Light‐Activation of DNA‐Methyltransferases

5‐Methylcytosine (5mC), the central epigenetic mark of mammalian DNA, plays fundamental roles in chromatin regulation. 5mC is written onto genomes by DNA methyltransferases (DNMT), and perturbation of this process is an early event in carcinogenesis. However, studying 5mC functions is limited by the inability to control individual DNMTs with spatiotemporal resolution in vivo. We report light‐control of DNMT catalysis by genetically encoding a photocaged cysteine as a catalytic residue. This enables translation of inactive DNMTs, their rapid activation by light‐decaging, and subsequent monitoring of de novo DNA methylation. We provide insights into how cancer‐related DNMT mutations alter de novo methylation in vivo, and demonstrate local and tuneable cytosine methylation by light‐controlled DNMTs fused to a programmable transcription activator‐like effector domain targeting pericentromeric satellite‐3 DNA. We further study early events of transcriptome alterations upon DNMT‐catalyzed cytosine methylation. Our study sets a basis to dissect the order and kinetics of diverse chromatin‐associated events triggered by normal and aberrant DNA methylation.