Identification of multiple transcription factors, HLF, FTF, and E4BP4, controlling hepatitis B virus enhancer II

Hepatitis B virus (HBV) enhancer II (EnII) is a hepatotropic cis element wh ich is responsible for the hepatocyte-specific gene expression of HBV. Mult iple transcription factors have been demonstrated to interact with this reg ion. In this study, the region from HBV nucleotides (nt) 1640 to 1663 in En II was demonstrated to be essential for enhancer activity and to be another target sequence of putative transcription factors. To elucidate the factor s which bind to this region, we used a yeast one-hybrid screening system an d cloned three transcription factors, HLF, FTF, and E4BP4, from a human adu lt liver cDNA library. All of these factors had binding affinity to the seq uence from nt 1640 to 1663. Investigation of the effects of these factors o n transcriptional regulation revealed that HLF and FTF had stimulatory acti vity on nt 1640 to 1663, whereas E4BP4 had a suppressing effect. FTF coordi nately activated both 3.5-kb RNA and 2.4/2.1-kb RNA transcription in a tran sient transfection assay with an HBV expression vector. ELF, however, activ ated only 3.5-kb RNA transcription, and in primer extension analysis, HLF s trongly stimulated the synthesis of pregenome RNA compared to precore RNA. Thus, FTF stimulated the activity of the second enhancer, while HLF stimula ted the activity of the core upstream regulatory sequence, which affects on ly the core promoter, and had a dominant effect on the pregenome RNA synthe sis.