Complementary constraints in germ and immune cells shape evolution of gene regulation and phenotype

New mutations arise in germ cells and must preserve fertility while altering somatic phenotype to confer a fitness advantage. To understand how whole organisms balance these constraints, we generated mice with humanized noncoding promoter sequence at Traf6, an NF-κB pathway component with recently diverged chromatin state in mouse germ cells. The humanized allele (Traf6^h) reinstated human-like expression in germ and immune cells, which we traced to a 13 base pair deletion that impairs CTCF binding. Traf6^h had no effect on fertility but induced a sensitized innate immune response to endotoxin. Other NF-κB pathway genes followed a similar pattern, suggesting that their expression has evolved in tandem and consistent with known mouse-human differences in inflammatory response. Our results reveal distinct roles for germ and immune cells in shaping gene regulatory evolution, where immune cells quickly adjust phenotype, enabling selection, while germ cells robustly maintain fertility despite changing expression levels, enabling inheritance.