Abstract
Regulatory landscapes drive complex developmentalgene expression,but it remains unclear howtheir integrityis maintained when incorporating novel genes and functions during evolution. Here, we investigated how aplacental mammal-specific gene, Zfp42, emerged in an ancient vertebrate topologically associated domain(TAD) without adopting or disrupting the conserved expression of its gene, Fat1. In ESCs, physical TAD partitioningseparatesZfp42 andFat1 withdistinct local enhancers thatdrivetheir independentexpression.This separationis driven by chromatin activity and not CTCF/cohesin. In contrast, in embryonic limbs, inactive Zfp42shares Fat1’s intact TADwithout responding to active Fat1 enhancers. However, neither Fat1 enhancer-incompatibilitynor nuclear envelope-attachment account for Zfp42’s unresponsiveness. Rather, Zfp42’s promoter isrendered inert to enhancers by context-dependent DNA methylation. Thus, diverse mechanisms enabled theintegration of independent Zfp42 regulation in the Fat1 locus. Critically, such regulatory complexity appearscommon in evolution as, genome wide, most TADs contain multiple independently expressed genes.