TRANSCRIPTIONAL REGULATION OF THE GLUR2 GENE - NEURAL-SPECIFIC EXPRESSION, MULTIPLE PROMOTERS, AND REGULATORY ELEMENTS

To understand how neurons control the expression of the AMPA receptor subunit GluR2, we cloned the 5' proximal region of the rat gene and in vestigated GluR2 promoter activity by transient transfection. RNase pr otection and primer extension of rat brain mRNA revealed multiple tran scription initiation sites from -340 to -481 bases upstream of the Glu R2 AUG codon. The relative use of 5' start sites was different in cort ex and cerebellum, indicating complexity of GluR2 transcript expressio n among different sets of neurons. When GluR2 promoter activity was in vestigated by plasmid transfection into cultured cortical neurons, cor tical glia, and C6 glioma cells, the promoter construct with the stron gest activity, per transfected cell, was 29.4-fold (+/- 3.7) more acti ve in neurons than in non-neural cells. Immunostaining of cortical cul tures showed that >97% of the luciferase-positive cells also expressed the neuronal marker MAP-2. Evaluation of internal deletion and substi tution mutations identified a functional repressor element I RE1-like silencer and functional Sp1 and nuclear respiratory factor-1 (NRF-1) e lements within a GC-rich proximal GluR2 promoter region. The GluR2 sil encer reduced promoter activity in glia and non-neuronal cell lines by two- to threefold, was without effect in cortical neurons, and could bind the RE1-silencing transcription factor (REST) because cotransfect ion of REST into neurons reduced GluR2 promoter activity in a silencer -dependent manner. Substitution of the GluR2 silencer by the homologou s Nail REI silencer further reduced GluR2 promoter activity in nonneur onal cells by 30-47%. Maximal positive GluR2 promoter activity require d both Spl and NRF-1 cis elements and an interelement nucleotide bridg e sequence. These results indicate that GluR2 transcription initiates from multiple sites, is highly neuronal selective, and is regulated by three regulatory elements in the 5' proximal promoter region.