Multiple elements regulate GAD65 transcription

GAD(65) and GAD(67), the two forms of GABA-synthesizing enzyme, are usually coexpressed, but their levels are regulated independently. The GAD(67) promoter has been described. We have now characterized transcriptional regulatory elements in the 5' flanking region of the GAD(65) gene, extending 2.4 kb from the ATG translation initiation site. Primer extension assays revealed that transcription begins at -228 in both adult rat brain and in P19 embryonal carcinoma cells, with additional start sites at -280 in brain and at -360 in P19 cells. These sites are in a GC-rich (72%) region lacking a TATA box. Transient transfection assays revealed that the basal promoter is between -740 and -60, and elements conferring cell-type specificity are further 5'. DNA sequences between -1652 and -1420 can 'silence' transcription from a heterologous promoter. GAD(65) and GAD(67) promoters share little sequence identity, consistent with differences in their transcriptional regulation.