Analysis of the molecular mechanisms regulating GAP-43 gene expression

Abstract

GAP-43 is a highly conserved neuronal protein whose expression is spatially and temporally regulated. Because this regulation may occur, at least in part, at the level of transcription we have characterized the regions upstream of the GAP-43 transcription unit which direct its neuronal specific expression. 230 bp of 5{dollar}\sp\prime{dollar} flanking DNA sequence defines a GAP-43 minimal promoter that is active in both neuronal and glial, but not in non-neuronal cell lines. In addition, we have demonstrated that two polypurine tracts within the 5{dollar}\sp\prime{dollar} flanking DNA sequence of the GAP-43 gene adopt a non-duplex configuration in plasmids; and, when studied in the context of chromosomal integration, these regions have a stimulatory effect on transcription.;The development of herpes simplex virus (HSV) amplicon vectors has provided a valuable tool for the introduction of foreign genes into a variety of mammalian cells including post-mitotic neurons which are normally refractory to gene transfer. To extend our analysis of the cell-type specific expression of the GAP-43 promoter to bona fide neurons, we have cloned different promoter fragments upstream of the {dollar}\beta{dollar}-galactosidase reporter gene in replication defective HSV amplicon vectors. To create a minimal origin of replication, these vectors were then modified by deleting regions of DNA within the origin of replication which are known to bind the transcriptional activator VP16. These vectors were packaged into viruses and used to infect cultured neurons to determine whether the promoter regions that we have defined in immortalized cell lines behaved analogously in neurons. The HSV amplicons which had employed the cytomegalovirus promoter region provided the highest level of {dollar}\beta{dollar}-galactosidase activity. Amplicons containing the neuronal promoters were moderately active, including those containing 1500 bp of GAP-43 5{dollar}\sp\prime{dollar} flanking DNA sequence which were able to direct expression in a neuronal specific manner when transduced into both nerve growth factor differentiated PC12 cells and primary neurons. These results indicate that HSV amplicon vectors are useful vehicles for the transfer of foreign genes into a variety of cell types.