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CELL BIOLOGY AND SIGNAL TRANSDUCTION

Methylated DNA-Binding Proteins from Arabidopsis¹

Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Nara 630–0192, Japan

The 5-methylcytosines (m⁵C) play a critical role in epigenetic control, often being recognized by proteins containing a methyl-CpG-binding domain (MBD). Database screening has identified at least 12 putative methyl-CpG-binding proteins from Arabidopsis; we have isolated corresponding cDNAs for seven of them. Despite variation in size and amino acid sequence, all seven proteins exclusively migrate into the nucleus as revealed by green fluorescent protein fusion protein assay, suggesting a relationship with chromatin structure. However, DNA-binding assays using bacterially expressed proteins and synthetic oligonucleotides containing m⁵C in CpGs showed only one to specifically bind, designated AtMBD5. Further analysis showed that AtMBD5 efficiently binds to m⁵C in CpNpN (N is A, T, or C) but not in CpNpG sequences, both frequently found in plant DNA. The other six proteins showed either nonspecific DNA binding or no ability to recognize m⁵C. RNA-blot hybridization and immunoblot analysis indicated AtMBD5 to be present essentially in all tissues. Using green fluorescent protein driven by the authentic promoter, AtMBD5 was found to be actively expressed in pistils and root tips. Because m⁵Cs in CpG and CpNpN are considered to function in gene expression and gene silencing, respectively, the present results suggest that AtMBD5 may have distinct functions in regulation and/or self defense of genes in actively proliferating cells.

¹ This work was supported by the Research for the Future Program (grant no. JSPS–RFTF00L01604) of the Japan Society for the Promotion of Science.

^* Corresponding author; e-mail sano{at}gtc.aist-nara.ac.jp; fax 81–743–72–5659.

Received May 10, 2003; returned for revision July 2, 2003; accepted July 27, 2003.

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