An in vitro Genome-wide Study of Chromatin Structure and Transcription Regulation

描述（由申请人提供）：染色质结构对真核生物基因表达的调控至关重要。染色质的基本单位核小体通过限制转录因子对启动子内特定DNA序列的可及性来调节转录。为了更好地了解真核生物基因是如何被调控的，需要在基因组DNA与正确组织的核小体包装的背景下进行重组转录。我们的实验室最近使用了最新的DNA测序技术，表明核小体的体内定位模式可以在体外通过染色质重塑器在酿酒葡萄球菌基因组中重现。在这里，我们建议开发一种全基因组固定化模板测定，这将使我们能够在适当的染色质结构的背景下，在全基因组范围内提出有关核小体动力学、转录因子占用和RNA转录的基本问题。与此分析方面的基因表达调控将研究转录因子结合和组蛋白修饰的转录要求。这些研究旨在阐明基因调控中的两个基本问题：转录因子能否内在地进入它们在天然染色质中的结合位点，或者是否需要重塑者的合作？活性组蛋白修饰的生理模式是在转录发生之前还是之后？该试验的发展将导致一种技术，在该技术中，该体外系统将被编程为包含特定的异常染色质状态，如在疾病状态中所见，以确定这些染色质结构的变化如何导致癌症的发展，以及心血管、神经退行性和自身免疫性疾病。

DESCRIPTION (provided by applicant): Chromatin structure is critical to the regulation of eukaryotic gene expression. The basic unit of chromatin, the nucleosome, regulates transcription by limiting the accessibility of transcriptional factors to specific DNA sequences within promoters. A better biochemical understanding of how eukaryotic genes are regulated will require reconstituted transcription in the context of genomic DNA packaged with properly organized nucleosomes. Our lab recently used the latest in DNA sequencing technology to show that the in vivo pattern of nucleosome positioning can be recapitulated in vitro across the S. cerevisiae genome by chromatin remodelers. Here we propose to develop a genome- wide immobilized template assay that will allow us to ask fundamental questions regarding nucleosome dynamics, transcription factor occupancy, and RNA transcription on a genome-wide scale in the context of proper chromatin structure. With this assay aspects of gene expression regulation will be investigated in regards to transcription factor binding and the transcriptional requirements of histone modifications. These studies are intended to shed light on two fundamental questions in gene regulation: Can transcription factors intrinsically access their binding sites in native chromatin, or is the cooperation of remodelers needed? Does the physiological pattern of active histone modifications arise before or after transcription occurs? The development of this assay will lead to a technique in which this in vitro system will be programmed to contain specific aberrant chromatin states as are seen in disease states in order to determine how these changes in chromatin structure lead to the development of cancer, along with cardiovascular, neurodegenerative, and autoimmune diseases. PUBLIC HEALTH RELEVANCE: Chromatin structure is critical to the regulation of eukaryotic gene expression, and alterations of chromatin structure are known to cause aberrant regulation of gene expression that can lead to cancer. Here we are proposing to develop a biochemical assay in yeast that will later be used to study the aberrant regulation of gene expression caused by specific alterations of chromatin structure to understand how it leads to the development of cancer.