在单拷贝基因的第二时间尺度上测量染色质相互作用动态
Kunal Poorey1, Ramya Viswanathan, Melissa N Carver
1Department of Biochemistry and Molecular Genetics, University of Virginia Health System, Charlottesville, VA 22908, USA.
Science (New York, N.Y.)
|October 5, 2013
概括
这项研究引入了一种修改的染色体免疫沉 (ChIP) 试验,以测量蛋白质与DNA的体内结合动态. 这种新方法量化了快速染色体相互作用,揭示了调节过程如何影响蛋白质结合和转录. 关键词:ChIP测定,染色素,蛋白质结合动态,转录.
科学领域:
- 分子生物学分子生物学
- 遗传学 是一个遗传学.
- 生物化学 生物化学
背景情况:
- 染色体免疫沉 (ChIP) 试验对于研究蛋白质-DNA相互作用至关重要,但缺乏时间分辨率来测量原生相互作用的稳定性.
- 现有的方法无法评估单拷贝基因的快速结合动态,从而限制了我们对动态调节过程的理解.
- 活细胞成像表明在重复的DNA元素中存在短暂的相互作用,但这并不反映独特的基因组位置的动态.
研究的目的:
- 开发一种经过修改的ChIP测定,能够测量蛋白质在特定DNA位点的体内结合和解离速率 (开启和关闭速率).
- 调查调控机制如何改变单复制基因中蛋白质-染色质相互作用的稳定性.
- 建立一种用于分析活细胞中染色质结合动态的定量方法.
主要方法:
- 通过分析甲交叉链接的时间依赖性,开发了一种修改后的ChIP试验,该试验包含了次秒时间分辨率.
- 该试验用于在体内测量局部特异性染色质相互作用的启动和关闭率,其动态范围为~100倍.
- 该方法用于研究在转录调节过程中TATA结合蛋白 (TBP) 的结合动态.
主要成果:
- 修改后的ChIP测定成功地量化了快速的,局部特定的色素相互作用 in vivo.
- 该研究表明,调节过程可以将弱结合蛋白,如TATA结合蛋白,转化为稳定的促进体相互作用.
- 蛋白质结合中的这些动态变化促进了转录复合物的形成,影响了基因表达.
结论:
- 开发的ChIP试验为染色质结合动态的系统和定量分析提供了一种新方法.
- 了解这些动态对于阐明分子层面的基因调节机制至关重要.
- 这种方法为研究各种生物环境中蛋白质-DNA相互作用的短暂性开辟了新的途径.
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相关概念视频
02:36Chromatin Immunoprecipitation- ChIP
Chromatin immunoprecipitation, or ChIP, is an antibody-based technique used to identify sites on DNA that bind to transcription factors of interest or histone proteins. It also helps determine the type of histone modifications such as acetylation, phosphorylation, or methylation.
Types of ChIP
ChIP can be divided into two types - X-ChIP and N-ChIP. X-ChIP involves in vivo cross-linking of histones and regulatory proteins to DNA, fragmenting the DNA by sonication, and isolating the protein-DNA...
Types of ChIP
ChIP can be divided into two types - X-ChIP and N-ChIP. X-ChIP involves in vivo cross-linking of histones and regulatory proteins to DNA, fragmenting the DNA by sonication, and isolating the protein-DNA...
02:05Duplication of Chromatin Structure
The process of chromosome duplication during cell division requires genome-wide disruption and re-assembly of chromatin. The chromatin structure must be accurately inherited, reassembled, and maintained in the daughter cells to ensure lineage propagation.
The basic unit of the chromatin is the nucleosome, consisting of DNA wrapped around octameric histone proteins and short stretches of linker DNA separating individual nucleosomes. The histone proteins within the nucleosome have their...
The basic unit of the chromatin is the nucleosome, consisting of DNA wrapped around octameric histone proteins and short stretches of linker DNA separating individual nucleosomes. The histone proteins within the nucleosome have their...
01:32Chromatin Packaging
Each human somatic cell contains 6 billion base pairs of DNA. Each base pair is 0.34 nm long, meaning each diploid cell contains a staggering 2 meters of DNA. This long DNA strand is packed inside a nucleus measuring only 10-20 microns in diameter with the help of specialized DNA-binding proteins called histones. Together they form a compact DNA-protein complex called chromatin. The chromatin is further compacted into higher-order structures. The highest level of compaction is achieved during...
02:21Chromatin Packaging
Each human somatic cell contains 6 billion base-pairs of DNA. Each base-pair is 0.34 nm long, which means that each diploid cell contains a staggering 2 meters of DNA. How is such a long DNA strand packed inside a nucleus measuring only 10 - 20 microns in diameter?
The chromatin
In combination with specialized DNA binding protein called Histones, the DNA double helix forms a compact DNA: protein complex called chromatin. The chromatin itself is further compacted into higher-order structures.
The chromatin
In combination with specialized DNA binding protein called Histones, the DNA double helix forms a compact DNA: protein complex called chromatin. The chromatin itself is further compacted into higher-order structures.