Methods for Determining Transcription Factor-DNA Kinetics In Vivo

体内转录因子-DNA 动力学测定方法

• 批准号: 8680869
• 负责人: David T. Auble
• 金额: $ 27.65万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8680869
• 关键词: Accounting; Algorithmic Software; Algorithms; Behavior; Binding; Binding Sites; Biological Assay; Biological Models; Cells; Chromatin; Competitive Binding; Complex; Computing Methodologies; DNA; DNA Binding; DNA Sequence; Data; Defect; Development; Disease; Elements; Gene Expression; Genes; Genetic Transcription; Genome; Genomics; Goals; Growth and Development function; Health; High-Throughput Nucleotide Sequencing; Hour; Human; Kinetics; Lead; Life; Link; Malignant Neoplasms; Measurement; Measures; Methods; Modeling; Normal Cell; Physics; Process; Protein Binding; Proteins; RNA; RNA Polymerase II; RNA chemical synthesis; Regulation; Relative (related person); Resolution; Role; Site; Staging; System; Testing; Time; Transcription Process; Transcriptional Regulation; Work; Yeasts; base; cell growth; cellular imaging; comparative; crosslink; genetic regulatory protein; genome-wide; histone modification; human disease; in vivo; innovation; insight; mathematical model; novel; physical model; promoter; public health relevance; transcription factor; tumor; tumorigenesis; yeast genome

DESCRIPTION: The broad, long-term objective of this proposal is to develop a comprehensive understanding of transcription factor-chromatin interaction kinetics, and to integrate this kinetic information with transcription rates and regulatory processes, in vivo, genome-wide. An understanding of transcription on this level is highly relevant for human health. Precise transcriptional control is essential for normal cell growth and development, and numerous transcriptional defects have been linked to human diseases including cancer. In many cases, however, how a defect in a broadly active transcriptional regulator or co-regulator actually gives rise to disease is unclear especially on a dynamic level. Transcription is an inherently dynamic process. Recent studies have shown that RNA synthesis is stochastic, occurring in infrequent bursts. The degree of transcriptional stochasticity varies across genes suggesting that it is regulated. Remarkably, no method exists with sufficient temporal resolution (seconds to minutes) to measure the dynamics of transcription factors, which regulate RNA synthesis and its stochasticity at specific DNA sites. Aim 1 will expand an innovative experimental and physics-based computational method, crosslinking kinetics, which extracts DNA binding rates for transcription factors at specific loci to each binding site across a genome in vivo. Currently, no method exists for obtaining in vivo binding rates at specific loci on the second time scale. While a locus-specific method with low temporal resolution (many minutes to hours), competition ChIP, has been developed, the analysis methods only estimate relative turnover dynamics of the transcription factor. Aim 2 will develop a novel physics-based method to extract transcription factor-DNA binding rates from competition ChIP data at loci where the transcription factor turnover takes many minutes or more. We will compare the transcription factor-DNA binding rates derived by the two methods at hundreds of sites. This will allow us to further assess the influence of competitive and co-factor binding on the derived parameters and suggest generalized models which account for these effects. The proposed work will develop two methods for determining transcription factor-DNA binding rates on a genomic scale in vivo. This will break a significant methodological bottleneck and enable the study of the dynamics of transcriptional regulation in the cell.

产品说明：广泛的，长期的目标，这一建议是发展一个全面的理解转录因子染色质相互作用的动力学，并整合这一动力学 信息与转录速率和调控过程，在体内，全基因组。在这个水平上理解转录与人类健康高度相关。精确的转录控制对于正常细胞生长和发育是必不可少的，并且许多转录缺陷与包括癌症在内的人类疾病有关。然而，在许多情况下，广泛活跃的转录调节因子或共调节因子中的缺陷如何实际上引起疾病尚不清楚，特别是在动态水平上。转录是一个内在的动态过程。最近的研究表明，RNA合成是随机的，发生在不频繁的爆发。转录随机性的程度因基因而异，表明它是受调控的。值得注意的是，没有方法存在足够的时间分辨率（秒到分钟）来测量转录因子的动态，这些转录因子调节RNA合成及其在特定DNA位点的随机性。目标1将扩展一种创新的实验和物理为基础的计算方法，交联动力学，提取DNA结合率的转录因子在特定位点的每个结合位点在体内的基因组。目前，没有方法存在获得在第二个时间尺度上的特定位点的体内结合率。虽然已经开发了具有低时间分辨率（许多分钟至数小时）的位点特异性方法，竞争ChIP，但分析方法仅估计转录因子的相对周转动力学。目的2将开发一种新的基于物理的方法，从竞争ChIP数据中提取转录因子-DNA结合率，其中转录因子周转需要数分钟或更长时间。我们将在数百个位点上比较这两种方法得出的转录因子-DNA结合率。这将使我们能够进一步评估竞争性和辅助因子结合对导出的参数的影响，并建议考虑这些影响的广义模型。拟议的工作将开发两种方法来确定转录因子-DNA结合率的基因组规模在体内。这将打破一个重要的方法瓶颈，使细胞中的转录调控的动力学研究。