MBD2-MBD3 complex binds to hemi-methylated DNA and forms a complex containing DNMT1 at the replication foci in late S phase

Background: In vertebrates and plants, DNA methylation is one of the major mechanisms regulating gene expression. Recently a family of methyl-CpG-binding proteins has been identified, and some members, such as MeCP2 and MBD2, were shown to mediate gene repression by recruiting histone deacetylase complexes to methylated genes. However, the function of another member of this family, MBD3, remained elusive. Results: It was shown that MBD2 and MBD3 form homo- and hetero-dimers (or multimers) in vitro and in vivo. Significantly, the MBD2-MBD3 complex showed an affinity to hemi-methylated DNAs, a property that has never been reported with any member of the family proteins. MBD2 and MBD3 were co-localized with DNMT1 at replication foci in 293 cell nuclei at late S phase. Moreover, by a coimmunoprecipitation experiment, DNMT1 was shown to form a complex with MBD2 and MBD3. Finally, the abundance of MBD3 was highest in the late S phase when the DNMT1 is also most abundant, whereas the MBD2 level was largely constant throughout the cell cycle. Conclusions: The results suggest that MBD3 may play an important role in the S phase. We hypothesize that the MBD2-MBD3 complex recognizes hemimethylated DNA concurrent with DNA replication and recruits histone deacetylase complexes, as well as DNMT1, to establish and/or maintain the transcriptionally repressed chromatin.