The 12th International Symposium on 3D Genomics

Precise control of gene expression through epigenetic modulation offers a reversible alternative to permanent genome editing, with broad relevance for biological processes requiring temporal flexibility and for therapeutic intervention. DNA methylation (DNAm) is a central component of the epigenetic landscape and operates within a coordinated network of histone modifications, chromatin accessibility, and three‑dimensional genome organization to regulate transcriptional programs, cell identity, and genomic stability. Although targeted epigenetic editing approaches have enabled site‑specific manipulation of DNAm in diverse disease contexts, the stability of engineered methylation marks, particularly following effector removal, remains poorly understood.

Here, we investigate the determinants of DNAm maintenance and recovery using temporally controlled epigenetic perturbation in mouse embryonic stem cells (mESCs). We combined the DNMT3A–DNMT3L–dCas9 system with a rapid degron‑based protein degradation strategy to establish a hit‑and‑run DNAm editing framework, allowing precise induction of methylation followed by acute effector clearance. This approach enabled direct analysis of residual DNAm dynamics during cell proliferation. To extend these findings genome‑wide, we introduced transient, sequence‑agnostic hypermethylation using an inducible MQ1–degron fusion protein, thereby probing how distinct genomic regions respond to acute methylation stress.

We identify pronounced heterogeneity in DNAm stability across the genome and define multiple classes of methylation‑dynamic regions with characteristic behaviors following methylation induction and withdrawal. These region‑specific dynamics are largely independent of active TET‑mediated demethylation and instead reflect intrinsic differences in DNAm maintenance fidelity that are strongly shaped by chromatin context, and histone modification patterns. Integration of chromatin accessibility and CUT&Tag profiling reveals coordinated epigenomic features that constrain or permit methylation persistence and turnover. Transient global hypermethylation induces reversible shifts in epigenetic age estimates, and region‑specific methylation patterns observed in mESCs parallel age‑associated methylation changes detected in mouse blood.

Together, our results demonstrate that DNAm maintenance is governed by a coordinated epigenomic network rather than uniform enzymatic activity, providing a mechanistic framework linking epigenetic editing outcomes to natural epigenetic drift. These findings establish principles for predicting DNAm stability and inform the design of safer and more durable epigenetic editing strategies.