Promoter region of the rat gene encoding ornithine aminotransferase: transcriptional activity, sequence, and DNase-I-hypersensitive sites

Abstract

In the rat, the gene ( rOAT) encoding ornithine aminotransferase (OAT) is expressed in all cell types examined; however, regulation of rOAT expression is complex and cell-type specific. Various regions of the rOAT 5' flanking domain were cloned upstream from the cat reporter gene, and the expression of these OAT:: cat fusions was examined following transfection into rat kidney epithelial cells (NRK-52E), human embryonic kidney cells (293), and rat hepatoma cells (H-4-II-E). Although these experiments suggested the presence of one or more positive regulatory elements between nucleotides −661 and −158, and one or more negative elements upstream from nt −897, none of these putative elements appeared to function in a cell-type-specific manner. The nt sequence of 2531 bp of the rOAT domain flanking the promoter revealed several putative promoter/enhancer elements in positions analogous to the human OAT gene, numerous AGGTCA-like motifs related to the binding sites for the estrogen and thyroid hormone receptors, and multiple motifs resembling a putative regulatory element associated with genes encoding enzymes of the urea cycle. Finally, sensitivity of the 5' end of rOAT to cleavage by DNase I was examined, as DNase-I-hypersensitive sites ( DHS) are often found in association with cis-acting regulatory elements. Two DHS were identified; one DHS approximately 140bp upstream, and the second DHS approximately 300 bp downstream, of the transcription start point ( tsp). These data provide the foundation upon which to base future studies aimed at elucidating the molecular mechanisms through which rOAT expression is regulated in a cell-type specific manner.