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GEO help: Mouse over screen elements for information. |
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| Status |
Public on May 30, 2013 |
| Title |
Bisulfite-Seq profiling of wild-type Arabidopsis with two-component transgene silencing (T+S) system |
| Organism |
Arabidopsis thaliana |
| Experiment type |
Methylation profiling by high throughput sequencing
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| Summary |
In plants, RNA polymerase II (Pol II) transcription of inverted DNA repeats produces hairpin RNAs that are processed by several DICER-LIKE enzymes into siRNAs that are 21-24-nt in length. When targeted to transcriptional regulatory regions, the 24-nt size class can induce RNA-directed DNA methylation (RdDM) and transcriptional gene silencing (TGS). In a forward genetic screen to identify mutants defective in RdDM of a target enhancer leading to TGS of a downstream GFP reporter gene in Arabidopsis thaliana, we recovered a structurally mutated silencer locus, named SΔ35S, in which the 35S promoter driving transcription of an inverted repeat of target enhancer sequences had been specifically deleted. Although Pol II-dependent, hairpin-derived 21-24-nt siRNAs were no longer generated at the newly created SΔ35S locus, the GFP reporter gene was nevertheless still partially silenced. Silencing was associated with methylation in a short tandem repeat in the upstream target enhancer and with low levels of 24-nt tandem repeat siRNAs. Introducing an nrpd1 mutation into the SΔ35S line fully released GFP silencing and eliminated both the tandem repeat methylation and associated 24-nt siRNAs, demonstrating their dependence on Pol IV. Deletion of the 35S promoter thus revealed a Pol IV-dependent pathway of 24-nt siRNA biogenesis that was previously inhibited or masked by the Pol II-dependent pathway in wild-type plants. Both Pol II- and Pol IV-dependent siRNAs accrued predominantly from cytosine (C)-containing segments of the tandem repeat monomer, suggesting that the local base composition influenced siRNA accumulation. Preferential accumulation of siRNAs at C-containing sequences was also observed at an endogenous tandem repeat comprising discrete C-rich and AT-rich sections. Our studies illuminate the potential complexity of siRNA generation at repeat-containing loci and show that Pol IV can act in siRNA biogenesis in the absence of a conventional Pol II promoter.
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| Overall design |
Examination of whole-genome DNA methylation status in transgenic T+S Arabidopsis plant
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| Contributor(s) |
Chen P, Matzke M, Sasaki T |
| Citation(s) |
24798377, 25819795 |
| Submission date |
May 29, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Pao-Yang Chen |
| Organization name |
Academia Sinica
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| Department |
Institute of Plant and Microbial Biology
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| Lab |
Pao-Yang Chen
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| Street address |
128 Sec. 2, Academia Rd, Nankang
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| City |
Taipei |
| State/province |
Taiwan |
| ZIP/Postal code |
11529 |
| Country |
Taiwan |
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| Platforms (1) |
| GPL13222 |
Illumina HiSeq 2000 (Arabidopsis thaliana) |
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| Samples (1) |
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| Relations |
| BioProject |
PRJNA205631 |
| SRA |
SRP023257 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE47453_RAW.tar |
216.5 Mb |
(http)(custom) |
TAR (of TXT) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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