Epidermal growth factor receptor gene promoter. Deletion analysis and identification of nuclear protein binding sites

Abstract

To determine the location of sites that may be important for the function of the promoter of the epidermal growth factor (EGF) receptor gene and to characterize the factors that bind to these sites, the promoter region was analyzed by deletion analysis, exonuclease III protection and gel retardation assays with crude and fractionated nuclear extracts and DNase I footprinting using purified Sp1. Transfection of chimeric chloramphenicol acetyltransferase plasmids containing various deletions of the EGF receptor gene promoter into CV-1 cells indicated that the region between -178 and -16 (initiator ATG is +1) is sufficient for promoter activity. Exonuclease III protection assays revealed the presence of eight specific nuclear protein binding sites in the region between -481 and -16. Gel retardation assays confirmed that multiple protein binding sites exist in this region (-481 to -16) and quantitatively agree with exonuclease III protection. DNase I footprinting using purified Sp1 showed that this transcription factor can bind to four sites (-457 to -440, -365 to -286, -214 to -200, and -110 to -84) in the EGF receptor gene promoter and therefore may play a role in its regulation.