Cloning and functional characterization of the 5 ' flanking region of the human mitochondrial malic enzyme gene - Regulatory role of Sp1 and AP-2

This work reports the molecular cloning and functional characterization of the 5' flanking region of the human mitochondrial malic enzyme (mME) gene. The proximal promoter region has features of housekeeping genes like high G + C-content and absence of TATA or CCAAT boxes. Deletion analysis of the 5 ' region of the mME showed that maximal transcriptional activity is located within the -205/+86 region. Footprinting analysis showed two protected reg ions, one comprising potential overlapped AP-1, CREB, and AP-4 sites and a second one encompassing AP-2 and several Sp1 ci-acting elements. Mutation o f putative AP-1/AP-4/CREB sites reduced basal promoter activity to less tha n 50%. Supershift assays demonstrated the specific binding of Sp1 and AP-2 proteins. Moreover, experiments in Drosophila SL2 cells lacking endogenous Sp1 demonstrated that the Sp1 site(s) is essential to maintain a normal bas al rate of transcription of this gene. A low-level expression of AP-2 enhan ced the activity of a mME promoter construct in HepG2 cells and this effect was prevented by disruption of the putative AP-2 element. In contrast, hig her levels of expression of AP-2 induced a DNA-independent inhibitory respo nse. A biphasic regulation of endogenous mME gene is also shown in HepG2 ce lls transfected with an AP-2 expression plasmid, suggesting that availabili ty of AP-2 protein may control this gene under physiological conditions. A recombinant lambda genomic clone containing a mME pseudogene was also isola ted. The high degree of sequence conservation seems to indicate a recent em ergency of this human pseudogene.