Light-Activatable TET-Dioxygenases Reveal Dynamics of 5-Methylcytosine Oxidation and Transcriptome Reorganization.
Shubhendu PaleiBenjamin BuchmullerJan WolffgrammÁlvaro Muñoz-LopezSascha JungPaul CzodrowskiDaniel SummererPublished in: Journal of the American Chemical Society (2020)
Ten-eleven-translocation (TET) dioxygenases catalyze the oxidation of 5-methylcytosine (5mC), the central epigenetic regulator of mammalian DNA. This activity dynamically reshapes the epigenome and transcriptome by depositing oxidized 5mC derivatives and initiating active DNA demethylation. However, studying this dynamic is hampered by the inability to selectively activate individual TETs with temporal control in cells. We report activation of TETs in mammalian cells by incorporation of genetically encoded 4,5-dimethoxy-2-nitrobenzyl-l-serine as a transient active-site block, and its subsequent deprotection with light. Our approach enables precise insights into the impact of cancer-associated TET2 mutations on the kinetics of TET2 catalysis in vivo, and allows time-resolved monitoring of target gene activation and transcriptome reorganization. This sets a basis for dissecting the order and kinetics of chromatin-associated events triggered by TET catalysis, ranging from DNA demethylation to chromatin and transcription regulation.
Keyphrases
- genome wide
- dna methylation
- gene expression
- circulating tumor
- single cell
- transcription factor
- cell free
- rna seq
- single molecule
- copy number
- induced apoptosis
- dna damage
- hydrogen peroxide
- visible light
- nucleic acid
- circulating tumor cells
- cell cycle arrest
- oxidative stress
- signaling pathway
- fluorescence imaging
- cell death
- cerebral ischemia