REPRESSION OF AFP TRANSCRIPTION IN THE LIVER AND GUT

在哺乳动物胎儿中，甲胎蛋白（AFP）基因转录于 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，AFP基因在转录上受到抑制， 所有这些细胞，除了肠内分泌的一个子集， 在成人小细胞中，占上皮细胞不到1%的细胞 肠子在成人肝脏中，AFP基因表达在 监测原发性肝细胞癌和血清AFP水平， 帮助诊断这种癌症。顺式和反式作用 负责新生儿AFP抑制的因素，以及 肝癌中的转录尚未被定义。最近的实验 在转基因小鼠中，已经鉴定出了一个区域， 位于AFP基因的-250碱基对（bp）和-838 bp之间的序列 这对新生儿肝脏中AFP基因的抑制很重要 还有内脏这些序列的缺失导致高水平的AFP转基因 转录在中央周围肝细胞和杯状细胞的小 肠子我们假设单个或多个顺式作用 转录抑制元件位于-250bp和-838bp之间 AFP基因，并且这些元件结合蛋白因子， 抑制AFP转录。为了测试，我们建议绘制 通过转染一系列AFP表达的阻遏物元件， 含有-250 bp至-250 bp的系统性5'和3'缺失的构建体， 838 bp的区域，并将一系列连接子扫描突变成两个 肠上皮细胞系，以及分化的非转化的 肝细胞系初步研究表明，Caco-2细胞，H2 细胞、Ht-29细胞系的亚克隆和肝AML 12细胞系， 将有助于定位阻遏物元件。使用最小 能够在这些细胞系中指导阻遏的序列区域， 将进行DNA迁移率变动试验以检测蛋白结合， DNA酶I足迹法将用于鉴定特异性蛋白结合 网站.含有蛋白结合突变或缺失的构建体 将产生位点并引入细胞系和转基因小鼠中 以证实这些序列的缺失消除了体内抑制。 评估阻遏元件在肝脏中的生物学作用， 再生和肝癌形成，我们将确定是否转录 当动物用以下药物治疗时， 化学致癌物和肝毒素。这将告诉我们是否阻遏物 当基因表达在发育过程中被重新诱导时， 肝细胞瘤如果我们证明蛋白质与新的顺式作用 元件，我们将尝试克隆和表征蛋白质介导 镇压在细胞分化过程中，特定的基因组被 在不同的细胞类型中激活和抑制。的识别 负责抑制AFP基因的新蛋白质可能提供 关于调节成熟和细胞分化的机制的见解， 肝脏和肠道此外，我们也许可以开始 剖析导致阻遏失活的分子信号 和原发性肝癌中AFP表达的起始。

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.