Human interleukin-1 alpha gene expression is regulated by Sp1 and a transcriptional repressor

The regulation of the human IL-1alpha gene was studied using a series of 5' deletion promoter chloramphenicol acetyltransferase (CAT) reporter constructs. The IL-1alpha promoter from -967 to +64 produced no significant expression of CAT. Progressive 5' deletion indicated the presence of a repressor binding site between -477 and -305 bp as deletion in this region resulted in CAT expression. Electrophoretic mobility shift assay (EMSA) analysis confirmed that protein(s) bound to this region and DNaseI footprinting localized the binding site to between -448 and -435. Deletion of the IL-1alpha promoter to -42 resulted in reduced CAT expression suggesting the presence of a positive regulatory element in this region. EMSA experiments using IL-1alpha promoter DNA from -163 to +64 demonstrated protein binding to this region and DNaseI footprinting demonstrated protection between -59 and -40. Transcriptional activity of the IL-1alpha promoter was also tested using an in vitro transcription assay. Reactions using -163, -100 and -52 promoter templates all produced a correctly sized transcript but deletion to -42 resulted in no transcript production. Analysis of the promoter indicated that a potential Sp1 binding site existed in the region from -52 to -45. An EMSA using an anti-Sp1 antibody indicated that Sp1 specifically bound to the -52 to +64 region.