VIP gene transcription is regulated by far upstream enhancer and repressorelements

SK-N-SH human neuroblastoma subclones differ widely in basal and second mes senger induction of the gene encoding the neuropeptide vasoactive intestina l peptide (VIP). These differences were recapitulated by a chimeric gene wh ich consisted of 5.2 kb of the human VIP gene 5 ' flanking sequence fused t o a reporter. Subsequent gene deletion experiments revealed several regulat ory regions on the gene, including a 645-bp sequence located approximately 4.0 upstream from the transcription start site. Here we examined this upstr eam region in detail. Inhibitory sequences were found to be present on each end of the 645-bp fragment. When removed, basal transcription increased mo re than 50-fold. Subsequent deletion/mutation analysis showed that the 213- bp fragment contained at least two enhancer elements. One of these was loca lized to an AT-rich 42-bp sequence shown by others to bind Oct proteins in neuroblastoma cells, while the other corresponded to a composite AP-1/ets e lement. In addition to these enhancers, a 28-bp sequence on the 213-bp frag ment with no apparent homology to known silencers inhibited transcription. The studies provide molecular details of a complex regulatory region on the VIP gene that is likely to be used to finely tune the level of gene transc ription in vivo. (C) 2001 Academic Press.