The three methyl-CpG-binding domains of AtMBD7 control its subnuclear localization and mobility

Three methyl-CpG-binding domain (MBD) proteins in Arabidopsis, AtMBD5, AtMBD6, and AtMBD7, are functional in binding methylated CpG dinucleotides in vitro and localize to the highly CpG-methylated chromocenters in vivo. These proteins differ, however, in their subnuclear localization pattern; AtMBD5 and AtMBD6, each containing a single MBD motif, show preference for two perinucleolar chromocenters, whereas AtMBD7, a naturally occurring poly-MBD protein containing three MBD motifs, localizes to all chromocenters. Here we studied the significance of multiple MBD motifs for subnuclear localization and mobility in living cells. We found that the number of MBD motifs determines the subnuclear localization of the MBD protein. Furthermore, live kinetic experiments showed that AtMBD7-green fluorescent protein (GFP) has lower mobility than AtMBD5-GFP and AtMBD6-GFP, which is conferred by cooperative activity of its three MBD motifs. Thus, the number of MBD motifs appears to affect not only binding affinity and mobility within the nucleus, but also the subnuclear localization of the protein. Our results suggest that poly-MBD proteins can directly affect chromatin structure by inducing intra- and inter-chromatin compaction via bridging over multiple methylated CpG sites.     

Different domains control the localization and mobility of LIKE HETEROCHROMATIN PROTEIN1 in Arabidopsis nuclei

Plants possess a single gene for the structurally related HETEROCHROMATIN PROTEIN1 (HP1), termed LIKE-HP1 (LHP1). We investigated the subnuclear localization, binding properties, and dynamics of LHP1 proteins in Arabidopsis thaliana cells. Transient expression assays showed that tomato (Solanum lycopersicum) LHP1 fused to green fluorescent protein (GFP; Sl LHP1-GFP) and Arabidopsis LHP1 (At LHP1-GFP) localized to heterochromatic chromocenters and showed punctuated distribution within the nucleus; tomato but not Arabidopsis LHP1 was also localized within the nucleolus. Mutations of aromatic cage residues that recognize methyl K9 of histone H3 abolished their punctuated distribution and localization to chromocenters. Sl LHP1-GFP plants displayed cell type-dependent subnuclear localization. The diverse localization pattern of tomato LHP1 did not require the chromo shadow domain (CSD), whereas the chromodomain alone was insufficient for localization to chromocenters; a nucleolar localization signal was identified within the hinge region. Fluorescence recovery after photobleaching showed that Sl LHP1 is a highly mobile protein whose localization and retention are controlled by distinct domains; retention at the nucleolus and chromocenters is conferred by the CSD. Our results imply that LHP1 recruitment to chromatin is mediated, at least in part, through interaction with methyl K9 and that LHP1 controls different nuclear processes via transient binding to its nuclear sites.