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

Abstract

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.

The Three Methyl-CpG-binding Domains of AtMBD7 Control Its Subnuclear Localization and Mobility

“…Purified HisAt-MBD6 was incubated with GST-AtMBDs and precipitated with glutathione-Sepharose beads. Western blot experiments using anti-His 6 antibody verified physical interaction of HisAtMBD6 and GST-AtMBD5, GST-AtMBD6, and GST-mbd 6 (Fig. 4C).…”

“…4B shows that among the GST-AtMBD proteins tested, GST-AtMBD5 (lane 4) and GST-AtMBD6 (lane 5) precipitated AtMBD5-GFP; slight interaction was also evident with the GST-AtMBD7 protein but not with the GST-AtMBD2 protein. Furthermore, the MBD motif of AtMBD6 (GST-mbd 6 , lane 8), but not of AtMBD2 (GST-mbd 2 , lane 7), was sufficient to pull down AtMBD5-GFP from nuclear extracts. To learn whether this interaction is direct or mediated by additional nuclear proteins, we performed pulldown experiments using His-tagged AtMBD6 recombinant protein.…”

“…Construction of AtMBD-GFP/mRFP Plasmids-Fusion of AtMBD5, AtMBD6, and AtMBD7 to GFP was described previously (6). To generate AtMBD5 and AtMBD7 derivatives fused to GFP, we amplified the various fragments by PCR using the following primers: for AtMBD5-(1-92), MBD5-S (5Ј-TGATA-TCAGATCTATGTCGAACGGCACGGATCAG-3Ј) and MBD5(1-92aa)-AS (5Ј-TCTCCCCGGGTTCCGCTGTCTT-GACGGAC-3Ј); for AtMBD7-(1-231), MBD7-S (5Ј-GAGAA-GATCTAGAATGCAGACGAGATCCTCTTCCTCTC-3Ј) and MBD7(231aa)-AS (5Ј-TCTCCCCGGGGTCAAGTGTA-ATGTTCCCG-3Ј); for AtMBD7-(103-231), MBD7(103aa)-S (5Ј-GAGGGATCCATGGATCACTGCGGAGTTGAAT-ATG-3Ј) and MBD7(231aa)-AS; for AtMBD7-(173-231), MBD7(173aa)-S (5Ј-GAGAAGATCTATGCGCCGTGGCCA-TTCCAAA-3Ј) and MBD7(231aa)-AS; and for AtMBD7-(103-175), MBD7(103aa)-S (5Ј-GAGGGATCCATGGATCACTGC-GGAGTTGAATATG-3Ј) and MBD7(175aa)-AS (5Ј-TCTCC-CCGGGGCCACGGCGATTCTGTAAAAC-3Ј).…”

“…Purified HisAtMBD6 (5 g) or nuclear extract (100 g) derived from transgenic Arabidopsis plants expressing AtMBD5-GFP was subjected to GST pulldown assay (9) using GST alone or the indicated GST-AtMBDs described previously (6,8). Precipitated proteins were resolved by SDS-PAGE and immunoblotted using either anti-His 6 monoclonal antibody (Covance) or anti-GFP (B-2) monoclonal antibody (Santa Cruz Biotechnology).…”

“…This chromocenter localization is CpG methylation-dependent inasmuch as it was disrupted in the hypomethylated mutants ddm1 and met1 (6). Yet, these three AtMBDs display different localization patterns within Arabidopsis nuclei; AtMBD7 is predominantly localized at all ChCs, whereas AtMBD5 and AtMBD6 show preference for two perinucleolar ChCs adjacent to the ribosomal DNA clusters (6). A major structural feature distinguishing AtMBD7 from AtMBD5 and AtMBD6 is the presence of three MBD motifs within its protein sequence.…”