IN VITRO RECONSTITUTION OF ALPHA FETOPROTEIN REGULATION

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These investigations focus on the regulation of alpha-fetoprotein (AFP) gene expression as a tumor-associated gene switch. During liver regeneration, hepatocarcinoma or germinal tumor generation, AFP transcription is specifically activated. Though widely applied as a diagnostic marker, the regulatory control mechanisms involved in tumorigenic expression of AFP remain largely undetermined, as does the importance of this reactivation for prognostic and therapeutic value. The specific aims of these proposed studies are as follows: Specific Aim 1: To determine the DNA domain(s) that mediates aberrant reactivation of AFP gene expression by: a) construction of a series of AFP deletion and hybrid gene insertion templates, and b) employing these templates in cell culture and transcription systems that reconstitute adult liver repression and hepatoma activation of AFP expression in vitro. Specific Aim 2: To model the stagewise progression of tumorigenesis by activating AFP gene expression within synthetic nuclei. We will undertake this by: a) assembly of chromatin templates that are repressed for AFP transcription in the presence of normal, adult liver extracts or purified, trans-acting factors; and, b) initiating in vitro, remodeling of the repressed AFP gene templates by presentation of cellular extracts or purified trans-activators from AFP expressing sources during semi-conservative DNA replication. c) Activation of chromatin-assembled templates will be determined by subsequent in vitro transcription analysis. Specific Aim 3: To employ identified DNA target elements and model assay systems in order to initiate isolation and identification of the protein factors present in hepatic cells and hepatoma cell cultures that confer the dramatic switch in regulated AFP gene expression between normal adult and tumorigenic liver cells. These studies will test the hypotheses that 1) an identified developmental repressor domain is a likely target for inappropriate activation during tumorigenesis and 2) that DNA replication plays an active role in remodeling DNA-protein interactions with dramatic repercussions for AFP gene expression. The long-term goal of our studies is to understand the initiation and progression of tumorigenesis at a molecular level. In pursuing this goal, we will develop an in vitro model of tumorigenic activation of the mouse AFP gene that reconstitutes many properties of the nuclear environment and regulation while remaining biochemically accessible.