Arabidopsis MBD Proteins Show Different Binding Specificities and Nuclear Localization

Plant Mol Biol. 2003 Nov;53(5):715-31. doi: 10.1023/B:PLAN.0000019118.56822.a9.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Arabidopsis / cytology
  • Arabidopsis / genetics
  • Arabidopsis / metabolism
  • Arabidopsis Proteins / genetics
  • Arabidopsis Proteins / metabolism*
  • Base Sequence
  • Binding Sites / genetics
  • Cell Cycle Proteins*
  • Cell Line
  • Cell Nucleus / metabolism*
  • Cytosine / metabolism
  • DNA Methylation
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • E2F Transcription Factors
  • Gene Expression Regulation, Plant
  • Green Fluorescent Proteins
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Microscopy, Confocal
  • Molecular Sequence Data
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • Oligonucleotides / genetics
  • Oligonucleotides / metabolism
  • Protein Binding
  • Protein Isoforms / genetics
  • Protein Isoforms / metabolism
  • RNA, Plant / genetics
  • RNA, Plant / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Homology, Amino Acid
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Transfection

Substances

  • Arabidopsis Proteins
  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • E2F Transcription Factors
  • Luminescent Proteins
  • Nuclear Proteins
  • Oligonucleotides
  • Protein Isoforms
  • RNA, Plant
  • Recombinant Fusion Proteins
  • Transcription Factors
  • Green Fluorescent Proteins
  • Cytosine