Biochemical and Functional Properties of a Palindromic Sequence Motif within the Hepatitis B Virus Enhancer 1

Abstract

The hepatitis B virus (HBV) enhancer 1 is a transcriptional element that contributes to the liver-specific regulation of HBV gene expression. We previously identified a novel protein binding site within the enhancer that contains an 8-bp palindromic sequence motif. This motif partially overlaps the binding sites for nuclear factor 1 and hepatocyte nuclear factor 3β (HNF3β). Moreover, we demonstrated that this novel site is recognized by a protein or proteins, tentatively designated as palindrome-binding factor (PBF), that cooperatively interact with HNF3β. In the present work, we have further examined the biochemical and functional attributes of PBF. Protein–DNA interaction studies indicate that three thymidine residues located at the 3′-end of the palindromic sequence motif are important for maximal PBF-binding activity. When protein–DNA complexes were photocrosslinked by exposure to ultraviolet (UV) light, a prominent polypeptide with an apparent molecular mass of 50 kDa was found to associate with the PBF-binding site. Furthermore, transient transfection studies support the hypothesis that PBF contributes to enhancer 1 activity by a combinatorial mechanism that involves at least one other cis-acting sequence motif, the HNF3β-binding site.