In previous reports, SELEX-seq profiling followed by DNA shape analyses of binding by heterodimers of all eight Drosophila melanogaster Hox proteins in complex with their common co-factor Extradenticle (Exd) revealed an important role for MGW readout [26, cuatro9]. More recently, an extension of the SELEX-seq method for methylated binding sites, EpiSELEX-seq, revealed that cytosine methylation modulates the affinity with which human orthologs (Pbx-Hox) of these heterodimers bind to CpG dinucleotide-containing sites . The DNA sequences associated with the largest binding affinity for the Exd-Hox and Pbx-Hox complexes matched the 12-bp sequence pattern NTGAYNNAYNNN, where Y represents pyrimidine (C or T) and N any nucleotide (Fig. 6a).
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 reduces binding, whereas methylation at offset 9/ten enhances binding. Methyl group readout was previously identified as underlying mechanism for the latter offset . 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 , respectively. Sequences containing CpG dinucleotides at other offsets (relatively weakly affected by methylation) are colored gray. c Alternative representation of the data in is once free b, showing the effect of methylation on binding free energy, denoted as ???G/RT. Positive (e.g., offsets 6/7 and ) 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 produce the same hexamer context for A4 and A8 (NNAYCG) 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. No significant change in MGW upon methylation was observed for offset 9/10
As previously reported , direct comparison of the relative binding affinities for unmethylated versus methylated sequences (Fig. 6b, c) shows that cytosine methylation can either have a stabilizing or destabilizing effect on Pbx-Hox binding, depending on the position of the CpG dinucleotide within the binding site. CGAYNNN; C6G7; green points/box in Fig. 6b, c) and offset (NTGAYNNAYCGN; C10Geleven; blue points/box in Fig. 6b, c) suppresses binding, whereas methylation at offset 9/10 (NTGAYNNACGNN; C9G10; magenta points/box in Fig. 6b, c) enhances binding by an order of magnitude. We previously proposed a plausible mechanism for the latter stabilizing effect, which we postulated to involve direct contacts to the methyl group in the major groove . However, an explanation of the suppressed binding at the CpG offsets 6/7 and was lacking (Fig. 6a).
Zero healthy protein–DNA get in touch with was noticed in new co-amazingly design (PDB ID: 1PUF) on offset 6/7. But not, the fresh new nucleotides on counterbalance 6/7 function an effective spacer located between a couple AY dinucleotides (Fig. 6a), which have been before demonstrated to showcase good contour choice. Especially, lesser groove narrowing during the AY ranks adjacent to the main spacer try been shown to be of improved binding in the event that nucleotide succession is ranged to own unmethylated DNA [twenty six, 49]. Therefore, we hypothesized one an excellent methylation-caused improvement in DNA profile near the CpG dinucleotide could affect joining attraction. We utilized the pentamer-situated profile dining tables you to definitely setting the foundation out-of DNAshape and methyl-DNAshape to research it perception systematically.