Aim: In order to curb the transmission of SARS-CoV-2, nation-wide travel restrictions at different levels were implemented in different countries. Country-specific mutational profile may exist and have an impact on vaccine efficacy. Materials & methods: We identified nonsynonymous mutations in approximately 215,000 SARS-CoV-2 sequences during the 1st year of the pandemic in 35 countries. Mutational profiles on a bimonthly basis were traced over time. We also examined the mutations that overlapped with the spike protein vaccine epitopes. Results: Several new mutations emerged over time and were dominating in specific countries. Many nonsynonymous mutations were within multiple spike protein epitopes that might impact the vaccine efficacy. Conclusion: Our study advocates requirement of active monitoring of country-specific mutations and vaccine efficacies in respective countries. As the number of SARS-CoV-2 isolates were sequenced and made publicly available, one can trace the evolution of the virus through analyzing its mutational landscape. Several nonsynonymous mutations have been characterized in the early months of the pandemic, chief among which are the substitutions D614G of the spike glycoprotein (S protein) and R203K and G204R of the viral nucleocapsid (N protein) [The novel coronavirus SARS-CoV-2 since its emergence in 2019 has been in a state of constant evolution toward attaining higher stability, increasing transmissibility and higher virulence. To minimize the transmission of this virus across the globe, international travel restrictions had been imposed toward the end of March 2020. China had been the first to implement travel restrictions across country and state borders as early as January 2020 [Since there is a dearth of medication and therapeutic agents available to combat the SARS-CoV-2 pathogen, engineering vaccines and drugs against this virus has gained top priority . SeveralIn this study, we have used 220,000 SARS-CoV-2 sequences and have analyzed for nonsynonymous substitutions in amino acid sequences throughout all viral proteins. We have segregated them into two categories based on the time around implementation of international travel restrictions and have compared the mutational profiles of 35 selected countries in the two time periods. We identified the mutations specific to certain countries, those that had diminished in abundance as well as those that were dominating with time. We assessed the temporal variation of highly mutated residues over the course of the year, to gain insight into the robustness of these variants. To shed light into the plausible effectiveness of the vaccines, country-specific frequently mutated residues were subsequently mapped to the predicted epitopes targeted toward the spike protein for potential vaccine development.www.epicov.org/epi3/), available as of 28 November 2020. For each of the open reading frames (ORFs) and proteins, partial sequences were filtered out, which accounted for less than 1% of the total isolates. The protein sequences were individually aligned using MUSCLE. The SARS-CoV-2 strain, originating in Wuhan, China (GenBank accession ID: NC_045512) was considered as the reference strain. Since countries started implementing international travel measures from the 3rd week of March to early April, the isolates were segregated into two groups, based on whether they were sequenced before May (group B for ‘before’) or after May (group A for ‘after’) 2020. We were left with a total of approximately 215,000 sequences, out of which 135,000 sequences are in group A and 80,000 sequences are in group B.A total of approximately 220,000 SARS-CoV-2 sequences were downloaded from GISAID .Nonsynonymous mutations were identified independently for each of the selected countries for group A and B. A complete summary with all the unique variants, each characterized by mutations in one or more amino acid positions, along with the frequency of occurrences are calculated. We have defined a substitutions having at least 2% occurrence in all the variants as a frequently mutated residue. An overall summary table depicting the frequencies for all the 35 selected countries, covering each of the identified frequently mutated residues are provided separately for the two groups (Supplementary Table 2). The variations in the mutation patterns for one or more countries from the global framework were observed.In order to obtain a bird’s eye view of the impact of the international travel measures in shaping the mutations in SARS-CoV-2, we have calculated a weighted average of the number of frequently mutated residues for each structural and nonstructural protein in 35 selected countries . The preThe mutated residues were grouped into three classes. The first of these occurred with a frequency of 10% and higher in at least one of the countries in group A and B were considered to have originated before the implementation of intercountry travel measures and continued to prevail, either in the previous location or in other regions around the globe. On the contrary, mutations which transpired in 10% of the strains in the early months of the pandemic but were observed to dwindle down after May were bracketed in the second class; whereas, mutations that were virtually nonexistent before May but emerged with higher frequencies during the ensuing months were placed in the third class. A heatmap depicting the frequencies of these mutations in their respective groups was generated using the R package ‘gplots’, where hierarchical clustering of the various substitutions was carried out using ‘ward.D2’ method .For each of the mutations comprising the three groups, we have calculated their frequency of occurrences in every 2 months, from January to October 2020. Since the amount of sequencing in each country is largely inconsistent, in order to capture the mutational frequencies with a fair degree of confidence, we chose to include the total sequences for every 2 months for frequency calculation instead of conducting the study on a monthly basis. The frequencies were calculated only for those countries in which the mutation was observed to occur with a minimum of 10% abundance in at least one of the timepoints considered.We carried out analysis of co-occurring amino acid substitutions, considering those that have occurred with 10% or higher frequencies in each country, encompassing all viral proteins and have analyzed for the residues which have co-occurred with higher abundance. This allowed us to identify the viral variants which were showing greater dominance in a certain region along with the co-occurring mutations in the respective variants. We have also discerned the substitutions which had occurred simultaneously in the countries prone to those substitutions, and have further corroborated the fact from the bimonthly frequencies of such variants.Supplementary Table 5A). Several studies have used in-silico approaches to predict T-cell and B-cell epitopes that could potentially be employed to trigger immune responses in the host. We have selected three such independent studies which used different methodologies to predict for putative epitopes that could be implemented in designing safe and effective vaccines [Supplementary Table 5B). These potential epitopes were intersected with all frequently mutated S-protein substitutions obtained in our study. The mutations found to be harbored within one or more of the epitopic sites were observed along with the respective countries in which they were dominant. To further ascertain the progression of these mutants over time, we studied the bi-monthly frequencies of these variants.A number of studies have been carried out for speedy engineering of vaccines against the SARS-CoV-2 virus. We selected eight studies which have experimentally determined potential immune-responsive epitopes owing to several viral proteins, of which we are interested especially with the epitopes delegated toward the spike protein ,28–34 . Among the structural proteins, the envelope (E) and membrane (M) proteins were the most resilient to nonsynonymous changes. On the other hand, the S and N proteins displayed greater plasticity, respectively accounting for 41 and 37 substitutions during the initial 4 months  and 80 and 69 substitutions during post travel restrictions . ORF3a, ORF8 and ORF7a were the most mutable in their respective phases. Expectedly, ORF1ab, which constitutes around 70% of the viral genome, represented by 16 nonstructural proteins (NSPs) had encountered the most number of substitutions, cumulatively equaling to 214 and 408 substitutions in group B and A, respectively. Probing into the substitutions sustained by the NSPs, NSPs 2, 3 and the replication transcription complex proteins NSP-12, 13 and 14 were the highest in the group. The summary tables containing the frequencies of each of the frequently mutated residues, as observed in 35 countries for both groups are presented in Supplementary Table 1A & B. Overall, we found that the number of mutations in group A had at least doubled compared with their status as observed in Group B.To assess the effect of international travel restrictions in shaping the mutational profile in 35 countries, all protein sequences of SARS-CoV-2 comprising approximately 215,000 isolates were analyzed. We identified 334 amino acid substitutions that were observed in at least 2% of the total number of isolates, for at least one country in the period from January to April 2020, while 656 such frequently mutated residues were obtained in the interval from May to November 2020  across all countries was calculated separately for both group B and A . For theSupplementary Table 2), some differences were observed. In the months leading to the travel curtailments where ORF8 had the highest mutation rates in 13 countries, followed by ORF3a and N protein in eight countries each. However, in the period from May to November, N protein displayed the highest mutation rates in 18 of the 35 countries, while in seven countries ORF3a had encountered the highest number of mutations (Supplementary Table 2). Country-wise mutation rates were most stark in case of ORF8, which had remained immune to changes in 21 countries while effecting fairly high rates of substitutions in the remaining countries. Apart from these, ORF7a in South Korea, ORF7b in UK and Latvia and ORF8 in Kenya and South Africa showed the highest rate of mutations.Upon examining the rate of mutations for each country  allowed us to perceive three distinct trends with which the mutations developed in the course of time. The substitution S_D614G was seen to have attained close to 100% concordance in most countries by October, with North American and European countries gaining the mutation at a faster rate compared with Middle East and Asian countries .Now that we had identified prominent mutations in diverse SARS-CoV-2 proteins, we wanted to find the substitutions which have co-occurred in different geographical locations. We have considered variants with a prevalence of at least 10% for a particular country to be the dominant variant. These prevailing variants arising from the merger of various proteins along with the corresponding frequencies are presented in Supplementary Table 5A & B). Among the studies concerned with experimentally determined epitopes, one study [Supplementary Table 6. Bimonthly frequencies were calculated for each of these mutations (Supplementary Table 7) to trace the course of their advancement through time and determine those mutants, which might seem innocuous in the early stages but could pose a threat to vaccine potency if left unchecked.We have mapped the frequently mutated S protein residues observed to be prevalent in one or more countries with Spike protein epitopes obtained from 11 studies . These mutants had not been observed with noteworthy frequencies in the preceding months, but only came into prominence toward the later period. As we cannot state with any degree of certainty the evolutionary course these variants will follow, countries should actively monitor these sites in the coming months. If any of these mutations are inadvertently incorporated among the epitopes of future vaccines, they may render them partly impotent in countries where those variants are thriving. Now that there have been considerable relaxations in inter-country migrations, other nations should also follow up the status of these mutations to confirm the fidelities of the associated amino acid residues. We conclude with the hope that the worst days of the pandemic are behind us, and await the timely disbursement of safe and effective vaccines while continuing to follow adequate social distancing norms around the world.The vaccines which recruit the whole virus in inactivated forms such as PicoVacc  and BBIBThe COVID-19 pandemic inflicted by the novel coronavirus SARS-CoV-2 continues to rage around the world in varying degrees. The virus is constantly in a state of evolution and reports of new viral strains continue to emerge in various geographical regions. We have found several novel variants of the virus in different countries, some of which were seen to have emanated toward the latter half of 2020. With inter-country travel restrictions being lifted, these novel variants could permeate the globe within a short span of time. As the administration of vaccines commences throughout the world, caution should be exercised that these novel mutations do not curb the effectiveness of the vaccines. We have already shown that several of the frequent spike mutations took residence within one or more of the predicted epitopic sites. Our study warrants a need of active monitoring of the spike protein mutations in the respective countries with regards to vaccine efficacy.In order to curb the spread of the COVID-19 pandemic, countries all over the world had implemented international travel restrictions.To examine the role of the travel measures in shaping the mutational profile of SARS-CoV-2, we tabulated the viral protein mutations encompassing 215,000 sequences across 35 countries before and after the travel restrictions.Three distinct classes of mutations were identified in the two time periods, namely those that had gained stability, those that had dwindled in abundance and others that had emerged post travel restrictions and were on the rise.Notable mutations included A222V and S477N in spike protein and A220V in the nucleocapsid, which were observed exclusive to European countries post travel curtailments.Bimonthly frequencies of the highly mutated residues revealed the evolutionary trends of these mutations over time.Spike protein mutations overlapped with several predicted epitopes, advocating close monitoring of the mutations for effective vaccine development.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.


Caenorhabditis elegans nematodes, small RNAs are transmitted across generations to establish a transgenerational memory trace of ancestral environments and distinguish self-genes from non-self-elements. Carryover of aberrant heritable small RNA responses was shown to be maladaptive and to lead to sterility. Here, we show that various types of stress  induce resetting of ancestral small RNA responses and a genome-wide reduction in heritable small RNA levels. We found that mutants that are defective in various stress pathways exhibit irregular RNAi inheritance dynamics even in the absence of stress. Moreover, we discovered that resetting of ancestral RNAi responses is specifically orchestrated by factors that function in the p38 MAPK pathway and the transcription factor SKN-1/Nrf2. Stress-dependent termination of small RNA inheritance could protect from run-on of environment-irrelevant heritable gene regulation.Transgenerational inheritance of small RNAs challenges basic concepts of heredity. In  Different human diseases, such as several imprinting-associated syndromes , arise due to the inheritance of parental information that is not encoded in the DNA sequence . FurtherCaenorhabditis elegans nematodes, much knowledge has been gained regarding the mechanisms that enable transgenerational gene regulation via inheritance of small RNAs . Feeding the worms with bacteria that express anti-gfp dsRNA induces heritable silencing of the gfp transgene for ±3–5 consecutive generations  and then exposed the next generation (F1) to three different types of stress. After examining several different stress regimes , we chose to either heat shock the worms for 2 hours   , culture stress) , or star stress)   and in the next generations that were not directly exposed to stress  and at the following generations which did not experience stress (F2-F6). We found that stress during the L1 stage led to a strong reduction of heritable oter see .We then tested whether the ability of stress to reset heritable small RNA responses depends on the developmental stage or the generation in which stress is applied: We found that, as was observed when stress was applied during the L1 stage, stress during adulthood leads to resetting of heritable silencing in the next generation  or PIWI-interacting small RNAs (piRNAs). For this purpose, we used (1) worms that carry a gfp transgene that contains an endo-siRNA-target sequence . The next generation was then grown under either similar mild stress conditions or transferred to regular growth conditions , even prior to the initiation of inheritance (for either one or three consecutive generations), leads to a similar resetting response as observed for stress that is applied in the next generation , induce small RNA changes that persist across generations and create a ‘transgenerational memory’ of past experiences . Such trneration . These rEndogenous small RNAs align to large parts of the genome, and their inheritance is especially important for transgenerational regulation of germline expressed transcripts . To bettC. elegans vary greatly in the initial (or ‘primary’) source of small RNAs, the processing steps toward small RNA maturation, the co-factors that function in each pathway, their effects on gene expression, heritability potential, and the actual genes that they target  Argonaute is an important regulator of transgenerational gene silencing by small RNAs and, while largely dispensable for silencing within the same generation, is explicitly required for transmission of silencing responses across generations  the targeting small RNAs were down-regulated, or ‘reset’, following stress. These endogenous small RNAs exhibited a strong enrichment for all RNAs   were almost exclusively CSR-1-dependent endo-siRNAs  or defective  RNAi inheritance  genes , and the skn-1 gene are required for resetting of RNAi inheritance in response to stress , while others were defective in RNAi inheritance (mek-1/sek-1 and kgb-1)  . Overallsek-1, which is required for stress-induced resetting of small RNAs, was shown to be expressed in neurons and in the intestine (REFs), two tissues which have a role in sensing and communicating environmental perturbations. We therefore used strains carrying a neuronal rescue or an intestinal rescue of the sek-1 gene (sek-1 on the silencing of the piRNA sensor (mCherry fluorescence) following stress. While this sensor provides indication of endogenous small RNAs silencing, we still observed that both exogenous and endogenous silencing responses are subjected to resetting following stress within the same generation (as described in the previous sections). Moreover, the rescue experiments were performed side by side with mutant lines that enabled a direct comparison of the general effects of the examined mutations on resetting of endogenous silencing following stress. These experiments included a particularly large quantity of worms tested over multiple biological and technical replicates over many conditions (N = 3193). This led to increased sensitivity during hypothesis testing and the detection of statistically significant effects that may not be biologically significant. We therefore proceed to report an effect size for each group in addition to corrected p-values   .sek-1 is sufficient to re-establish a strong resetting of silencing in response to stress , but did regenerate a strong resetting response following osmotic stress (Cliff’s Delta = 0.4108). However, we note that the general levels of silencing in worms that carry an intestinal rescue of sek-1 were reduced even in the absence of stress and thus we cannot exclude that stress-induced resetting in these worms is masked by generally low levels of silencing . We found that this group of genes is significantly enriched for epigenetic genes and p-granule factors  were the putative RNA-dependent RNA polymerase rrf-3 and the putative H3K9 histone methyltransferase met-2   .met-2 mutants (FDR = 2.1e-05) . Interestingly, we also observed that MET-2 is essential for stress-induced resetting of heritable RNAi   and thatble RNAi , even whg stress . OverallNon-DNA-based inheritance could be involved in multiple human disorders . It is sStandard culture techniques were used to maintain the nematodes. The worms were grown on Nematode Growth Medium (NGM) plates and fed with OP50 bacteria. Except when otherwise noted, all experiments were performed at 20°C. The worms were kept fed for at least five generations before the beginning of each experiment. Extreme care was taken to avoid contamination or starvation. Contaminated plates were discarded from the analysis. All experiments were performed with at least three biological replicates. The nematode strains used in this study are indicated in the Key Resources Table.gfp, or control HT115 bacteria that only contain an empty vector plasmid, were grown in LB containing 100 ng/ml Carbenicillin, and were then seeded on NGM plates that contain 100 ng/ml Carbenicillin and 1mM IPTG . In the generation of worms that were exposed to RNAi (P0), worms were cultivated on those plates.The standard assay for RNAi by feeding was carried out as previously described : HT115 bIn experiments where the concentration of RNAi bacteria was controlled, a spectrophotometer was used to measure optical density at OD600. HT115 bacteria were diluted with empty vector bacteria to 2 OD and 0.5 OD.For each stress condition, multiple durations and/or intensities of stress were tested see : for heaThe chosen conditions for each stress, which are described below, were chosen to be the longest/most intense conditions that most of the worm population would survive. This was done in order to avoid potential epigenetic selection that would bias the mean small RNA inheritance of the stressed population.Synchronized eggs were obtained by bleaching gravid adult worms. The progeny was then subjected to the various stress conditions.Heat shock treatment was performed at 37°C. The nematodes were heat-shocked 24 hr after the bleach treatment, for 120 min. After heat shock, the treated worms were placed back in an incubator set to 20°C, with each plate placed in a separate location (not in a pile) to avoid temperature differences between the top/bottom plates and the middle plates.After bleach, the obtained eggs were seeded on high-salt growth media plates (350 mM NaCl) and were grown on these plates for 48 hr. After 48 hr, the worms were washed off with M9 buffer onto regular NGM plates.aak-1/2 double mutants, both the wild-type and mutant worms were starved for 3 days instead of 6 days, due to starvation-sensitivity of the mutants.For starvation conditions, the nematodes were grown on empty NGM plates and were maintained this way for a total of 6 days. After 6 days, the worms were washed off with M9 onto regular NGM plates seeded with OP50 bacteria. In the experiment with In the experiments described in In the experiments described in In the adult stress assays , stress In both assays (L1 and adults stress), the next generation of worms was cultured by randomly picking several mothers from each plate to a fresh plate and allowing them to lay eggs for few hours (8–24 hr). The number of picked worms and the length of the egg-laying period were constant within each experiment across all plates and conditions.To obtain expression of the piRNAs-silenced mCherry and the endo-siRNAs-silenced GFP, worms were grown in 25°C for three generations and then transferred back to recovery at 20°C. After two generations, worms were bleached and assayed for the different stress conditions, as described above.sek-1(km4) mutation. Additionally, ZD202 carries a neuron-specific rescue of sek-1 tagged with GFP (unc-119p::sek-1(cDNA)::GFP::unc-54–3' UTR), and ZD193 carries an intestine-specific rescue of sek-1 tagged with GFP (ges-1p::sek-1(cDNA)::GFP::unc-54–3' UTR) (see Key Resources Table). Due to the inherent GFP expression of these tagged rescue strains, we crossed these strains to the piRNA sensor strain, and the experiment was performed as described above. When analyzing the tissue-specific rescue groups in this experiment, special care was taken to quantify only worms that carry the rescue array.We used strains ZD202 and ZD193, which both carry the Olympus BX63 microscope for fluorescence microscopy assays. Experiments were filmed with a 10X objective lens, with an exposure time of 750 ms.We used an Since exposure to stress leads to developmental arrest and delays, worms in different conditions reached adulthood in different days. To avoid age bias between the different conditions, the developmental stages of all worms were tracked, and the worms under all conditions were imaged when they reached ‘day one’ adulthood.gfp RNAi trigger to enable the phenotypic detection of resetting in the next generations. All sequencing experiments were done in triplicates .Total RNA was extracted from ‘day one’ adults. As stress induces variability in development even in isogenic worms’ populations, tight synchronization of the worms’ populations was achieved by picking L4 worms 1 day prior to collecting the worms for sequencing. Each experiment started by exposure of the generation before stress to an anti-Worms were lysed using the TRIzol reagent (Life Technologies) followed by repetitive freezing, thawing, and vortex. The total RNA samples were treated with tobacco acid pyrophosphatase , to ensure 5′ monophosphate-independent capturing of small RNAs. Libraries were prepared using the NEBNext Small RNA Library Prep Set for Illumina according to the manufacturer’s protocol. The resulting cDNAs were separated on a 4% agarose E-Gel , and the 140–160 nt length species were selected. cDNA was purified using the MinElute Gel Extraction kit (QIAGEN). Libraries were sequenced using an Illumina NextSeq500 instrument.Fiji ‘measure’ function , with an FDR of 0.05. The q-values reported in this study are adjusted to multiple comparisons. In the figures, not significant (ns) indicates q ≥ 0.05, (*) indicates q < 0.05, (**) indicate q < 0.01, and (***) indicate q < 0.001. Unless marked otherwise in the figures, comparisons were made between each stressed group and its corresponding unstressed control group.In the tissue-specific rescue experiments , where aThe Cliff’s Delta non-parametric effect size measure was used to estimate effect sizes for each comparison . Cliff’sData are the individual worms’ mean germline CTCF, with each dot representing the measurement of a single worm. Data are represented as medians, with box limits representing the 25th (Q1) and 75th (Q3) percentiles, notch representing a 95% confidence interval, and whiskers indicating Q1-1.5*IQR and Q3+1.5IQR. The minimal value of each experiment was set at 0 and the other values were adjusted accordingly.AGATCGGAAGAGCACACGTCT input.fastq > output.fastq -m 15 discard reads which are shorter than 15 nucleotides after the adapter clipping process -a AGATCGGAAGAGCACACGTCT the 3’ adapter sequence used as a query. The clipped reads were then aligned against the ce11 version of the C. elegans genome using ShortStack  and extracted the signal along the flanking regions of 500 base pairs upstream and downstream of the TSS. In the case of genes with two transcripts or more, we averaged the histone modification signal of all the corresponding transcripts. The signal of individual genes was determined as the averaged signal of the two published replicates of each chromatin modification.Chip-Seq data were downloaded from GSE87522. The transcription start site (TSS) of all the protein-coding genes in  public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below.Our editorial process produces two outputs: (i) [Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your work entitled "Stress Resets Transgenerational Small RNA Inheritance" for consideration by eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in The individual reviews are appended below and I am also summarizing the main issues that all reviewers agreed would be the minimum set of experiments/rewriting that would be necessary for the manuscript to be reconsidered as a new submission.1) Expand on the connection among MET-2/MAPK/SNK-1 and RNAi pathways.2) Establish tissue specificity (gut? neurons?)3) Expand on the insulin signaling pathway – perform the transgene expression assay in daf-2; daf-16 and daf-2; skn-1 double mutants4) Show as controls in the P0 generation what the effect of the mapk, skn-1 and met-2 mutations is on the GFP reporter strain5) Establish whether the phenomenon is terminated in the F4 or F5 generation6) Writing revision to make the paper more cohesive and integrate the findings conceptually with published literature (including your 2016 Cell paper), and explain how the different regulators identified are connected to each other. All reviewers found the paper to be somewhat disjointed.7) Expand on how different stressors may affect small RNA pools in different ways.Reviewer #1:Here, Houri-Ze'evi, et al. treated progeny of parents that had inherited small RNA response  with 3 stresses – heat shock, hyperosmolarity, or starvation – starting at L1, and examined gfp silencing. All three treatments reduced silencing (visible as increased GFP fluorescence) in subsequent generations (F1-F3).The authors tested resetting of endogenous (endo-siRNA) and piRNAs using an endo-siRNA sensor target sequence and piRNA recognition sites, respectively. Again, all 3 stressors reset both in the same generation, but did not reset the effect transgenerationally, suggesting that exogenous RNAi resetting functions through a different mechanism than endogenous.Next, they tested adults, which also led to resetting. However, only the F1 generation, not F2, is susceptible to resetting (how? Why?), revealing a critical period for resetting susceptibility.Reversal of the stress with RNAi treatment does not result in resetting, nor does simply changing conditions.The authors then went on to examine mutants that might be defective in stress responses or in resetting; MAPK genes and skn-1 are required for resetting.Small RNA-seq from stressed worms and their progeny showed a decrease overall with stresses, and reveals some potential classes of genes, including targets of the mutator genes, and overlap with classic stress response pathways .Overall, this work presents some interesting phenomena and moves towards explaining how it might work through the identification of a critical period and some genes that are required. However, several large gaps remain to be addressed.The major gaps that need to be addressed are in the MAPK/SKN-1 function (how? Connections? Where?) and in general, the route of communication of these resetting process, particularly in light of the lab's most recent findings in neurons.1) It seems that the ultimate conclusions regarding MAPK/SKN-1 were left hanging. Does MAPK pathway regulate SKN-1?  How do MAPK and SKN-1 function to reset? And where?2) It would be good to compare transcription in the skn-1 mutants than can't reset with wt under the same resetting conditions, to identify possible mechanisms by which resetting occurs.3) What is the connection between MAPK, SKN-1, and mutator genes? Does SKN-1 regulate these mutator genes directly? Is there a functional connection with MET-2?4) Perhaps most critically, where (in which tissues) are these effects taking place? Does the stress response happen in somatic tissues (and which ones? intestine? neurons?) and communicate to the germline? The authors cite papers that discuss gene functions in the nervous system  but have not shown that these genes function in the nervous system to reset. Tissue-specific rescues of the required genes should be done to address this question.5) Do the answers to these questions explain why there are differences between the exogenous and endogenous siRNA and piRNA pathways? or critical period/generation? This aspect was also left unresolved.Reviewer #2:C. elegans, transgenerational inheritance often lasts 3-5 generations, and increasing evidence suggests that the inherited gene expression states are mediated by RNA interference pathways. How this inheritance is maintained and "reset" after approximately three generations has been an open question in the small RNA field. Understanding how environmental stress can propagate gene expression states across generations has become an important field of study with regard to human disease, particularly for diseases with fetal origins such as metabolic programming disorders. In a previous publication, this lab showed that the termination of transgenerational gene expression states is a regulated process. In this follow-up manuscript, the authors show that environmental stresses- starvation, high osmolarity, and high temperature- can disrupt the inheritance of gene expression through changes in small RNA levels. The authors also demonstrate that the stress-induced disruption of inherited gene expression is dependent upon the MAPK pathway and the MET-2 histone H3K9 methyltransferase.Transgenerational inheritance of gene expression states has been demonstrated in plants and animals. In While I think this manuscript describes an important finding with regards to small RNA inheritance, I think the results need to be more conceptually integrated with previous work. For example, numerous studies have examined transgenerational inheritance after heat stress and the role of MET-2 in establishing H3K9me2 marks in the early embryo and in the adult germ line. How does this study complement or disagree with previous findings? The answer to this question might help in developing a more testable model of how MAPK and MET-2 function are mechanistically linked to small RNA production. Related to this comment, the authors do not address how this work relates to their previous paper , which showed that dsRNA-induced RNAi triggered the RNAi-mediated downregulated of RNAi genes. Does stress in L1 progeny of adults treated with dsRNA further down-regulate these RNAi genes, allowing the "reset" of gene expression to happen earlier? In addition, I found the term "reset" to be somewhat of a misnomer, which led to some confusion while reading the manuscript. Dr. Rechavi demonstrated as a postdoc that L1 starvation leads to transgenerational inheritance of a small RNA response to stress. Thus, it seems that stress does not exactly "reset" the small RNA populations to a "normal" state, but rather the worms re-prioritize their small RNA repertoire for a greater need, i.e. starvation/osmolarity/temperature stress trumps silencing a single transgene.The final major comment about the manuscript is that the authors have not fully explored the potential contribution of the insulin signaling pathway to this process, despite reporting an effect on small RNA "resetting" in the daf-2 mutant and a significant overlap of insulin regulated genes with their identified 281 stress-affected genes. Insulin signaling regulates SKN-1 function, which could potentially explain how the authors observe a generalized small RNA response to different stresses (this review might be helpful: doi: 10.1016/j.freeradbiomed.2015.06.008). The authors should perform their transgene expression assay in daf-2;daf-16 and daf-2;skn-1 double mutants to examine the potential role of insulin signaling and DAF-16 in this process. It would also be useful to examine the overlap of their 281 stress-responsive genes with DAF-16 class I and class II genes.Reviewer #3:In the paper entitled "Stress Resets Transgenerational Small RNA Inheritance" Houri-Ze'evi L, Teichman G et al. examine the interaction between multiple heritable phenotypes by knocking down a heritable GFP reporter and examining its interaction with other stresses, such as starvation and high temperature, which cause transgenerationally heritable phenotypes. They demonstrate that exposing worms to stresses inhibits the transgenerational silencing of the GFP reporter strain they use. They further demonstrate that deletion of genes involved in the MAPK pathway, the skn-1 transcription factor and the putative H3K9 methyltranferase met-2 eliminate the differential response in the F1 and F2 generations after exposure to stress and the GFP reporter silencing. They also sequence the small RNAs in the P0 and F1 generation with and without the added stresses. This is an extremely exciting finding but I feel that it should be a little more flushed out before publication. How are these different pathways connected? The system itself should also be a little more rigorously probed. Some detailed suggestions to help ameliorate the paper are listed below.1) For Figure 1 How long does the phenotype persist (what happens in F4 and F5)? If worms are persistently stressed (across many generations in advance of the experiment) does this blunt the effect of the GFP knockdown in the first generation or its transmission? Basically is the reverse true as well?2) For Figure 3 it suggests to me that there is only so much bandwidth that the heritable silencing can occupy so if it is activated to respond to a dramatic stress than it isn't going to be as efficient at having heritable silencing of some GFP transgene. It seems like testing the magnitude of the different stresses would be good to help tease this apart. Is there a certain amount of heat shock or starvation which doesn't elicit a heritable response? Can you tune that down to such an extent that there is no effect on the heritable GFP knock-down? Also I couldn't really figure it out from the text if this was done with one GFP reporter strain or multiple ones? If it was done with multiple strains maybe explain that a little more thoroughly, if it was only done with one strain it would strengthen the paper to do it with another one.3) For Figure 4 it would be nice to delve a little deeper. Does something happen to the MAPK protein levels in response to stress in one generation? It seems as though there isn't much connection between the different mediators of the stress response. Can the authors connect them more either experimentally or at least by describing in more detail their theories.4) Again in Figure 5 when discussing different stressors affecting small RNA pools in different ways, it's unclear as to whether this is due to a different magnitude of stress or different pathways. Can the authors delve deeper into this? What is different about the small RNAs? Maybe more description of what the small RNAs are rather than the numbers and types GO analysis of enrichment in each category and the statistics and figure should represent that info.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kevin Struhl as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Stress Resets Ancestral Heritable Small RNA Responses" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Reviewer #1 (Recommendations for the authors):It was unclear whether the germline rescue of sek-2 was tested for its contribution to the observed effects.Some but not all of the RNA-seq data are described in terms of enrichment and significance; it would be preferable to see this in all the discussions of transcriptional data.Reviewer #2 (Recommendations for the authors):1) The figures show that stress treatment of F1 progeny of P0 adults treated with dsRNA resulted in de-silencing of the GFP transgene, presumably through changes in exo-siRNA levels. In Figure 1C, the transgene de-silencing appears complete by the F4 generation, compared to control progeny that do not completely de-silence the transgene until F6 generation.  In contrast, when endo-siRNA responses were examined using an endo-siRNA and piRNA transgene sensor , the de-silencing was only observed F1 generation that experienced the stress. These differences indicate two different mechanisms regulating siRNAs during and after stress. Exo-siRNAs appear to be “turned over” much faster due to stress conditions, whereas siRNAs were only affected during the stressed generation. These two mechanisms are lumped together in the manuscript as one mechanism, although the authors do somewhat make distinctions in parts of the Results. This creates problems later in Figure 5D, when rescue of sek-1 is examined using a piRNA sensor strain, while Figure 5C examines the role of mutants regulating exo-siRNAs. For consistency, the experiment in Figure 5D should use a transgene reporter regulated by exo-siRNAs as well. I also think it would be more helpful to the reader to discuss these mechanisms separately in the manuscript.2) In Figure 4, the small RNA levels were examined in stressed adults and their non-stressed progeny. They show an overall decrease in sRNAs for all three stresses of stressed adults, but show variable changes for each stress in the non-stressed progeny. This data is largely consistent with the results of Figure 2. Since Figure 3 explored the dynamics of how sRNA populations could be altered by different types of stress, I think it would be informative in Figure 4 to also show the levels of exo-RNAs targeting GFP in these populations. The dynamics of how exo-RNAs and endo-siRNAs may compete for resources during a stress response could be explored more here. In addition, the authors mention the lack of reproducibility among the replicates in Figure 4C. The control lanes look very similar, yet replicates of same stress do not cluster together. Are the replicates for each stress collected at the same time clustering together instead? Perhaps another environmental influence was contributing to these sRNA differences.3) The nomenclature of a subset of the siRNAs identified in Figure 4C as "stress-reset" I find confusing. I think of a "reset" as return to baseline, whereas in this case, the worms are exhibiting a decrease from baseline for those siRNAs that are maintained over generations to protect germ line integrity.4) Figure 1 is blurry and pixelated, which may have occurred during the conversion process.5) In Figure 1C, 2B, (and others)- it would be helpful to have GFP levels of the transgene with empty vector treatment to compare if populations were actually "reset" or if the populations were gradually de-silencing over generations.6) In Figure 5, the mutant names do not line up with the boxes.7) In Figure 5C- mek-1;sek-1 F2 looks like wildtype F6 in Figure 1. Perhaps this strain has a failure to inherit small RNAs in addition to failure to reset.8) Introduction – nuclear small RNAs “promote modification of” chromatin9) Subsection “Stress, and not any change in growth conditions, leads to theresetting of ancestral small RNA silencing”/Figure 3: it would be helpful to indicate "mild stress" to distinguish it from the harsher stresses used in other figures for clarity10) Are RNAi genes targeted by MET-2, resulting in maintained strong silencing of the transgene?11) Results final paragraph – : should be changed to period to start new sentence12) In the Discussion, it would be helpful to compare the results presented in this manuscript to some of the studies that have examined transgenerational phenotypes resulting from similar stresses.Reviewer #3 (Recommendations for the authors):The authors for the most part answered my questions and I think the paper is basically ready for publication. I would make a few minor changes as suggested below. Additionally I still feel that the portion with MET-2 is not sufficiently fleshed out nor linked to the rest of the manuscript. I would suggest removing this final figure as the connection to skn-1 etc is tenuous still.– First paragraph in subsection “Stress-induced resetting of target-specific small RNAs” ends prematurely "rapamycin exposure and changes in…"– Third paragraph of the same section miscited as Figure 5E should be 4E.– Something messed up happened in the citations. There is a citation Chen Qi, Yan Menghong, etc. which seems to just include a long list of researchers not a specific paper. Similarly the citation Kishimoto S, Uno M,.… is a list of people not in the paper that is cited.– If the authors insist on including the MET-2 portion of the manuscript (again which I feel is out of place and doesn't yet belong in this paper), when discussing MET-2 and citing Kerr et al., 2014 and work from your own lab it seems appropriate to also cite work from Susan Gasser, Susan Mango, Yang Shi, and Susan Strome, all of whose work came out either before or at the same time. [Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]Reviewer #1:Here, Houri-Ze'evi, et al. treated progeny of parents that had inherited small RNA response  with 3 stresses – heat shock, hyperosmolarity, or starvation – starting at L1, and examined gfp silencing. All three treatments reduced silencing (visible as increased GFP fluorescence) in subsequent generations (F1-F3).The authors tested resetting of endogenous (endo-siRNA) and piRNAs using an endo-siRNA sensor target sequence and piRNA recognition sites, respectively. Again, all 3 stressors reset both in the same generation, but did not reset the effect transgenerationally, suggesting that exogenous RNAi resetting functions through a different mechanism than endogenous.Next, they tested adults, which also led to resetting. However, only the F1 generation, not F2, is susceptible to resetting (how? Why?), revealing a critical period for resetting susceptibility.Reversal of the stress with RNAi treatment does not result in resetting, nor does simply changing conditions.The authors then went on to examine mutants that might be defective in stress responses or in resetting; MAPK genes and skn-1 are required for resetting.Small RNA-seq from stressed worms and their progeny showed a decrease overall with stresses, and reveals some potential classes of genes, including targets of the mutator genes, and overlap with classic stress response pathways .Overall, this work presents some interesting phenomena and moves towards explaining how it might work through the identification of a critical period and some genes that are required. However, several large gaps remain to be addressed.We thank the reviewer for finding our work interesting, for the concise summary of our results, and for the constructive remarks that help improve the manuscript. We resolved all the reviewer’s suggestions and requests with new experiments and extensive analyses. We also made major structural changes in the manuscript that should improve the flow and clarity of the revised manuscript, in particular in the sections that explore the regulation of stress-induced resetting of heritable responses.The major gaps that need to be addressed are in the MAPK/SKN-1 function (how? Connections? Where?) and in general, the route of communication of these resetting process, particularly in light of the lab's most recent findings in neurons.1) It seems that the ultimate conclusions regarding MAPK/SKN-1 were left hanging. Does MAPK pathway regulate SKN-1?  How do MAPK and SKN-1 function to reset? And where?We made great efforts in the revision process to address this remark. The p38 MAPK pathway was previously shown to regulate SKN-1 through MAPK-dependent phosphorylation, which leads to nuclear localization of the transcription factor SKN-1 . We elaborate on this in the revised manuscript.To investigate the reviewer’s question, we ran multiple experiments and analyses:sek-1, but not an intestinal rescue, was sufficient for worms to reset small RNAs in response to stress. While this shows that SEK-1 can affect resetting non-cell autonomously (via neurons), we note that the general levels of silencing in worms which carry an intestinal rescue of sek-1 were reduced even in the absence of stress and thus we cannot exclude that stress-induced resetting in these worms is masked by generally low levels of silencing. These results are congruent with our lab’s recent findings about neuronal small RNAs , which the reviewer mentioned . We elaborate on those results further in the revised manuscript .1) To assess whether stress-induced resetting of small RNAs depends on a MAPK response in a particular tissue, we used worms that express the MAPK protein SEK-1, which we showed is crucial for stress-induced resetting of small RNAs, specifically in neurons or in the intestine (using tissue-specific promoters). We found that a neuronal rescue of skn-1 knockdown worms that were exposed to either control conditions or hyperosmotic stress . We found that exposure to stress in general represses the expression of a considerable group of epigenetic and small RNA factors. We elaborate on those results further in the revised manuscript. More details regarding the specific factors can be found below in this letter (in response to comment #2 of Reviewer #1).2) To discover the downstream effectors of SKN-1 and the MAPK pathway in the stress-induced resetting response, we analyzed mRNA sequencing data from wild-type and skn-1 knockdown, on its own, did not affect the expression of any notable epigenetics-related genes, the stress-induced repression of multiple small RNA factors depends upon skn-1. We identified a group of small RNA factors (N=35) that are repressed in response to stress in a skn-1-dependent manner. These include, in particular, the argonaute protein nrde-3, and the epigenetic factors rrf-3, rde-8 and nyn-2. We elaborate on those results further in the revised manuscript.Furthermore, we found that while skn-1 that function in resetting, we examined the putative promoter regions of known small RNA factors for the established binding motif sequence of SKN-1 . We found that the promoters of multiple (N=63) small RNA factors contain SKN-1 binding sites (including met-2). We elaborate on those results further in the revised manuscript. SKN-1 regulation might work directly or indirectly, affecting MET-2 via any of its many target genes. However, further work is required to fully capture the regulatory role of SKN-1 over MET-2 and other small RNA factors.3) To identify other potential downstream effectors of 4) In addition, while we were revising this manuscript, additional evidence that SKN-1 functions in transgenerational inheritance was published, strengthening the link that we identified between SKN-1 and regulation of transgenerational epigenetic inheritance . SKN-1 functions were also found to be regulated transgenerationally by heritable small RNA .2) It would be good to compare transcription in the skn-1 mutants than can't reset with wt under the same resetting conditions, to identify possible mechanisms by which resetting occurs.skn-1 knockdown worms that were exposed to either control conditions or hyperosmotic stress . We elaborated about the results of this analysis in response to the comment above, as well as in the revised manuscript. In summary, we found that a considerable group of small RNA factors that are significantly downregulated in response to stress in a skn-1-dependent manner. In particular, this group includes the argonaute protein nrde-3 and the epigenetic factors rrf-3, rde-8 and nyn-2.We thank the reviewer for this great suggestion. We analyzed mRNA sequencing data from wild-type and 3) What is the connection between MAPK, SKN-1, and mutator genes? Does SKN-1 regulate these mutator genes directly? Is there a functional connection with MET-2?We thank the reviewer for these important questions. We took multiple approaches to answer these questions:1) As we elaborated in response to Comment #1 of the reviewer, previous works have shown that the p38 MAPK pathway regulates the activity of SKN-1 through MAPK-dependent phosphorylations, which in turn leads to nuclear localization of SKN-1 .skn-1 knockdown worms which were exposed to either control conditions or hyperosmotic stress. We elaborate on those results further in the revised manuscript, and in the responses above.2) As we elaborated in response to Comment #1 of the reviewer, we analyzed mRNA sequencing data from wild-type and rde-8 and nyn-2 are both repressed in response to stress in a skn-1 dependent manner. The two proteins that these genes encode for were shown to localize to the Mutator foci and have a crucial role in the cleavage of mRNA targets and the recruitment of the RNA-dependent RNA polymerase complex . These results indicate that skn-1 has a role in regulating components of the Mutator foci.Notably, we found that the genes met-2 contains multiple SKN-1 binding motifs. We elaborate on those results further in the revised manuscript.3) As we elaborated in response to Comment #1 of the reviewer, we examined the putative promoter regions of epigenetic and small RNA factors for the SKN-1 binding motif . We found that the promoter region of mut-2 and mut-16, as well as the Mutator-interacting factor rde-8, are significantly downregulated in response to stress. We elaborate on those results further in the revised manuscript.4) We analyzed mRNA sequencing data from worms that were exposed to heat shock, hyperosmotic stress, or starvation . Among other findings, we discovered that a notable group of epigenetic and small RNA factors are repressed in response to any of those stress conditions. In particular, we found that the Mutator genes 4) Perhaps most critically, where (in which tissues) are these effects taking place? Does the stress response happen in somatic tissues (and which ones? intestine? neurons?) and communicate to the germline? The authors cite papers that discuss gene functions in the nervous system  but have not shown that these genes function in the nervous system to reset. Tissue-specific rescues of the required genes should be done to address this question.sek-1, a MAPK gene that we found to be required for stress-induced resetting of small RNAs, either in neurons or in the intestine, as suggested by the reviewer. We elaborated on these results in response to Comment #1 of the reviewer, but in short, we found that only neuron-specific expression of sek-1 was sufficient to rescue stress-induced resetting, while intestinal-specific expression was not. While this shows that SEK-1 can affect resetting non-cell autonomously (via neurons), we note that the general levels of silencing in worms which carry an intestinal rescue of sek-1 were reduced even in the absence of stress and thus we cannot exclude that stress-induced resetting in these worms is masked by generally low levels of silencing. We elaborate on those results further in the revised manuscript .We thank the reviewer for these excellent ideas. To address this point, we used worms that express 5) Do the answers to these questions explain why there are differences between the exogenous and endogenous siRNA and piRNA pathways? or critical period/generation? This aspect was also left unresolved.We thank the reviewer for these interesting questions. Regarding the differences between the exogenous and endogenous small RNA pathways – we explained our hypothesis for the difference between exo-siRNAs and endo-siRNAs/piRNA in the manuscript and chose to elaborate on it further: unlike exogenous primary small RNAs which cannot be re-synthesized in the progeny, primary endo-siRNAs and piRNAs are encoded in the genome and do not depend on exogenous sources for their existence . The re-establishment of endo-siRNAs and piRNAs-mediated silencing in the next generations after stress indicates that these small RNAs can be transcribed de novo at each generation, and thus can compensate for stress-induced erasure of parental small RNA molecules, suggesting a fundamental difference in the “memory programs” of exogenous and endogenous transgenerational small RNA responses. We hope our explanation is now clearer.Regarding the critical period: this is a very good question. We do not know the exact mechanism that explains why F2 worms are unable to reset small RNAs in response to stress. However, it could be related to the presence of primary small RNAs, which are only produced in response to the dsRNA trigger, and become diluted over generations, and should only be available to the worms during the P0-F1 generations . We encountered a similar “critical period” in a previous paper from our lab , where we found that external activation of the RNAi machinery can extend the inheritance of an ancestral RNAi response, but only of the external activation occurs within one generation of the RNAi trigger. Similarly, in a recently-published paper from our lab, we found that worms can occupy different epigenetic “states” only at the early generations, and that this state, once established, determines their capability to inherit the small RNA responses across the lineage . The epigenetic state of the worms could also explain the ability of worms to reset an RNAi trigger only within one generation of the RNAi trigger.Reviewer #2:C. elegans, transgenerational inheritance often lasts 3-5 generations, and increasing evidence suggests that the inherited gene expression states are mediated by RNA interference pathways. How this inheritance is maintained and "reset" after approximately three generations has been an open question in the small RNA field. Understanding how environmental stress can propagate gene expression states across generations has become an important field of study with regard to human disease, particularly for diseases with fetal origins such as metabolic programming disorders. In a previous publication, this lab showed that the termination of transgenerational gene expression states is a regulated process. In this follow-up manuscript, the authors show that environmental stresses- starvation, high osmolarity, and high temperature- can disrupt the inheritance of gene expression through changes in small RNA levels. The authors also demonstrate that the stress-induced disruption of inherited gene expression is dependent upon the MAPK pathway and the MET-2 histone H3K9 methyltransferase.Transgenerational inheritance of gene expression states has been demonstrated in plants and animals. In While I think this manuscript describes an important finding with regards to small RNA inheritance, I think the results need to be more conceptually integrated with previous work.For example, numerous studies have examined transgenerational inheritance after heat stress and the role of MET-2 in establishing H3K9me2 marks in the early embryo and in the adult germ line. How does this study complement or disagree with previous findings? The answer to this question might help in developing a more testable model of how MAPK and MET-2 function are mechanistically linked to small RNA production.We thank the reviewer for raising this point. In the revised manuscript we delved further into the potential links of our results with the existing literature about MET-2 and H3K9me2 in the early embryo (see details below).Related to this comment, the authors do not address how this work relates to their previous paper , which showed that dsRNA-induced RNAi triggered the RNAi-mediated downregulated of RNAi genes. Does stress in L1 progeny of adults treated with dsRNA further down-regulate these RNAi genes, allowing the "reset" of gene expression to happen earlier?We thank the reviewer for raising this interesting comment.hrde-1, rde-1, ergo-1, nrde-3, wago-1, and alg-2, the mutator genes mut-2 and mut-16, and the small RNA factors rde-4, rrf-3, hrde-4, and rde-8, were all significantly downregulated following exposure to stress. Some of those repressed factors, such as rde-1, rde-4, rrf-3, and wago-1, are epigenetic genes which we previously showed were differentially regulated by exposure to an RNAi trigger. We elaborate on these results further in the revised manuscript.To address it, we analyzed mRNA sequencing data from worms that underwent either heat shock, hyperosmotic stress, or starvation. Interestingly, we found that a considerable group of small RNA factors, epigenetic factors, and p-granule proteins, are significantly repressed in response to all stress conditions. In particular, we found that the argonaute genes Moreover, we further discuss the connection between this manuscript and our previous paper , as well as more recent findings from our lab  in our revised manuscript.In addition, I found the term "reset" to be somewhat of a misnomer, which led to some confusion while reading the manuscript. Dr. Rechavi demonstrated as a postdoc that L1 starvation leads to transgenerational inheritance of a small RNA response to stress. Thus, it seems that stress does not exactly "reset" the small RNA populations to a "normal" state, but rather the worms re-prioritize their small RNA repertoire for a greater need, i.e. starvation/osmolarity/temperature stress trumps silencing a single transgene.This is a good point.The main method we used in our paper to observe and quantify the effects of stress on heritable small RNAs was monitoring of ancestral silencing responses that affect fluorescent transgenes, and therefore we refer to these effects throughout the paper as “resetting”. However, the reviewer has a good point and therefore to make the title more precise and to avoid confusion, we changed the title of the manuscript to “Stress Resets Ancestral Heritable Small RNA Responses”.It is indeed a possibility that worms do not exactly “reset” their small RNA populations but readjust them. Previous studies have shown that the different small RNA populations in the worm are competing over the same silencing and bio-synthesis machinery . Therefore, it is plausible that synthesis of new small RNAs in response to a stimulus such as stress could come at the expense of other small RNAs, leading to a visible “resetting” of the other small RNA populations.A similar hypothesis was raised by reviewer #3, and we were glad to investigate it.We performed an experiment which supports the reviewer’s hypothesis and our 2014 paper. In this experiment, worms were starved either one generation before RNAi treatment, or one generation after RNAi treatment. We observed that starvation blunted the heritable GFP knockdown in both the F1 worms and their progeny, whether the starvation happened before the RNAi trigger or after it.This result suggests that the resetting we observed may be a result of competition between different populations of small RNAs, and not necessarily the direct removal of small RNAs.We elaborate on this experiment in Figure 3 and in the text.The final major comment about the manuscript is that the authors have not fully explored the potential contribution of the insulin signaling pathway to this process, despite reporting an effect on small RNA "resetting" in the daf-2 mutant and a significant overlap of insulin regulated genes with their identified 281 stress-affected genes. Insulin signaling regulates SKN-1 function, which could potentially explain how the authors observe a generalized small RNA response to different stresses (this review might be helpful: doi: 10.1016/j.freeradbiomed.2015.06.008). The authors should perform their transgene expression assay in daf-2;daf-16 and daf-2;skn-1 double mutants to examine the potential role of insulin signaling and DAF-16 in this process. It would also be useful to examine the overlap of their 281 stress-responsive genes with DAF-16 class I and class II genes.We thank the reviewer for these suggestions. We were glad to address all of them:daf-2;daf-16 double mutants. We observed that those mutants reset siRNAs normally following stress. Their response to RNAi appears to be weaker (RNAi resistant). See Figure 5—figure supplement 2.1) We performed the transgene expression experiment in daf-2;skn-1 double mutants. We observed that those mutants behaved very similarly to skn-1 single mutants – they were unable to reset siRNAs in response to stress, and showed a significantly stronger response to RNAi (Eri). See Figure 5—figure supplement 2.2) We performed the transgene expression experiment in 3) When examining the siRNA levels of the 281 stress-responsive genes, we observed no significant overlap or lack thereof between them and the DAF-16 class I or class II genes . See the summarized result in Reviewer #3:In the paper entitled "Stress Resets Transgenerational Small RNA Inheritance" Houri-Ze'evi L, Teichman G et al. examine the interaction between multiple heritable phenotypes by knocking down a heritable GFP reporter and examining its interaction with other stresses, such as starvation and high temperature, which cause transgenerationally heritable phenotypes. They demonstrate that exposing worms to stresses inhibits the transgenerational silencing of the GFP reporter strain they use. They further demonstrate that deletion of genes involved in the MAPK pathway, the skn-1 transcription factor and the putative H3K9 methyltranferase met-2 eliminate the differential response in the F1 and F2 generations after exposure to stress and the GFP reporter silencing. They also sequence the small RNAs in the P0 and F1 generation with and without the added stresses. This is an extremely exciting finding but I feel that it should be a little more flushed out before publication. How are these different pathways connected? The system itself should also be a little more rigorously probed. Some detailed suggestions to help ameliorate the paper are listed below.1) For Figure 1 How long does the phenotype persist (what happens in F4 and F5)?gfp siRNAs.This is a good question. Following this question, we performed an experiment in which we examined the worms for 6 consecutive generations following stress and updated the paper in accordance. We found that the resetting effect lasted as long as the RNAi response was still heritable (5 generations in our case). since some inheritance of the small RNAs is lost in every generation, after 3-5 generations the silencing is completely lost, and the control animals are indistinguishable from animals that were never treated with RNAi. Therefore, after 6 generations there is no observable distance between the stressed and unstressed worms, because both have completely lost the inheritance of the anti-We elaborate on those results further in the revised manuscript .If worms are persistently stressed (across many generations in advance of the experiment) does this blunt the effect of the GFP knockdown in the first generation or its transmission? Basically is the reverse true as well?We thank the reviewer for this interesting suggestion. A similar hypothesis was raised by reviewer #2, and we were glad to investigate it.We performed an experiment in which worms were starved either three generations before RNAi treatment, one generation before RNAi treatment, or one generation after RNAi treatment. We observed that starvation blunted the heritable GFP knockdown in all F1 worms that were exposed to starvation and their progeny, whether the starvation happened before the RNAi trigger or after it. We did not observe a cumulative effect when worms were starved for multiple generations before RNAi.This result suggests that the resetting we observed may be a result of competition between different populations of small RNAs, and not necessarily the direct removal of small RNAs.We elaborate on this experiment in Figure 3 and discuss it further in the text.2) For Figure 3 it suggests to me that there is only so much bandwidth that the heritable silencing can occupy so if it is activated to respond to a dramatic stress than it isn't going to be as efficient at having heritable silencing of some GFP transgene. It seems like testing the magnitude of the different stresses would be good to help tease this apart. Is there a certain amount of heat shock or starvation which doesn't elicit a heritable response? Can you tune that down to such an extent that there is no effect on the heritable GFP knock-down?We thank the reviewer for this interesting suggestion.For each stress condition, we tested multiple durations and magnitudes. Our general finding was that increasing or decreasing the magnitude of the stress conditions does not increase or decrease their effect on the heritable GFP knockdown, but when stress conditions went below a certain magnitude the effect became less consistent . We elaborated further on our methodology of choosing stress conditions in the Materials and methods section, and show the stress conditions that lead to resetting in Figure 1—figure supplement 1.Also I couldn't really figure it out from the text if this was done with one GFP reporter strain or multiple ones? If it was done with multiple strains maybe explain that a little more thoroughly, if it was only done with one strain it would strengthen the paper to do it with another one.We used a total of 4 fluorescent reporters in the paper: two reporters of RNAi-induced silencing (strains SX1263 and EG6089), a reporter of endo-siRNA-silencing (GR1720), and a reporter of piRNA-silencing (JA1527). We now state it more clearly in the Materials and methods and Results sections.daf-2 mutants was performed using the EG6089 reporter strain instead of the SX1263 transgene that was used in other assays.In particular, the experiment in which we tested 3) For Figure 4 it would be nice to delve a little deeper. Does something happen to the MAPK protein levels in response to stress in one generation? It seems as though there isn't much connection between the different mediators of the stress response. Can the authors connect them more either experimentally or at least by describing in more detail their theories.We thank the reviewer for this question.To answer this question, we analyzed mRNA sequencing data from worms that were exposed to either heat shock, hyperosmotic stress, or starvation . We did not observe any significant changes in MAPK transcription levels in response to stress. This is to be expected since the MAPK pathway was shown to activate in response to stress through a phosphorylation cascade . This is also conserved across multiple species . We elaborate further on this analysis in the text.To better connect the different mediators of stress-induced resetting of small RNAs, we took multiple approaches:1) The p38 MAPK pathway was previously shown to regulate SKN-1 through MAPK-dependent phosphorylation, which leads to nuclear localization of the transcription factor SKN-1 . We elaborate on this in the revised manuscript.2) To discover the downstream effectors of SKN-1 and the MAPK pathway in the stress-induced resetting response, we analyzed mRNA sequencing data from wild-type and skn-1 knockdown worms that were exposed to either control conditions or hyperosmotic stress. We found that exposure to stress represses the expression of a considerable group of epigenetic and small RNA factors. We elaborate on those results further in the revised manuscript.skn-1 knockdown on its own did not affect the expression of any notable epigenetic genes, the stress-induced repression of multiple small RNA factors depends upon skn-1. We identified a group of small RNA factors that are repressed in response to stress in a skn-1 dependent manner, which includes, in particular, the argonaute nrde-3 and the epigenetic factors rrf-3, rde-8 and nyn-2. We elaborate on those results further in the revised manuscript.3) Furthermore, we found that while skn-1 that function in resetting, we examine the putative promoter regions of known small RNA factors for the established binding motif sequence of SKN-1. We found that the promoters of multiple small RNA factors contain SKN-1 binding sites, including the RNA-dependent RNA polymerase rrf-3 and the putative H3K9 histone methyltransferase MET-2. We elaborate on those results further in the revised manuscript.To identify other potential downstream effectors of hsf-1 mutants. We recently showed that HSF-1 determines the “inheritance state” of worms when initiating a heritable RNAi response . This “inheritance state” determines the fate of the heritable response (its persistence across generations). Our new experiment showed that hsf-1 mutants are capable of resetting RNAi inheritance in response to stress. This result stress indicates that HSF-1’s role is likely in the initiation, and not maintenance, of heritable RNAi responses.4) To delve deeper into the mediators of stress response, and to connect the finding in this manuscript to other recent findings from our lab, we examined an additional mutant strain, 4) Again in Figure 5 when discussing different stressors affecting small RNA pools in different ways, it's unclear as to whether this is due to a different magnitude of stress or different pathways. Can the authors delve deeper into this? What is different about the small RNAs? Maybe more description of what the small RNAs are rather than the numbers and types GO analysis of enrichment in each category and the statistics and figure should represent that info.This is a big question, which is very difficult to address experimentally. Any major stress condition, other than affecting small RNA factors and pathways, affects also many different pathways, and it is difficult to disentangle the different effects. For example, it was shown that even 15-minute starvation of L1 larvae, leads to dramatic changes in expression of 27% in expression  . Additional studies to clearly understand the difference between the stress conditions will be required in the future, but this is outside the scope of this particular work.[Editors’ note: what follows is the authors’ response to the second round of review.]Reviewer #1 (Recommendations for the authors):It was unclear whether the germline rescue of sek-2 was tested for its contribution to the observed effects.Thank you for pointing this out. Germline rescue was not tested in this paper:1) Since transgene silencing in the germline is also mediated by small RNAs, introducing a transgene rescue might affect the normal function of the RNAi system and will not allow a clear conclusion regarding the role of the rescued gene in stress-induced resetting.2) The endogenous SEK-1 does not seem to be expressed in the germline and artificially expressing it in the germline would probably generate ectopic germline expression rather than “rescue” of germline expression.We further clarified in the paper about the tissue-specific rescues that were tested.Some but not all of the RNA-seq data are described in terms of enrichment and significance; it would be preferable to see this in all the discussions of transcriptional data.We added enrichment and significance scores to all discussions of transcriptional data.Reviewer #2 (Recommendations for the authors):1) The figures show that stress treatment of F1 progeny of P0 adults treated with dsRNA resulted in de-silencing of the GFP transgene, presumably through changes in exo-siRNA levels. In Figure 1C, the transgene de-silencing appears complete by the F4 generation, compared to control progeny that do not completely de-silence the transgene until F6 generation.  In contrast, when endo-siRNA responses were examined using an endo-siRNA and piRNA transgene sensor , the de-silencing was only observed F1 generation that experienced the stress. These differences indicate two different mechanisms regulating siRNAs during and after stress. Exo-siRNAs appear to be “turned over” much faster due to stress conditions, whereas siRNAs were only affected during the stressed generation. These two mechanisms are lumped together in the manuscript as one mechanism, although the authors do somewhat make distinctions in parts of the Results. This creates problems later in Figure 5D, when rescue of sek-1 is examined using a piRNA sensor strain, while Figure 5C examines the role of mutants regulating exo-siRNAs. For consistency, the experiment in Figure 5D should use a transgene reporter regulated by exo-siRNAs as well. I also think it would be more helpful to the reader to discuss these mechanisms separately in the manuscript.We appreciate these comments.ANOVA:It could have been interesting to perform ANOVA analysis on out data. However, after looking into the matter, we came to the conclusion that performing ANOVA on our fluorescence data would be statistically inappropriate and could yield meaningless or even misleading results. The fluorescence data fails to meet the basic requirements for a two/three-way ANOVA analysis .Unfortunately, as far as we are aware, there is no appropriate a-parametric alternative to a two-way ANOVA that does not require data homoscedasticity and normal distribution of residuals.Exogenous RNAi vs. endogenous silencing:Indeed, we find that exogenous RNAi responses show permanent resetting dynamics compared to endogenous responses which seem to “reestablish” the silencing response in the next generations after stress (most likely due to their ability to be transcribed off the genome). We tried to separate the two phenomena in a clear way in the original version of the manuscript but clarified it further now. We also describe the rescue experiments in a clearer manner. As mentioned in the manuscript, we chose to use the endogenous silencing assay for these rescue experiments since the only established transgene for exogenous silencing  overlaps with the transgene expression of the rescue lines . However, we made sure that the rescue experiments were performed side by side with mutant lines to validate the importance of the examined mutations – and rescues – in endogenous small RNA silencing as well.2) In Figure 4, the small RNA levels were examined in stressed adults and their non-stressed progeny. They show an overall decrease in sRNAs for all three stresses of stressed adults, but show variable changes for each stress in the non-stressed progeny. This data is largely consistent with the results of Figure 2. Since Figure 3 explored the dynamics of how sRNA populations could be altered by different types of stress, I think it would be informative in Figure 4 to also show the levels of exo-RNAs targeting GFP in these populations. The dynamics of how exo-RNAs and endo-siRNAs may compete for resources during a stress response could be explored more here. In addition, the authors mention the lack of reproducibility among the replicates in Figure 4C. The control lanes look very similar, yet replicates of same stress do not cluster together. Are the replicates for each stress collected at the same time clustering together instead? Perhaps another environmental influence was contributing to these sRNA differences.We added further discussion regarding the dynamics and possible competition between exogenously and endogenously derived small RNAs.Regarding the additional environmental factor that might contribute to the variability among different stress replicates: to generate the “cleanest” data, each replicate (including the control groups) in these experiments was collected separately in different days and over the course of three months, with no overlap between different replicates. Thus, we conclude that the observed variability that is observed in the stress groups – but is not observed in the control groups – rises due to the nature of recovery from stress and not due to any batch effects.3) The nomenclature of a subset of the siRNAs identified in Figure 4C as "stress-reset" I find confusing. I think of a "reset" as return to baseline, whereas in this case, the worms are exhibiting a decrease from baseline for those siRNAs that are maintained over generations to protect germ line integrity.We changed the term to “stress-reduced”.4) Figure 1 is blurry and pixelated, which may have occurred during the conversion process.Thank you for pointing this out. We made sure to have a clear version uploaded.5) In Figure 1C, 2B, (and others)- it would be helpful to have GFP levels of the transgene with empty vector treatment to compare if populations were actually "reset" or if the populations were gradually de-silencing over generations.Thank you for this comment. We added the basal level of GFP expression with no RNAi treatment to Figure 1—figure supplement 4.see Figure 1—figure supplement 4). Interestingly, in the same experiment, we observed that GFP expression in worms which were stressed several generations ago reached a plateau around the F4 generation, and that progeny of unstressed worms reach said plateau at the F6 generation.In more detail: we observed that even six generations after the initiation of RNAi, a large portion of the worm population did not entirely re-express the GFP transgene, and some worms even maintained complete silencing of the transgene , which showed that, following an RNAi treatment, some lineages of worms tend to completely silence the targeted gene for multiple number of generations – while others “lose” the silencing quite rapidly. The results which were now added to Figure 1—figure supplement 4 suggest that stress is more likely to “reset” heritable silencing in worms that were predisposed to inherit the silencing response for shorter duration.6) In Figure 5, the mutant names do not line up with the boxes.Fixed.7) In Figure 5C- mek-1;sek-1 F2 looks like wildtype F6 in Figure 1. Perhaps this strain has a failure to inherit small RNAs in addition to failure to reset.This strain is indeed RNAi resistant, as described in Figure 5A and in the manuscript. We added further clarification regarding it in the manuscript.8) Introduction – nuclear small RNAs *promote modification of* chromatinWe changed the text in accordance.9) Subsection “Stress, and not any change in growth conditions, leads to theresetting of ancestral small RNA silencing”/Figure 3: it would be helpful to indicate "mild stress" to distinguish it from the harsher stresses used in other figures for clarityWe changed the text and figure in accordance.10) Are RNAi genes targeted by MET-2, resulting in maintained strong silencing of the transgene?hrde-1) gene. However, the general role of MET-2 in silencing and inheritance was not our focus in this study and we hope that future work will explore this possibility further.It’s a very plausible explanation which was explored to some degree at  in which they show H3K9me2 peaks at the promoter region of the Heritable RNAi Deficient-1 (11) Results final paragraph – : should be changed to period to start new sentenceFixed.12) In the Discussion, it would be helpful to compare the results presented in this manuscript to some of the studies that have examined transgenerational phenotypes resulting from similar stresses.We added more discussion about comparisons to previous stress-induced transgenerational inheritance studies.13) Some references incorrectly have too many authorsFixed.Reviewer #3 (Recommendations for the authors):The authors for the most part answered my questions and I think the paper is basically ready for publication. I would make a few minor changes as suggested below. Additionally I still feel that the portion with MET-2 is not sufficiently fleshed out nor linked to the rest of the manuscript. I would suggest removing this final figure as the connection to skn-1 etc is tenuous still.We appreciate this concern. We removed the met-2 part from the main Results section and instead describe these results in the discussion as an opening for future studies.– First paragraph in subsection “Stress-induced resetting of target-specific small RNAs” ends prematurely "rapamycin exposure and changes in…"– Third paragraph of the same section miscited as Figure 5E should be 4E.– Something messed up happened in the citations. There is a citation Chen Qi, Yan Menghong, etc. which seems to just include a long list of researchers not a specific paper. Similarly the citation Kishimoto S, Uno M,.… is a list of people not in the paper that is cited.All fixed.– If the authors insist on including the MET-2 portion of the manuscript (again which I feel is out of place and doesn't yet belong in this paper), when discussing MET-2 and citing Kerr et al., 2014 and work from your own lab it seems appropriate to also cite work from Susan Gasser, Susan Mango, Yang Shi, and Susan Strome, all of whose work came out either before or at the same time.We added a fuller reference list to the met-2 results in the discussion part.