Probing the three-dimensional organization of enhancer-promoter communication

项目概要 该提案的目标是确定基因调控是以下三个因素的原因还是结果： 三维（3-D）基因组组织。增强子是驱动时空基因的顺式调控元件 从其目标启动子表达。增强子-启动子 (E-P) 相互作用的破坏可能导致严重的 发育障碍和先天畸形。 增强子通常在基因组折叠的 3D 特征内与其同源启动子进行通信，称为 拓扑关联域（TAD）。这些特征最初是通过邻近连接来表征的 测序技术（即：Hi-C）。两种结构蛋白（CTCF 或粘连蛋白）的耗尽导致 Hi-C 溶解 TAD；然而，基于成像的方法显示 3D 结构仍然存在。 此外，结构蛋白缺失对基因表达的影响相对轻微，表明E- P 通信对于 TAD 传播具有很强的鲁棒性。这些令人费解的发现已经离开了基因组领域 组织对 TAD 的形成和功能存在分歧。一种假设认为 E-P 相互作用给出 上升为TAD结构。另一个假设是结构蛋白形成 TAD 以促进 E-P 内的相互作用。我假设基因调控元件和结构蛋白都有助于 3-D 拓扑。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。 为了以单细胞分辨率保留体内时空信息，我将研究 3-D 使用原位荧光技术在小鼠胚胎脑组织中组织音刺猬 (Shh) TAD 杂交（FISH）方法。我设计了小型（10 kilobase）DNA-FISH 探针来测量物理 E-P 元素之间的距离。我对一对 E-P 的初步数据揭示了增强子依赖性和 发育中大脑特定区域的增强子独立接近度。虽然增强子依赖性 邻近度支持主动增强子介导 3-D 结构的模型，我假设增强子- 独立的接近度是由结构蛋白介导的。 在目标 1 中，我将使用连续 DNA-FISH 绘制 Shh 基因座的所有 E-P 相互作用图谱。然后我会确定 通过使用缺乏增强子和结构蛋白的突变体，增强子和结构蛋白对基因座配置的贡献 分别是增强子和特定的 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增强器的通信。我将分析转录输出和空间组织 冗余增强子，并确定在没有互惠增强子的情况下这些指标是否会改变。 总而言之，该提案中的数据将为理解组合基因如何 调控与 3D 基因组组织相交叉。重要的是，保留体内发育背景 对于解释 E-P 沟通不畅如何导致发育障碍和疾病具有不可估量的价值。

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.