RESUMO
The cytosolic aspartate aminotransferase (cAspAT) gene is ubiquitously expressed but it is regulated by hormones in a tissue-specific manner. In vitro DNase I footprinting studies of a 260-base pair fragment carrying the basal promoter activity revealed that three CCAAT sequences bind liver nuclear proteins (protected regions P2, P3, P4). Competition studies, the heat resistance of these proteins, and identical footprints obtained using a recombinant CCAAT/enhancer-binding protein (C/EBP) alpha fragment indicate that they belong to the C/EBP family of transcription factors. A fourth protected region P1, overlapping the P2 region, was observed in the liver in the presence of competing oligonucleotides containing the C/EBP site. In cotransfection experiments, the C/EBP beta protein trans-activated 10-15-fold the cAspAT gene promoter in HepG2 cells. Deletion studies revealed that regions P2 and P4 are critical for promoter activity. In gel retardation experiments, the P4 region bound different C/EBP-related proteins in different tissues: the brain protein is heat sensitive in contrast to the liver protein. The synthetic oligonucleotide OL 1-2, which covers the P1 and P2 regions, binds C/EBP-like proteins as well as NF1 and CP1 (or NFY) transcription factors, but the preferential binding of one of these protein is tissue-specific. In summary, using the cAspAT gene promoter, we have shown that ubiquitously active promoters may be recognized by different proteins in different tissues.
