The rat α-fetoprotein (afp) gene is controlled by three enhancers whose function depends on their interaction with liver-enriched transcription factors. The afp enhancer III, located at −6kb, is composed of three regions that act in synergy. Two of these regions, called s1 and s2, contain a putative binding site for hepatocyte nuclear factor-6 (HNF-6). This factor is the prototype of the ONECUT family of cut-homoeodomain proteins and is a known regulator of liver gene expression in adults and during development. We show here that the two splicing isoforms of HNF-6 bind to a site in the s1 region and in the s2 region. The core sequence of the s1 site corresponds to none of the known HNF-6 binding sites. Nevertheless, the binding properties of the s1 site are identical with those of the s2 site and of previously characterized HNF-6 binding sequences. The HNF-6 consensus should therefore be rewritten as DRRTCVATND. Binding of HNF-6 to the s1 and s2 sites requires both the cut and the homoeo domains, is co-operative and induces DNA bending. HNF-6 strongly stimulates the activity of the afp enhancer III in transient transfection experiments. This effect requires the stereo-specific alignment of the two HNF-6 sites. Moreover, HNF-6 stimulates the enhancer in synergy with the retinoic-acid-receptor-related orphan receptor α (RORα), which binds to a neighbouring site in the s1 region. Thus expression of the afp gene requires functional interactions between HNF-6 molecules and between HNF-6 and RORα.

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