ANALYSIS OF COMBINATORIAL CIS-REGULATION IN SYNTHETIC AND GENOMIC PROMOTERS

描述（由申请人提供）：本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。正常的生长和发育依赖于TFBS在时间和空间上对基因表达水平的严格控制，并且基因表达的异常调节是许多遗传疾病的基础。虽然已经取得了很大的进展，确定TFBS，和转录因子（TF）结合到他们，少得多是关于TFBS如何相互作用，以产生特定的基因表达模式。这种知识的缺乏表现在无法预测TFBS的新组合所指定的表达模式，以及无法区分基因组中的真正调控区与TFBS的假簇。该提案通过旨在揭示TFBS相互作用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，并测定其表达和TF占有率。来自这些文库的数据将用描述TFBS、TF和RNA聚合酶之间的物理相互作用的热力学模型进行分析。由合成启动子产生的模型将在基因组启动子上明确测试。在目的1中，该系统将被用来研究TFBS的简单占用TF决定基因表达的复杂模式的程度。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控的影响，包括TATA盒的强度、染色体位置和染色质修饰。成功完成本提案的目标将导致定量和分子的理解的规则组合顺式调控。这样的理解对于增强基于操纵基因表达模式的生物医学应用（例如干细胞工程）是必要的。从这个提议中产生的结果也将有助于指导基因组中指定基因表达模式的非编码DNA大区域的注释。最后，对TFBS相互作用的清楚理解将有助于识别和解释影响顺式调节的致病遗传变异。 公共卫生相关性：除了作为基因的“部分列表”，基因组还编码精确控制基因在何处、何时以及以何种水平产生（表达）的信息。严格控制基因表达对正常生长和发育至关重要，而异常基因表达是许多遗传疾病（包括癌症）的基础。本提案中的实验的成功完成将阐明基因组中的信息控制基因表达的精确模式的过程，并将帮助我们解释导致疾病的遗传变异，这些变异改变了基因表达的正常模式。

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. PUBLIC HEALTH RELEVANCE: In addition to serving as a "parts list" of genes, the genome also encodes information that controls precisely where, when, and to what levels genes are produced (expressed). Strict control of gene expression is critical for normal growth and development, and aberrant gene expression underlies many genetic diseases, including cancer. Successful completion of the experiments in this proposal will illuminate the processes through which information in the genome controls precise patterns of gene expression, and will help us interpret disease causing genetic variants that alter normal patterns of gene expression.