"Calling Cards" for DNA-binding proteins: A tool for their genome-wide mapping

描述（由申请人提供）：拟议的实验旨在填补我们绘制蛋白质-DNA相互作用能力的主要空白：我们可以鉴定任何DNA结合蛋白的靶基因（通过“染色质免疫沉淀”方法），但我们不能做匡威的事情：鉴定与给定启动子结合的所有蛋白质。我们将尝试获得酵母S的这种能力。通过开发用于绘制DNA-蛋白质相互作用的体内方法来研究酿酒酵母。该方法使用Ty 5逆转录转座子，其将被工程化以提供酵母的每个DNA结合蛋白与“呼叫卡”，其沉积在其结合的基因组区域。回收这些“名片”并确定它们所包含的信息（与它们相关的蛋白质的DNA序列标识符）将揭示访问基因组特定区域的蛋白质。我们将尝试采用一种高通量DNA测序方法，该方法有望在一个实验中定位所有酵母转录因子的结合位点。我们打算应用该方法来产生一个转录因子-基因相互作用的地图酵母生长在各种条件下，这应该有助于定义所有功能元件的基因组的实验生物体，提供了一个模型，为人类生物学和疾病。基因表达的适当调节是生物体正常发育所必需的;异常的基因表达可导致癌症等疾病。基因表达的调控受与基因附近结合的DNA结合蛋白的影响，为了理解这一过程以及为什么会出错，我们需要知道哪些DNA结合蛋白与哪些基因附近结合。我们建议开发一种简便的方法，在酵母S。酿酒酵母是一种流行的实验生物，为人类生物学和疾病提供了模型，如果成功，可能能够适应其他生物体的实验，包括人类。

DESCRIPTION (provided by applicant): The proposed experiments aim to fill a major gap in our ability to map protein-DNA interactions: we can identify the target genes of any DNA-binding protein (by the "chromatin immunoprecipitation" method), but we cannot do the converse: identify all the proteins that bind to a given promoter. We will attempt to gain this capability for the yeast S. cerevisiae by developing an in vivo method for mapping DNA-protein interactions. The method employs the Ty5 retrotransposon, which will be engineered to provide each DNA-binding protein of yeast with a "calling card" that it deposits at regions of the genome where it binds. Recovery of these "calling cards" and determination of the information they contain (a DNA sequence identifier of the protein they are associated with) will reveal the proteins that visit particular regions of the genome. We will attempt to employ a high-throughput DNA sequencing method that promises to enable mapping of the sites of binding of all yeast transcription factors in one experiment. We intend to apply the method to produce a transcription factor-gene interaction map for yeast grown under a variety of conditions, which should contribute to defining all functional elements of the genome of this experimental organism that provides a model for human biology and disease. Proper regulation of gene expression is necessary for normal development of organisms; abnormal gene expression can lead to diseases such as cancer. Regulation of gene expression is effected by DNA-binding proteins that bind near genes, and to understand this process and why it goes wrong we need to know which DNA-binding proteins bind near which genes. We propose to develop a facile method for doing this in the yeast S. cerevisiae, a popular organism for experimentation that provides a model for human biology and disease, and if successful, may be able to be adapted for experimentation with other organisms, including humans.