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。特别是，我们缺乏对大基因组中如何实现特异性的理解，在大基因组中，细胞必须区分“真正的”顺式调控元件和数千个相似但无功能的序列。该提案通过旨在揭示控制顺式调控元件特异性的机制的实验来解决这个问题。将构建简化的合成启动子的大型文库，并测定其表达、TF占用率和染色质状态。来自这些文库的数据将用描述TFBS、TF、RNA聚合酶和决定顺式调节活性的其他元件之间的物理相互作用的热力学模型进行分析。我们从合成启动子产生的模型将在基因组启动子上进行明确测试。该提案的三个目标是围绕确定具体性的三个不同信息来源组织的。在目标1，我们测试的假设，功能TFBS指定的相互作用与其他附近的TFBS。在Aim2中，我们探讨了除了TFBS之外的局部序列特征在多大程度上有助于指定功能性顺式调控元件。最后，在Aim3中，我们将测试顺式调节元件的染色体位置和染色质状态影响TFBS之间的相互作用的程度，TFBS支配它们的活性。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。这样的理解是必要的授权生物医学应用，如干细胞工程，是基于操纵基因表达模式。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。

 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 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. In particular we lack an understanding of how specificity is achieved in large genomes where the cell must distinguish between "true" cis-regulatory elements and thousands of similar, but non-functional sequences. The proposal addresses this problem through experiments designed to unravel the mechanisms that govern cis-regulatory element specificity. Large libraries of simplified synthetic promoters will be constructed and assayed for expression, TF occupancy, and chromatin state. The data from these libraries will be analyzed with a thermodynamic model that describes the physical interactions between TFBS, TFs, RNA polymerase, and other elements that determine cis- regulatory activity. The models we produce from synthetic promoters will be explicitly tested on genomic promoters. The three aims of the proposal are organized around three different sources of information that determine specificity. In Aim1 we test the hypothesis that functional TFBS are specified by their interactions with other nearby TFBS. In Aim2 we explore the extent to which local sequence features besides TFBS help specify functional cis-regulatory elements. Finally, in Aim3 we will test the extent to which the chromosomal position and chromatin state of cis-regulatory elements influence the interactions between TFBS that govern their activity. 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.