Direct DNA methylation profiling using methyl binding domain proteins

Abstract

Methylation of DNA is responsible for gene silencing by establishing heterochromatin structure that represses transcription, and studies have shown that cytosine methylation of CpG islands in promoter regions acts as a precursor to early cancer development. The naturally occurring methyl binding domain (MBD) proteins from mammals are known to bind to the methylated CpG dinucleotide (mCpG) and subsequently recruit other chromatin-modifying proteins to suppress transcription. Conventional methods of detection for methylated DNA involve bisulfite treatment or immunoprecipitation prior to performing an assay. We focus on proof-of-concept studies for a direct microarray-based assay using surface-bound methylated probes. The recombinant protein 1xMBD-GFP recognizes hemimethylation and symmetric methylation of the CpG sequence of hybridized dsDNA, while displaying greater affinity for the symmetric methylation motif, as evaluated by SPR. From these studies, for symmetric mCpG, the K(D) for 1xMBD-GFP ranged from 106 to 870 nM, depending upon the proximity of the methylation site to the sensor surface. The K(D) values for nonsymmetrical methylation motifs were consistently greater (>2 muM), but the binding selectivity between symmetric and hemimethylation motifs ranged from 4 to 30, with reduced selectivity for sites close to the surface or multiple sites in proximity, which we attribute to steric effects. Fitting skew normal probability density functions to our data, we estimate an accuracy of 97.5% for our method in identifying methylated CpG loci, which can be improved through optimization of probe design and surface density.

Figure 1

Illustration of the two-step methylation profiling assay. Capture probe oligonucleotides are singly-methylated at all CpG loci. The first step is similar to a conventional DNA assay in that sample ssDNA, of unknown methylation status, is hybridized to the capture array. Hybridization may be quantified by fluorescence detection or SPR, for example. In the second step, binding of MBD protein is performed, with quantification via fluorescence (under excitation from a second laser) or SPR.MBD preferentially binds to symmetric methylation sites (i.e. where the methylation states of the target and probe overlap). By comparing the (calibrated) sensor response from the MBD protein to that of the dsDNA at each capture spot, the methylation status can be determined at each CpG location.

Figure 2

SPR sensorgrams for 1xMBD-GFP binding to the dsDNA matrix for the MGMT gene sequence, with normalized RU (y-axis) versus time (x-axis). Each column represents a different probe methylation pattern, and each row, a different target methylation pattern.

Figure 3

SPR sensorgrams for 1xMBD-GFP binding to the dsDNA matrix for the MGMT promoter sequence, with normalized RU (y-axis) versus time (x-axis). Each column represents a different probe methylation pattern, and each row, a different target methylation pattern.

Figure 4

Histogram of the K_D population obtained from SPR experiments for both the gene and promoter sequences. Each group, symmetric methylation and non-symmetric methylation, was fitted to a skew-normal probability density function. These pdfs were scaled before plotting in order to match the histogram scale; prior to any error calculations, the pdfs were normalized to integrate to 1.