DIRECT DETECTION OF DNA METHYLATION USING SPLIT-PROTEIN SENSORS

Andrew Ma , Pui Wing Mok, Ahmed H. Badran, Alexander J. Riemen, and Indraneel Ghosh

DIRECT DETECTION OF DNA METHYLATION USING SPLIT-PROTEIN SENSORS

Gene expression is controlled chiefly through DNA sequences but can also be modulated through epigenetics.  DNA methylation is one of the most prominent epigenetic markers and through recruitment of methyl binding proteins and additional chromatin-modifying enzymes, can alter protein expression.  Methylation of DNA occurs when methyltransferases catalyze a reaction that results in the addition of a methyl group from s-adenosylmethionine to the 5’ position of cytosine in CpG dinucleotides. Previous research has demonstrated a correlation between hypermethylation in the promoter regions of tumor suppressors and a subsequent cancerous state of cells.  Such a relationship is of particular interest because it implies that specific instances of DNA methylation are precursors to cancer.  Consequently, detection of specific DNA methylation can provide a method for cancer diagnostics.

DNA methylation can currently be detected through methods such as ChIP-chip analysis and bisulfite sequencing, both of which are time-consuming, labor-intensive, and error-prone.  Direct detection of DNA methylation can be achieved through the use of split-protein complementation, which takes advantage of the eponymous capabilities of the family of Methyl-CpG Binding Domain (MBD) proteins and the reassembly properties of proteins that readily generate detectable signal.  Previously, we evalulated the effectiveness of various proteins with methyl-CpG binding capabilities in the split-protein system and identified several that were particularly effective at detecting DNA methylation.  Our goal now is to study these proteins to understand the mechanism of methyl-CpG binding in order to create an even more selective detector for DNA methylation.

In order to study the mechanism of methyl-CpG binding, we performed a scan of MBD1 and MBD2 utilizing site-directed mutagenesis to create MBD mutants that each have an alanine mutation at a different position.  Our in vitro translation system allows us to perform rapid evaluation of these mutational effects, and these split-protein assays have shed light on the importance of certain residues in recognizing mCpG sites. We are currently creating a library of randomized MBD2 sequences from which we seek to identify a better sensor for DNA methylation using phage display selections with the overall goal of engineering a protein to be used for DNA diagnostics.

The authors would like to thank the Arnold and Mabel Beckman Foundation for their generous funding.

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