Abstract
Although highly homologous to other methylcytosine-binding domain (MBD) proteins, MBD3 does not selectively bind methylated DNA, and thus the functional role of MBD3 remains in question. To explore the structural basis of its binding properties and potential function, we characterized the solution structure and binding distribution of the MBD3 MBD on hydroxymethylated, methylated, and unmethylated DNA. The overall fold of this domain is very similar to other MBDs, yet a key loop involved in DNA binding is more disordered than previously observed. Specific recognition of methylated DNA constrains the structure of this loop and results in large chemical shift changes in NMR spectra. Based on these spectral changes, we show that MBD3 preferentially localizes to methylated and, to a lesser degree, unmethylated cytosine-guanosine dinucleotides (CpGs), yet does not distinguish between hydroxymethylated and unmethylated sites. Measuring residual dipolar couplings for the different bound states clearly shows that the MBD3 structure does not change between methylation-specific and nonspecific binding modes. Furthermore, residual dipolar couplings measured for MBD3 bound to methylated DNA can be described by a linear combination of those for the methylation and nonspecific binding modes, confirming the preferential localization to methylated sites. The highly homologous MBD2 protein shows similar but much stronger localization to methylated as well as unmethylated CpGs. Together, these data establish the structural basis for the relative distribution of MBD2 and MBD3 on genomic DNA and their observed occupancy at active and inactive CpG-rich promoters.
Highlights
Highly homologous to MBD2, the functional role of MBD3 remains in question
Highly homologous to other methylcytosine-binding domain (MBD) proteins, MBD3 does not selectively bind methylated DNA, and the functional role of MBD3 remains in question
It has been established that MBD3 binds DNA with lower affinity and much less specificity for mCpG dinucleotides than MBD2, the functional role of MBD3 has not been well defined
Summary
Highly homologous to MBD2, the functional role of MBD3 remains in question. Results: MBD3 preferentially localizes to methylated and, to a lesser degree, unmethylated CpG dinucleotides. Specific recognition of methylated DNA constrains the structure of this loop and results in large chemical shift changes in NMR spectra Based on these spectral changes, we show that MBD3 preferentially localizes to methylated and, to a lesser degree, unmethylated cytosine-guanosine dinucleotides (CpGs), yet does not distinguish between hydroxymethylated and unmethylated sites. Residual dipolar coupling (RDC) analysis confirms our findings by showing that MBD3 preferentially localizes to mCpG sites and that MBD3 adopts a very similar structure on mCpG, CpG, and hmCpG DNA. The tendency to localize on a specific site does not necessarily translate into a global binding affinity preference These data are consistent with recent studies showing that both MBD2 and MBD3 localize to transcription start sites associated with CGIs (16, 17). The presence of MBD3 at unmethylated CGIs could modify the distribution of MBD2 and potentially mitigate strong silencing by the high affinity, more strongly localizing MBD2 protein, thereby preserving bivalency with respect to transcription
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