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First published online April 1, 2005; 10.1105/tpc.105.031567

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The Plant Cell 17:1549-1558 (2005)
© 2005 American Society of Plant Biologists

DDM1 Binds Arabidopsis Methyl-CpG Binding Domain Proteins and Affects Their Subnuclear Localization{boxw}

Assaf Zemacha, Yan Lia, Bess Wayburna, Hagit Ben-Meira, Vladimir Kissb, Yigal Avivia, Vyacheslav Kalchenkoc, Steven E. Jacobsend and Gideon Grafia,1

a Department of Plant Sciences, Weizmann Institute of Science, Rehovot 76100, Israel
b Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
c Department of Veterinary Resources, Weizmann Institute of Science, Rehovot 76100, Israel
d Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, California 90095

1 To whom correspondence should be addressed. E-mail gideon.grafi{at}weizmann.ac.il; fax 972-8-934-4181.

Methyl-CpG binding domain (MBD) proteins in Arabidopsis thaliana bind in vitro methylated CpG sites. Here, we aimed to characterize the binding properties of AtMBDs to chromatin in Arabidopsis nuclei. By expressing in wild-type cells AtMBDs fused to green fluorescent protein (GFP), we showed that AtMBD7 was evenly distributed at all chromocenters, whereas AtMBD5 and 6 showed preference for two perinucleolar chromocenters adjacent to nucleolar organizing regions. AtMBD2, previously shown to be incapable of binding in vitro–methylated CpG, was dispersed within the nucleus, excluding chromocenters and the nucleolus. Recruitment of AtMBD5, 6, and 7 to chromocenters was disrupted in ddm1 and met1 mutant cells, where a significant reduction in cytosine methylation occurs. In these mutant cells, however, AtMBD2 accumulated at chromocenters. No effect on localization was observed in the chromomethylase3 mutant showing reduced CpNpG methylation or in kyp-2 displaying a reduction in Lys 9 histone H3 methylation. Transient expression of DDM1 fused to GFP showed that DDM1 shares common sites with AtMBD proteins. Glutathione S-transferase pull-down assays demonstrated that AtMBDs bind DDM1; the MBD motif was sufficient for this interaction. Our results suggest that the subnuclear localization of AtMBD is not solely dependent on CpG methylation; DDM1 may facilitate localization of AtMBDs at specific nuclear domains.




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