A complex regulatory landscape involved in the development of external genitals [RNA-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. RNA-seq analysis of genital tubercle tissue from E16.5 and E18.5 mouse embryos

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）规模相当的大型调控景观所调控。在多种个体发育语境中，相关的组成型染色质骨架会通过增强子-增强子与增强子-启动子互作图谱的精准特异性调控变化而发生修饰。本研究以侧翼环绕HoxD基因簇的一个TAD作为研究范式，旨在解析这些复杂调控结构的形成机制，以及当其完成功能后如何被解聚。本研究认为，该TAD本身可被视为一个连贯的功能单元，多条调控序列协同作用以触发转录应答。除一个显著例外外，单独删除其中任意一条序列均未对该系统的整体转录结果造成显著改变，这一结果与长距离增强子功能的传统认知相悖。同样地，位于此类序列富集区域的一个CCCTC结合因子（CTCF）位点的缺失与倒位，均未影响靶基因的转录。然而在后者（即倒位）的实验中，尽管未观察到明显的功能后果，但体内互作图谱出现了细微变化，且这一变化与环挤出模型（loop extrusion model）的预测相符。本研究针对这些意外结果展开讨论，既考量了传统解释，也提出了一种替代假说：即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。对E16.5与E18.5阶段小鼠胚胎的生殖结节组织进行的RNA测序（RNA-seq）分析