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:6574817
- 负责人:
- 金额:$ 35.32万
- 依托单位:
- 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 2007-11-30
- 项目状态:已结题
- 来源:
- 关键词:DNA footprinting RNase protection assay albumins alpha fetoprotein gene deletion mutation gene expression gene induction /repression genetic enhancer element genetic library genetic mapping genetic promoter element genetic regulatory element immunoprecipitation liver molecular genetics polymerase chain reaction recombinant DNA transcription factor transfection
项目摘要
DESCRIPTION (provided by applicant): Alpha-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 Alb promoter, HNF1 is the critical enhancer-activating factor. Aim 1 experiments will define the specific HNF1 domains 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.
描述(由申请人提供):甲胎蛋白(AFP)和白蛋白(AIb)是密切相关的血清蛋白,由协调调节的染色体位点编码。 AFP 受发育调节,在胎儿肝脏中表达,出生时沉默,但在肝癌中重新激活。 AIb 表征了肝细胞的特性,其表达在整个生命过程中持续存在。研究人员很早就认识到,调节 AIb-AFP 基因座的机制将定义正常、胎儿和肿瘤肝细胞表型。这两个基因主要由远端增强子调控,而 AFP 沉默则由启动子控制介导。先前的实验已经证明每个启动子通过不同的机制与增强子相互作用。在 Alb 启动子中,HNF1 是关键的增强子激活因子。目标 1 实验将定义介导这种相互作用的特定 HNF1 结构域以及这些结构域招募的辅助因子。进一步的研究将表征启动子其余部分中未定义的元件,特别是促进增强子激活的“结构”因子。目标 2 将重点关注 AFP 启动子,通过使用易于修饰的合成启动子来促进。消音器将采用本地化;其结合因素和机制将得到表征。还将评估 6 个新转录因子的潜在作用。这些是通过微阵列研究中与 AFP 的共调节来识别的。目标 3 将把整个基因座整合到一个新的细胞转染系统中,以研究增强子和启动子的正确染色体关系。整个 70 kb 基因座经过轻微修改,可将其克隆到一组易于操作的质粒中,这些质粒可以连接在一起或通过实验进行更改。这些已被放入重组质粒中,该质粒将把构建体插入人肝癌细胞系中。 Cre 介导的位点特异性重组会将单个基因拷贝整合到特定的染色体位置。该系统将用于定义各个增强子和启动子的关系、序列分析中确定的一组新的调控元件以及特定的转录机制。
项目成果
期刊论文数量(0)
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JOSEPH D LOCKER其他文献
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{{ truncateString('JOSEPH D LOCKER', 18)}}的其他基金
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- 批准号:
6317727 - 财政年份:2000
- 资助金额:
$ 35.32万 - 项目类别: