IN VITRO RECONSTITUTION OF ALPHA FETOPROTEIN REGULATION

这些研究集中在甲胎蛋白的调节上。 甲胎蛋白(AFP)基因表达作为肿瘤相关基因开关。在肝脏中 再生、肝癌或生殖细胞肿瘤生成 转录是专门被激活的。虽然被广泛用作 诊断标记物，参与的调控机制 甲胎蛋白的致瘤性表达在很大程度上仍未确定， 这种重新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： 特定目的1：确定介导异常的DNA域(S) 通过以下方式重新激活AFP基因的表达：a)构建一系列 AFP缺失和杂交基因插入模板，以及b)使用 细胞培养和转录系统中的这些模板 甲胎蛋白重建成人肝抑制与肝癌激活 体外表达。 具体目标2：建立肿瘤发生的阶段性进展模型 通过激活AFP基因在人工合成核内的表达。我们会 通过以下方式进行：a)组装符合以下条件的染色质模板 正常成人肝脏中甲胎蛋白转录被抑制 提取或纯化的反式作用因子；以及，b)在 体外重组抑制的甲胎蛋白基因模板 表达甲胎蛋白的细胞提取物或纯化的反式激活剂 半保守DNA复制过程中的来源。C)激活 染色质组装的模板将由随后的 体外转录分析。 具体目标3：使用已确定的DNA目标元素和模型 检测系统，以启动分离和鉴定 肝细胞和肝癌细胞培养中存在的蛋白质因子 这使得调控的AFP基因表达发生了戏剧性的变化 正常成人肝细胞和致瘤肝细胞之间。 这些研究将检验以下假设：1)已确定的 发育抑制域可能是不适当的靶标 肿瘤发生过程中的激活和2)DNA复制在 在重塑DNA-蛋白质相互作用中的积极作用 对甲胎蛋白基因表达的影响。我们的长期目标是 研究是为了了解 分子水平上的肿瘤发生。为了实现这一目标，我们将 小鼠甲胎蛋白体外致瘤活化模型的建立 基因重组了核环境的许多性质，并 监管，同时保持生物化学可获得性。

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.