Non-additive control of gene expression by long-range interactions between multiple regulatory elements

通过多个调控元件之间的长程相互作用对基因表达进行非加性控制

• 批准号: 9976553
• 负责人: Manu Manu
• 金额: $ 20.49万
• 依托单位: UNIVERSITY OF NORTH DAKOTA
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9976553
• 关键词: 3-Dimensional; ATAC-seq; Adult; Architecture; Behavior; Biological Models; CRISPR/Cas technology; Carbon; Cell Line; Cells; Centers of Research Excellence; ChIP-seq; Chromatin; Chromatin Loop; Chromatin Structure; Complement; Complex; DNA Sequence; Data; Development; Developmental Gene; Disease; Distal; Enhancers; Epigenetic Process; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genomics; Homeostasis; Individual; Knowledge; Location; Malignant Neoplasms; Measurement; Measures; Mechanics; Mediating; Mediator of activation protein; Modeling; Molecular; Molecular Conformation; Mutate; Mutation; Myeloid Cells; Nucleosomes; Pathology; Positioning Attribute; Regulation; Regulatory Element; Scheme; Signal Transduction; Site; Testing; Thermodynamics; Time; Transgenic Organisms; Variant; chromosome conformation capture; cohesin; design; epigenomics; experimental study; functional genomics; mathematical model; mutant; neutrophil; novel; predictive modeling; promoter; response; success; synergism; synthetic biology; synthetic construct; transcription factor; tumorigenesis

Summary This project has two broad objectives. The first is to discover the rules by which genes are co-regulated by mul- tiple enhancers in complex mammalian loci. Multiple enhancers produce non-additive responses by interfering with each other's function. The project seeks to uncover the epigenetic and chromatin-structure basis of enhancer interference. The second objective is to develop a new class of mathematical models capable of simulating the regulation of multi-enhancer loci by simulating enhancer interference resulting from enhancer-promoter looping. The studies will utilize Cebpa, which encodes a transcription factor necessary for neutrophil development, as a model system. The approach combines aspects of synthetic biology, functional genomics, and mathematical modeling. In Aim 1, several transgenic myeloid cell lines, each carrying a variant of a synthetic locus in a heterol- ogous genomic location, will be constructed. The synthetic loci will be composed of the Cebpa promoter and two enhancers, whose strength will be varied by altering their sequence composition with the aid of a predictive math- ematical model of gene regulation. The response of the co-regulated gene to varying combinations of enhancer inputs will be measured to ascertain the rules of co-regulation. The hypothesis that enhancers interfere with each other via chromatin looping will be tested using chromosome conformation capture and the measurement of Mediator/Cohesin occupancy at the regulatory elements. An alternative hypothesis, that enhancers interfere by directly modifying the epigenetic state of other enhancers, will be tested by profiling chromatin accessibility and nucleosome positioning. These experiments will allow a causal analysis of enhancer interference by measuring alterations in the epigenetic state of one enhancer as a result of mutations introduced into another. Aim 2 is to build a mathematical model that can predict the expression of a two-enhancer locus. The proposed model will explicitly incorporate chromatin looping using a statistical mechanical framework and the chromatin conformation data acquired in Aim 1. The synthetic biology approach of Aim 1 will be complemented in Aim 3 by investigating enhancer interference in the endogenous Cebpa locus. Whether Cebpa enhancers modify the global chromatin conformation of the locus will be determined by editing an enhancer using CRISPR/Cas9 and profiling 3D chro- matin architecture and epigenetic state. Success in these objectives will help advance our understanding of mis-regulation of genes during oncogenesis and, in the long-term, allow us to computationally predict aberrant gene expression patterns driven by mutated DNA sequence.

总结 该项目有两大目标。首先是发现基因与穆尔共同调节的规则， 在复杂的哺乳动物基因座中的三个增强子。多个增强子通过干扰产生非加性反应， 与彼此的功能。该项目旨在揭示增强子的表观遗传和染色质结构基础 干扰第二个目标是开发一种新的数学模型，能够模拟 通过模拟由增强子-启动子成环引起的增强子干扰来调节多增强子基因座。 这些研究将利用编码中性粒细胞发育所必需的转录因子的Cebpa， 一个模型系统该方法结合了合成生物学，功能基因组学和数学 建模在目的1中，几种转基因骨髓细胞系，每种都携带异源基因座中合成基因座的变体， 基因组定位，将被构建。合成基因座将由Cebpa启动子和两个 增强子，其强度将通过借助于预测数学改变其序列组成而变化- 基因调控的数学模型共调节基因对不同增强子组合的反应 将对投入进行衡量，以确定共同监管的规则。增强子干扰 将使用染色体构象捕获和测量来测试通过染色质成环的相互作用。 调节元件处的介体/粘附素占据。另一种假设是，增强子干扰 直接修饰其他增强子的表观遗传状态，将通过分析染色质可及性进行测试， 核小体定位这些实验将允许通过测量增强子干扰的因果分析。 一个增强子的表观遗传状态由于引入另一个增强子的突变而发生改变。目标二是 建立一个数学模型，可以预测两个增强子位点的表达。拟议的模式将 使用统计力学框架和染色质构象明确地并入染色质成环 目标1中获得的数据。目标1的合成生物学方法将在目标3中得到补充， 增强子干扰内源性Cebpa基因座。Cebpa增强子是否修饰全局染色质 该基因座的构象将通过使用CRISPR/Cas9编辑增强子并分析3D chro- matin架构和表观遗传状态。这些目标的成功将有助于我们进一步了解 在肿瘤发生过程中基因的错误调节，从长远来看，使我们能够通过计算预测异常的 由突变的DNA序列驱动的基因表达模式。