Promoter repression and 3D-restructuring resolves divergent developmental gene expression in TADs [WGBS]

Cohesin loop extrusion facilitates precise gene expression by continuously driving promoters to sample all enhancers located within the same topologically-associated domain (TAD). However, many TADs contain multiple genes with divergent expression patterns, thereby indicating additional forces further refine how enhancer activities are utilised. Here, we unravel the mechanisms enabling a new gene, Rex1, to emerge with divergent expression within the ancient Fat1 TAD in placental mammals. We show that such divergent expression is not determined by a strict enhancer-promoter compatibility code, intra-TAD position or nuclear envelope-attachment. Instead, TAD-restructuring in embryonic stem cells (ESCs) separates Rex1 and Fat1 with distinct proximal enhancers that independently drive their expression. By contrast, in later embryonic tissues, DNA methylation renders the inactive Rex1 promoter profoundly unresponsive to Fat1 enhancers within the intact TAD. Combined, these features adapted an ancient regulatory landscape during evolution to support two entirely independent Rex1 and Fat1 expression programs. Thus, rather than operating only as rigid blocks of co-regulated genes, TAD-regulatory landscapes can orchestrate complex divergent expression patterns in evolution. We performed whole genome bisuphite sequencing on wildtype mouse ESCs and E11.5 limbs and mutant E11.5 limbs with a complete loss of function of DNMT3B.

黏连蛋白环挤出（Cohesin loop extrusion）通过持续驱动启动子采样位于同一拓扑关联结构域（topologically-associated domain, TAD）内的所有增强子，助力实现精准的基因表达调控。然而，诸多TAD包含多个表达模式迥异的基因，这表明存在额外作用力进一步优化增强子活性的利用方式。本研究解析了胎生哺乳动物古老Fat1 TAD内新基因Rex1何以呈现差异化表达的调控机制。我们发现，此类差异化表达并非由严格的增强子-启动子兼容性编码、TAD内位置或核被膜附着所决定。取而代之的是，胚胎干细胞（embryonic stem cells, ESCs）中的TAD结构重塑，将Rex1与Fat1通过各自独立的近端增强子分隔开，分别调控二者的表达。相比之下，在晚期胚胎组织中，DNA甲基化使失活的Rex1启动子对完整TAD内的Fat1增强子产生显著的不应答性。综合来看，这些特征在进化过程中重塑了古老的调控景观，得以支撑两套完全独立的Rex1与Fat1表达程序。由此可见，TAD调控景观并非仅作为共调控基因的刚性模块发挥作用，还可在进化过程中调控复杂的差异化表达模式。我们对野生型小鼠ESCs、E11.5胚胎肢体，以及完全丧失DNMT3B功能的突变型E11.5胚胎肢体开展了全基因组亚硫酸氢盐测序（whole genome bisulfite sequencing）。