Methylation of CpG dinucleotides in regulatory regions of genes is an important mark for epigenetic regulation of transcription (2
). Since DNA methylation is passed on to daughter cells during cell division, these methyl CpG marks can be maintained during development and provide epigenetic memory (31
). A number of proteins have been identified in the human genome that can specifically bind to methylated CpG residues via a methyl CpG binding domain (MBD) (15
). Recruitment of these proteins to promoters containing methylated CpG-rich stretches—CpG islands—is thought to result in modulation of chromatin structure and repression of transcription. The human genome encodes five MBD proteins: MeCP2 and MBD1 to -4 (6
). Apart from MBD3, these proteins have been shown to have specific methyl CpG binding activity. Recently a novel protein, Kaiso, was identified as a methyl CpG binding protein even though this protein lacks a classical MBD but appears to bind specifically to methylated DNA via a zinc finger domain (30
Several MBD proteins have been reported to interact with histone deacetylases (HDACs) as well as histone methyltransferases. MeCP2 has been described to interact with the Sin3/HDAC corepressor complex (18
) and Brahma (13
), as well as with the histone H3 lysine-9 methyltransferase Suvar 3-9 (12
), although these interactions may not be stable since MeCP2 is mostly present inside the cell as a monomer (12
). MBD2 and MBD3 have been identified as core subunits of the Mi-2/NuRD complex (9
), whereas Kaiso is part of the HDAC-containing N-CoR complex that plays an important role in transcription regulation by nuclear hormone receptors (27
). Collectively, these findings suggest a functional link between DNA methylation, histone deacetylation, and histone methylation and indicate that these epigenetic events functionally cooperate to regulate transcription and cellular memory.
MBD2 and MBD3 have both been described as subunits of the Mi-2/NuRD complex. It has been proposed that MBD2, which exhibits methyl CpG binding activity, serves to recruit the MBD3-containing Mi-2/NuRD complex to methylated promoters (44
). Knockout studies in mice, however, suggest that MBD2 and MBD3 have distinct nonoverlapping functions: whereas knocking out MBD3 results in embryonic lethality, MBD2-knockout mice are viable and display relatively subtle defects (16
). Interestingly, Sansom and coworkers recently showed that the absence of MBD2 protects against intestinal tumorigenesis (32
). Thus, although biochemical evidence suggests that MBD2 and MBD3 are part of the same complex, the knockout studies suggest that both proteins have specific or maybe partially overlapping functions.
To gain insights into the protein composition and function of MBD2 and MBD3, we generated stable cell lines expressing tagged versions of these proteins. Purification of the protein complexes revealed that MBD2 and MBD3 are not copurifying but are mutually exclusive. In addition to known Mi-2/NuRD subunits, a 12-kDa protein called DOC-1 was identified as a novel core subunit of both the MBD3 and MBD2 complexes. Furthermore, PRMT5 and its associated cofactor MEP50 were found to copurify with and methylate MBD2 in vitro. Finally, PRMT5 and its H4R3 histone methyltransferase activity were shown to be recruited with MBD2 to CpG islands in a methylation-sensitive manner in vivo, suggesting an unexpected role for an arginine methyltransferase in repression by MBD2. Collectively, these findings provide evidence that MBD2/NuRD and MBD3/NuRD define two distinct protein complexes with different biochemical and functional properties.