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

The 5'-flanking region of the murine dihydrolipoamide dehydrogenase (Dld) g ene was characterized for its promoter activity. DNase I footprinting analy sis of the promoter region (-545 bp to +41 bp) revealed six major protein-b inding domains (termed P1 to P6) that were protected by NIH3T3 fibroblast n uclear extracts. Transient transfection assays, using a series of nested de letions of the 2.5 kb 5'-flanking region ligated to the chloramphenicol ace tyltransferase reporter gene, identified that the -42-bp to +41-bp region, which harbours the P1, P2, and P3 domains, had minimal transcriptional acti vity. When the 5'-flanking region was extended from -42 bp to -82 bp, there was an approx. 5-fold increase in promoter activity. To identify further t he cis elements involved in transcription of the Did gene (-82 bp to +41 bp ), a series of mutations were introduced into this region and evaluated for functional effects using transient transfection and electrophoretic mobili ty shift assays. Mutation or deletion pf the CACGAC direct repeat, located from -61 bp to -46 bp, resulted in minimal promoter activity. Mutation of t he Ets motif, located from -37 bp to -32 bp, reduced the minimal promoter a ctivity by approx. 50 %, whereas the deletion of this motif almost abolishe d the promoter activity. These results indicate that: (i) the Ets motif is required for the minimal promoter activity and (ii) the CACGAC direct repea t enhances promoter activity. Database searches failed to identify the dire ct repeat with the CACGAC motif and hence the CACGAC sequence may represent a novel motif. The requirement of both the Ets motif and the direct repeat element for optimal promoter activity represents a unique combination for gene transcription.