Initial characterization of a potential transcriptional enhancer for the human c-K-ras gene

Abstract

The nucleotide sequence of the 5' end distal region of the human c-K-ras gene promoter was determined. This region, coincident with a variable DNAse I hypersensitive site in native chromatin, contains sequence similarities with known enhancers. A 400 bp MstII DNA fragment of this region stimulated in cis the correctly initiated transcription of the human beta-globin gene in transfected Hela cells. The stimulation of beta-globin transcription (5-6 fold) was dependent on the distance and orientation of the c-K-ras sequences and on the presence of the CCAAT and CACCC elements in the beta-globin promoter. Interaction of nuclear factors with these c-K-ras sequences was analysed by DNAase I footprinting assays using Hela nuclear extracts. A protein binding to these sequences was identified as nuclear factor 1 (NF-1) by DNAase I competition footprinting experiments. However, disruption of the c-K-ras NF-1 binding site by insertion mutagenesis had no effect on the transcriptional activity of the c-K-ras element.