Molecular mechanisms regulating cell type specific expression of BMP/RA Inducible Neural-specific Protein-1 that suppresses cell cycle progression: roles of NRSF/REST and DNA methylation.

We have recently identified a novel protein family, BMP/RA-Inducible Neural-specific Protein (BRINP) including BRINP1, 2, 3. Among BRINP family genes, BRINP1 is most highly and widely expressed in various regions of the mammalian nervous system, although its expression is also found in some non-neural tissues and cell types at low levels. We have previously suggested that BRINPs are involved in the suppression of cell-cycle progression in post-mitotic neuronal cells. In the present study, we investigated the transcriptional mechanisms regulating the cell type-specific expression of BRINP1. First, bisulfite analysis of the methylation status revealed hypermethylation of the CpG island surrounding BRINP1 exon 1 in a non-neural cell line, NIH 3T3, which expresses low but detectable levels of BRINP1, while methylation levels of the BRINP1 CpG island in either non-neural or neural tissues are very low. Treatment of NIH 3T3 cells with a demethylating agent, 5-azacytidine, upregulated the expression of BRINP1 remarkably. Then, we analyzed the promoter activity of 7 kb region surrounding BRINP1 exon 1 in neuronal and non-neuronal cells. Consequently, we found a basic promoter region and a non-neural-specific silencing region which contains neuron-restrictive silencing element/repressor element 1 (NRSE/RE-1) like element (BRINP1-NRSE). Mutation of BRINP1-NRSE recovered the BRINP1 promoter activity in non-neuronal cells. Furthermore, proteins in nuclear extract from non-neural cells bound to the BRINP1-NRSE. These results strongly suggest that BRINP1-NRSE determines neural-specific expression of BRINP1, while hypermethylation of the BRINP1-CpG island suppresses BRINP1 expression in NIH 3T3 cells.