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

摘要 这个项目有两个广泛的目标。fi的第一个任务是发现多个基因共同调控的规则。 复杂哺乳动物基因座中的滴度增强剂。多种增强剂通过干扰产生非相加反应 与彼此的功能有关。该项目试图揭示增强子的表观遗传学和染色质结构基础。 干扰。第二个目标是开发一类新的数学模型，能够模拟 通过模拟增强子-启动子环引起的增强子干扰来调节多个增强子基因座。 这项研究将利用CEBPA，它编码中性粒细胞发育所必需的转录因子，作为 一个模范系统。该方法结合了合成生物学、功能基因组学和数学 模特儿。在目标1中，几个转基因髓系细胞系，每个都携带异源基因中一个合成基因的变体- OGUS基因组位置，将被构建。合成的基因座将由CEBPA启动子和两个 增强子，其强度将通过在预测数学的帮助下改变其序列组成而变化- 基因调控的数学模型。共调控基因对不同增强剂组合的响应 将对投入进行衡量，以确定共同监管的规则。增强剂干扰的假说 通过染色质循环的彼此将使用染色体构象捕获和测量来测试 在监管要素的调解人/粘附素占有率。另一种假设是，增强剂通过 直接修改其他增强子的表观遗传状态，将通过ProfiLing染色质可及性和 核小体定位。这些实验将允许通过测量来分析增强子干扰的原因 由于一个增强子被引入另一个增强子而导致的表观遗传状态的改变。目标2是 建立一个数学模型，可以预测两个增强子基因座的表达。建议的模式将 使用统计机械框架和染色质构象显式合并染色质环路 在目标1中获得的数据。目标1的合成生物学方法将在目标3中通过调查 内源性CEBPA基因座中的增强子干扰。CEBPA增强子是否修改全局染色质 通过使用CRISPR/CAS9和PROfiLIN3D chro编辑增强子来确定该基因的构象。 配对结构和表观遗传状态。这些目标的成功将有助于增进我们对 在肿瘤发生过程中基因的错误调控，从长远来看，使我们能够通过计算预测异常 突变DNA序列驱动的基因表达模式。

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.