Abstract
Neural-specific expression of the mouse regulatory type-I beta (RI beta) subunit gene of cAMP-dependent protein kinase is controlled by a fragment 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 recognized by proteins found in brain nuclear extract. To identify sequences critical for RI beta expression in functional neurons, we performed a deletion 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) of 5'-upstream sequence was functional in mouse brain. One likely regulatory element coincides with the start of transcription and includes an EGR-1 motif and 3 consecutive SP1 sites within a 21-bp interval. Maximal RI beta promoter activity required the adjacent 663 bp of 5'-upstream DNA where most, but not all, of the regulatory activity was localized between position -663 and -333. A 37-bp direct repeat lies within this region that contains 2 basic helix-loop-helix binding sites, each of which are overlapped by two steroid hormone receptor half-sites, and a shared AP1 consensus sequence. Intron I sequences were also tested, and deletion of a 388-bp region containing numerous Sp1-like sequences lowered transgene activity significantly. These results have identified specific regions of the RI beta promoter that are required for the expression of this signal transduction protein in mouse neurons.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) S72345
Previous results demonstrated that 3.5 kb of DNA encompassing the RI promoter was sufficient to direct lac z gene expression in the central nervous system of transgenic mice [20]
In contrast to many genes, the 3.5 kb of DNA that flank the RI promoter contain a rather complex arrangement of transcription factor binding sites that includes 18 SP1 sites, 15 bHLH E-boxes, 40 steroid hormone receptor half-sites, and multiple EGR-1, AP2, AP1, and POU sequence motifs (Fig. 1, Ref. 21). The redundancy of these binding sites has presented an interesting challenge in identifying the sequences responsible for RI gene expression, and rather than systematically mutating specific classes of binding sites, we prepared a series of constructs in which varying amounts of DNA were removed from the 5Ј-upstream region and from intron 1
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) S72345. The diversity of neuronal phenotypes is further enhanced during the life of the organism by stimulusdependent modifications in differentiated neurons This adaptive plasticity, controlled by various neurotransmitters and cytokines, elicits new patterns of gene expression and long-term changes in cell behavior [9, 10]. Relative to the other R subunits, RI makes the holoenzyme more sensitive to cAMP [18], and recent gene disruption experiments indicate that mice lacking RI show deficits in long-term depression and depotentiation in hippocampal neurons [19]. The neural-specific component of RI gene expression was localized to 3.5 kb of genomic DNA that includes 1.5 kb upstream of a GC-rich (TATA-less) promoter, exon I, and a 1.8-kb intron. The results indicate that the sequences required for neuronal expression lie within the GC-rich proximal promoter and that flanking regions upstream of transcription start and within intron I are required for full transgene promoter activity
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