Synergistic insulation of regulatory domains by developmental genes and clusters of CTCF sites

The specificity of gene expression during development requires the insulation of regulatory domains to avoid inappropriate enhancer-gene interactions. In vertebrates, this insulator function is mostly attributed to clusters of CTCF sites located at topologically associating domain (TAD) boundaries. However, TAD boundaries allow certain level of physical crosstalk across regulatory domains, which is at odds with the highly specific and precise expression of developmental genes. Here we show that developmental genes and nearby clusters of CTCF sites synergistically foster the robust insulation of regulatory domains. Firstly, we found that the TADs containing developmental genes have distinctive features, including the sequential organization of developmental genes and CTCF clusters near TAD boundaries. Most importantly, by genetically dissecting representative loci in mouse embryonic stem cells, we showed that developmental genes and CTCF sites synergistically strengthened the insulation capacity of nearby boundaries through different mechanisms. Namely, while CTCF sites prevent undesirable enhancer-gene contacts (i.e. physical insulation), developmental genes preferentially contribute to regulatory insulation through non-structural mechanisms involving promoter competition rather than enhancer blocking. Overall, our work provides important insights into the specificity of gene regulation, which in turn might help interpreting the pathological consequences of certain structural variants. Capture-C experiments were performed to investigate enhancer-gene contacts within specific loci (i.e. Gbx2/Asb18 or Six3/Six2) in WT mouse embryonic stem cells (mESC) as well as in transgenic mESC lines containing different genomic re-arrangements (i.e. deletions or inversions). Where indicated the Capture-C experiments were performed upon differentiation of the mESC lines into anterior neural progenitors (NPC).

发育进程中基因表达的特异性，要求调控结构域实现绝缘效应，以规避异常的增强子-基因互作。在脊椎动物中，这类绝缘功能主要归因于位于拓扑关联结构域（topologically associating domain, TAD）边界处的CTCF结合位点簇。然而，TAD边界会允许调控结构域间存在一定程度的物理串扰，这与发育基因高度特异性和精准的表达模式相悖。本研究表明，发育基因与其邻近的CTCF结合位点簇可协同增强调控结构域的高效绝缘能力。首先，本研究发现携带发育基因的TAD具有独特特征，包括发育基因与CTCF位点簇在TAD边界附近的顺次排布。最为关键的是，通过对小鼠胚胎干细胞中的代表性基因座进行遗传解析，本研究证实发育基因与CTCF结合位点可通过不同机制协同强化邻近边界的绝缘能力。具体而言，CTCF结合位点可阻断异常的增强子-基因互作（即物理绝缘），而发育基因则主要通过启动子竞争而非增强子阻断这类非结构性机制实现调控绝缘。总体而言，本研究为基因调控的特异性提供了重要见解，或可助力解析特定结构变异的病理后果。本研究通过捕获测序（Capture-C）实验，探究了野生型小鼠胚胎干细胞（mouse embryonic stem cells, mESC）以及携带不同基因组重排（即缺失或倒位）的转基因mESC系中，特定基因座（如Gbx2/Asb18或Six3/Six2）内的增强子-基因互作情况。如需注明，本研究还在将mESC系分化为前脑神经前体细胞（anterior neural progenitors, NPC）后开展了Capture-C实验。