Stress resets ancestral heritable small RNA responses

  1. Leah Houri-Zeevi  Is a corresponding author
  2. Guy Teichman  Is a corresponding author
  3. Hila Gingold
  4. Oded Rechavi  Is a corresponding author
  1. Department of Neurobiology, Wise Faculty of Life Sciences & Sagol School of Neuroscience, Tel Aviv University, Israel
6 figures, 1 table and 8 additional files

Figures

Figure 1 with 5 supplements
Stress resets heritable small RNA silencing.

(AExperimental scheme. Heritable small RNA responses are initiated at the first generation, and the F1 progeny are then subjected to three different stress types (heat shock {HS}, hyperosmotic …

Figure 1—figure supplement 1
Multiple durations and magnitudes of stress can induce resetting of small RNAs.

(A) Both 3 day long and 6 day long starvation lead to resetting of a heritable RNAi response. Each condition was tested separately, and they are displayed side-by-side for readability. The graph …

Figure 1—figure supplement 2
Starvation and hyperosmotic stress do not affect the basal expression level of the GFP reporter.

(A) Hyperosmotic stress does not affect the basal expression level of the GFP reporter. The graph displays the measured germline GFP fluorescence levels of wild-type worms which were not exposed to …

Figure 1—figure supplement 3
stress expedites the typical diminishment of heritable small RNA responses.

(AHeritable exogenous small RNA silencing is reset by stress. The graph displays the measured germline GFP fluorescence levels of worms (y-axis) across generations (x-axis). Shown are the median …

Figure 1—figure supplement 4
Stress resets heritable small RNAs even when applied during adulthood.

Stress applied during adulthood leads to resetting of heritable silencing in the next generation. The graph displays the measured germline GFP fluorescence levels of individual worms (y-axis) across …

Figure 1—figure supplement 5
Resetting of heritable small RNA responses can only occur during the F1 generation.

Stress applied two generations after the initiation of the heritable small RNAs response does not reset the inheritance. Upper panel: Experimental scheme. A heritable anti-gfp small RNA response is …

Stress resets endo-siRNAs and piRNAs-induced silencing.

(A) Representative images of worms expressing endo-siRNAs sensor (left) or the piRNAs sensor (right) under control or stress (HS) conditions. (B) The graph displays the measured GFP (left, …

Resetting of heritable small RNA responses is induced specifically by stress, also when experienced prior to initiation of RNAi.

(a) Shifting from stress to non-stress conditions fails to reset small RNA inheritance. Upper panel: Experimental scheme. Worms grown in regular growth conditions (20°C, control) or in high …

Genome-wide small RNA changes following stress.

(A) Experimental scheme. Worms were exposed to stress during their first larval stage and were collected for RNA extraction and small RNA sequencing on the first day of adulthood. The next …

Figure 5 with 3 supplements
The MAPK pathway and the SKN-1 transcription factor regulate small RNAs resetting in response to stress.

(A) Mutants defective in stress-responsive genes display altered heritable RNAi dynamics. Heatmap representing the log2-fold change of GFP fluorescence levels (color coded and indicated values) in …

Figure 5—figure supplement 1
Multiple stress signaling and processing pathways affect heritable RNAi dynamics and stress-induced resetting of heritable silencing.

The graphs display the measured germline GFP fluorescence levels of mutant worms (y-axis) across generations under the indicated condition (x-axis). Each dot represents the value of an individual …

Figure 5—figure supplement 2
Mutants of the daf-16, aak-1/2 and daf-2 genes are capable of stress-induced resetting of heritable responses.

The graphs display the measured germline GFP fluorescence levels of mutant worms (y-axis) across generations under the indicated condition (x-axis). Each dot represents the value of an individual …

Figure 5—figure supplement 3
Mutants in the MAPK pathway, the transcription factor SKN-1, and the putative H3K9 methyltransferase MET-2 do not affect the basal expression of the GFP reporter.

The graphs display the measured germline GFP fluorescence levels of worms which were not exposed to RNAi (y-axis) under the indicated genotype (x-axis). Each dot represents the value of an …

Figure 6 with 1 supplement
Stress-induced small RNA resetting depends on the H3K9 methyltransferase MET-2.

(ATargets of stress-affected small RNAs show significantly increased H3K9me2 marks. An analysis of H3K9me2 signal (based on published data from McMurchy et al., 2017). Presented is the averaged …

Figure 6—figure supplement 1
Targets of stress-affected small RNAs show unique H3K9 marks and the H3K9 methyltransferase met-2 is required for the execution of small RNA resetting.

Refers to Figure 6. (A) Targets of stress-affected small RNAs show significantly different H3K9me2 and H3K9me3 marks. An analysis of H3K9me2 and H3K9me3 signals (based on published data from McMurchy…

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiers Additional
information
Chemical compound, drugLevamisole hydrochlorideSigmaL0380000
Chemical compound, drugTrizol ReagentLife Technologies15596026
Chemical compound, drugPhenol Chloroform IsoamylSigmaP2069
OtherHeavy Phase Lock tubeQuantaBio23028330
Chemical compound, drugUltra Pure GlycogenThermoFisher10814010
Peptide, recombinant proteinRNA 5' PolyphosphataseEpicenterRP8092H
Commercial assay, kitNEBNext Multiplex Small RNA Library Prep Set for IlluminaNew England BiolabsE7300
Commercial assay, kit
TapeStation screen tapes
Agilent
5067–5582
5067–5588
Commercial assay, kit
TapeStation reagents
Agilent
5067–5583
5067–5589
Chemical compound, drugE-Gel 4% agaroseLife TechnologiesG401004
Commercial assay, kitMinElute DNA purification kitQiagen28006
Chemical compound, drugRNase free Nuclease-free waterAmbionAM9932
Peptide, recombinant proteinNlaIIINew Englang BioLabs (NEB)631207
Commercial assay, kitTG NextSeq 500/550 High Output Kit v2 (75 cycles)IlluminaTG-160–2005
Strain, strain background (Caenorhabditis elegans)C. elegans: Strain SX1263: unc-119(ed3) III; mex-5::gfp::h2b::tbb-2 IIThe Eric Miska lab (Sapetschnig et al., 2015)SX1263
Strain, strain background (C. elegans)C. elegans: Strain EG6089: unc-119(ed3) III; oxTi38[cb-unc-119(+) Ppie-1::GFP] IVThe Eric Miska lab (Sapetschnig et al., 2015)EG6089
Strain, strain background (C. elegans)C. elegans: Strain JA1527: weSi14 [Pmex-5::mCherry::(Gly)5Ala/his-58/tbb-2 3′UTR; cb-unc-119(+)] IVThe Julie Ahringer lab (Zeiser et al., 2011)JA1527
Strain, strain background (C. elegans)C. elegans: Strain GR1720: mgSi4 [(pCMP2) ubl-1p::GFP::siR-1-sensor-ubl-1–3'UTR + Cbr-unc-119(+)] IVCaenorhabditis Genetics CenterWB strain: GR1720
Strain, strain background (C. elegans)C. elegans: Strain KB3: kgb-1 (um3) IVCaenorhabditis Genetics CenterKB3
Strain, strain background (C. elegans)C. elegans: Strain KB3: kgb-1 (um3) IV crossed with Strain SX1263This study
Strain, strain background (C. elegans)C. elegans: Strain FK171: mek-1(ks54); sek-1(qd127)Caenorhabditis Genetics CenterFK171
Strain, strain background (C. elegans)C. elegans: Strain FK171: mek-1(ks54); sek-1(qd127) X crossed with Strain SX1263This study
Strain, strain background (C. elegans)C. elegans: Strain CF1038: daf-16(mu86) ICaenorhabditis Genetics CenterCF1038
Strain, strain background (C. elegans)C. elegans: Strain CF1038: daf-16(mu86) I, crossed with Strain SX1263This study
Strain, strain background (C. elegans)C. elegans: Strain KU4: sek-1(km4) XCaenorhabditis Genetics CenterKU4
Strain, strain background (C. elegans)C. elegans: Strain KU4: sek-1(km4) X, crossed with Strain SX1263This study
Strain, strain background (C. elegans)C. elegans: Strain KU25: pmk-1(km25) IVCaenorhabditis Genetics CenterKU25
Strain, strain background (C. elegans)C. elegans: Strain KU25: pmk-1(km25) IV, crossed with Strain SX1263This study
Strain, strain background (C. elegans)C. elegans: Strain MR1175: aak-1(tm1944) III, aak-2(ok524) XThe Richard Roy lab (Demoinet et al., 2017)MR1175
Strain, strain background (C. elegans)C. elegans: Strain MR1175: aak-1(tm1944) III, aak-2(ok524) X, crossed with Strain SX1263This study
Strain, strain background (C. elegans)C. elegans: Strain PS355: hsf-1(sy441) ICaenorhabditis Genetics CenterPS355
Strain, strain background (C. elegans)C. elegans: Strain PS355: hsf-1(sy441) I, crossed with Strain SX1263This study
Strain, strain background (C. elegans)C. elegans: Strain CB1370: daf-2(e1370) IIICaenorhabditis Genetics CenterCB1370
Strain, strain background (C. elegans)C. elegans: Strain CB1370: daf-2(e1370) III, crossed with Strain EG6089This study
Strain, strain background (C. elegans)C. elegans: Strain QV225: skn-1(zj15) IVCaenorhabditis Genetics CenterQV225
Strain, strain background (C. elegans)C. elegans: Strain QV225: skn-1(zj15) IV, crossed with Strain SX1263This study
Strain, strain background (C. elegans)C. elegans: Strain MT13293: met-2(n4256) IIICaenorhabditis Genetics CenterMT13293
Strain, strain background (C. elegans)C. elegans: Strain MT13293: met-2(n4256) III, crossed with Strain SX1263This study
Strain, strain background (C. elegans)C. elegans: Strain RB1789: met-2(ok2307) IIICaenorhabditis Genetics CenterRB1789
Strain, strain background (C. elegans)C. elegans: Strain RB1789: met-2(ok2307) III, crossed with Strain SX1263This study
Strain, strain background (C. elegans)C. elegans: Strain KU4: sek-1(km4) X, crossed with strain JA1527This study
Strain, strain background (C. elegans)C. elegans: Strain ZD202: sek-1(km4) X; qdEx8[unc-119p::sek-1(cDNA)::GFP::unc-54–3' UTR + myo-2p::mStrawberry::unc-54–3' UTR], crossed with strain JA1527This study
Strain, strain background (C. elegans)C. elegans: Strain ZD193: sek-1(km4) X; qdEx4 [ges-1p::sek-1(cDNA)::GFP::unc-54–3' UTR + myo-2p::mStrawberry::unc-54–3' UTR], crossed with strain JA1527This study
Strain, strain background (C. elegans)C. elegans: Strain OH14221: met-2(ot861[met-2::mKate2]) IIICaenorhabditis Genetics Center (Patel and Hobert, 2017)OH14221
Strain, strain background (C. elegans)C. elegans: Strain OH14221: met-2(ot861[met-2::mKate2]) III crossed with strain QV225This study
Sequence-based reagentPCR 1-FWD: mek-1/sek-1IDTTTTCCATCAACTCAGTCGCCG
Sequence-based reagentPCR 1-REV1: mek-1/sek-1IDTTTCATTAGTCAATTGGGTCAG
Sequence-based reagentPCR 1-REV2: mek-1/sek-1IDTCACTTTTCAATTAAGGTACAAC
Sequence-based reagentPCR 2-FWD: kgb-1IDTCCCTACTTTATAATGAGATGC
Sequence-based reagentPCR 2-REV1: kgb-1IDTTTCATTAGTCAATTGGGTCAG
Sequence-based reagentPCR 2-REV2: kgb-1IDTCACTTTTCAATTAAGGTACAAC
Sequence-based reagentPCR 3-FWD: daf-16IDTGTTCAGTAGACGGTGACCATCT
Sequence-based reagentPCR 3-REV1: daf-16IDTGCTTCGGCTTGAAAGATCAGTG
Sequence-based reagentPCR 3-REV2: daf-16IDTGTACGCCGTGGTCCGACTA
Sequence-based reagentPCR 4-FWD: skn-1IDTGAAGAGAATGCTCGATATGAAG
Sequence-based reagentPCR 4-REV: skn-1IDTTTTCAGTCGTTTATAAGAGAGC
Sequence-based reagentPCR 5-FWD: aak-1IDTATCGATACGGAACCAACTG
Sequence-based reagentPCR 5-REV: aak-1IDTGGGTATGGTAGTACCAATAGG
Sequence-based reagentPCR 6-FWD: aak-2IDTCGATAGCACAGACAACAGTTCG
Sequence-based reagentPCR 6-REV: aak-2IDTGATGGTGGCCCTCTTCATC
Sequence-based reagentPCR 7-FWD1: daf-18IDTAGGGTAATGCATTTCAGCAC
Sequence-based reagentPCR 7-FWD2: daf-18IDTCCCGCATATAAACTGGAAATGTG
Sequence-based reagentPCR 7-REV: daf-18IDTCAAATACGTCAGTTTCAACGTG
Sequence-based reagentPCR 8-FWD: pmk-1IDTCTATAAGTTGCCATGACCTC
Sequence-based reagentPCR 8-REV: pmk-1IDTGCTCCCATCAACATTGATAC
Sequence-based reagentPCR 9-FWD1: sek-1IDTCTAGAATAAGTGCTATGCTAG
Sequence-based reagentPCR 9-FWD2: sek-1IDTGTTGTCTAAGTATAATTGTCC
Sequence-based reagentPCR 9-REV: sek-1IDTTGATTGATTATAACTACGAGG
Sequence-based reagentPCR 10-FWD1: met-2IDTTTTACTGTCACATCACCTGC
Sequence-based reagentPCR 10-FWD2: met-2IDTAAGCAGATGTTTGTCAGAATCC
Sequence-based reagentPCR 10-REV: met-2IDTAGCAGCATTCATCTTCGC
Software, algorithmFastQCAndrews, 2010
Software, algorithmCutadaptMartin, 2011
Software, algorithmShortstackShahid and Axtell, 2014
Software, algorithmHTSeq countAnders et al., 2014
Software, algorithmR Deseq2Love et al., 2014
Software, algorithmRNAlysisTeichman, 2019Version 1.3.5
Software, algorithmFijiSchindelin et al., 2012
Software, algorithmMATLAB R2018bMATLABVersion R2018b
Software, algorithmIoSR-Surry MatlabToolbox (BoxPlot function)Institute of Sound Recording, University of Surreyhttps://github.com/IoSR-Surrey/MatlabToolboxVersion 2.8
Software, algorithmGraphPad Prism 8GraphPad Softwarehttp://www.graphpad.com/Version 8.0.0

Additional files

Supplementary file 1

A total of 281 targets of small RNAs which were affected across all stress conditions at the stress generation.

Table presents DESeq2 comparison of Control vs. Stress samples. Related to Figure 4.

https://cdn.elifesciences.org/articles/65797/elife-65797-supp1-v2.csv
Supplementary file 2

Ten targets of small RNAs which were affected across all stress conditions at the next generation.

Table presents DESeq2 comparison of Control vs. Stress samples. Related to Figure 4.

https://cdn.elifesciences.org/articles/65797/elife-65797-supp2-v2.csv
Supplementary file 3

Enrichment table for the 281 targets of stress-affected genes, generated using WormExp (Yang et al., 2016).

Related to Figure 4.

https://cdn.elifesciences.org/articles/65797/elife-65797-supp3-v2.csv
Supplementary file 4

Seventy-three epigenetic-related genes significantly downregulated under all stress conditions.

https://cdn.elifesciences.org/articles/65797/elife-65797-supp4-v2.csv
Supplementary file 5

Thirty-five epigenetic-related skn-1-dependent stress-dependent genes.

https://cdn.elifesciences.org/articles/65797/elife-65797-supp5-v2.csv
Supplementary file 6

Enrichment table for skn-1-dependent stress-dependent genes, generated using RNAlysis (Teichman, 2019).

https://cdn.elifesciences.org/articles/65797/elife-65797-supp6-v2.csv
Supplementary file 7

Sixty-three epigenetic-related genes whose putative promoter regions contain the binding motif sequence of SKN-1, generated using TF2DNA (Pujato et al., 2014).

https://cdn.elifesciences.org/articles/65797/elife-65797-supp7-v2.csv
Transparent reporting form
https://cdn.elifesciences.org/articles/65797/elife-65797-transrepform-v2.docx

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