Temporal constraints on enhancer usage and enhancer-promoter connectivity shape the regulation of limb gene transcription [ChIP-seq]

The repertoire of enhancers orchestrating gene expression during embryonic development shapes the forms and functions of organs. Within regulatory landscapes, enhancers control their target genes through spatially distinct or overlapping activities. However, the temporal specificity of enhancers repertoires—how their activities change over time to dynamically influence gene expression—is less well characterized. In this study, we introduce the regulatory trajectory framework to monitor how enhancer landscapes govern gene transcription in vivo across extensive developmental periods. This paradigm conceptually involves transcriptional initiation, marking the beginning of gene expression, followed by its maintenance over time, and ultimately decommissioning, leading to gene repression. To track and sort cells undergoing these distinct phases, we devised a transgenic recorder approach at the Shox2 model locus. Through this method, we discovered that cells maintaining Shox2 transcription in early and late limb development relies on distinct, temporally restricted enhancer repertoires. We demonstrate that eliminating early- or late-acting enhancers only transiently affects Shox2 expression indicating that these enhancer repertoires function independently. Additionally, we found that changes in the locus' 3D topology associate with enhancer activities and that a rapid loss of enhancer-promoter contacts occurs during decommissioning. Finally, we show that the decommissioning of the Shox2 locus can be actively driven by Hoxd13, a gene which expression is known to antagonize Shox2. Overall, our work uncovers the dependency of developmental genes on enhancers with temporally restricted activities to generate complex expression patterns over time and shed light on the dynamics of enhancer-promoter interactions. Overall design: We devised a dual-reporter system to monitor Shox2 regulatory trajectories and sort cells undergoing these distinct phases. First, we inserted in mouse embryonic stem cells (mESCs), 1kb upstream of the Shox2 transcriptional start site, a regulatory sensor cassette constituted by a minimal ß-globin promoter, a mCherry reporter gene followed by a destabilized PEST sequence, a P2A self-cleavage sequence and a CRE recombinase gene. These cells cells were then retargeted to integrate, at the Rosa26 locus, a cassette with a splice acceptor followed by a floxed 3x SV40pA STOP signal and the EYFP gene open reading frame. We studied the transcriptional identity of these cells by scRNA-seq. We the profile the identity of sorted cells by RNA-seq and their regulatory profile by CHiC and ChIP-seq

在胚胎发育过程中，调控基因表达的增强子（enhancer）库塑造了器官的形态与功能。在调控景观中，增强子通过空间特异性或重叠的活性调控其靶基因。然而，增强子库的时间特异性——即其活性随时间变化以动态影响基因表达的方式——尚未得到充分表征。
本研究引入调控轨迹框架，以监测体内广泛发育阶段中增强子景观如何调控基因转录。该范式在概念上包含转录起始（标志着基因表达的启动）、随后的表达维持阶段，以及最终的沉默阶段（最终导致基因抑制）。为了追踪并分选经历这些不同阶段的细胞，我们在Shox2模式基因座上设计了一种转基因记录器方法。
通过该方法，我们发现维持肢体发育早期和晚期Shox2转录的细胞依赖于不同的、受时间限制的增强子库。我们证实，敲除早期或晚期作用的增强子仅会短暂影响Shox2的表达，表明这些增强子库功能相互独立。此外，我们发现该基因座的三维拓扑结构变化与增强子活性相关，且在沉默阶段增强子-启动子接触会快速丧失。最后，我们证明Shox2基因座的沉默可由Hoxd13主动驱动，而Hoxd13的表达已知会拮抗Shox2的功能。
总体而言，本研究揭示了发育基因依赖具有时间限制性活性的增强子库，以随时间产生复杂的表达模式，并阐明了增强子-启动子相互作用的动态机制。
总体实验设计：我们设计了双报告系统以监测Shox2的调控轨迹，并分选经历这些不同阶段的细胞。首先，我们在小鼠胚胎干细胞（mESCs）的Shox2转录起始位点上游1kb处插入了一个调控传感器盒，该盒由最小β-珠蛋白启动子、带有不稳定PEST序列的mCherry报告基因、P2A自剪切序列以及CRE重组酶基因组成。随后，我们将这些细胞再次靶向整合，在Rosa26基因座处插入一个包含剪接受体、带loxP位点的3x SV40pA终止信号以及EYFP基因开放阅读框的盒式结构。我们通过单细胞RNA测序（scRNA-seq）分析了这些细胞的转录组身份，通过RNA测序（RNA-seq）解析了分选细胞的表达特征，并通过CHiC和染色质免疫沉淀测序（ChIP-seq）分析了其调控景观。