title: Arabidopsis MBD proteins show different binding specificities and nuclear localization.
abstract: Recent results in animals and plants have shown a strong link between DNA methylation, chromatin structure and epigenetic control. In plants DNA methylation affects both symmetric and asymmetric cytosines by means of different DNA-methyltransferases. In vertebrates these modifications are interpreted by a group of proteins (methylated DNA-binding domain proteins, MBDs) able to specifically bind methylated CpG. In plants several genes sharing structural homology to mammalian MBD have been identified in Arabidopsis and maize, but their characterization is still to be completed. Here we present the characterization of six different MBDs from Arabidopsis. As judged by semi-quantitative RT-PCR, their expression proved to be differentially modulated in different organs. All the corresponding polypeptides, expressed in Escherichia coli as His-tagged recombinant proteins, have been functionally tested on gel shift experiments but only two of them (namely MBD5, 6) were able to specifically bind methylated CpG oligonucleotides. A third protein, AtMBD11, showed a strong affinity for DNA independently from the level of methylation. Moreover we were able to differentiate MBD5 and 6, despite their high homology, for their ability to recognize methylated asymmetrical sites. The binding specificity of these three AtMBD proteins was tested not only on arbitrarily chosen probes but also on the Arabidopsis E2F recognition sequence containing a single CpG site. Protoplasts transient expression experiments of GFP-fusion proteins showed for AtMBD5 and AtMBD6 a heterochromatic localization which was affected by 5-azacytidine treatment. These data demonstrate that AtMBD5 and AtMBD6 bind methylated DNA in vitro and in vivo with different specificity and might therefore have different roles in methylation-mediated transcriptional silencing.
authors.affiliation: Laboratorio di Terapia Genica e Molecolare-Istituto di Fisiologia Clinica-Area della Ricerca CNR, via Moruzzi 1, 56100 Pisa, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Scebba
authors.forename: Giovanni
authors.initials: G
authors.lastname: Bernacchia
authors.forename: Morena
authors.initials: M
authors.lastname: De Bastiani
authors.forename: Monica
authors.initials: M
authors.lastname: Evangelista
authors.forename: Rita Maria
authors.initials: RM
authors.lastname: Cantoni
authors.forename: Rino
authors.initials: R
authors.lastname: Cella
authors.forename: Maria Tereasa
authors.initials: MT
authors.lastname: Locci
authors.forename: Letizia
authors.initials: L
authors.lastname: Pitto
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018797:Cell Cycle Proteins; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003596:Cytosine / Q000378:metabolism; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D050684:E2F Transcription Factors; D018506:Gene Expression Regulation, Plant; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D029681:Arabidopsis Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D009841:Oligonucleotides; D020033:Protein Isoforms; D018749:RNA, Plant; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D003596:Cytosine