Project description:DNA methylation is an epigenetic modification that plays critical roles in gene silencing, development, and the maintenance of genome integrity. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) and is targeted by 24 nt small interfering RNAs (siRNAs) through a pathway termed RNA-directed DNA methylation (RdDM)1. This pathway requires two plant-specific RNA polymerases: Pol-IV, which functions to initiate siRNA biogenesis and Pol-V, which functions in the downstream DNA methyltransferase targeting phase of the RdDM pathway to generate scaffold transcripts that recruit downstream RdDM factors1,2. To understand the mechanisms controlling Pol-IV targeting we investigated the function of SAWADEE HOMEODOMAIN HOMOLOG 1 (SHH1)3,4, a Pol-IV interacting protein3. Here we show that SHH1 acts upstream in the RdDM pathway to enable siRNA production from a large subset of the most active RdDM targets and that SHH1 is required for Pol-IV occupancy at these same loci. We also show that the SHH1 SAWADEE domain is a novel chromatin binding module that adopts a unique tandem Tudor-like fold and functions as a dual lysine reader, probing for both unmethylated K4 and methylated K9 modifications on the histone 3 (H3) tail. Finally, we show that key residues within both lysine binding pockets of SHH1 are required in vivo to maintain siRNA and DNA methylation levels as well as Pol-IV occupancy at RdDM targets, demonstrating a central role for methyl H3K9 binding in SHH1 function and providing the first insights into the mechanism of Pol-IV targeting. Given the parallels between methylation systems in plants and mammals1,5, a further understanding of this early targeting step may aid in our ability to control the expression of endogenous and newly introduced genes, which has broad implications for agriculture and gene therapy. For wild type plants (ecotype Columbia) and RdDM mutants whole-genome small RNA (sRNA-seq) and bisulfite sequencing (BS-seq) was performed. The Col and nrpe1 BS-seq libraries were previously reported (GSE39247) and so are not part of this submission. In addition, two replicates of whole genome chromatin immunoprecipitation (ChIP-seq) was performed on wild type (ecotype Columbia) plants as a negative control with experimentals consiting of nrpd1 mutant plants carrying a C-terminally epitope tagged (3XFLAG) NRPD1. Whole-genome bisulfite sequencing and small RNA sequencing was also performed on shh1 mutant plants transformed with the wild-type SHH1 protein-coding construct as well as multiple constructs containing point mutations. For these complementation libraries a separate shh1 mutant and Col control line were sequenced (“complementation replicates”).

Project description:Abstract: Cytosine DNA methylation plays crucial roles in gene regulation, transposon silencing, and diverse developmental processes. While methylation patterns are known to differ between cell-types, tissues, and disease states, how these differences arise remains poorly understood. In plants, DNA methylation is established via the RNA-directed DNA methylation pathway (RdDM), wherein 24-nucleotide small interfering RNAs (siRNAs) guide methylation at cognate genomic loci1. RNA POLYMERASE-IV (Pol-IV), a plant-specific polymerase, initiates the biogenesis of these methylation-targeting RNAs, thus understanding how Pol-IV is regulated is critical in determining how specific patterns of DNA methylation are generated. Here we show roles for four Pol-IV-associated factors, CLASSY (CLSY) 1-42,3, in both locus-specific and global regulation of Pol-IV function. Individually, each CLSY protein controls siRNA production and Pol-IV chromatin association at unique set of loci. This translates into locus-specific DNA methylation losses and the release of silencing. In addition to locus-specific effects, several layers of redundancy were identified: The clsy1,2 and clsy3,4 mutants act synergistically, regulating thousands more siRNA loci than the single mutants. Furthermore, the clsy1,2- and clsy3,4-dependent loci are mutually exclusive and geographically distinct, revealing a striking division of labor amongst the CLSY family. Finally, the clsy quadruple mutant causes global siRNA losses, demonstrating that Pol-IV is completely dependent on the CLSY family. Investigation into the mechanisms underlying the CLSY specificity revealed connections between clsy1,2- and clsy3,4-dependent loci and either SAWADEE HOMEODOMAIN HOMOLOG 1 or DNA METHYLTRANSFERASE 1, demonstrating a reliance on different chromatin modifications, H3K9 or CG DNA methylation, respectively4,5. Together, these findings not only shed light on Pol-IV function, but also reveal an additional layer of complexity to the RdDM pathway that enables the locus-specific control of DNA methylation patterns. Given the parallels between methylation systems in plants and mammals1, these findings will be informative for analogous processes in a broad range of organisms.

Dataset Information

Polymerase-IV occupancy at RNA-directed DNA methylation sites requires SHH1

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure