Cloning and functional expression of the rabbit transferrin gene promoter

The transferrin gene is expressed in all mammals, mainly in the liver. A rabbit genomic library was screened using cDNA probes, and 8kb of 5' flanking sequence of the rabbit transferrin gene was cloned upstream of the cat reporter gene. The first 200 nucleotides of this promoter were sequenced. The rabbit transferrin promoter is highly homologous to the human and murine ones. Its functional activity was tested in the human hepatic cell line HepG2. Using transitory transfections in these cells, a proximal positive region, a negative region and a distal positive region located between -3.6 and -4.0kb were identified. This distal positive region sequence is highly conserved with the the human gene enhancer sequence, and contains an HNF3alpha binding site, the mutation of which totally abolished its effect in HepG2 and HuH7 cell lines. The rabbit transferrin 5' flanking sequence thus shows a promoter organization very similar to that of the human gene, and the HNF3alpha binding site in the distal positive region presents the same functional importance in both genes.