The MBD of MDU binds methylated CpA motifs

MBD proteins bind methylated DNA and play important roles in epigenetic silencing (Ng et al., 2000; Bienvenu and Chelly, 2006). Our experiments revealed that the MBD of MDU is a functional methylated DNA binding domain and binds methylated CpA motifs. Unlike mammalian MeCP2, the MBD of MDU does not bind methylated CpG-motifs, which suggests that MBD proteins in invertebrates contact methylated DNA motifs other than CpG-motifs. That hypothesis is supported by recent studies suggesting that the MBD does not exclusively bind methylated CpG motifs but rather is a functional module, whose binding affinities to methylated DNA have adopted in response to the various DNA methylation patterns present in various species (Pitto et al., 2000; Scebba et al., 2003; Marhold et al., 2004). Vertebrates methylate their genomes mainly at symmetrical CpG sequences, and human MBD proteins such as MeCP2 and MBD1 showed a preference for CpG-methylated DNA. In carrot, two classes of MBD proteins have been identified. The first shows high affinity for sequences containing 5-methyl cytosine in a canonical CpG methylation context, whereas the second efficiently binds 5-methyl cytosine within both CpXpX and CpXpG (X=A, T, or C) tri-nucleotides (Pitto et al., 2000). The Arabidopsis MBD protein AtMBD5 can bind both symmetrically methylated CpG and asymmetrically methylated CpXpX sequences (Scebba et al., 2003). The Drosophila genome is predominantly methylated at asymmetrical CpA and CpT sequences (Lyko et al., 2000a; Gowher et al., 2003).

Similar to MDU, Drosophila dMBD2/3, the ortholog of vertebrate MBD2 and MBD3, binds methylated CpT/A-motifs (Marhold et al., 2004). Thus, MBD proteins in Drosophila may preferentially bind methylated CpA and/or CpT-motifs, which indicates that the binding specificity of fly MBD proteins corresponds well with the DNA methylation pattern of Drosophila.

How does MDU bind methylated CpA motifs? Why does MDU bind methylated CpA and not other methylated motifs?

The comparison of the primary sequence of the MBD of MDU and other proteins, especially the MBD of MeCP2, reveals that key amino acids involved in the binding of MeCP2 to methylated CpG-motifs such as R106 and R133 are conserved in the MBD of MDU. Only the position of amino acid residue corresponding to R111 of MeCP2 is not conserved but was replaced with a glutamine-residue (Q) in the MBD of MDU. Several MBD proteins (e.g., MBD4, ARBP) contain a QR111 motif, whereas MDU contains a RQ motif. The association of the MBD

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of MDU with methylated CpA motifs indicates that the reversal of the QR111 motif to RQ does not affect the ability of the MBD to bind methylated DNA. However, the switch of the RQ motif in the MBD of MDU may allow MDU to bind methylated CpA instead of CpG motifs.

Another conserved amino acid in the MBD of MDU is R436, which corresponds to R133 in MeCP2. In our study, the mutation R436C greatly attenuated the binding of the MBD of MDU to methylated DNA (3.3), which suggests that R436, like R133 of MeCP2, plays a key role in the association of MDU with methylated DNA.

The structures of three different MBD motifs (human MeCP2 and MBD1, chicken MeCp2) have been solved (Ohki et al., 1999; Wakefield et al., 1999; Heitmann et al., 2003. Figure 35.

Structure of human MeCP2’s MBD).

Figure 35. Structure and conserved residues in the MBD domain of human MeP2. (Top) Schematic drawing of MeP2 indicating the location of the MBD and the secondary structures of the MBD. (Bottom) NMR-based model of the structure of the MBD with DNA-binding residues marked with asterisks (Ghosh et al., 2008).

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The MBD forms a wedge-shaped structure composed of a β-sheet superimposed over a α-helix and loop. Amino acid side chains in two of the β-strands along with residues immediately NH2 -terminal to the α-helix interact with the cytosine methyl groups within the major groove, providing the structural basis for selective recognition of methylated CpG dinucleotides (Ohki et al., 2001;

Wade and Wolffe 2001) (Figure 35).

Amino acid residues R106, R133, F155 and T158 in the MBD domain of MeCP2 are involved in the binding of MBD to methylated DNA. Mutations in those amino acid residues have been associated with Rett syndrome, a severe X-linked neurodevelopmental disorder in humans (Amir et al., 1999). Another important residue for methyl DNA binding activity of MeCP2 is R111.

Mutations in R111G, R106W and R133C greatly decrease the binding of MeCP2’s MBD to methyl DNA (Free et al., 2001)(Figure 36).

Figure 36. Amino acids involved in interaction of MBD of MeCP2 with methylated CpG-motifs. Mutated MBDs with single amino acid exchange mutations were assayed for binding to a methylated 27-bp duplex oligonucleotide containing a single, symmetrically methylated CpG dinucleotide by using EMSA. Each mutated MBD was assayed at three concentrations (20, 200 and 2000 nM). DNA binding activity was calculated as the percentage of DNA retained by mutated or wild type MBD to total DNA applied to the assay. Amino acids mutated are indicated by their single letter codes with numbering corresponding to their position in MeCP2. Substituted amino acids are indicated in parentheses, and residues mutated in cases of Rett syndrome are indicated by asterisks (Free et al., 2001).

Discussion 107

Among the three mutations R111G, R106W and R133C in the MeCP2 MBD domain, the R111G resulted in the most severe impairment in methylated DNA binding activity. R133C is the second strongest mutation (Free et al., 2001). Although the mutation R133C resulted in greatly decreased methyl DNA binding activity, the mutation is not believed to affect the structure of MBD domain (Free et al., 2001).

Another study demonstrated that all 4 mutations (R106W, R133C, F155S, T158M) located in the MBD domain of MeCP2 have profound and diverse effects on the structure, stability, and DNA-binding properties of the MBD (Ghosh et al., 2008). The mutations R133C, F155S, and T158M reduce the thermal stability of the MBD (Ghosh et al., 2008). Thermal stability of the wild-type protein is increased in the presence of unmethylated DNA, and further enhanced by DNA methylation. DNA-induced thermal stability was also observed for mutant proteins but to a lesser extent (Ghosh et al., 2008). According to this study, the mutant R133C causes structural changes in the MBD. Both the full-length mutant and the MBD mutant of R133C show reduced thermal stability as compared with the wild type, and the EMSA data show reduced binding to methylated DNA (Ghosh et al., 2008).

The crystal structure of the MBD of MeCP2 complexed to methylated DNA (Ho et al., 2008) revealed that contrary to the traditional model proposing that the binding specificity of the MBD depends on hydrophobic interactions between cytosine methyl groups and a hydrophobic patch within the MBD, the methyl groups predominantly contact hydrophilic surfaces that include tightly bound water molecules. The only amino acid residues of the MBD of MeCP, which directly interact with DNA, are D121, R111, and R133 (Ho et al., 2008). Of the 25 interactions occurring between the methyl cytosine groups of the DNA and the MBD, only two interactions between methyl cytosine and R133 are classically hydrophobic in character. Also, hydrogen bonds are formed between the symmetrical arginine fingers (R111 and R133) and each guanine of the methyl CpG pair (Ho et al., 2008). Thus, R133 in MeCP2 has unique and indispensable functions for the structure and function of the MBD.

Our results are consistent with other studies indicating that R436 of MDU (R133 of MeCP2) is important for the methyl DNA binding activity of the MBD. Further functional studies of other amino acid residues in the MBD of MDU and the study of the crystal structure of the MBD are necessary to demonstrate the mechanism of MBD of MDU recognizing and binding methylated CpA motifs in target DNA.

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