HBx-mediated disruption ofp53 tumor suppressor protein function leading to re-activation of a silenced tumor marker gene

Chronic infection with Hepatitis B Virus (HBV) is a predominant risk factor associated with development of hepatocellular carcinoma (HCC). Individuals who are chronically infected with HBV are over 200-times more likely to develop HCC than those who are not infected. Multiple studies have implicated the virally encoded X protein (HBx) as the candidate oncoprotein responsible for cellular transformation. HBx forms a complex with cellular p53 tumor suppressor protein to result in modification of p53-mediated gene regulation, DNA damage detection and modulation of apoptosis. The developmentally silenced α-fetoprotein (AFP) tumor marker gene, which is transcriptionally repressed by p53, is tightly correlated with HCC development: it is reactivated in over 80% of all liver carcinomas. p53 mediates transcriptional repression of AFP through an overlapping HNF-3/Smad4/p53 binding element located within the developmental repressor domain of the AFP promoter. Here, using AFP as a model gene, we have examined the mechanism by which p53 facilitates transcriptional repression, and how this repression is disrupted upon p53-HBx interaction. In vitro chromatin assembled transcription analysis and enzyme accessibility studies demonstrate that p53 association at the overlapping binding element is required during chromatin assembly for reorganization of AFP promoter chromatin structure to result in occlusion of restriction enzymes and general transcription factors from the transcription start site. Protein-DNA binding assays show that p53 association at this element is required to recruit mSin3A co-repressor and stabilize association of a putative Smad4 and SnoN containing co-repressor complex with AFP chromatin templates. HBx-mediated reactivation of AFP is achieved through direct p53-HBx interaction resulting in disruption of SnoN co-repressor binding to AFP chromatin templates.