Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 1.02

ARABIDOPSIS MBD PROTEINS SHOW DIFFERENT BINDING SPECIFICITIES AND NUCLEAR LOCALIZATION

F. SCEBBA*, G. BERNACCHIA**, M. DE BASTIANI**, A. ANDREUCCI***, M.T. LOCCI****, L. PITTO*

*) Laboratory of Gene and Molecular Terapy, Institute of Clinical Fisiology, CNR, Pisa, Italy

**) Department of Biological Sciences University of Ferrara, Italy

***) Department of Botanical Sciences, University of Pisa, Italy

****) Department of Human Morphology and Applied Biology, University of Pisa, Italy

DNA-binding proteins, symmetric and asymmetric methylation, GFP tagged proteins

Recent results in animals and plants have shown a strong link between DNA methylation, chromatin structure and epigenetic control (Rice and Allis, 2001, Nature,414:258-61). In plants DNA methylation affects both symmetrical and asymmetrical cytosines by means of different DNA-methyltransferases. In vertebrates it is known that these modifications are interpreted by a group of proteins (methylated DNA binding domain proteins, MBDs) able to specifically bind methylated CpG (Hendrich and Bird, 1998, Mol Cell Biol. 18:6538-47). All MBDs, except MBD4, a thymine glycosylase that forms a complex with the DNA mismatch-repair protein MLH1 (Bellacosa et al, 1999, Proc Natl Acad Sci U S A. 96:3969-74) form complexes with histone deacetylases (HDAC) which they recruit to methyl CpG enriched regions to repress transcription (Ballestar and Wolffe,2001. Eur J Biochem. 268:1-6.). In plants several genes sharing structural homology to mammalian MBD have been identified in Arabidopsis and maize, but their characterization is yet to be completed. Here we present results concerning (six) different MBDs from Arabidopsis chosen on the basis of differences in their expression profiles as judged by semi-quantitative RT-PCR. All the corresponding polypeptides, expressed in E. coli as His-tagged recombinant proteins, have been functionally tested on gel shift experiments but only three of them (namely AtMBD5, AtMBD6 and AtMBD11) were able to bind methylated oligonucleotide duplexes. Moreover we were able to differentiate AtMBD5 and AtMBD6, despite their high homology, for their specific affinities: AtMBD5 is able to specifically bind cytosine methylated in symmetrical and asymmetrical contexts while AtMBD6 efficiently complexes only sequences methylated in the canonical CpG site. AtMBD11 differs from the other tested AtMBD proteins for its capacity to strongly bind methylated and unmethylated DNA. The binding specificity of these proteins was tested not only on arbitrarily chosen probes but also on Arabidopsis E2F-recognition sequence containing two CpG sites. Our data indicate that methylation can trasform a sequence target for the members of the AtE2F transcription factors family in a binding site for AtMBD5 and AtMBD6. Transient expression experiments of AtMBD5, AtMBD6 and AtMBD11 tagged with green fluorescent protein in Arabidopsis protoplasts showed that AtMBD5, AtMBD6 and AtMBD11 are nuclear protein. Moreover our data indicate that AtMBD5 and AtMBD6 localize in heterochromatin and this localization is affected by 5-azacytidine treatment. These data demonstrate that AtMBD5 and AtMBD6 bind methylated DNA in vitro and in vivo with different specificities and distribution, therefore they might have different roles in methylation-mediated transcriptional silencing.