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 interleu kin-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 tha t is required for activation of an IL-1 beta promoter-chloramphenicol acetyltransferase (CAT) fusion gene in the murine macrophage line RAW2 64.7. 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 t o 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 specif ic retarded complex formed with RAW264.7 nuclear extract was not detec ted under similar conditions with nuclear extracts from RLM-11, a muri ne 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 g ene by lipopolysaccharide (LPS) in transfected RAW264.7 cells. Multime rization of the 15-bp sequence containing the core wild type 6-bp sequ ence 5' of minimal homologous or heterologous promoters in CAT reporte r plasmids resulted in significant enhancement of CAT expression compa red with parallel constructs containing the mutant 6-bp core sequence. This element was LPS independent and position and orientation depende nt. The multimerized 15-bp sequence did not enhance expression in RLM- 11 cells. Methylation interference revealed contact residues from -228 1 to -2271, CCAAAAAGGAA. Because a search of the NIH TFD data bank wit h the 11-bp binding site sequence found no homology to known nuclear f actor binding sites, we have designated this sequence the IL1 beta-ups tream nuclear factor 1 (IL1 beta-UNF1) target. UV cross-linking and so dium dodecyl sulfate-polyacrylamide electrophoresis identified an IL1 beta-UNF1-specific binding factor approximately 85 to 90 kDa in size.