Probing the three-dimensional organization of enhancer-promoter communication

项目摘要 这项提案的目标是确定基因调控是三个方面的原因还是结果- 三维基因组结构。增强子是驱动时空基因表达的顺式调控元件 从其靶启动子表达。增强子-启动子（E-P）相互作用的破坏可导致严重的 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特征内通信，称为 拓扑关联域（TADs）。这些特征最初是由邻位连接所表征的 测序技术（即：Hi-C）。两种结构蛋白，CTCF或粘附素的缺失导致 然而，基于成像的方法显示，三维结构仍然存在。 此外，结构蛋白缺失对基因表达的影响相对温和，表明E- P传播对P2P传播具有鲁棒性。这些令人困惑的发现已经离开了基因组领域 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 上升到结构。另一种假说是，结构蛋白形成TADs，以促进E-P 内部的互动。我假设基因调控元件和结构蛋白都有助于三维 topology.我将在一个统一的系统和定义的开发环境中测试每个模型的贡献。 为了在单细胞分辨率下保留体内时空信息，我将研究3D 小鼠胚胎脑组织中Sonic hedgehog（Shh）神经元的荧光原位组织学研究 杂交（FISH）方法。我设计了小的（10个酶）DNA-FISH探针来测量 E-P元素之间的距离。我对一个E-P对的初步数据显示，增强子依赖性和 在发育中的大脑的特定区域的增强子独立的接近。而增强子依赖性 邻近支持模型的积极增强子介导的3-D结构，我假设，增强子- 独立的邻近性由结构蛋白介导。 在目标1中，我将使用顺序DNA-FISH绘制Shh位点的所有E-P相互作用。然后我会决定 增强子和结构蛋白对基因座构型的贡献， 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 两个冗余Shh增强子的通信。我将分析转录输出和空间组织 的冗余增强子，并确定这些指标是否在不存在互补增强子的情况下改变。 总的来说，这项提议的数据将为理解组合基因是如何产生的创造一个范例。 调控与3D基因组组织交叉。重要的是，保持体内发育环境 对于解释E-P错误沟通如何导致发育障碍和疾病将是非常宝贵的。

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.