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 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 195 bp 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)-kappa B-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-kappa B. We also found that one base in the NE-1 sequence (the underlined G residue in GGGATTCGCGGA) is critical for binding specificity to discriminate between NEBP and NF-kappa B. By UV cross-linking analysis, we found that NEBP is an approx. 104 kDa nuclear protein.
Yoo
J
J
Cell Biology Laboratory, Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yusung, Taejon 305-600, Korea.
Lee
S S
SS
Jeong
M J
MJ
Lee
K I
KI
Kwon
B M
BM
Kim
S H
SH
Park
Y M
YM
Han
M Y
MY
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA; D034284:Dynamin I; D034281:Dynamins; D020558:GTP Phosphohydrolases / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D016328:NF-kappa B; D016329:Sp1 Transcription Factor; D004247:DNA; D034284:Dynamin I; D020558:GTP Phosphohydrolases; D034281:Dynamins