AP2-like cis element is required for calretinin gene promoter activity in cells of neuronal phenotype differentiated from multipotent human cell line DEV.

Calretinin (CR) is an EF-hand calcium binding protein expressed at high level in neurons. To identify regulatory elements in CR gene promoter, cultured rat cortical cells were transfected with constructs containing its 5'-end deletion mutants and the luciferase reporter gene. A fragment ending at -115 bp upstream of the transcription start site had high promoter activity and was able to induce expression of luciferase specifically in neuronal cells of cortical cultures. The wild type sequence of -115/+54 CR promoter fragment preferentially drove the expression of green fluorescent protein analog in cells of neuronal phenotype differentiated from multipotent human cell line DEV. Electrophoretic mobility shift assays (EMSA) revealed that the -115/-71 CR gene promoter region contains a binding site for a factor present in brain nuclear extract. Among oligonucleotides containing consensus binding sites for transcription factors within this region, the one representing AP2 binding site was able to compete formation of a protein complex. Mutations of this site prevented the binding between brain protein(s) and the -115/+54 CR gene promoter region and abolished the preferential expression of reporter gene in neuronal cells of DEV line. Thus, the AP2-like element seems to be essential for the neuron-specific activity of the CR gene promoter.