Methyl-donor inhibitors relax 3D genome structures to promote totipotent reprogramming.

The 3D genome safeguards cell identity while shaping developmental plasticity, yet its coordination with metabolism during cell fate transitions remains unclear. Here, we use a mouse totipotent stem cell induction system to dissect the role of methyl-donor metabolism in 3D genome remodeling and cell fate transitions. We show that inhibition of methyl-donor pathways enhances reprogramming toward a totipotent-like state and drives widespread relaxation of 3D genome architecture, characterized by reduced chromatin compaction and attenuated higher-order chromatin organization. Mechanistically, methyl-donor inhibitors induce pronounced DNA hypomethylation and modest reductions in histone methylation, accompanied by decreased chromatin binding of DNA methylation-associated chromatin regulators, with limited effects at CTCF-enriched regions, suggesting that methyl-donor inhibition enhances reprogramming by disrupting pre-existing 3D genome organization through largely CTCF-independent mechanisms. Together, our findings reveal metabolic-3D genome cooperation as a mechanism for lowering the barrier to totipotency and point to regenerative targets beyond canonical chromatin architectural proteins.