Biotechnological Applications of MBD Domain Proteins for DNA Methylation Analysis

Abstract

5-Methylcytosine binding domain (MBD) family proteins are essential readers of DNA methylation. Their methylation specific DNA binding has been exploited in the context of two main groups of important biotechnological applications. In the first, an MBD domain is used to bind methylated DNA in vitro. This can be employed for global DNA methylation analysis in MBD-seq assays, where methylated DNA is purified from fragmented genomic DNA by MBD pulldown or capture, followed by next-generation sequencing (NGS) and downstream data analysis as established for ChIP-seq applications. In addition, the ability of MBD domains to bind methylated DNA can be used for in vitro DNMT activity and inhibition assays. In the second type of applications, MBD domains are used to bind methylated DNA in cells. In MBD imaging, these domains are fused to fluorophores and expressed in cells, where they bind to methylated DNA allowing the readout of DNA methylation by fluorescence microscopy. This approach recently has been further developed to allow the locus-specific readout of DNA methylation using bimolecular fluorescence complementation-based bimolecular anchor detector sensors. These tools, which are compatible with live cell imaging, combine the sequence-specific DNA binding of anchor domains and the 5-methylcytosine-specific binding of an MBD domain to chromatin. Depending on the individual assay, MBD-based detection systems for DNA methylation provide important advantages, ranging from cost efficiency and easy workflows to unique opportunities for the readout of methylation levels in living cells with locus-specific resolution during organismic development.

Keywords: BiAD sensor; DNA methylation analysis; MBD imaging; MBD pulldown.