(610c) A Novel Platform of Monitoring DNA Hydroxymethylation (5-hmC) Change in Live Cells

DNA methylation is one of the most important epigenetic modifications that play an essential role in development, aging and disease onset. Methylation of cytosine (5mC) is the most abundant form of DNA methylation found in humans and was assumed to be fairly persistent during the life course. Recent studies have shown that the ten-eleven translocation (TET) proteins can catalyze the oxidation of 5mC to form 5-hydroxymethylcytosine (5-hmC) followed by a DNA demethylation process suggesting dynamic nature of DNA methylation. Moreover, 5-hmC is more abundant in central nervous system and has been implicated in many crucial brain activities, including long-term potentiation, synaptic transmission and etc. This exhilarating discovery has motivated engineering efforts to track dynamic changes in 5-hmC in live cells and potentially animals. We have designed and optimized a recombinant protein sensor for probing DNA hydroxymethylation in situ. The probe is based on the native 5hmC “reader” domains from SRA and utilized the Bimolecular Fluorescence Complementation (BiFC) technique to improve signal to noise ratio. The probe is shown to have equivalent sensitivity to commercial antibodies but is live-cell compatible. We adopted the probe to track changes in 5hmC during neuron differentiation and recorded 5hmC dynamics at a single cell resolution. 5hmC changes when neurons are exposed to PD-risk factors are also recorded to reveal the potential contributions of 5hmC to the disease development.