Trans-regulation of heterochromatin underlies genetic variation in 3D genome contacts in mouse embryonic stem cells

Genetic variation drives phenotypic diversity and disease susceptibility. Trans -acting genetic variation coordinates genome-wide chromatin changes, yet the molecular mechanisms underlying this distal regulation remain unclear. Here, we use the power of mouse genetics to investigate how genetic variation at trans -acting loci regulates three-dimensional (3D) chromatin interactions. Using HiChIP to map chromatin contacts among regulatory elements in C57BL/6J and DBA/2J embryonic stem cells (ESCs), we identify 4,962 strain-differential interactions. Of these, 71% overlap chromatin accessibility quantitative trait loci (QTL), establishing that interaction variation is predominantly heritable. These differential interactions show coordinated changes in chromatin state and gene expression, with stronger interactions associated with increased accessibility and transcription. Notably, loci regulated in trans exhibit a unique chromatin signature where weaker interactions are enriched for H3K9me3-marked heterochromatin. Analysis of F1 hybrids reveal dominant repressive effects, consistent with heterochromatin-mediated trans -regulation. To causally test this mechanism, we generate reciprocal congenic mouse strains swapping a trans -QTL region. Integrated multiomic profiling of congenic ESC lines demonstrates that this locus coordinates H3K9me3, H3K27ac, chromatin accessibility, and 3D contacts at hundreds of regulatory elements, with 73–94% validating directional predictions from QTL mapping. The functional impact of heterochromatin deposition is confirmed by corresponding changes in trans -regulated gene expression. This work establishes heterochromatin formation as a defining feature of trans -regulation in mouse embryonic stem cells, providing a framework for understanding how genetic variation in early developmental chromatin states could generate phenotypic diversity while preserving essential developmental programs.