Roles of an Ets motif and a novel CACGAC direct repeat in transcription of the murine dihydrolipoamide dehydrogenase (Dld) gene

Abstract

The 5h-flanking region of the murine dihydrolipoamide dehydrogenase (Dld) gene was characterized for its promoter activity. DNase I footprinting analysis of the promoter region (k545 bp to j41 bp) revealed six major protein-binding domains (termed P1 to P6) that were protected by NIH3T3 fibroblast nuclear extracts. Transient transfection assays, using a series of nested deletions of the 2.5 kb 5h-flanking region ligated to the chloramphenicol acetyltransferase reporter gene, identified that the k42-bp to j41-bp region, which harbours the P1, P2, and P3 domains, had minimal transcriptional activity. When the 5h-flanking region was extended from k42 bp to k82 bp, there was an approx. 5-fold increase in promoter activity. To identify further the cis elements involved in transcription of the Dld gene (k82 bp to j41 bp), a series of mutations were introduced into this region and evaluated for functional effects using transient transfection and electrophoretic mobility shift assays. Mutation