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首页> 外文学位 >Characterization of the c-kit promoter: Participation of the Sp -family of transcription factors in modulating hematopoietic c-kit expression.
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Characterization of the c-kit promoter: Participation of the Sp -family of transcription factors in modulating hematopoietic c-kit expression.

机译：c-kit启动子的表征：转录因子Sp家族参与调节造血c-kit表达。

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摘要

The c-kit proto-oncogene encodes a tyrosine kinase receptor that plays an important role in mammalian hematopoiesis. Expression of the human gene in hematopoietic cell types is regulated at the level of transcription initiation. Previous experiments have implicated a region 183 base pairs upstream from the translational initiation site to be critical for promoter activity. This proximal region is GC-rich, contains no TATA-element, and harbors multiple transcription initiation sites. The focus of this dissertation was to define promoter element(s) in the proximal promoter and identify trans-acting factors that potentially modulate c-kit expression in hematopoietic cell types.;The approach taken was first to identify protein-DNA interaction(s) in c-kit expressing human hematopoietic cell lines. A protected region between base pairs -112 and -158 was defined by in vitro DNase I footprinting analysis of nuclear extracts from HEL and MEG-01 cell lines. This region contains consensus sites for the Sp-family of transcription factors, and purified Sp1 protects a similar region. Electrophoretic mobility shift assays reveal that Sp1 as well as isoforms of Sp3, independently interacted with this protected region in vitro. These interactions are dependent on two Sp-specific consensus sites located in this region. Additionally, mutation or deletion of these sites reduces c-kit proximal promoter activity in transient expression analysis. Cotransfection of c- kit proximal promoter-luciferase constructs with either Sp1 or Sp3 into Sp-deficient Drosophila SL2 cells demonstrated that both Sp1 and Sp3 affect promoter activity. However, cotransfection of a small isoform of Sp3 inhibited both Sp1 and Sp3-mediated activation.;To substantiate the in vitro findings, nuclear protein interactions on the c-kit proximal promoter were investigated in human hematopoietic cell lines. In vivo footprinting by LM-PCR identified occupancy of the c-kit proximal promoter by nuclear protein(s) in both c-kit expressing and non-expressing hematopoiefic cell lines. No interactions were observed in the c-kit non-expressing HeLa cell line. These studies imply that proximal promoter occupancy, presumably by the Sp-family of transcription factors, modulate c-kit expression in hematopoietic cell types.
机译：c-kit原癌基因编码酪氨酸激酶受体，在哺乳动物的造血过程中起重要作用。人基因在造血细胞类型中的表达受转录起始水平的调节。先前的实验暗示了翻译起始位点上游的183个碱基对区域对启动子活性至关重要。该近端区域富含GC，不包含TATA元素，并且具有多个转录起始位点。本论文的重点是在近端启动子中定义启动子元件，并鉴定可能调节造血细胞类型中c-kit表达的反式作用因子。该方法首先用于鉴定蛋白质-DNA相互作用。表达人类造血细胞系的c-kit中的表达。通过对HEL和MEG-01细胞系的核提取物进行体外DNase I足迹分析，确定了碱基对-112和-158之间的保护区。该区域包含转录因子Sp家族的共有位点，纯化的Sp1保护类似区域。电泳迁移率变动分析表明，Sp1和Sp3的同工型在体外与该保护区独立相互作用。这些相互作用取决于该区域中两个特定于Sp的共有位点。另外，在瞬时表达分析中，这些位点的突变或缺失降低了c-kit近端启动子活性。将c-kit近端启动子荧光素酶构建体与Sp1或Sp3共转染到Sp缺陷果蝇SL2细胞中，证明Sp1和Sp3都影响启动子活性。然而，Sp3的一个小亚型的共转染同时抑制了Sp1和Sp3介导的激活。为了证实体外发现，在人类造血细胞系中研究了c-kit近端启动子上的核蛋白相互作用。通过LM-PCR进行的体内足迹鉴定了在表达c-kit的和非表达的造血细胞系中核蛋白对c-kit近端启动子的占用。在未表达c-kit的HeLa细胞系中未观察到相互作用。这些研究表明，大概是转录因子的Sp家族占据了近端启动子，从而调节了造血细胞类型中的c-kit表达。

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  • 作者

    Pavlick, Kevin Paul.;

  • 作者单位

    Louisiana State University Health Sciences Center - Shreveport.;

  • 授予单位 Louisiana State University Health Sciences Center - Shreveport.;
  • 学科 Molecular biology.
  • 学位 Ph.D.
  • 年度 2000
  • 页码 199 p.
  • 总页数 199
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物物理学;
  • 关键词

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