ANALYSIS OF COMBINATORIAL CIS-REGULATION IN SYNTHETIC AND GENOMIC PROMOTERS

描述(由申请人提供)：该项目的长期目标是了解控制转录因子结合位点(TFBS)之间相互作用的组合规则。通过这些规则，TFBS的组合指定了复杂基因表达模式的巨大多样性。正常的生长发育依赖于TFBS在时间和空间上对基因表达水平的严格控制，而基因表达的异常调控是许多遗传性疾病的基础。尽管识别TFBS以及与其结合的转录因子(TF)已经取得了很大进展，但TFBS如何相互作用产生特定的基因表达模式还知之甚少。这种知识的缺乏表现在无法预测TFBS的新组合指定的表达模式，以及无法区分基因组中真实的调节区和虚假的TFBS簇。该提案通过旨在揭示管理TFBS相互作用的机制的实验来解决这些问题。将构建简化的合成启动子的大型文库，并对其进行表达和Tf占有率的检测。来自这些文库的数据将用热力学模型进行分析，该模型描述了TFBS、TFS和RNA聚合酶之间的物理相互作用。由合成启动子产生的模型将在基因组启动子上进行明确的测试。在目标1中，这一系统将被用来研究TFS对TFBS的简单占据在多大程度上决定了基因表达的复杂模式。在目标2中，该系统将被扩展以确定其他变量对组合顺式调节的影响，包括TATA框的强度、染色体位置和染色质修饰。这项提案的目标的成功完成将导致对组合顺式调控基础规则的定量和分子理解。这样的理解对于增强生物医学应用是必要的，例如基于操纵基因表达模式的干细胞工程。这一提议产生的结果也将有助于指导对基因组中指定基因表达模式的大片非编码DNA区域的注释。最后，清楚地了解TFBS的相互作用将有助于识别和解释影响顺式调控的引起疾病的基因变异。

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the combinatorial rules that govern the interactions between transcription factor binding sites (TFBS). Through these rules, combinations of TFBS specify an enormous diversity of complex gene expression patterns. Normal growth and development depends on the tight control of TFBS over levels of gene expression in both time and space, and aberrant regulation of gene expression underlies many genetic diseases. Although much progress has been made identifying TFBS, and the transcription factors (TFs) that bind to them, much less is known about how TFBS interact with each other to generate specific patterns of gene expression. This lack of knowledge is manifested in the inability to predict the expression patterns specified by novel combinations of TFBS, and the inability to distinguish true regulatory regions in the genome from spurious clusters of TFBS. The proposal addresses these problems through experiments designed to unravel the mechanisms that govern TFBS interactions. Large libraries of simplified synthetic promoters will be constructed and assayed for expression and TF occupancy. The data from these libraries will be analyzed with a thermodynamic model that describes the physical interactions between TFBS, TFs and RNA polymerase. The models produced from synthetic promoters will be explicitly tested on genomic promoters. In Aim 1 this system will be used to study the extent to which the simple occupancy of TFBS by TFs determines complex patterns of gene expression. In Aim 2 the system will be extended to determine the effects of additional variables on combinatorial cis-regulation, including the strength of the TATA box, chromosomal location, and chromatin modifications. The successful completion of the aims of this proposal will result in a quantitative and molecular understanding of the rules underlying combinatorial cis-regulation. Such an understanding is necessary to empower biomedical applications, such as stem cell engineering, that are based on manipulating gene expression patterns. The results produced from this proposal will also help guide the annotation of the large regions of non-coding DNA in the genome that specify gene expression patterns. Finally, a clear understanding of TFBS interactions will help the identification and interpretation of disease causing genetic variants that affect cis-regulation.