Fig. 6
CpG methylation induces a DNA shape change that explains its effect on Pbx-Hox binding. a Schematic representation of Pbx-Hox heterodimer bound to DNA (PDB ID 1PUF), and of the effect of CpG methylation on binding. Pbx (green) and Hox (blue) homeodomains bind up- and downstream of the central spacer region (indicated in red), respectively. CpG methylation at offsets 6/7 and 10/11 reduces binding, whereas methylation at offset 9/10 enhances binding. Methyl group readout was previously identified as underlying mechanism for the latter offset [13]. b Scatter-plot representation of relative binding affinities of methylated versus unmethylated sequences for Pbx-HoxA1 complex. Sequences carrying a single methylation event and their corresponding unmethylated part were considered. Green, magenta, and blue points correspond to methylation at offsets 6/7, 9/10, and 10/11, respectively. Sequences containing CpG dinucleotides at other offsets (relatively weakly affected by methylation) are colored gray. c Alternative representation of the data in b, showing the effect of methylation on binding free energy, denoted as ΔΔΔG/RT. Positive (e.g., offsets 6/7 and 10/11) and negative (e.g., offset 9/10) shifts from the dashed line (indicating no methylation effect) reflect reduced and enhanced binding (on logarithmic scale) due to methylation. CpG dinucleotides at offsets 6/7 and 10/11 produce the same hexamer context for A4 and A8 (NNAYCG/NGAYCG) and, hence, were assigned a common color, dark-cyan. d Analysis of the methylation-induced change in MGW at positions A4 and A8 within the Pbx-Hox binding site (NNGAYNNAYNNN), for the different hexameric/pentameric contexts that the Pbx-Hox heterodimer may encounter within its binding sequence. Coloring corresponds to that of labels and rectangular patches in c. Statistically significant widening of minor groove (first two boxes) plausibly explains the observed reduced binding due to methylation at CpG offsets 6/7 and 10/11. No significant change in MGW upon methylation was observed for offset 9/10