Cell Type-Dependent Regulation of the Activity of the Negative Regulatory Element of the Hepatitis B Virus Core Promoter

Abstract

The Hepatitis B virus core promoter regulates the expression of the core protein, the precore protein, and the viral DNA polymerase. This promoter is transactivated by HNF4, a liver-enriched transcription factor, through an HNF4 binding site located upstream of the core promoter. The transactivation activity of HNF4 on the core promoter is antagonized by a negative regulatory element (NRE) located upstream of the HNF4 binding site. While the NRE can effectively antagonize HNF4 to suppress the core promoter in HeLa cervical carcinoma cells, it has only a marginal suppressing activity on the core promoter in Huh7 hepatoma cells. By performing deletion-mapping experiments, we have found that the NRE contains at least three independent subregions named NREα, NREβ, and NREγ. Each of these three subregions possesses a weak suppressing activity, but together they generate a strong synergistic suppressing effect on the core promoter. The NREγ subregion is active in both HeLa and Huh7 cells and is bound by a protein factor slightly less than 130 kDa in molecular mass. The NREα and NREβ subregions are active in HeLa cells but not in Huh7 cells. Thus, the marginal suppressing effect of the NRE observed in Huh7 cells was mostly due to the activity of the NREγ subregion. No clear protein factor binding sites could be identified in the NREα and NREβ subregions when the HeLa nuclear extract was used for the DNaseI-footprinting analysis, indicating weak or no protein association with these two subregions in this cell type. However, extensive protein factor binding sites could be identified throughout the sequences of these two subregions when the Huh7 nuclear extract was used for the analysis. These results indicate that a different set of protein factors binds to the NREα and NREβ subregions in Huh7 cells and may account for the inactivity of these two subregions in this cell type. Thus, our results indicate that the cell type-dependent activity of the NRE is due to differential regulation of the activities of the NREα and NREβ subregions by the cell types. This regulation is most likely mediated by cell type-dependent protein factors.