Novel dual repressor elements for neuronal cell-specific transcription of the rat 5-HT1A receptor gene

The level of expression of the 5-HT1A receptor in the raphe and limbic syst ems is implicated in the etiology and treatment of major depression and anx iety disorders. The rat 5-HT1A receptor gene is regulated by a proximal TAT A-driven promoter and by upstream repressors that inhibit gene expression. Deletion of a 71-base pair (bp) segment between -1590/-1519 bp of the 5-HT1 A receptor gene induced over 10-fold enhancement of transcriptional activit y in both 5-HT1A receptor-expressing (RN46A raphe and SN48 septal) cells an d receptor-negative (L6 myoblast and C6 glioma) cells. A 31-bp segment of t he repressor was protected from DNase I digestion by RN46A or L6 nuclear ex tracts. Within the 31-bp segment, a single protein complex was present in r eceptor-expressing cells that bound a novel 14-bp DNA element; in receptor- negative: cells, an additional complex bound an adjacent 12-bp sequence. In receptor-positive but not receptor-negative cells, mutation of the 14-bp e lement to eliminate protein binding abrogated repression to nearly the same extent as deletion of the -1590/-1519 bp segment. Additional mutation of b oth 14-bp and 12-bp elements abolished protein binding and repressor activi ty in receptor-negative cells. Thus a single protein-DNA complex at the 14- bp element represses the 5-HT1A receptor gene in 5-HT1A receptor-positive n euronal cells, whereas adjacent DNA elements provide a dual repression mech anism in 5-HT1A receptor-negative cells.