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Small RNA profiling of WT, ros1-4, nrpd1 single mutants and ros1-4/nrpd1doubble mutant [small RNA-seq]

ABSTRACT: Arabidopsis ROS1 is the first genetically characterized DNA demethylase in eukaryotes. Dysfunction of ROS1 leads to increase in DNA methylation level at thousands of genomic loci. However, the features of ROS1 targets are not well understood. In this study, we identified and characterized ROS1 target loci in Arabidopsis Col-0 and C24 ecotypes. Most ROS1 targets are transposable elements (TEs) and intergenic regions. Compared to other TEs, ROS1-targeted TEs are closer to protein coding genes, suggesting a role for ROS1 in preventing the spreading of DNA methylation from highly methylated TEs to nearby genes. Interestingly, we found that unlike general TEs, ROS1 targets are associated with an enrichment of H3K18ac and H3K27me3, and depletion of H3K27me and H3K9me2. We investigated the antagonism between ROS1 and RNA-directed DNA methylation (RdDM) by identifying and characterizing thousands of genomic regions regulated by both ROS1 and RdDM. Unexpectedly, we uncovered thousands of previously unidentified RdDM targets by analyzing the DNA methylome of ros1/nrpd1 double mutant plants. In addition, we show that ROS1 also antagonizes RdDM-independent DNA methylation at more than a thousand genomic loci. Our results provide significant insights into the genome-wide effects of both ROS1-mediated active DNA demethylation and RNA-directed DNA methylation as well as their interaction in plants. Using small RNA-Seq(sRNA-Seq) to get small RNA profiling of WT, ros1-4, nrpd1 single mutants and ros1-4/nrpd1doubble mutant

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE83800 | GEO | 2016/11/01

SECONDARY ACCESSION(S): PRJNA327041

REPOSITORIES: GEO

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Project description:DNA cytosine methylation is a hallmark of epigenetic gene silencing. DNA demethylation requires ROS1, a bifunctional DNA glycosylase/lyases and open chromatin status mediated by IDM1. HDA6 is a RDP3-like histone deacetylase and was confirmed to mediate DNA methylation. In previous screening for ros1 suppressor, we identified two hda6 mutants reverting ros1-caused hypermethylation at RD29A and 35S promoters respectively, indicating the antagonization of DNA methylation between HDA6 and ROS1. To learn antagonized effects between HDA6 and ROS1 at DNA cytosine methylation genome-wide, we performed whole genome bisulfite sequencing to search antagonized targets of HDA6 and ROS1 and their specific targets to evaluate their roles on DNA methylation. To evaluate HDA6’s roles in sRNA biogenesis and nucleosome positioning, we also performed small RNA sequencing and genome-wide mapping of nucleosome positioning of C24, ros1, hda6-9 and hda6-10. Our results indicate that around 43% ros1-caused CG hypermethylation, 74.5% and 84.5% ros1-caused CHG and CHH hypermethylation were reverted by the two hda6 alleles in the ros1 background respectively. These results indicate that most of ROS1-demethylated targets are also HDA6-mediated DNA methylated targets. In addition, we observed that HDA6-affected DNA methylation targets are far more than ROS1-demethylation targets at CHG and CHH context, but not at CG context. sRNA analysis showed that HDA6 inhibits LTR/Gypsy and TE gene 24nt siRNA accumulation, while promotes RC/Helitron 24nt siRNA accumulation. Our Mono-nucleosome positioning data further showed that the two hda6 alleles have striking difference on nucleosome positioning, hda6-10 obviously have different nucleosome positioning patterns with hda6-9. Our results indicate HDA6 not only mediates overall DNA methylation at CG, CHG and CHH context, and antagonizes with ROS1 mediated DNA demethylation, but also regulates nucleosome positioning and small RNA accumulation at some genome specific regions.

Project description:In plants, RNA-directed DNA methylation (RdDM) pathway is a well-known de novo DNA methylation pathway that is involved in many important biological processes, such as pathogen defense, stress response and plant development. RdDM involves two plant-specific RNA polymerases, Pol IV and Pol V. In previous studies, several genetic screen systems, including a release of silencing 1 (ros1) suppressor screen, have been designed and used to discover new RdDM components. Through the ros1 suppressor screen, several new RdDM components, such as RDM1 and KTF1, have been reported. In this study, we isolated a new ros1 suppressor mutant, rdm15. Through DNA methylome and whole-genome siRNA analyses, we showed that RDM15 was required for RdDM-dependent DNA methylation and siRNA accumulation at a subset of RdDM target loci. Our further investigation revealed that RDM15 mainly contributes to Pol V-dependent downstream siRNA accumulation, and can interact with NRPE3B, a subunit specific to Pol V. DNA methylation and histone modifications are known to interact with each other to determine DNA methylation and chromatin states. Here, we showed that RDM15 can specifically recognize the histone 3 lysine 4 monomethylation (H3K4me1) mark by its C-terminal tudor domain. The structure of RDM15 tudor domain in complex with an H3K4me1 peptide highlights the specific recognition of H3K4me1 by an aromatic cage and specific hydorgen bonding interactions, which is different from all previously reported lower methyllysine recognition mechanisms. In addition, at the genome wide level, RDM15 and H3K4me1 showed similar distribution patterns at RDM15-dependent RdDM loci, suggesting that RDM15 recognition of H3K4me1 is associated with RDM15 function in RdDM. In summary, we identified and characterized a histone H3K4me1 specific binding protein as a new RdDM component, and the structural analysis of RDM15 revealed a new type of chemical feature-based lower methyllysine recognition mechanism.

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Small RNA profiling of WT, ros1-4, nrpd1 single mutants and ros1-4/nrpd1doubble mutant [small RNA-seq]

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