Identification of a novel regulatory element in the human interleukin 1 alpha (IL-1alpha) gene promoter.

Human interleukin 1alpha (IL-1alpha) is secreted by a variety of cell types including epithelial cells, endothelial cells, keratinocytes, adipocytes and activated monocytes/macrophages. IL-1alpha is a major player in immune and inflammatory responses, yet very little is known about its regulation. We have identified a novel regulatory sequence at -65 to -41 of the human IL-1alpha promoter using DNase I footprint studies. A triple GCC repeat is present within the footprint region. In order to study the importance of this GCC motif in the regulation of IL-1alpha, we mutated the GCC to a CAA at positions -60 to -58 and -52 to -50. In transfection studies using the human epithelial (HEp-2) and human erythroleukemia (HEL/DAMI) cell lines, the mutated promoter fragment showed a 90% decrease in basal activity and was not inducible by external stimuli. To characterize transcription factor binding to the footprint sequence, we used both electrophoretic mobility shift assays (EMSA) and DNase I protection techniques. Both of these studies confirmed that the GCC motif is also essential for DNA/protein complex formation. The NCBI and TESS databases were utilized to search for known transcripiton factor binding sites with homolgy to the footprint sequence. The only element identified with any homology was an NFkappaB binding sequence. However, competition assays using cold NFkappaB consensus sequence DNA had no effect on the mobility shift signal. This data strongly suggests that a novel regulatory element associated with the GCC motif is located in the footprint sequence. These results will contribute to understanding of the regulatory mechanisms governing induction of the IL-1alpha gene and may lead to the isolation of a novel transcription factor.