Characterization of the mouse dynamin I gene promoter and identification of sequences that direct expression in neuronal cells

Abstract

Dynamin I is expressed at high levels in brain and its expression is regulated during the developmental stages of brain. To elucidate the molecular mechanism by which the expression is tissue-specifically regulated, we cloned the 5′-flanking region of the mouse dynamin I gene and determined the nucleotide sequence of 1036 bases upstream from the translation start site. Transient transfection studies with a chloramphenicol acetyltransferase reporter gene in neuroblastoma NS20Y and Lewis lung cells demonstrated that the 5′-flanking region has a cell-type-specific promoter activity. Deletion analyses demonstrated that the minimal promoter activity was detected in the proximal region 195bp upstream of the translation initiation codon (-90 to +105). The minimal promoter was embedded in a GC-rich region (75% GC content), in which an Sp1-binding motif and a nuclear factor (NF)-κB-like element (NE-1) were found, but it lacked TATA and CAAT boxes. Mutational analysis and electrophoretic mobility-shift assay analysis revealed that Sp1 binds to the Sp1 site and that this element is critical for the promoter activity of the dynamin I gene. We found that the NE-1 sequence is required for the expression of the dynamin I gene but NEBP (NE-1-binding protein), which binds to the NE-1 sequence, is not NF-κB. We also found that one base in the NE-1 sequence (the underlined G residue in GGGATTCG̲CGGA) is critical for binding specificity to discriminate between NEBP and NF-κB. By UV cross-linking analysis, we found that NEBP is an approx. 104kDa nuclear protein.