Probing the three-dimensional organization of enhancer-promoter communication

项目总结 这项提案的目标是确定基因调控是三个因素的原因还是结果- 三维(3D)基因组组织。增强子是驱动时空基因的顺式调控元件 从它们的目标启动子中表达。增强子-启动子(E-P)相互作用的中断可导致严重的 发育障碍和先天畸形。 增强子通常与它们的同源启动子在基因组折叠的3-D特征内进行通信，称为 拓扑关联域(TADS)。这些特征最初以邻近结扎为特征。 测序技术(即：HI-C)。两种结构蛋白，CTCF或粘附素的耗尽，导致 在Hi-C对TADS的溶解中；然而，基于成像的方法显示三维结构仍然存在。 此外，建筑蛋白缺失对基因表达的影响相对较小，表明E- P传播对于TAD传播是稳健的。这些令人困惑的发现离开了基因组领域 组织对TADS的形成和功能存在分歧。一种假设认为，E-P相互作用给 上升到TAD结构。另一种假设是，建筑蛋白形成TADS是为了促进E-P 内部的相互作用。我假设基因调控元件和结构蛋白都对3D有贡献 拓扑学。我将在统一的系统和定义的开发环境中测试每个模型的贡献。 为了在体内保留单细胞分辨率的时空信息，我将研究3D 小鼠胚胎脑组织中Sonic Hedgehog(Shh)TAD的原位荧光组织 杂交(FISH)方法。我设计了小的(10千碱基)DNA-FISH探针来测量物理 E-P元素之间的距离。我对一个E-P对的初步数据显示，增强子依赖和 在发育中的大脑特定区域增强非依赖于启动子的亲近性。而依赖于增强子的 邻近性支持活性增强子在介导三维结构中的模型，我假设增强子- 独立的亲和力是由建筑蛋白介导的。 在目标1中，我将使用序列DNA-FISH绘制Shh基因座的所有E-P相互作用图。到时候我会决定 增强子和建筑蛋白对基因座构型的贡献 分别为增强子和特异性CTCF结合位点。目标2中的实验将探索复杂的E-P 两个多余的Shh增强器的通信。我将分析转录输出和空间组织 并确定在缺少互易增强器的情况下这些度量是否被改变。 综上所述，来自这项提议的数据将创建一个理解组合基因如何 调控与3-D基因组组织相交。重要的是，保护体内的发育环境 对于解释E-P沟通错误是如何导致发育障碍和疾病的，将是非常宝贵的。

PROJECT SUMMARY The goal of this proposal is to determine whether gene regulation is the cause or consequence of three- dimensional (3-D) genome organization. Enhancers are cis-regulatory elements that drive spatiotemporal gene expression from their target promoter. Disruption of enhancer-promoter (E-P) interactions can result in severe developmental disorders and congenital malformations. Enhancers typically communicate with their cognate promoter within 3-D features of genome folding called topologically associating domains (TADs). These features were originally characterized by proximity ligation sequencing techniques (ie: Hi-C). The depletion of two architectural proteins, either CTCF or cohesin, resulted in the dissolution of TADs by Hi-C; however, imaging-based approaches revealed that 3-D structures remained. Moreover, the effect of architectural protein depletion on gene expression was relatively mild, suggesting that E- P communication is robust to TAD dissemination. These perplexing findings have left the field of genome organization divided about the formation and function of TADs. One hypothesis posits that E-P interactions give rise to TAD structure. The other hypothesis is that architectural proteins form TADs in order to facilitate the E-P interactions within. I hypothesize that both gene regulatory elements and architectural proteins contribute to 3-D topology. I will test the contribution of each model in a unified system and defined developmental context. In order to retain in vivo spatiotemporal information at single-cell resolution, I will investigate the 3-D organization of the Sonic hedgehog (Shh) TAD in mouse embryonic brain tissue using a fluorescence in situ hybridization (FISH) approach. I designed small (10 kilobase) DNA-FISH probes to measure the physical distances between E-P elements. My preliminary data for one E-P pair, revealed both enhancer-dependent and enhancer-independent proximity in specific regions of the developing brain. While the enhancer-dependent proximity supports the model of active enhancers in mediating 3-D structure, I hypothesize that the enhancer- independent proximity is mediated by architectural proteins. In Aim 1, I will map all E-P interactions for the Shh locus using sequential DNA-FISH. I will then determine the contribution of enhancers and architectural proteins to the locus’ configuration by using mutants devoid of enhancers and specific CTCF binding sites, respectively. The experiments in Aim 2 will explore complex E-P communication of two redundant Shh enhancers. I will analyze the transcriptional output and spatial organization of the redundant enhancers and determine if these metrics are altered in the absence of the reciprocal enhancer. Taken together, the data from this proposal will create a paradigm for understanding how combinatorial gene regulation intersects with 3-D genome organization. Importantly, preserving the in vivo developmental context will be invaluable for translating how E-P miscommunication results in developmental disorders and disease.