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

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. HIGHLIGHTS ### Competing Interest Statement The authors have declared no competing interest.