Abstract
RNA-directed DNA methylation (RdDM) is a plant-specific de novo DNA methylation mechanism that requires long noncoding RNA (lncRNA) as scaffold to define target genomic loci. While the role of RdDM in maintaining genome stability is well established, how it regulates protein-coding genes remains poorly understood and few RdDM target genes have been identified. In this study, we obtained sequences of RdDM-associated lncRNAs using nuclear RNA immunoprecipitation against ARGONAUTE 4 (AGO4), a key component of RdDM that binds specifically with the lncRNA. Comparison of these lncRNAs with gene expression data of RdDM mutants identified novel RdDM target genes. Surprisingly, a large proportion of these target genes were repressed in RdDM mutants suggesting that they are normally activated by RdDM. These RdDM-activated genes are more enriched for gene body lncRNA than the RdDM-repressed genes. Histone modification and RNA analyses of several RdDM-activated stress response genes detected increased levels of active histone mark and short RNA transcript in the lncRNA-overlapping gene body regions in the ago4 mutant despite the repressed expression of these genes. These results suggest that RdDM, or AGO4, may play a role in maintaining or activating stress response gene expression by directing gene body chromatin modification preventing cryptic transcription.
Highlights
RNA-directed DNA methylation (RdDM) can result in transcriptional gene silencing in plants [1,2,3].Of the three major RNA silencing pathways in plants, RdDM is the sole pathway capable of mediatingDNA methylation [4,5,6,7,8]
Western blot analysis showed that FLAG:ARGONAUTE 4 (AGO4) protein accumulated in a tissue-specific manner despite being driven by the 35S promoter, with the highest level in flowers (Figure S2A) where 24-nt siRNA levels are relatively abundant [32,33]
This is consistent with the previous report suggesting that AGO4 protein accumulation depends on 24-nt siRNAs [13], and implies that the pattern of FLAG:AGO4 accumulation mimics that of the endogenous AGO4 protein, despite being expressed from the heterologous 35S promoter
Summary
RNA-directed DNA methylation (RdDM) can result in transcriptional gene silencing in plants [1,2,3].Of the three major RNA silencing pathways in plants, RdDM is the sole pathway capable of mediatingDNA methylation [4,5,6,7,8]. RNA-directed DNA methylation (RdDM) can result in transcriptional gene silencing in plants [1,2,3]. Of the three major RNA silencing pathways in plants, RdDM is the sole pathway capable of mediating. DNA methylation [4,5,6,7,8]. RdDM plays a fundamental role in plant defense against invasive DNA and in maintaining genome stability by silencing transposons and repetitive sequences. RdDM has been shown to regulate protein-coding genes [9,10], but this aspect of RdDM function has not been well studied, and very few RdDM-targeted genes have been identified. Much effort over the past decade has been attributed to the characterization of the components and the molecular mechanism of RdDM allowing a canonical pathway to be well established.
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