Identification and Characterization of a Family of Mammalian Methyl-CpG Binding Proteins

ABSTRACT

Methylation at the DNA sequence 5′-CpG is required for mouse development. MeCP2 and MBD1 (formerly PCM1) are two known proteins that bind specifically to methylated DNA via a related amino acid motif and that can repress transcription. We describe here three novel human and mouse proteins (MBD2, MBD3, and MBD4) that contain the methyl-CpG binding domain. MBD2 and MBD4 bind specifically to methylated DNA in vitro. Expression of MBD2 and MBD4 tagged with green fluorescent protein in mouse cells shows that both proteins colocalize with foci of heavily methylated satellite DNA. Localization is disrupted in cells that have greatly reduced levels of CpG methylation. MBD3 does not bind methylated DNA in vivo or in vitro. MBD1, MBD2, MBD3, and MBD4 are expressed in somatic tissues, but MBD1 and MBD2 expression is reduced or absent in embryonic stem cells which are known to be deficient in MeCP1 activity. The data demonstrate that MBD2 and MBD4 bind specifically to methyl-CpG in vitro and in vivo and are therefore likely to be mediators of the biological consequences of the methylation signal.

ACKNOWLEDGMENTS

We thank En Li for the DNMT-deficient ES cells, Andrew Smith for the 129 genomic library, Jonathan Pines for the pCMXGFP vector, Christer Höög for the MMTEST clone, John Huntriss and Marilyn Monk for sending oligonucleotide probes, and the HGMP for providing numerous clones and libraries. We also thank members of the Bird lab; Cathy Abbott and Beth Sullivan for advice; Kevin Hardwick for use of the imaging system; Vicky Clark for sequencing; Aileen Greig and Joan Davidson for technical assistance; and Beth Sullivan, Cathy Abbott, Susan Tweedie, Sally Cross, and Huck-Hui Ng for critical reading of the manuscript.

B. H. was the recipient of a Long Term Postdoctoral Fellowship from the Human Frontiers Science Program. The work was also supported by a program grant to A.B. from the Wellcome Trust.