Complete Profiling of Methyl-CpG-Binding Domains for Combinations of Cytosine Modifications at CpG Dinucleotides Reveals Differential Read-out in Normal and Rett-Associated States

5-Methylcytosine (mC) exists in CpG dinucleotides of mammalian DNA and plays key roles in chromatin regulation during development and disease. As a main regulatory pathway, fully methylated CpG are recognized by methyl-CpG-binding domain (MBD) proteins that act in concert with chromatin remodelers, histone deacetylases and methyltransferases to trigger transcriptional downregulation. In turn, MBD mutations can alter CpG binding, and in case of the MBD protein MeCP2 can cause the neurological disorder Rett syndrome (RTT). An additional layer of complexity in CpG recognition is added by ten-eleven-translocation (TET) dioxygenases that oxidize mC to 5-hydroxymethyl-, 5-formyl- and 5-carboxylcytosine, giving rise to fifteen possible combinations of cytosine modifications in the two CpG strands. We report a comprehensive, comparative interaction analysis of the human MBD proteins MeCP2, MBD1, MBD2, MBD3, and MBD4 with all CpG combinations and observe individual preferences of each MBD for distinct combinations. In addition, we profile four MeCP2 RTT mutants and reveal that although interactions to methylated CpGs are similarly affected by the mutations, interactions to oxidized mC combinations are differentially affected. These findings argue for a complex interplay between local TET activity/processivity and CpG recognition by MBDs, with potential consequences for the transcriptional landscape in normal and RTT states.