Characterization of the role of Arabidopsis methyl-readers MBD5 and MBD6 in transcriptional silencing
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Characterization of the role of Arabidopsis methyl-readers MBD5 and MBD6 in transcriptional silencing

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DNA methylation is a well-conserved epigenetic mark that regulates gene expression in many organisms, including animals and plants. Methyl-reader proteins can recognize 5-methylcytosines (mCs) and convert this epigenetic mark into specific gene regulatory outcomes. The molecular mechanisms involved in this process are not fully elucidated, especially in the model plant Arabidopsis thaliana, in which the function of several methyl-readers is unknown. We investigated the family of Arabidopsis methyl-CpG-binding domain (MBD) proteins, and we discovered that two members, MBD5 and MBD6 (MBD5/6), are CG-specific methyl-readers in vitro and in vivo, and they redundantly silence a subset of promoter-methylated genes and transposable elements (TEs). MBD5/6 act via a novel mechanism of transcriptional repression involving a previously uncharacterized J-domain protein that we named SILENZIO. We found that SILENZIO’s conserved J-domain motif was required for its interaction with HSP70s and for its silencing function. By performing single-nucleus RNA-seq (snRNA-seq) we also found that loss of silencing in mbd5 mbd6 and silenzio mutants occurs in the vegetative nucleus (VN) of developing pollen grains. The VN is a very peculiar cell that functions to support the sperm cell during fertilization. It is characterized by a relaxed chromatin state which facilitates access of DNA demethylases to their targets, to remove methylation and allow expression of some genes required for fertilization. We found that MBD5/6 counteract the activity of DNA demethylases in the VN to prevent demethylation and aberrant expression of peri-centromeric genes and TEs. Indeed, simultaneous analysis of cytosine methylation and RNA via single-nucleus methylcytosine and transcriptome sequencing (snmCT-seq) revealed that loss of methylation in mbd5/6 occurs only in the VN of pollen and precedes transcriptional derepression. MBD5/6 act in part by interfering with the function of MBD7, another CG-specific methyl-reader that works as an antisilencer through DNA demethylases. Indeed, both CG methylation and RNA expression at MBD5/6’ targets are decreased in mbd5 mbd6 mutant pollen but largely restored in mbd5 mbd6 mbd7 mutants. Overall, these results highlight the discovery of a role for MBD5 and MBD6 in transcriptional silencing, through the novel J-domain chaperone SILENZIO. We propose that cell types in which chromatin is decondensed, like the pollen VN, require MBD5/6 to protect accessible DNA from the activity of DNA demethylases.

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This item is under embargo until November 1, 2024.