IN VITRO RECONSTITUTION OF ALPHA FETOPROTEIN REGULATION

这些研究集中在甲胎蛋白的调节 (AFP)基因表达作为肿瘤相关基因开关。 在肝 再生，肝癌或生殖器肿瘤生成，AFP 转录被特别激活。 虽然被广泛用作 诊断标志物，参与的调控机制， AFP的致瘤性表达在很大程度上仍不确定， 这种再激活对于预后和治疗价值的重要性。 这些拟议研究的具体目标如下： 具体目的1：确定介导异常表达的DNA结构域 AFP基因表达的再激活，通过：a）构建一系列 AFP缺失和杂合基因插入模板，和B）采用 细胞培养和转录系统中的这些模板， 重组AFP的成人肝抑制和肝癌激活 体外表达。 具体目标2：对肿瘤发生的阶段性进展进行建模 通过激活合成核内的AFP基因表达。 我们将 通过以下方式进行：a）组装染色质模板， 在正常成人肝脏中抑制AFP转录 提取物或纯化的反式作用因子;和，B）在 在体外，通过呈递来重塑被抑制的AFP基因模板 从表达AFP的细胞提取物或纯化的反式激活因子中 在半保守DNA复制过程中。 C.激活 染色质组装的模板将通过随后的测定来确定。 体外转录分析 具体目标3：使用已鉴定的DNA靶元件和模型 分析系统，以启动分离和鉴定 存在于肝细胞和肝癌细胞培养物中的蛋白质因子 它赋予了AFP基因表达的戏剧性转变， 正常成人和致瘤肝细胞之间的差异。 这些研究将检验以下假设：1） 发育抑制域是一个可能的目标， 2）DNA复制在肿瘤发生过程中起着重要作用， 在重塑DNA-蛋白质相互作用中的积极作用， AFP基因表达的影响。 我们的长期目标是 研究的目的是了解 在分子水平上的肿瘤发生。 为了实现这一目标，我们将 建立小鼠AFP体外致瘤激活模型 基因，重建核环境的许多特性， 同时保持生物化学可及性。

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.