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Delineation of the Genome-wide Recruitment of Hepatitis B Virus Trans-activator Protein HBx, in Primary Hepatocytes and Liver Cancer Cells Open Access

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Hepatocellular carcinoma (HCC) is among the top five cancers worldwide, with Hepatitis B virus (HBV)-associated HCC accounting for the majority of all cases. The cancer promoting activity of HBV is derived from one of its gene products, HBx, the primary trans-activator of cellular genes with roles in carcinogenesis. Although the contribution of HBx to HCC is firmly established, the nature of comparative genome-wide targets of HBx in primary human hepatocytes (PHH) and liver cancer cells, remains unknown, and is being investigated in the present work. Using a genome-wide chromatin immunoprecipitation (ChIP) approach, we characterized the patterns of global recruitment (and consequently, affected genes) of HBx or its mutant, deficient in binding to p65/RelA, of the NF-kB complex, in the presence or experimental depletion of a master coregulator MTA1. We found that the overall recruitment of HBx increases following an interruption in the involvement of MTA1 and/or p65/RelA, suggesting that the levels or activities of MTA1 and p65/RelA represent two major modifiers of HBx recruitment to the human genome. Special attention was placed on targets with recruitment to transcription start sites (TSS), suggesting a probable contribution of the trans-regulatory processes, in influencing the expression of the putative HBx target genes. Representative target genes with TSS recruitment were validated by PCR and DNA gel electrophoresis and with quantitative PCR for analysis of resulting altered expression in ChIP samples. This is the first high-throughput analysis of the genome-wide recruitment of HBx to characterize the involvement of a master chromatin modifier as well as a transcription factor, which act to alter the ability of HBx to trans-regulate genes that may be involved in the development of HBV-associated HCC. This analysis provides novel insight into the identity of targeted genes that are trans-regulated by this viral protein and elucidates how these factors may contribute to the functions of HBx, a major risk factor for the development of HCC.

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