REPRESSION OF AFP TRANSCRIPTION IN THE LIVER AND GUT

在哺乳动物胎儿中，甲胎蛋白(AFP)基因在 卵黄囊、所有肝细胞和大多数小肠中的高水平 上皮细胞。在出生时，甲胎蛋白基因在转录上受到抑制 除一部分肠内分泌细胞外，所有这些细胞 代表不到1%的上皮细胞的成体小细胞 肠子。在成人肝脏中，AFP基因的表达在 对原发性肝细胞癌和血清AFP水平进行监测 帮助诊断这类癌症。顺式和反式 导致新生儿甲胎蛋白抑制和AFP重新激活的因素 在肝癌中的转录还没有被定义。最近的实验 在转基因小鼠中发现了一个区域 甲胎蛋白基因-250个碱基对和-838个碱基之间的序列 这对于抑制AFP基因在新生儿肝脏中的表达具有重要意义。 还有勇气。这些序列的缺失导致高水平的AFP转基因 小鼠中心区周围肝细胞和杯状细胞中的转录 肠子。我们假设单个或多个顺式作用 转录抑制元件位于-250和-838个碱基之间。 甲胎蛋白基因，并认为这些元件结合蛋白因子(S)的中介 抑制甲胎蛋白转录。为了进行测试，我们建议绘制位置图 通过一系列甲胎蛋白表达对抑制元件的影响 含有系统的5‘和3’缺失的-250个碱基对- 838个碱基区，以及一系列连接子扫描突变成两个 肠上皮细胞株，并分化为未转化 肝细胞系。初步研究表明，Caco-2细胞H2 Ht-29细胞亚克隆和肝AML12细胞系， 将对映射抑制因子元素非常有用。使用最低限度的 能够在这些细胞系中直接抑制的序列区域， 将进行DNA迁移率变化分析以检测蛋白质结合，以及 DNase I足迹将用于识别特定的蛋白质结合 网站。含有蛋白质结合突变或缺失的构建体 将产生位点并将其引入细胞系和转基因小鼠 以确认这些序列的缺失在体内取消了抑制。 评估抑制因子在肝脏中的生物学作用 再生和肝癌的形成，我们将确定是否转录 当动物被处理时，抑制物减去转基因会受到影响 化学致癌物质和肝毒素。这会告诉我们如果抑制者 当基因表达在发育过程中被重新诱导时，元件被靶向 得了肝癌。如果我们证明蛋白质与新的顺式作用结合 元素，我们将尝试克隆和鉴定这些蛋白。 压抑。在细胞分化过程中，特定的基因集合 在不同的细胞类型中激活和抑制。身份的鉴定 负责抑制AFP基因的新蛋白可能提供 细胞成熟和分化调控机制的研究进展 肝脏和肠道。此外，我们或许能够开始 剖析导致抑制失活的分子信号 AFP在原发性肝癌中的启动表达。

In the mammalian fetus, the alpha-fetoprotein (AFP) gene is transcribed at high levels in the yolk sac, all hepatocytes, and most small intestinal epithelial cells. At birth the AFP gene is transcriptionally repressed in all of these cells, with the exception of a subset of enteroendocrine cells representing less than 1% of the epithelial cells in the adult small intestine. In the adult liver, AFP gene expression is reactivated in primary hepatocellular carcinomas, and serum AFP levels are monitored to aid in the diagnosis of this type of cancer. The cis and trans-acting factors responsible for AFP repression in the neonate, and reactivation of transcription in hepatomas have not yet been defined. Recent experiments in transgenic mice have resulted in the identification of a region of sequence lying between -250 basepairs (bp) and -838 bp of the AFP gene that is important for the repression of the AFP gene in the neonatal liver and gut. Deletion of these sequences results in high level AFP transgene transcription in pericentral hepatocytes and in goblet cells of the small intestine. We hypothesize that single or multiple cis-acting transcriptional repressor elements lie between -250 bp and -838 bp of the AFP gene, and that these elements bind protein factor(s) that mediate repression of AFP transcription. To test, we propose to map the position of the repressor elements by transfection of a series of AFP expression constructs containing systematic 5' and 3' deletions of the -250 bp to - 838 bp region, and a series of linker scanning mutations into two intestinal epithelial cell lines, and a differentiated nontransformed hepatocyte cell line. Preliminary studies indicate that Caco-2 cells, H2 cells, a subclone of the Ht-29 cell line, and the liver AML12 cell line, will be useful for mapping the repressor elements. Using the minimal region of sequence that is able to direct repression in these cell lines, DNA mobility shift assays will be performed to detect protein binding, and DNase I footprinting will be used to identify specific protein binding sites. Constructs containing mutations or deletions of protein binding sites will be generated and introduced into cell lines and transgenic mice to confirm that deletion of these sequences abrogates repression in vivo. To assess the biological role of the repressor elements during liver regeneration and hepatoma formation, we will determine if transcription of the repressor minus transgenes is influenced when animals are treated with chemical carcinogens and hepatotoxins. This will tell us if the repressor elements are targeted when gene expression is reinduced during development of hepatomas. If we demonstrate protein binding to novel cis-acting elements, we will attempt to clone and characterize the proteins mediating repression. During cell differentiation, specific sets of genes are activated and repressed in different cell types. The identification of novel proteins responsible for repression of the AFP gene may provide insight about mechanisms regulating maturation and cell differentiation in the liver and the intestine. In addition, we may be able to begin to dissect the molecular signals that lead to the inactivation of repression and the initiation of AFP expression in primary liver cancers.