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Single-molecule tracking in living cells reveals dynamics of DNMT1 through the cell cycle and its redistribution upon drug treatment

ABSTRACT: DNMT1 is a methyltransferase that restores 5-methylcytidine marks on newly replicated DNA and is required for maintaining epigenetic inheritance. Using Halo-tagged DNMT1 and highly inclined thin illumination (HiLo) microscopy, we show that DNMT1 mobility in living human cells changes under a variety of conditions. Consistent with its role in methylating DNA, DNMT1 molecules become increasingly bound to chromatin in the S-phase of the cell cycle, but surprisingly a 4% increase in chromatin bound DNMT1 is sufficient to maintain DNA methylation. In G2 phase, however, DNMT1 is released from DNA and becomes even more dynamic. Upon treatment with small molecule inhibitors, GSK-3484862 (GSK) and 5-azacytidine (5-azaC), the in vivo DNMT1 dynamics are greatly altered. Unexpectedly, treatment of cells with GSK, a non-covalent inhibitor, causes binding of DNMT1 to chromatin similar to that observed upon treatment with 5-azaC, a covalent inhibitor. 5-azaC inhibition of DNMT1 dynamics occurs during the S phase of the cell cycle. Collectively, our work using live cell single molecule imaging quantifies the molecular dynamics of DNMT1 and how this relates to its function under physiological (different phases of the cell cycle) and non-physiological (drug inhibition) conditions. Understanding the dynamics of DNMT1 in vivo provides a framework for developing better therapeutics that target DNMT1.

ORGANISM(S): Homo sapiens

PROVIDER: GSE299486 | GEO | 2026/02/11

REPOSITORIES: GEO

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