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| Status |
Public on Jan 01, 2017 |
| Title |
Decoupling the downstream effects of germline nuclear RNAi reveals that transcriptional repression and heritable RNAi are independent of the H3K9me3 response in C. elegans [RNA-seq] |
| Organism |
Caenorhabditis elegans |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Germline nuclear RNAi in C. elegans is a transgenerational gene-silencing pathway that leads to the H3K9 trimethylation (H3K9me3) response and transcriptional repression of target genes. The H3K9me3 response induced either by exogenous dsRNA or endogenous siRNA (endo-siRNA) is highly specific to the target loci and transgenerationally heritable. Despite these features, the role of H3K9me3 in transcriptional repression and heritable gene silencing at native target genes has not been tested. To resolve this gap, we first determined that the combined activities of three H3K9 histone methyltransferases (HMTs), MET-2, SET-25, and SET-32, are responsible for virtually all of the detectable level of germline nuclear RNAi-dependent H3K9me3 at native genes, triggered either by exogenous dsRNA or endo-siRNAs. By performing RNA Polymerase II ChIP-seq and pre-mRNA-seq analyses, we found that the loss of the H3K9me3 response at germline nuclear RNAi targets in the met-2;set-25;set-32 mutant does not lead to any defect in transcriptional repression or heritable RNAi. Therefore, H3K9me3 is not required for exogenous dsRNA-induced heritable RNAi or the maintenance of endo siRNA-mediated transcriptional silencing in C. elegans germline. This study provides a unique paradigm in which transcriptional silencing and heterochromatin, triggered by the same upstream pathway, can be decoupled.
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| Overall design |
In this study we tested if RNAi-mediated H3K9me3 is required for the heritable RNAi and transcriptional silencing at native endogenous and exogenous RNAi targets. Using genetic approach we generated nearly completely deficient H3K9me3 worm strain (met-2;set-25;set-32). Using Pol II ChIP-seq, pre-mRNA-seq and mRNA-seq we validated transcriptional changes at the endogenous targets in the H3K9me3 deficient condition (met-2;set-25;set-32). We performed oma-1 dsRNA feeding and heritable RNAi experiment and using H3K9me3 ChIP-seq measured level of RNAi-triggered H3K9me3 contribution by set-32 or met-2;set-25 or met-2;set-25;set-32 HMTs at the oma-1 gene. Using oma-1 mRNA and pre-mRNA qRT-PCR we tested heritable RNAi effect at oma-1 genomic locus in these HMT mutants.
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| Contributor(s) |
Kalinava N, Ni JZ, Peterman K, Chen E, Gu SG |
| Citation(s) |
28228846 |
| NIH grant(s) |
| Grant ID |
Grant title |
Affiliation |
Name |
| R01 GM111752 |
RNA-mediated Chromatin Regulation and Epigenetic Inheritance in C. elegans |
RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY |
Guoping Gu |
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| Submission date |
Sep 07, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Sam Guoping Gu |
| E-mail(s) |
ggu@dls.rutgers.edu
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| Organization name |
Rutgers University
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| Department |
Molecular Biology and Biochemistry
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| Street address |
604 Allison Road
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| City |
Piscataway |
| State/province |
New Jersey |
| ZIP/Postal code |
08854 |
| Country |
USA |
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| Platforms (1) |
| GPL13657 |
Illumina HiSeq 2000 (Caenorhabditis elegans) |
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| Samples (5)
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| This SubSeries is part of SuperSeries: |
| GSE86517 |
Decoupling the downstream effects of germline nuclear RNAi reveals that transcriptional repression and heritable RNAi are independent of the H3K9me3 response in C. elegans |
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| Relations |
| BioProject |
PRJNA342126 |
| SRA |
SRP087443 |
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