Promoter Repression and 3D-Restructuring Resolves Divergent Developmental Gene Expression in TADs

Topologically-associated domains (TADs) facilitate precise gene expression by continuously driving promoters to sample all enhancers located within their boundaries. However, many TADs contain multiple divergently expressed genes indicating additional forces must refine how enhancer activities are utilised. Here, we examine mechanisms enabling a new gene, Rex1, to emerge with divergent expression within Fat1’s ancient TAD. We show 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 local enhancers that independently drive their expression. By contrast, in later embryos, DNA methylation renders Rex1’s inactive promoter unresponsive to Fat1 enhancers within the intact TAD. Combined, these features adapted an ancient regulatory landscape to support two independent Rex1 and Fat1 expression programs. Thus, TAD-regulatory landscapes can operate beyond rigid blocks of co-regulated genes to orchestrate complex divergent expression patterns in evolution.