THE ROLE OF NUCLEAR FACTOR NF-Y CP1 IN THE TRANSCRIPTIONAL REGULATIONOF THE HUMAN ALDEHYDE DEHYDROGENASE 2-ENCODING GENE/

Mitochondrial aldehyde dehydrogenase (ALDH2) activity is produced at l ow levels in many tissues, with highest production in liver. Transfect ion assays using the first 600 bp of upstream DNA provided evidence fo r both positive and negative regulatory elements in the proximal promo ter. A region from -79 to -116 bp was protected in DNase I footprintin g assays and bound in electrophoretic mobility shift assays (EMSA) by a nuclear factor found in all cell lines and tissues tested. This regi on, denoted FP160, contained the consensus recognition sites for Sp1 a nd AP2, and a CCAAT box. The CCAAT box was specifically protected by a nuclear factor in methylation interference assays. Mutagenesis of spe cific bp within the CCAAT box eliminated protein binding in vitro and decreased transcriptional activity from the ALDH2 promoter approximate ly 50% in reporter gene assays. Competition experiments showed that th e nuclear factor binding to the FP160 oligodeoxyribonucleotide (oligo) was competed by oligos corresponding to an NF-Y/CP1-binding site to a greater extent than by those containing sites for CTF/NF1, C/EBP or C P2. The heat stability, resistance to proteinase K digestion, sensitiv ity to inhibition of DNA binding by o-phenanthroline, and immunologica l properties of the liver factor binding to FP160 were very similar to the corresponding properties of NF-Y/CP1, Thus, the proximal ALDH2 pr omoter was bound by NF-Y/CP1 and this transcription factor may be resp onsible for the basal expression of the gene observed in most tissues. The NF-Y/CP1 present in rat liver has similar properties to that prev iously characterized in M12 B-lymphoma cells and LMTK mouse fibroblast s.