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Long distance control of liver gene expression

肝脏基因表达的远距离控制

基本信息

批准号：
7152868
负责人：
JOSEPH D LOCKER
金额：
$ 34.3万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-07-01 至 2009-11-30
项目状态：
已结题

项目摘要

a-Fetoprotein (AFP) and Albumin (AIb) are closely related serum proteins, encoded by a coordinately regulated chromosomal locus. AFP is developmentally regulated, expressed in fetal liver, silenced at birth, but reactivated in liver cancer. AIb characterizes hepatocyte specification and its expression persists throughout life. Investigators have long understood that the mechanisms that regulate the AIb-AFP locus will define the normal, fetal, and neoplastic hepatocyte phenotype. Both genes are primarily regulated by distant enhancers, while AFP silencing is mediated by promoter controls. Previous experiments have demonstrated that each promoter interacts with enhancers through a different mechanism. In the AIb _romoter, HNF1 is the critical enhancer-activating factor. Aim 1 experiments will define the specific HNF1 "lomains that mediate this interaction and the cofactors that these domains recruit. Further studies will characterize undefined elements in the rest of the promoter, especially "architectural" factors that facilitate enhancer activation. Aim 2 will focus on the AFP promoter, facilitated by use of a synthetic promoter designed for easy modification. The silencer will be localized; its binding factors and mechanism will be characterized. The potential roles of 6 new transcription factors will also be assessed. These were identified by their coregulation with AFP in microarray studies. Aim 3 will integrate the entire locus into a novel cell transfection system to study enhancers and promoters in their proper chromosomal relationships. The entire 70-kb locus has been slightly modified to clone it into a set of easily manipulated plasmids that can be joined together or experimentally changed. These have been placed into a recombining plasmid that will insert constructs into human HCC cell lines. Cre-mediated site-specific recombination will incorporate a single gene copy into a specific chromosomal location. The system will be used to define the relationships of individual enhancers and promoters, a set of new regulatory elements identified in sequence analysis, and specific transcriptional mechanisms.

甲胎蛋白（AFP）和白蛋白（Alb）是两种密切相关的血清蛋白，由一个协同编码的蛋白质，调节染色体位点。AFP受发育调节，在胎儿肝脏中表达，出生时沉默，但在肝癌中被重新激活。AIb表征肝细胞特化并且其表达持续存在在生活中。研究人员早就了解到，调节AIb-AFP基因座的机制将定义正常、胎儿和肿瘤性肝细胞表型。这两种基因主要由远距离增强子，而AFP沉默由启动子控制介导。之前的实验表明，证明了每个启动子通过不同的机制与增强子相互作用。在AIb 此外，HNF 1是关键的增强子激活因子。目标1实验将定义特定的HNF 1 “介导这种相互作用的lomains和这些域招募的辅因子。进一步的研究将在启动子的其余部分中表征未定义的元件，特别是促进启动子的“结构”因素。增强子激活目标2将集中在甲胎蛋白启动子，促进使用合成启动子易于修改的设计。沉默者将被定位，其约束因素和机制将被确定。表征了还将评估6种新转录因子的潜在作用。这些是通过微阵列研究中与AFP的共调节来鉴定。目标3将整个轨迹整合到一个研究增强子和启动子在其固有染色体中的新细胞转染系统关系。整个70-kb的基因座已被轻微修改，将其克隆到一组易于操作的可以连接在一起或通过实验改变的质粒。这些已被放置在一个重组质粒将插入构建体到人HCC细胞系中。Cre介导的位点特异性重组将单个基因拷贝掺入特定的染色体位置。该系统将用于定义单个增强子和启动子的关系，一组新的调控元件在序列分析中确定，和特定的转录机制。