CGSENS: Visualization of CG methylation using a fluorescence protein biosensor
This project aims to design and validate a fluorescent protein biosensor for DNA methylation.
Cytosine DNA methylation is an epigenetic mark critical in diverse biological functions, such as gene regulation or genome stability. So far, the cellular epigenetic landscape has been pictured using in-vitro approaches that provide information about DNA methylation at specific loci or genome-wide, but lacking an in vivo spatiotemporal resolution. The aim of this proof-of-concept project is to design and validate a fluorescent protein biosensor for DNA methylation in CG context. The validation of this fluorescent biosensor will facilitate its use as a tool to monitor fluctuations in DNA methylation in living organisms.
We propose the design of a new generation of biosensor (CGSENS) with higher SNR based on bimolecular fluorescence complementation (BiFC) to visualize protein-DNA interactions to study the global CG DNA methylation dynamics. CGSENS will consist of a methyl-CpG binding domain (MBD) linked to the amino (CGSENS-N) or carboxyl (CGSENS-C) end of a split VENUS. When two methylated cytosines in a CG context are in proximity, they will be recognized by the MBD domains that will bring in close proximity the amino and carboxyl ends of Venus, resulting in a reassembly of the protein and concomitant fluorescence signal (Figure 1). To assay the interaction of the biosensor with DNA we will use a cell-free system, where the CGSENS will be incubated with DNA with different levels of methylation. The level of restored VENUS activity can be measured using a plate reader, and the fluorescence levels will be indicative of the percentage of DNA methylation.
Summary of the project's achievements and future plans
Heading banner image of DNA methylation is by Christoph Bock, Max Planck Institute for Informatics (Own work). Shared under CC BY-SA 3.0 via Wikimedia Commons.