Three SRA-Domain Methylcytosine-Binding Proteins Cooperate to Maintain Global CpG Methylation and Epigenetic Silencing in Arabidopsis

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Abstract
Methylcytosine-binding proteins decipher the epigenetic information encoded by DNA methylation and provide a link between DNA methylation, modification of chromatin structure, and gene silencing. VARIANT IN METHYLATION 1 (VIM1) encodes an SRA (SET- and RING-associated) domain methylcytosine-binding protein in Arabidopsis thaliana, and loss of VIM1 function causes centromere DNA hypomethylation and centromeric heterochromatin decondensation in interphase. In the Arabidopsis genome, there are five VIM genes that share very high sequence similarity and encode proteins containing a PHD domain, two RING domains, and an SRA domain. To gain further insight into the function and potential redundancy among the VIM proteins, we investigated strains combining different vim mutations and transgenic vim knock-down lines that down-regulate multiple VIM family genes. The vim1 vim3 double mutant and the transgenic vim knock-down lines showed decreased DNA methylation primarily at CpG sites in genic regions, as well as repeated sequences in heterochromatic regions. In addition, transcriptional silencing was released in these plants at most heterochromatin regions examined. Interestingly, the vim1 vim3 mutant and vim knock-down lines gained ectopic CpHpH methylation in the 5S rRNA genes against a background of CpG hypomethylation. The vim1 vim2 vim3 triple mutant displayed abnormal morphological phenotypes including late flowering, which is associated with DNA hypomethylation of the 5′ region of FWA and release of FWA gene silencing. Our findings demonstrate that VIM1, VIM2, and VIM3 have overlapping functions in maintenance of global CpG methylation and epigenetic transcriptional silencing. Methylation of cytosine bases provides one layer of epigenetic information that is superimposed on the nucleotide sequence of a genome. Proteins that bind methylated cytosines and also help maintain that DNA modification are important linchpins in a self-propagating system mediating memory of epigenetic states. We previously demonstrated that the VIM1 (VARIANT IN METHYLATION 1) protein from the flowering plant Arabidopsis thaliana binds DNA that contains methylated cytosine and is required for complete methylation and compaction of centromeric DNA. In this study, we show that VIM1 works in concert with two related proteins, VIM2 and VIM3, to maintain cytosine methylation not only at centromeres but throughout the genome. VIM proteins act specifically in the DNA methylation pathway that targets CpG dinucleotides, which plants share with animals, rather than the plant-specific non-CpG methylation pathways. Loss of VIM1, VIM2, and VIM3 function also causes a reduction in transcriptional gene silencing at a variety of sequences, and leads to abnormal developmental phenotypes, including late flowering associated with loss of FWA gene silencing. Our results demonstrate that these three related VIM family proteins have overlapping functions in the MET1-mediated CpG methylation pathway.

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