Structural Basis of MeCP2 Distribution on Non-CpG Methylated and Hydroxymethylated DNA

The Rett-syndrome-associated methyl-CpG-binding protein 2 (MeCP2) selectively binds methylated DNA to regulate transcription during the development of mature neurons. Like other members of the methyl-CpG-binding domain (MBD) family, MeCP2 functions through the recognition of symmetrical 5-methylcytosines in CpG (mCG) dinucleotides. Advances in base-level resolution epigenetic mapping techniques have revealed, however, that MeCP2 can bind asymmetrically methylated and hydroxymethylated CpA dinucleotides and that this alternative binding selectivity modifies gene expression in the developing mammalian brain. The structural determinants of binding to methylated CpA (mCA) and hydroxymethylated DNA have not been previously investigated. Here, we employ isothermal titration calorimetry and NMR spectroscopy to characterize MeCP2 binding to methylated and hydroxymethylated mCG and mCA DNA, examine the effects of Rett-syndrome-associated missense mutations, and make comparisons to the related and evolutionarily most ancient protein, MBD2. These analyses reveal that MeCP2 binds mCA with high affinity in a strand-specific and orientation-dependent manner. In contrast, MBD2 does not show high affinity or methyl-specific binding to mCA. The Rett-associated missense mutations (T158M, R106W, and P101S) destabilize the MeCP2 MBD and disrupt the recognition of mCG and mCA equally. Finally, hydroxymethylation of a high-affinity mCA site does not alter the binding properties, whereas hemi-hydroxylation of the equivalent cytosine in an mCG site decreases affinity and specificity. Based on these findings, we suggest that MeCP2 recognition of methylated/hydroxymethylated CpA dinucleotides functions as an epigenetic switch redistributing MeCP2 among mCG and mCA loci.