Cloning and functional characterization of the 5' flanking region of the human mitochondrial malic enzyme gene

11 páginas, 10 figuras, 1 tabla -- PAGS nros. 3017-3027This 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 regions, one comprising potential overlapped AP-1, CREB, and AP-4 sites and a secondone encompassing AP-2 and several Sp1 ciacting elements. Mutationof putative AP-1/AP-4/CREB sites reduced basal promoter activity to less than 50%. Supershift assays demonstrated the specific binding of Sp1 and AP-2 proteins. Moreover, experiments in DrosophilaSL2 cells lacking endogenous Sp1 demonstrated that the Sp1 site(s) is essential to maintain a normal basal rate of transcription of this gene. A low-level expression of AP-2 enhanced 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, higher levels of expression of AP-2 induced a DNA-independent inhibitory response. A biphasic regulation of endogenous mMEgene is also shown in HepG2 cells transfected with an AP-2 expression plasmid, suggesting that availability of AP-2 protein may control this gene under physiological conditions. A recombinant genomic clone containing a mME pseudogene was also isolated.The high degree of sequence conservation seems to indicate arecent emergency of this human pseudogene. Keywords: AP-2; Sp1; mitochondrial malic enzyme; promoter. . Abbreviations: AP-1, activating protein-1; AP-2, activating protein-2, AP-4, activating protein-4; bp, base pair(s); CAT, chloramphenicol acetyl transferase; CREB, cAMP responsive element binding factor; DMEM, Dulbecco’s modified Eagle’s medium; EMSA, electrophoretic mobility shift assays; fpu, footprinting units; mME, mitochondrial malic enzyme; RACE, rapid amplification of cDNA ends; RTPCR, reverse transcriptase-PCR; SOE, splicing by overlap extensionThis work has been supported in part by grants from the Dirección General de Investigación Científica y Técnica (DGICYT PB97-1240, and DGICYT PM97-0016, and SAF 2000–0127) and Comunidad Autónoma de Madrid (08.4/0031/1998). N. Butta was recipient of a research contract from the Consejo Superior de Investigaciones Científicas (CSIC)Peer reviewe