Single-cell chromatin state transitions during epigenetic memory formation

Repressive chromatin modifications compact chromatin and mediate heritable gene silencing, but how structural changes quantitatively relate to epigenetic memory remains unclear. Using targeted recruitment of the KRAB repressor to induce H3K9me3 at a reporter gene, combined with single-molecule 3D chromatin imaging, we show that irreversible silencing is associated with large-scale chromatin compaction across tens of kilobases. In contrast, histone deacetylation produces reversible silencing without such compaction. Despite substantial single-cell heterogeneity, average compaction at the end of silencing quantitatively predicts epigenetic memory weeks after KRAB removal. Here, memory arises not through stable H3K9me3 domains but rather through a dynamic handoff in which H3K9me3 is gradually lost and replaced by DNA methylation. Stochastic simulations recapitulating these dynamics suggest that compaction enhances read-write feedback to promote this transition. Similar compaction is observed at endogenous loci during differentiation and fate commitment, suggesting that spatial organization may be predictive of epigenetic memory in other systems.