A complex regulatory landscape involved in the development of external genitals [ChIP-Seq]. A complex regulatory landscape involved in the development of external genitals [ChIP-Seq]

In vertebrates, developmental genes are often controlled by large regulatory landscapes matching the dimensions of topologically associating domains (TADs). In various ontogenic contexts, the associated constitutive chromatin backbone is modified by fine-tuned specific variations in enhancer-enhancer and enhancer-promoter interaction profiles. In this work, we use a TAD flanking the HoxD gene cluster as a paradigm to address the question of how these complex regulatory architectures are formed and how they are de-constructed once their function has been achieved. We suggest that this TAD can be considered as a coherent functional unit in itself, with several regulatory sequences acting together to elicit a transcriptional response. With one notable exception, the deletion of each of these sequences in isolation did not produce any substantial modification in the global transcriptional outcome of the system, a result at odds with a conventional view of long-range enhancer function. Likewise, both the deletion and inversion of a CTCF site located in a region rich in such sequences did not affect transcription of the target gene. In the latter case, however, slight modifications were observed in interaction profiles in vivo in agreement with the loop extrusion model, despite no apparent functional consequences. We discuss these unexpected results by considering both conventional explanations and an alternative possibility whereby a rather unspecific accumulation of particular factors within the TAD backbone may have a global impact upon transcription. Overall design: ChIP-seq analysis of H3K27ac and H3K27me3 in the genital tubercle (GT) or the cloaca region (CR) of wildtype mouse embryos. CTCF ChIP-seq analysis of wildtype or del(V) mouse GT.

在脊椎动物中，发育基因通常受与拓扑关联结构域（topologically associating domains, TADs）尺度匹配的大型调控景观调控。在多种发育进程中，相关的组成型染色质骨架会因增强子-增强子与增强子-启动子互作图谱的精准特异性变化而发生修饰。本研究以HoxD基因簇侧翼的一个拓扑关联结构域为研究范式，旨在解析这类复杂调控结构的形成机制，以及其完成功能后是如何被解构的。我们认为该拓扑关联结构域本身可作为一个连贯的功能单元，多个调控序列协同作用以触发转录响应。除一个显著例外外，单独缺失这些序列中的任意一个均未使该系统的整体转录结局发生显著改变，这一结果与远程增强子功能的传统认知相悖。同样地，对位于此类序列富集区域的一个CTCF结合位点进行缺失或倒位操作，均未影响靶基因的转录。然而在后一种（倒位）情形中，尽管未观察到明显的功能后果，但体内互作图谱出现了轻微改变，这与环挤出模型（loop extrusion model）的理论预测一致。我们针对这些意外结果展开讨论，既考量了传统解释路径，也提出了一种替代可能性：TAD骨架内特定因子的非特异性积累可能会对转录产生全局性影响。实验整体设计：对野生型小鼠胚胎的生殖结节（genital tubercle, GT）或泄殖腔区域（cloaca region, CR）开展H3K27ac与H3K27me3的染色质免疫共沉淀测序（ChIP-seq）分析；对野生型或del(V)小鼠的生殖结节开展CTCF染色质免疫共沉淀测序分析。