Genomic Approaches to DNA-binding Specificity in vivo

描述(由申请人提供)：描述(由申请人提供)：尽管已有数百种蛋白质的精确共识DMA结合序列，以及数十种生物的完整基因组序列，但无法预测给定的DNA结合蛋白将在体内与基因组的位置相关联。DNA结合蛋白如何识别和结合基因组DNA序列的一个子集，同时又不与数千个计算上无法区分的序列结合，仍然是生物学中一个重大的悬而未决的问题。使用酿酒酵母作为模型系统，我们开发了一套独特的遗传、生化和基因组工具来解决这个问题。。 目的一：我们先前的工作表明，在体内，转录因子优先与基因上游的DNA结合，而不是编码区，尽管这两个区域都有很强的共识结合部位。协同蛋白质-蛋白质-DNA相互作用和不同染色质的可及性被假设为介导上下文相关的结合。为了量化依赖于体内因素的特异性程度，以及蛋白质和DNA固有的程度，将使用纯化的蛋白质和裸酵母基因组DNA在体外确定Raplp和LeuSp的全基因组特异性，并与体内的特异性进行比较。还将确定RAPLP的分布因环境条件的变化而发生的变化。目的二：我们以前的工作表明，在活的酵母细胞中，核小体在整个基因组中的占有率是不同的。我们建议用实验来确定差异核小体占有率的分子基础，以及它是如何在酵母中建立、调节和维持的。目的三：我们的数据和来自其他小组的数据表明，通过DMA结合蛋白(在目标1中解决)、全球染色质组织(在目标2中解决)和转录活性进行的靶标选择之间存在密切关系。第三个目标具体测试这三个过程之间的关系。我们将分析以下菌株的Rap1p靶标选择：(I)Raplp结合位点的上下文发生变化；(Ii)特定位点的转录被禁用；(Iii)RNA PolII CTD发生突变。我们将绘制转录偶联染色质修饰图，并执行高通量定点突变，以将组蛋白结构和修饰与生物学结果联系起来。 人类健康：转录因子在缺失表达或突变时，是人类疾病的普遍原因。对它们体内靶点的更好预测可能会导致抑制与不适当靶点结合的治疗。FIRE是我们开发的一种新的染色质分析方法，它有可能成为影响或引起染色质或转录缺陷的疾病(包括癌症)的预后或诊断工具。

DESCRIPTION (provided by applicant): DESCRIPTION (provided by applicant): Despite the availability of precise consensus DMA-binding sequences for hundreds of proteins, and the complete genome sequence of dozens of organisms, it is not possible to predict where a given DNA-binding protein will associate with a genome in vivo. How DNA-binding proteins recognize and bind to a subset of genomic DNA sequences, while at the same time not binding to thousands of computationally indistinguishable sequences, remains a major unsolved problem in biology. Using Saccharomyces cerevisiae as a model system, we have developed a unique set of genetic, biochemical, and genomic tools to attack this problem. . Aim one: Our previous work has shown that in vivo, transcription factors bind to DNA upstream of genes in preference to coding regions, even though both regions contain strong consensus binding sites. Cooperative protein-protein-DNA interactions and differential chromatin accessibility are hypothesized to mediate context-dependent binding. To quantitate the degree of specificity dependent on in vivo factors, and how much is inherent to the protein and DNA, the genome-wide specificities of Raplp and LeuSp will be determined in vitro using purified proteins and naked yeast genomic DNA, and compared to specificity in vivo. Changes in the distribution of Raplp in response to changes in environmental conditions will also be determined. Aim two: Our previous work has shown that nucleosome occupancy throughout the genome is heterogeneous in living yeast cells. We propose experiments to determine the molecular basis for differential nucleosome occupancy, and how it is established, regulated, and maintained in yeast. Aim three: Our data and data from other groups suggest an intimate relationship between target selection by DMA binding proteins (addressed in Aim 1), global chromatin organization (addressed in Aim 2), and transcriptional activity. The third aim specifically tests relationships between these three processes. We will assay Rap1p target selection in strains in which (i) the context of Raplp binding sites has been changed (ii) transcription at specific loci has been disabled, and (iii) the RNA Pol II CTD is mutated. We will map transcription-coupled chromatin modifications and perform high-throughput site-directed mutagenesis to link histone structure and modification to biological outcomes. Human Health: Transcription factors, when missexpressed or mutated, are a prevalent cause of human disease. Better prediction of their in vivo targets may lead to therapies that inhibit binding to inappropriate targets. FAIRE, a new chromatin assay we have developed, has potential as a prognostic or diagnostic tool for diseases (including cancer) that affect, or arise from defects in, chromatin or transcription.