Genomic Approaches to DNA-binding Specificity in vivo

描述（由申请人提供）：描述（由申请人提供）：尽管可获得数百种蛋白质的精确共有DNA结合序列，以及数十种生物体的完整基因组序列，但不可能预测给定DNA结合蛋白将在体内与基因组结合的位置。DNA结合蛋白如何识别并结合到基因组DNA序列的子集，而同时不结合到数千个计算上无法区分的序列，仍然是生物学中未解决的主要问题。使用酿酒酵母作为模型系统，我们已经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。. 目标一：我们以前的工作表明，在体内，转录因子优先于编码区与基因上游的DNA结合，即使这两个区域都含有强的共有结合位点。协同蛋白质-蛋白质-DNA相互作用和差异染色质可及性被假设为介导上下文依赖性结合。为了定量依赖于体内因素的特异性程度，以及多少是蛋白质和DNA固有的，将使用纯化的蛋白质和裸酵母基因组DNA在体外测定Raplp和LeuSp的全基因组特异性，并与体内特异性进行比较。还将确定响应于环境条件变化的Raplp分布的变化。目的二：我们以前的工作表明，在活的酵母细胞中，整个基因组的核小体占有率是异质的。我们提出的实验，以确定差异核小体占用的分子基础，以及它是如何建立，调节和维持在酵母中。目标三：我们的数据和来自其他组的数据表明，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）和转录活性的目标选择之间的密切关系。第三个目标具体测试这三个过程之间的关系。我们将在菌株中测定Raplp靶选择，其中（i）Raplp结合位点的背景已经改变，（ii）在特定基因座的转录已经被禁用，和（iii）RNA Pol II CTD突变。我们将绘制转录偶联染色质修饰图，并进行高通量定点诱变，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，当错误表达或突变时，是人类疾病的普遍原因。更好地预测它们的体内靶点可能会导致抑制与不适当靶点结合的疗法。FAIRE是我们开发的一种新的染色质检测方法，具有作为影响染色质或转录缺陷或由染色质或转录缺陷引起的疾病（包括癌症）的预后或诊断工具的潜力。

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.