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Series GSE45368 Query DataSets for GSE45368
Status Public on May 13, 2013
Title Polymerase-IV occupancy at RNA-directed DNA methylation sites requires SHH1
Organism Arabidopsis thaliana
Experiment type Non-coding RNA profiling by high throughput sequencing
Genome binding/occupancy profiling by high throughput sequencing
Methylation profiling by high throughput sequencing
Summary 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.
 
Overall design 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 (‚Äúcomplementation replicates‚ÄĚ).
 
Contributor(s) Hale CJ
Citation(s) 23636332
Submission date Mar 20, 2013
Last update date May 15, 2019
Contact name Christopher Joel Hale
E-mail(s) chris.joel.hale@gmail.com
Organization name University of Washington
Department Pathology
Lab Center for Precision Diagnostics
Street address 1959 NE Pacific St., HSC H-458
City Seattle
State/province WA
ZIP/Postal code 98195
Country USA
 
Platforms (1)
GPL13222 Illumina HiSeq 2000 (Arabidopsis thaliana)
Samples (28)
GSM1103230 Columbia ChIP
GSM1103231 PolIV ChIP
GSM1103232 Columbia ChIP replicate 2
Relations
SRA SRP019835
BioProject PRJNA193689

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Supplementary file Size Download File type/resource
GSE45368_RAW.tar 779.7 Mb (http)(custom) TAR (of WIG)
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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