Studies on the Mechanisms of Neurogenesis using Gene Transfer Techniques

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Neurons in the brain withdraw from the cell cycle immediately after differentiation from their proliferative precursors (reuroepithelial stem cells) , and enter the postmitotic state, in which the cells remain undivided throughout the lifetime. We have investigated the mechanisms underlying the permanent arrest of cell growth displayd by differentiated neurons using gene transfer techniques. Necdin is a 325 amino acid residue protein localized to the muclei of postmitotic neurons, which withdraw permanently form the cell cycle. To examine whether necdin confers the postmitotic phenotype, necdin cDNA was stably transfected into NIH3T3 fibroblasts using a eukaryotic lac repressor-operator expression system. When the transfectants were induced to express ectopic necdin, cell growth was arrested without appreciable reduction in cell viability. In order to elucidate the mechanisms underlying necdin gene expression, we have isolated and characterized the mouse necdin gene. The necdin gene contains no intron, and its upstream region lacks canonical TATA and CAAT boxes. Deletion analysis of the promoter region using neurally differentiated P19 cells transfected with luciferase reporter genes demonstrated that a neuron-restrictive core promoter is localized to positions -80 to -35, in which a G+C-rich domain and a putative binding site for transcription factors with PAS (per, arnt, and single-minded) dimerization domain are comprised. These results suggest that necdin induces growth arrest during neuronal differntiation of the neuroepithelial stem cells in which expression of this gene is mediated by the specific cis-acting elements located at the 5' upstream region.