URE, an initiator (Inr)-like site, suppresses the promoter of the rat dynorphin gene

Abstract

We previously identified a DNA binding element termed the upstream regulatory element (URE) that contains the consensus initiator sequence (Inr) in the upstream promoter of the rat prodynorphin gene. The URE displays specific binding to the upstream regulatory element binding protein (UREB1), a novel transcription regulator. Here, we report that the URE functions as a suppressor element. A series of chloramphenicol acetyltransferase reporters (pCAT) were constructed by subcloning either 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 dynorphin promoter (pCAT 0.2) has robust transcriptional activity in Chinese hamster ovary (CHO) cells but not in rat pheochromocytoma PC12 cells. This robust transcriptional activity was completely inhibited in the presence of wildtype URE, whereas the mutations of the URE had no effect. Gel mobility shift assays showed that the complex formed by the URE and nuclear protein extracts can be competed by addition of wildtype URE oligonucleotide but not by specific mutations of the URE, defining particular bases required for protein interaction with the URE. The identical URE sequence is also found upstream in the promoter of human macrophage inflammatory protein 1β (hMIP 1β). The suppressive activity of the rat dynorphin URE can be replaced by the hMIP 1β URE. These data suggest that the URE may serve as a suppressor element in the regulation of dynorphin and hMIP 1β gene transcription.