Cloning of the Human Promoter: Differential Regulation by Hypoxia and Prolyl-Hydroxylase Inhibitors.

MAPK-organizer 1 (MORG1) is a molecular scaffold for prolyl-hydroxylase-3 containing a domain (PHD3) protein linking MORG1 to mechanisms of adaptation in hypoxic conditions. In this paper, we report the cloning of the promoter region of the murine and human gene. Among other transcriptional factors binding sites, we identified that both (mouse and human) promoter regions contained several putative hypoxia-inducible factor binding motifs. Analyses of the human promoter by reporter assays revealed that hypoxia and pharmacological inhibitors of prolyl-hydroxylases under in vitro conditions in HEK 293 cells differentially regulate the promoter reporter activity. The exposure of the cells to 10% hypoxia showed inhibition of MORG1 promotor activity at 6 and 12 h, but stimulation after 24 h while treated with prolyl-hydroxylase inhibitors led to a time-independent MORG1 promoter activation. Mutational analyses of the individual HIF binding sites on human promoter suggest that the binding sites work in a complex corporation because single mutations were not sufficient to abolish completely the reporter activation by PHD inhibitors. Our data provide the first evidence that not only MORG1 regulate HIF stabilization through a PHD complex, but also that, vice versa, HIFs control MORG1 expression directly or indirectly by a complex regulatory mechanism.