Chronic hepatitis B virus (HBV) infection remains a major problem, with 400 million chronic carriers in the world for whom, to date, there is no reliable treatment. The HBV X gene is a major factor in hepato-carcinogenesis and is essential for viral replication and infection in vivo. The X gene product deregulates various cellular functions, including the Ca2+dependent signal transduction pathways, transcription and apoptosis pathways, and eventually leads to transformation of liver cells. However, detection of X gene expression is technically difficult and thus the regulatory mechanisms of X gene transcription have been unclear. The goal of this project is to identify and characterize the transcription factors important for X gene transcription. Recent investigations revealed the importance of general transcription factor (GTF) diversity and core promoter-enhancer specificity in transcriptional regulation. Therefore, features of the X gene core promoter that may explain the unique pattern of the X gene transcription will be particularly studied. X mRNA transcription starts at multiple sites, and the promoter region contains no commonly used core promoter elements such as the TATA box. The X gene promoter may use previously unknown core promoter elements, core promoter-binding factors, and a nontraditional set of GTFs. The X core promoters may preferentially interact with particular classes of transcriptional regulators and coregulators to establish unique expression patterns. In this work, the core promoter elements will be defined by extensive mutagenesis and their binding proteins will be purified by using DNA-protein interaction (Specific Aim 1). The GTFs required for X gene transcription will be determined by immunodepletion and in vitro reconstituted transcription (Specific Aim 2). Regulation of the X gene promoter will be investigated by transient transfection, in vitro transcription, and various biochemical interaction assays (Specific Aim 3). By thoroughly characterizing the transcription factors involved in X gene transcription, this research will serve as a foundation for identifying potential targets for therapeutics and strategies to control HBV gene expression. It will also contribute to the general understanding of transcription from TATA-less promoters, which covers about two thirds of protein-coding genes in humans. Combined with the functional studies of the X protein, our research will improve understanding of the role that the X gene plays in hepatocarcinogenesis.
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