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Abstract
DNA methylation is important in regulation of gene expression and normal development because it alters the interplay between protein and DNA. Experiments have shown that a single 5-methylcytosine at different CpG sites (mCpG) might have different effects on specific recognition, but the atomistic origin and dynamic details are largely unclear. In this work, we investigated the mechanism of monomethylation at different CpG sites in the cognate motif and the cooperativity of full methylation. By constructing four models of c-Jun/Jun protein binding to the 5[Formula: see text]-XGAGTCA-3[Formula: see text] (X represents C or methylated C) motif, we characterized the dynamics of the contact interface using the all-atom molecular dynamics method. Free energy analysis of MM/GBSA suggests that regardless of whether the C12pG13 site of the bottom strand is methylated, the effects from mC25 of the top strand are dominant and can moderately enhance the binding by [Formula: see text] 31 kcal/mol, whereas mC12 showed a relatively small contribution, in agreement with the experimental data. Remarkably, we found that this spatial-specific influence was induced by different regulatory rules. The influence of the mC25 site is mainly mediated by steric hindrance. The additional methyl group leads to the conformational changes in nearby residues and triggers an obvious structural bending in the protein, which results in the formation of a new T-Asn-C triad that enhances the specific recognition of TCA half-sites. The substitution of the methyl group at the mC12 site of the bottom strand breaks the original H-bonds directly. Such changes in electrostatic interactions also lead to the remote allosteric effects of protein by multifaceted interactions but have negligible contributions to binding. Although these two influence modes are different, they can both fine-tune the local environment, which might produce remote allosteric effects through protein-protein interactions. Further analysis reveals that the discrepancies in these two modes are primarily due to their location. Moreover, when both sites are methylated, the major determinant of binding specificity depends on the context and the location of the methylation site, which is the result of crosstalk and cooperativity.
Highlights
In mammalian DNA, methylation of the fifth position of cytosine (5-methylcytosine, 5mC) is a highly conserved epigenetic modification of DNA that frequently occurs at the CpG dinucleotides and can be read by a set of transcription factors (TFs) known as methyl CpG-binding proteins (MBPs) [1, 2]
Evidence exists that each transcription factor might work independently, and the most important features of a cis-regulatory sequence should be the number of binding sites and how tightly it binds to those sites, which explains the hypomethylation or hypermethylation of DNA methylation observed in cancer [18]
Conclusions two DNA strands, protein-DNA interactions and proteinprotein interactions occur as well, which adds an additional layer of complexity in determining the TF-DNA interaction dynamics
Summary
In mammalian DNA, methylation of the fifth position of cytosine (5-methylcytosine, 5mC) is a highly conserved epigenetic modification of DNA that frequently occurs at the CpG dinucleotides (mCpG) and can be read by a set of transcription factors (TFs) known as methyl CpG-binding proteins (MBPs) [1, 2]. Certain experiments have shown that selected mCpGs at different positions of the target motif have different effects on the binding of protein, such as Kaiso, and the major determinants of its remarkable specificity for methylated DNA are the interactions with the particular first single site mC8pG in the 5 C8GC10G motif [1]
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