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

Dynamin I is expressed at high levels in brain and its expression is regula ted during the developmental stages of brain. To elucidate the molecular me chanism by which the expression is tissue-specifically regulated, we cloned the 5'-flanking region of the mouse dynamin I gene and determined the nucl eotide sequence of 1036 bases upstream from the translation start site. Tra nsient transfection studies with a chloramphenicol acetyltransferase report er gene in neuroblastoma NS20Y and Lewis lung cells demonstrated that the 5 '-flanking region has a cell-type-specific promoter activity. Deletion anal yses demonstrated that the minimal promoter activity was detected in the pr oximal region 195 bp upstream of the translation initiation codon (-90 to 105). The minimal promoter was embedded in a GC-rich region (75 % GC conte nt), in which an Sp1-binding motif and a nuclear factor (NF)-kappaB-like el ement (NE-1) were found, but it lacked TATA and CAAT boxes. Mutational anal ysis and electrophoretic mobility-shift assay analysis revealed that Sp1 bi nds to the Sp1 site and that this element is critical for the promoter acti vity 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), whic h binds to the NE-1 sequence, is not NF-kappaB. We also found that one base in the NE-1 sequence (the underlined G residue in GGGATTCGCGGA) is critica l for binding specificity to discriminate between NEBP and NF-kappaB. By UV cross-linking analysis, we found that NEBP is an approx. 104 kDa nuclear p rotein.