PROMOTER SEQUENCES IN THE RI-BETA SUBUNIT GENE OF CAMP-DEPENDENT PROTEIN-KINASE REQUIRED FOR TRANSGENE EXPRESSION IN MOUSE-BRAIN

Neural-specific expression of the mouse regulatory type-I beta (RI bet a) subunit gene of cAMP-dependent protein kinase is controlled by a fr agment of genomic DNA comprised of a TATA-less promoter flanked by 1.5 kilobases of 5'-upstream sequence and a 1.8-kilobase intron. This DNA contains a complex arrangement of transcription factor binding motifs , and previous experiments have shown that many of these are recognize d by proteins found in brain nuclear extract, To identify sequences cr itical for RIP expression in functional neurons, we performed a deleti on analysis in transgenic mice. Evidence is presented that the GC-rich proximal promoter is responsible for cell type-specific expression in vivo because RI beta DNA containing as little as 17 base pairs (bp) o f 5'-upstream sequence was functional in mouse brain. One likely regul atory element coincides with the start of transcription and includes a n EGR-1 motif and 3 consecutive SP1 sites within a 21-bp interval, Max imal RI beta promoter activity required the adjacent 663 bp of 5'-upst ream DNA where most, but not all, of the regulatory activity was local ized between position -663 and -333. A 37-bp direct repeat lies within this region that contains 2 basic helix-loop-helix binding sites, eac h of which are overlapped by two steroid hormone receptor half-sites, and a shared AP1 consensus sequence, Intron I sequences were also test ed, and deletion of a 388-bp region containing numerous Sp1-like seque nces lowered transgene activity significantly, These results have iden tified specific regions of the RIP promoter that are required for the expression of this signal transduction protein in mouse neurons.