Mutation of the 6-bp sequence (-2280 to -2275) in the chimeric IL-1 beta promoter -4093 +I CAT plasmid virtually eliminated the activation of this reporter gene by lipopolysaccharide (LPS) in transfected RAW264.7 cells. Multimerization of the 15-bp sequence containing the core wild-type 6-bp sequence 5' of minimal homologous or heterologous promoters in CAT reporter plasmids resulted in significant enhancement of CAT expression compared with parallel constructs containing the mutant 6-bp core sequence. This element was LPS independent and position and orientation dependent. The multimerized 15-bp sequence did not enhance expression in RLM-11 cells. Methylation interference revealed contact residues from -2281 to -2271, CCAAAAAGGAA. Because a search of the NIH TFD data bank with the 11-bp binding site sequence found no homology to known nuclear factor binding sites, we have designated this sequence the IL1 beta -upstream nuclear factor 1 (IL1 beta -UNF1) target. UV cross-linking and sodium dodecyl sulfate-polyacrylamide electrophoresis identified an IL1 beta -UNF1-specific binding factor approximately 85 to 90 kDa in size.
Godambe
S A
SA
Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Chaplin
D D
DD
Takova
T
T
Read
L M
LM
Bellone
C J
CJ
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D007375:Interleukin-1 / Q000235:genetics*; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic / Q000187:drug effects
D017931:DNA Primers; D004268:DNA-Binding Proteins; D007375:Interleukin-1; D008070:Lipopolysaccharides; D009687:Nuclear Proteins; D012333:RNA, Messenger