Uniting disparate fields to explore transcription factor binding dynamics

描述(申请人提供)：存储在每个生物的DNA中的信息必须被阅读和解释，这主要是由蛋白质完成的。一类调节蛋白控制DNA转录成信使RNA，信使RNA然后翻译成结构蛋白和酶。适当调节转录的能力缺陷是许多人类疾病的基础，其中一些疾病，如癌症和许多与衰老相关的疾病，非常明显地根源于基因组功能障碍。为了参与发育和对环境做出反应，细胞对周围环境的反应非常迅速，部分是通过制定特定的转录反应。因此，转录调控必然是一个从根本上动态的过程。然而，我们所知道的几乎所有关于转录调控的机制都来自静态分析，如足迹或染色质免疫沉淀(CHIP)。这项资助的主要目的是结合不同学科的元素来探索体内结合动力学，这是一个在标准芯片实验中完全丢失的基本参数。我们的目标是(1)测量酵母中几乎每个转录因子的转录因子结合动力学，每个转录因子在基因组的每个位置同时进行；(2)在酵母中创建既能进行FRAP又能进行顺序芯片实验的实验系统，以便我们和其他专家实验室可以在完全相同的系统上使用他们的方法；以及(3)测量重组染色质模板上纯化的转录因子的靶向性和动力学。然后，我们可以使用这些系统来测试关于染色质成分和转录因子之间竞争的特定假设，测试周转在调节转录方面的生物学功能，并确定适当调节周转动力学所需的细胞成分。 与公共健康相关：转录调控中与人类健康相关的缺陷是许多人类疾病的基础，其中一些疾病，如癌症和许多与衰老相关的疾病，非常明显地根源于基因组功能障碍。我们建议结合不同学科的元素来探索体内转录因子-DNA结合动力学，这是一个在标准芯片-芯片或芯片-序列实验中完全丢失的基本参数。我们将进行实验，测试有关染色质成分和转录因子之间竞争的特定假设，并确定适当调节结合动力学所需的细胞成分。由于酵母中可用的强大遗传工具，这些实验目前只能在酵母中进行，但我们获得的结果以及我们开发的方法和技术将是本质上的基础，并适用于更复杂的基因组，包括人类。

DESCRIPTION (provided by applicant): The information stored in the DNA of every living thing must be read and interpreted, and this is accomplished chiefly by proteins. One class of regulatory proteins control the transcription of DNA into messenger RNAs, which are then translated into structural proteins and enzymes. Defects in the ability to properly regulate transcription are at the foundation of many human diseases, with some, such as cancer and many aging-related maladies, very clearly rooted in genomic dysfunction. To take part in development and to respond to their environment, cells respond extremely rapidly to their surroundings, in part by enacting specific transcriptional responses. Therefore transcriptional regulation is by necessity a fundamentally dynamic process. However, almost everything we know about the mechanisms underlying transcriptional regulation are derived from static assays like footprinting or Chromatin Immunoprecipitation (ChIP). The major thrust of this grant is to combine elements from distinct disciplines to explore in vivo binding dynamics, a fundamental parameter that is lost completely in standard ChIP experiments. We aim to (1) measure transcription factor binding dynamics for nearly every transcription factor in yeast, each at every position the genome simultaneously, (2) to create experimental systems in yeast amenable to both FRAP and sequential ChIP experiments, so that we and other expert laboratories can use their methods on the exact same system, and (3) to measure purified transcription factor targeting and dynamics on reconstituted chromatin templates. We can then use these systems to test specific hypotheses regarding competition between chromatin components and transcription factors, to test the biological function of turnover in regulating transcription, and to determine the cellular components required for proper regulation of turnover dynamics. PUBLIC HEALTH RELEVANCE: Human health relevance Defects in transcriptional regulation are at the foundation of many human diseases, with some, such as cancer and many aging-related maladies, very clearly rooted in genomic dysfunction. We propose to combine elements from distinct disciplines to explore in vivo transcription factor- DNA binding dynamics, a fundamental parameter that is lost completely in standard ChIP-chip or ChIP-seq experiments. We will conduct experiments that test specific hypotheses regarding competition between chromatin components and transcription factors, and that determine the cellular components required for proper regulation of binding dynamics. Due to the powerful genetic tools available in yeast, these experiments can only be conducted in yeast at this time, but the results we obtain and perhaps just as importantly the approaches and technologies we develop will be fundamental in nature and applicable to more complex genomes, including humans.