No Effect of Expression of Neuron-Restrictive Silencer Factor (NRSF) Protein on N-Type Ca²⁺ Channel α_1B Gene Promoter Activity in NS20Y Cells

The expression of the N-type voltage-sensitive calcium channel α_1B gene is restricted to neurons by an 5′-upstream region (-3992 to -1788) containing negative regulation element(s) active in non-neuronal cells (Kim et al., 1997). The neuron-restrictive silencer factor (NRSF) represses the transcription of several neuronal genes in non-neuronal cells by binding to a 21 bp DNA element, termed the neuron-restrictive silencer element (NRSE). To analyze the involvement of NRSF in the neuron-specific expression of the α_1B gene, the coding region of NRSF cDNA was cloned by PCR and the exogenous NRSF cDNA was transiently cotransfected with tester plasmids into NS20Y neuronal cells which do not contain the endogenous NRSF protein. The luciferase activity of a positive control plasmid NRSEL containing a single copy of the NRSE sequence of the SCG10 gene was repressed 5 fold in HeLa cells containing the endogenous NRSF protein, and its activity was repressed to 44-27% of the control with an increasing amount of exogenous DNA in NS20Y cells. Unlike NRSEL, the promoter activity of the α_1B subunit-luciferase fusion construct (-3992L) was about 15 fold repressed in HeLa cells compared to NS20Y cells, while any remarkable changes was undetectable in the NRSF expressed NS20Y. These results suggest that the repression of the α_1B gene in non-neuronal cells may not be mediated by the NRSF function.