Temporal constraints on enhancer usage and enhancer-promoter connectivity shape the regulation of limb gene transcription [CUT&Run]. Temporal constraints on enhancer usage and enhancer-promoter connectivity shape the regulation of limb gene transcription [CUT&Run]

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 β-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组库（enhancer repertoire）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，增强子组库的时间特异性——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（regulatory trajectory framework），以监测体内广泛发育周期中增强子景观如何调控基因转录。该范式在概念上涵盖转录起始（标志着基因表达的启动）、随后的表达维持，以及最终的增强子失活过程，该过程最终导致基因阻遏。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（Shox2 model locus）上设计了一种转基因记录器方法。通过该方法，我们发现，在肢体发育早期和晚期维持Shox2转录的细胞，依赖于不同的、具有时间限制性的增强子组库。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，表明这些增强子组库发挥着独立的功能。此外，我们发现该基因座的三维拓扑结构（3D topology）变化与增强子活性相关，且在失活过程中增强子-启动子接触（enhancer-promoter contacts）会快速丧失。最后，我们证明Shox2基因座的失活可由Hoxd13主动驱动——该基因的表达已知会拮抗Shox2的功能。总体而言，我们的研究揭示了发育基因依赖具有时间限制性活性的增强子组库，以随时间产生复杂的表达模式，并为增强子-启动子相互作用的动态机制提供了新见解。总体实验设计：我们构建了双报告系统（dual-reporter system）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）中，于Shox2转录起始位点（transcriptional start site）上游1kb处插入了一个调控传感器盒（regulatory sensor cassette）：该盒包含最小β-珠蛋白启动子（minimal β-globin promoter）、带有不稳定PEST序列（destabilized PEST sequence）的mCherry报告基因（mCherry reporter gene）、P2A自剪切序列（P2A self-cleavage sequence）以及CRE重组酶基因（CRE recombinase gene）。随后，我们将这些细胞重新靶向整合至Rosa26基因座（Rosa26 locus），整合的盒包含剪接受体（splice acceptor）、带flox位点的3x SV40pA终止信号（floxed 3x SV40pA STOP signal）以及EYFP基因开放阅读框（EYFP gene open reading frame）。我们通过单细胞RNA测序（scRNA-seq）分析了这些细胞的转录身份，并通过RNA测序（RNA-seq）表征了分选细胞的身份，同时通过CHiC与染色质免疫沉淀测序（ChIP-seq）分析了其调控特征。