Structural Insights into Methylated DNA Recognition by the Methyl-CpG Binding Domain of MBD6 from Arabidopsis thaliana.

Yutaka Mahana,Daichi Morimoto,Masahiro Shirakawa,Erik Walinda,Izuru Ohki,Kenji Sugase

doi:10.1021/acsomega.1c04917

Abstract

Cytosine methylation is an epigenetic modification essential for formation of mature heterochromatin, gene silencing, and genomic stability. In plants, methylation occurs not only at cytosine bases in CpG but also in CpHpG and CpHpH contexts, where H denotes A, T, or C. Methyl-CpG binding domain (MBD) proteins, which recognize symmetrical methyl-CpG dinucleotides and act as gene repressors in mammalian cells, are also present in plant cells, although their structural and functional properties still remain poorly understood. To fill this gap, in this study, we determined the solution structure of the MBD domain of the MBD6 protein from Arabidopsis thaliana and investigated its binding properties to methylated DNA by binding assays and an in-depth NMR spectroscopic analysis. The AtMBD6 MBD domain folds into a canonical MBD structure in line with its binding specificity toward methyl-CpG and possesses a DNA binding interface similar to mammalian MBD domains. Intriguingly, however, the binding affinity of the AtMBD6 MBD domain toward methyl-CpG-containing DNA was found to be much lower than that of known mammalian MBD domains. The main difference arises from the absence of positively charged residues in AtMBD6 that supposedly interact with the DNA backbone as seen in mammalian MBD/methyl-CpG-containing DNA complexes. Taken together, we have established a structural basis for methyl-CpG recognition by AtMBD6 to develop a deeper understanding how MBD proteins work as mediators of epigenetic signals in plant cells.

Highlights

A certain percentage of cytosine bases in eukaryotic DNA exist in an epigenetically modified form, as 5-methylcytosine
While we found that the L1 loop of MBDAtMBD6 shows high structural flexibility prior to DNA binding, interestingly, previous structural and chemical shift analyses indicated that the specific binding of human Methyl-CpG binding domain (MBD) domains to the methylCpG site stabilizes the dynamic L1 loop, thereby reducing its conformational flexibility.[18,25]
Our analysis further indicates that the DNA binding affinity of AtMBD5 is likely to be even lower than that of AtMBD6, as the important tyrosine (Y104 in AtMBD6) is substituted to phenylalanine (Figures 2C and S4) and, AtMBD5 could not form the water-mediated hydrogen bond network that has been implicated in DNA base recognition by HsMeCP2.19 The sequence homology of the MBD domains of AtMBD7 with mammalian MBD domains is slightly lower than that of AtMBD6

Summary

Introduction

A certain percentage of cytosine bases in eukaryotic DNA exist in an epigenetically modified form, as 5-methylcytosine. Cytosine methylation plays essential roles in numerous vital functions such as repression of gene expression, organization of the chromatin structure, and inactivation of transposons.[1,2] In animal cells, cytosine methylation occurs mostly at symmetrical CpG sequences and is achieved by the action of DNA methyltransferases.1,3 5-Methylcytosine may be further oxidized to 5-hydroxymethylcytosine. These two modifications are examples of distinct epigenetic marks, which can be recognized (“read-out”) with specific reader domains in many proteins. As another example of a canonical MBD protein, the HsMBD4 MBD domain preferably recognizes (mismatch) TpG/methyl-

Methods

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Discussion

Conclusion