Characterization of the active part of the human transferrin gene enhancer and purification of two liver nuclear factors interacting with the TGTTTGC motif present in this region.

The human transferrin gene enhancer is composed of two functional domains (A and B). We have previously shown that domain A is able to mediate enhancer activity in transient expression experiments. Here, we show that multimers of the domain A single enhanson coupled to a canonical TATA box are sufficient to promote transcription in vitro with liver nuclear extracts. Gel mobility shift assays reveal the binding of two liver nuclear factors to this enhanson, and methylation interference experiments show that the motif 5'-TGTTTGCTTT-3' is the target site for these factors. This was confirmed by the use of mutants in gel retardation assays and transient expression experiments. The two proteins interacting with the decanucleotide have been purified from rat liver nuclear extracts by DNA affinity chromatography. The purified proteins named enhancer-binding protein (EBP)-45 and EBP-40 appear as single polypeptide bands with respective molecular masses of 45 and 40 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The TGTTTGC motif was found to be important for the hepatocyte-specific expression of several genes; and in some cases, it was demonstrated that the transcription factor CCAAT/enhancer-binding protein (C/EBP) is able to bind to this sequence. In vitro experiments show that EBP-45 and EBP-40 are different from C/EBP; they also show that the two proteins interact with the TGTTTGC motif present in control regions of other hepatic genes, such as the mouse albumin enhancer eH and hepatitis B virus enhancer E elements.