URE, AN INITIATOR (INR)-LIKE SITE - SUPPRESSES THE PROMOTER OF THE RAT DYNORPHIN GENE

We previously identified a DNA binding element termed the upstream reg ulatory element (URE) that contains the consensus initiator sequence ( Inr) in the upstream promoter of the rat prodynorphin gene, The URE di splays specific binding to the upstream regulatory element binding pro tein (UREB1), a novel transcription regulator, Here, we report that th e URE functions as a suppressor element, A series of chloramphenicol a cetyltransferase reporters (pCAT) were constructed by subcloning eithe r wildtype or mutated URE sequences into a pCAT reporter plasmid 5' of bases -135 to +58 of the prodynorphin gene, The basal -135 to +58 dyn orphin promoter (pCAT 0.2) has robust transcriptional activity in Chin ese hamster ovary (CHO) cells but not in rat pheochromocytoma PC12 cel ls, This robust transcriptional activity was completely inhibited in t he presence of wildtype URE, whereas the mutations of the URE had no e ffect, Gel mobility shift assays showed that the complex formed by the URE and nuclear protein extracts can be competed by addition of wild- type URE oligonucleotide but not by specific mutations of the URE, def ining particular bases required for protein interaction with the URE, The identical URE sequence is also found upstream in the promoter of h uman macrophage inflammatory protein 1 beta (hMLP 1 beta), The suppres sive activity of the rat dynorphin URE can be replaced by the hMIP 1 b eta URE, These data suggest that the URE may serve as a suppressor ele ment in the regulation of dynorphin and hMIP 1 beta gene transcription , (C) 1997 Academic Press.