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• W4304196160 abstract "•Gene expression changes for the duration of starvation-induced developmental arrest•Somatic transcription is required early, but not late, to support starvation survival•The germline remains transcriptionally quiescent, supporting future reproduction•Germline transcripts are exceptionally stable compared to somatic transcripts Nutrient availability governs growth and quiescence, and many animals arrest development when starved. Using C. elegans L1 arrest as a model, we show that gene expression changes deep into starvation. Surprisingly, relative expression of germline-enriched genes increases for days. We conditionally degrade the large subunit of RNA polymerase II using the auxin-inducible degron system and analyze absolute expression levels. We find that somatic transcription is required for survival, but the germline maintains transcriptional quiescence. Thousands of genes are continuously transcribed in the soma, though their absolute abundance declines, such that relative expression of germline transcripts increases given extreme transcript stability. Aberrantly activating transcription in starved germ cells compromises reproduction, demonstrating important physiological function of transcriptional quiescence. This work reveals alternative somatic and germline gene-regulatory strategies during starvation, with the soma maintaining a robust transcriptional response to support survival and the germline maintaining transcriptional quiescence to support future reproductive success. Nutrient availability governs growth and quiescence, and many animals arrest development when starved. Using C. elegans L1 arrest as a model, we show that gene expression changes deep into starvation. Surprisingly, relative expression of germline-enriched genes increases for days. We conditionally degrade the large subunit of RNA polymerase II using the auxin-inducible degron system and analyze absolute expression levels. We find that somatic transcription is required for survival, but the germline maintains transcriptional quiescence. Thousands of genes are continuously transcribed in the soma, though their absolute abundance declines, such that relative expression of germline transcripts increases given extreme transcript stability. Aberrantly activating transcription in starved germ cells compromises reproduction, demonstrating important physiological function of transcriptional quiescence. This work reveals alternative somatic and germline gene-regulatory strategies during starvation, with the soma maintaining a robust transcriptional response to support survival and the germline maintaining transcriptional quiescence to support future reproductive success. IntroductionDevelopment requires favorable environmental conditions, and diverse animals enter a state of developmental arrest in response to unfavorable conditions (MacRae, 2010MacRae T.H. Gene expression, metabolic regulation and stress tolerance during diapause.Cell. Mol. Life Sci. 2010; 67: 2405-2424Crossref PubMed Scopus (162) Google Scholar). Starvation causes cellular quiescence in cells ranging from yeast to human, and some animals arrest development in response to inadequate nutrition (Su et al., 1996Su S.S. Tanaka Y. Samejima I. Tanaka K. Yanagida M. A nitrogen starvation-induced dormant G0 state in fission yeast: the establishment from uncommitted G1 state and its delay for return to proliferation.J. Cell Sci. 1996; 109: 1347-1357Crossref PubMed Google Scholar; Yao, 2014Yao G. Modelling mammalian cellular quiescence.Interface Focus. 2014; 4 (20130074)Crossref PubMed Scopus (95) Google Scholar). C. elegans nematodes hatch as L1 larvae, and in the absence of food, they arrest development, providing a valuable model of starvation resistance and developmental arrest (Baugh, 2013Baugh L.R. To grow or not to grow: nutritional control of development during Caenorhabditis elegans L1 arrest.Genetics. 2013; 194: 539-555Crossref PubMed Scopus (134) Google Scholar). L1 arrest (or L1 diapause) has garnered attention because time spent in arrest does not shorten lifespan upon recovery, as if it is an “ageless” state (Johnson et al., 1984Johnson T.E. Mitchell D.H. Kline S. Kemal R. Foy J. Arresting development arrests aging in the nematode Caenorhabditis elegans.Mech. Ageing Dev. 1984; 28: 23-40Crossref PubMed Scopus (106) Google Scholar). However, worms in L1 arrest actually exhibit signs of aging, but most are reversible upon recovery (Roux et al., 2016Roux A.E. Langhans K. Huynh W. Kenyon C. Reversible age-related phenotypes induced during larval quiescence in C. elegans.Cell Metabol. 2016; 23: 1113-1126Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Nonetheless, extended L1 starvation impacts many life-history traits, including brood size and growth rate (Jobson et al., 2015Jobson M.A. Jordan J.M. Sandrof M.A. Hibshman J.D. Lennox A.L. Baugh L.R. Transgenerational effects of early life starvation on growth, reproduction, and stress resistance in Caenorhabditis elegans.Genetics. 2015; 201: 201-212Crossref PubMed Scopus (87) Google Scholar), and some effects persist across generations (Jobson et al., 2015Jobson M.A. Jordan J.M. Sandrof M.A. Hibshman J.D. Lennox A.L. Baugh L.R. Transgenerational effects of early life starvation on growth, reproduction, and stress resistance in Caenorhabditis elegans.Genetics. 2015; 201: 201-212Crossref PubMed Scopus (87) Google Scholar; Webster et al., 2018Webster A.K. Jordan J.M. Hibshman J.D. Chitrakar R. Baugh L.R. Transgenerational effects of extended dauer diapause on starvation survival and gene expression plasticity in Caenorhabditis elegans.Genetics. 2018; 210: 263-274Crossref PubMed Scopus (39) Google Scholar). These observations suggest that starvation takes a toll on both somatic and germline cells. These cells have different metabolic demands, developmental constraints, and organismal functions, but how their starvation responses are tailored is unknown.L1 arrest is accompanied by changes in transcriptional regulation and gene expression. Gene expression profiles of mRNA change rapidly early in L1 arrest (within hours), as the starvation response is mounted (Baugh et al., 2009Baugh L.R. Demodena J. Sternberg P.W. RNA Pol II accumulates at promoters of growth genes during developmental arrest.Science. 2009; 324: 92-94Crossref PubMed Scopus (129) Google Scholar). A number of transcriptional regulators, including transcription factors, are required to support starvation survival, suggesting a critical role of transcriptional regulation (Baugh and Sternberg, 2006Baugh L.R. Sternberg P.W. DAF-16/FOXO regulates transcription of cki-1/Cip/Kip and repression of lin-4 during C. elegans L1 arrest.Curr. Biol. 2006; 16: 780-785Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar; Zhong et al., 2010Zhong M. Niu W. Lu Z.J. Sarov M. Murray J.I. Janette J. Raha D. Sheaffer K.L. Lam H.Y.K. Preston E. et al.Genome-wide identification of binding sites defines distinct functions for Caenorhabditis elegans PHA-4/FOXA in development and environmental response.PLoS Genet. 2010; 6: e1000848Crossref PubMed Scopus (131) Google Scholar; Fukuyama et al., 2012Fukuyama M. Sakuma K. Park R. Kasuga H. Nagaya R. Atsumi Y. Shimomura Y. Takahashi S. Kajiho H. Rougvie A. et al.C. elegans AMPKs promote survival and arrest germline development during nutrient stress.Biol. Open. 2012; 1: 929-936Crossref PubMed Scopus (62) Google Scholar; Baugh, 2013Baugh L.R. To grow or not to grow: nutritional control of development during Caenorhabditis elegans L1 arrest.Genetics. 2013; 194: 539-555Crossref PubMed Scopus (134) Google Scholar; Cui et al., 2013Cui M. Cohen M.L. Teng C. Han M. The tumor suppressor Rb critically regulates starvation-induced stress response in C. elegans.Curr. Biol. 2013; 23: 975-980Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar; O'Rourke and Ruvkun, 2013O'Rourke E.J. Ruvkun G. MXL-3 and HLH-30 transcriptionally link lipolysis and autophagy to nutrient availability.Nat. Cell Biol. 2013; 15: 668-676Crossref PubMed Scopus (202) Google Scholar; Kaplan et al., 2015Kaplan R.E.W. Chen Y. Moore B.T. Jordan J.M. Maxwell C.S. Schindler A.J. Baugh L.R. dbl-1/TGF-beta and daf-12/NHR signaling mediate cell-nonautonomous effects of daf-16/FOXO on starvation-induced developmental arrest.PLoS Genet. 2015; 11: e1005731Crossref PubMed Scopus (28) Google Scholar; Murphy et al., 2019Murphy J.T. Liu H. Ma X. Shaver A. Egan B.M. Oh C. Boyko A. Mazer T. Ang S. Khopkar R. et al.Simple nutrients bypass the requirement for HLH-30 in coupling lysosomal nutrient sensing to survival.PLoS Biol. 2019; 17: e3000245Crossref PubMed Scopus (10) Google Scholar; Baugh and Hu, 2020Baugh L.R. Hu P.J. Starvation responses throughout the Caenorhabditis elegans life cycle.Genetics. 2020; 216: 837-878Crossref PubMed Scopus (24) Google Scholar). However, gene expression dynamics that occur beyond 24 h of starvation, and time of action of transcriptional regulation for supporting survival, are largely unknown.In C. elegans, zygotic mRNA transcription begins in somatic blastomeres at the two- to four-cell stage of embryogenesis (Seydoux and Fire, 1994Seydoux G. Fire A. Soma-germline asymmetry in the distributions of embryonic RNAs in Caenorhabditis elegans.Development. 1994; 120: 2823-2834Crossref PubMed Google Scholar; Baugh et al., 2003Baugh L.R. Hill A.A. Slonim D.K. Brown E.L. Hunter C.P. Composition and dynamics of the Caenorhabditis elegans early embryonic transcriptome.Development. 2003; 130: 889-900Crossref PubMed Scopus (210) Google Scholar), whereas PIE-1 represses transcription in the P lineage, which produces primordial germ cells (PGCs) (Mello et al., 1996Mello C.C. Schubert C. Draper B. Zhang W. Lobel R. Priess J.R. The PIE-1 protein and germline specification in C. elegans embryos.Nature. 1996; 382: 710-712Crossref PubMed Scopus (266) Google Scholar; Seydoux et al., 1996Seydoux G. Mello C.C. Pettitt J. Wood W.B. Priess J.R. Fire A. Repression of gene expression in the embryonic germ lineage of C. elegans.Nature. 1996; 382: 713-716Crossref PubMed Scopus (249) Google Scholar; Seydoux and Dunn, 1997Seydoux G. Dunn M.A. Transcriptionally repressed germ cells lack a subpopulation of phosphorylated RNA polymerase II in early embryos of Caenorhabditis elegans and Drosophila melanogaster.Development. 1997; 124: 2191-2201Crossref PubMed Google Scholar). Global repression of transcription in the early embryonic germline is conserved among metazoa, apparently preventing specification of somatic fates (Wang and Seydoux, 2013Wang J.T. Seydoux G. Germ cell specification.Adv. Exp. Med. Biol. 2013; 757: 17-39Crossref PubMed Scopus (39) Google Scholar). Zygotic mRNA transcription begins in PGCs during mid-embryogenesis in C. elegans (Wang and Seydoux, 2013Wang J.T. Seydoux G. Germ cell specification.Adv. Exp. Med. Biol. 2013; 757: 17-39Crossref PubMed Scopus (39) Google Scholar). Upon hatching, L1 larvae have 558 cells, two of which are the PGCs, Z2 and Z3. Z2 and Z3 are transcriptionally repressed during L1 arrest, which depends on chromatin compaction genes CEC-4 and HPL-2/HP1 as well as the kinase AMPK and phosphatase DAF-18/PTEN (Demoinet et al., 2017Demoinet E. Li S. Roy R. AMPK blocks starvation-inducible transgenerational defects in Caenorhabditis elegans.Proc. Natl. Acad. Sci. USA. 2017; 114: E2689-E2698Crossref PubMed Scopus (35) Google Scholar; Belew et al., 2021Belew M.D. Chien E. Wong M. Michael W.M. A global chromatin compaction pathway that represses germline gene expression during starvation.J. Cell Biol. 2021; 220 (e202009197)Crossref PubMed Scopus (3) Google Scholar; Fry et al., 2021Fry A.L. Webster A.K. Burnett J. Chitrakar R. Baugh L.R. Hubbard E.J.A. DAF-18/PTEN inhibits germline zygotic gene activation during primordial germ cell quiescence.PLoS Genet. 2021; 17 (e1009650)Crossref PubMed Scopus (4) Google Scholar). However, the physiological significance of germline transcriptional repression during L1 arrest, and whether this repression is maintained throughout arrest, has not been addressed. It is also unclear if the soma remains transcriptionally active after establishing the starvation response, and the contribution of transcript stability to global gene expression dynamics has not been determined for the soma or germline.Here, we performed mRNA sequencing (mRNA-seq) on whole, starved L1 larvae over 12 time points spanning the entirety of L1 arrest. We found that gene expression changes throughout starvation, affecting the majority of genes. We used selective degradation of the large subunit of RNA polymerase II (RNA Pol II) AMA-1 in the soma and germline to show that transcription during the first 2 days of starvation in the soma, but at no point in the germline, is required to support starvation survival. Late in starvation, transcription of thousands of genes continues in the soma as a way of maintaining the initial starvation response while the overall amount of mRNA per animal declines. In contrast, the germline is transcriptionally inactive throughout arrest, but germline transcripts are remarkably stable. Critically, disruption of cec-4 shows that germ cell transcriptional quiescence supports reproductive success upon recovery. Collectively, our results suggest that the soma and germline use distinct regulatory strategies to support organismal fitness during starvation-induced developmental arrest.ResultsWe performed mRNA-seq on whole, starved L1 larvae over time to determine gene expression dynamics throughout L1 arrest. The time series starts approximately 2 h prior to hatching (−2 h) to capture the onset of starvation and extended 12 days beyond that (Figure 1A ). Because expression dynamics slow within 12 h (Baugh et al., 2009Baugh L.R. Demodena J. Sternberg P.W. RNA Pol II accumulates at promoters of growth genes during developmental arrest.Science. 2009; 324: 92-94Crossref PubMed Scopus (129) Google Scholar), we collected time points densely early in arrest and sparsely late in arrest (Figure 1A). ∼50% of larvae hatched between 0 and 2 h, and ∼80% were hatched by 4 h (Figure 1B), reflecting consistent synchrony and staging. About 80% of larvae were still alive at 8 days, but survival dropped to about 30% at 12 days (Figure 1C). Thus, the time series spans the entirety of L1 arrest, from hatch to death.Gene expression dynamics throughout starvationWe performed principal-component analysis (PCA) as an initial evaluation of expression dynamics. Biological replicates clustered together, as expected, and time points were ordered based on the duration of L1 arrest (Figure 1D). A rapid response to starvation was evident in the early hours after hatching, as expected (Baugh et al., 2009Baugh L.R. Demodena J. Sternberg P.W. RNA Pol II accumulates at promoters of growth genes during developmental arrest.Science. 2009; 324: 92-94Crossref PubMed Scopus (129) Google Scholar). However, time points including day 1 and beyond are clearly distinct from earlier time points, revealing that gene expression continues changing late in starvation. We measured the rate of change between adjacent time points and found that it decreases dramatically during starvation, with a major inflection near 24 h (Figure 1E). These results suggest a rapid early response to starvation followed by a much slower late response extending until death.Strikingly, the vast majority of genes are differentially expressed during starvation. Over 84% of detected genes (14,034 genes) are differentially expressed at a false discovery rate (FDR) of 0.05, and over 35% (6,027 genes) are differentially expressed at a highly stringent cutoff of 10−30 (Figure 1F; Table S1). We generated an RShiny app for users to generate plots of differentially expressed genes of interest over time throughout starvation (https://awebster.shinyapps.io/shinyapp/). This app can also be downloaded and run locally (https://github.com/amykwebster/StarvationTimeSeriesPlots). These results demonstrate the profound effect of starvation on gene expression.Despite pervasive effects of starvation, cluster analysis revealed relatively simple temporal patterns. The clustering algorithm used produced 129 clusters for the 6,027 most significantly affected genes, but many of them are distinguished by relatively minor differences in timing or include only a few genes (Figure S1). The 10 largest clusters include about two-thirds of the genes, and these clusters show the predominant expression patterns present in the full dataset. Broadly, these patterns consist of genes monotonically increasing or decreasing in relative expression, even deep into starvation (Figure 2A ). The most complex common pattern is an increase followed by a decrease with a single peak early in starvation (e.g., clusters 5, 9, and 10), and other more complex patterns are either very rare or absent (Figures 2B and S1). These observations suggest that the gene-regulatory network controlling the starvation response is relatively shallow compared with developmental regulatory networks.Figure 2Expression analysis reveals temporal patterns of regulatory activityShow full caption(A) Z score-normalized expression dynamics over time for the 10 largest clusters.(B) Heatmap of all clustered genes (FDR <10−30), sorted by cluster similarity and color coded by Z score. Colored bars to the left correspond to genes in clusters shown in (A).(C–F) Gene expression dynamics for known targets of important transcriptional regulators. Number of genes included is inset on each graph. Lines indicate the mean Z score and 99% confidence intervals for all genes in each group. To the right of each graph, −log10 enrichment p values are plotted for the top 10 clusters.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Known transcriptional regulators mostly act early in starvationPrevious studies have identified regulators of L1 starvation survival, including transcription factors and signaling molecules that affect transcription factor activity (Baugh and Sternberg, 2006Baugh L.R. Sternberg P.W. DAF-16/FOXO regulates transcription of cki-1/Cip/Kip and repression of lin-4 during C. elegans L1 arrest.Curr. Biol. 2006; 16: 780-785Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar; Zhong et al., 2010Zhong M. Niu W. Lu Z.J. Sarov M. Murray J.I. Janette J. Raha D. Sheaffer K.L. Lam H.Y.K. Preston E. et al.Genome-wide identification of binding sites defines distinct functions for Caenorhabditis elegans PHA-4/FOXA in development and environmental response.PLoS Genet. 2010; 6: e1000848Crossref PubMed Scopus (131) Google Scholar; Fukuyama et al., 2012Fukuyama M. Sakuma K. Park R. Kasuga H. Nagaya R. Atsumi Y. Shimomura Y. Takahashi S. Kajiho H. Rougvie A. et al.C. elegans AMPKs promote survival and arrest germline development during nutrient stress.Biol. Open. 2012; 1: 929-936Crossref PubMed Scopus (62) Google Scholar; Baugh, 2013Baugh L.R. To grow or not to grow: nutritional control of development during Caenorhabditis elegans L1 arrest.Genetics. 2013; 194: 539-555Crossref PubMed Scopus (134) Google Scholar; Cui et al., 2013Cui M. Cohen M.L. Teng C. Han M. The tumor suppressor Rb critically regulates starvation-induced stress response in C. elegans.Curr. Biol. 2013; 23: 975-980Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar; O'Rourke and Ruvkun, 2013O'Rourke E.J. Ruvkun G. MXL-3 and HLH-30 transcriptionally link lipolysis and autophagy to nutrient availability.Nat. Cell Biol. 2013; 15: 668-676Crossref PubMed Scopus (202) Google Scholar; Kaplan et al., 2015Kaplan R.E.W. Chen Y. Moore B.T. Jordan J.M. Maxwell C.S. Schindler A.J. Baugh L.R. dbl-1/TGF-beta and daf-12/NHR signaling mediate cell-nonautonomous effects of daf-16/FOXO on starvation-induced developmental arrest.PLoS Genet. 2015; 11: e1005731Crossref PubMed Scopus (28) Google Scholar; Murphy et al., 2019Murphy J.T. Liu H. Ma X. Shaver A. Egan B.M. Oh C. Boyko A. Mazer T. Ang S. Khopkar R. et al.Simple nutrients bypass the requirement for HLH-30 in coupling lysosomal nutrient sensing to survival.PLoS Biol. 2019; 17: e3000245Crossref PubMed Scopus (10) Google Scholar; Baugh and Hu, 2020Baugh L.R. Hu P.J. Starvation responses throughout the Caenorhabditis elegans life cycle.Genetics. 2020; 216: 837-878Crossref PubMed Scopus (24) Google Scholar). We determined expression profiles of known targets (direct and indirect) of critical regulators to shed light on when they are most active. We focused our analysis on DAF-16/FoxO, DAF-18/PTEN, LIN-35/Rb, and HLH-30/TFEB because loss of each severely compromises starvation survival and genome-wide expression data for each mutant in L1 arrest are available (Cui et al., 2013Cui M. Cohen M.L. Teng C. Han M. The tumor suppressor Rb critically regulates starvation-induced stress response in C. elegans.Curr. Biol. 2013; 23: 975-980Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar; Tepper et al., 2013Tepper R.G. Ashraf J. Kaletsky R. Kleemann G. Murphy C.T. Bussemaker H.J. PQM-1 complements DAF-16 as a key transcriptional regulator of DAF-2-mediated development and longevity.Cell. 2013; 154: 676-690Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar; Kaplan et al., 2015Kaplan R.E.W. Chen Y. Moore B.T. Jordan J.M. Maxwell C.S. Schindler A.J. Baugh L.R. dbl-1/TGF-beta and daf-12/NHR signaling mediate cell-nonautonomous effects of daf-16/FOXO on starvation-induced developmental arrest.PLoS Genet. 2015; 11: e1005731Crossref PubMed Scopus (28) Google Scholar; Murphy et al., 2019Murphy J.T. Liu H. Ma X. Shaver A. Egan B.M. Oh C. Boyko A. Mazer T. Ang S. Khopkar R. et al.Simple nutrients bypass the requirement for HLH-30 in coupling lysosomal nutrient sensing to survival.PLoS Biol. 2019; 17: e3000245Crossref PubMed Scopus (10) Google Scholar; Fry et al., 2021Fry A.L. Webster A.K. Burnett J. Chitrakar R. Baugh L.R. Hubbard E.J.A. DAF-18/PTEN inhibits germline zygotic gene activation during primordial germ cell quiescence.PLoS Genet. 2021; 17 (e1009650)Crossref PubMed Scopus (4) Google Scholar). Positively regulated targets (genes down-regulated in the mutant) are expressed at their highest levels at different times after hatching, with DAF-16, DAF-18, and some HLH-30 targets peaking between 6 and 12 h of arrest, and LIN-35 and other HLH-30 targets peaking between 2 and 4 days of L1 arrest (Figures 2C–2F), consistent with relatively early and late function, respectively. SKN-1/Nrf and AMPK targets were identified in later developmental stages without starvation (Steinbaugh et al., 2015Steinbaugh M.J. Narasimhan S.D. Robida-Stubbs S. Moronetti Mazzeo L.E. Dreyfuss J.M. Hourihan J.M. Raghavan P. Operaña T.N. Esmaillie R. Blackwell T.K. Lipid-mediated regulation of SKN-1/Nrf in response to germ cell absence.Elife. 2015; 4Crossref PubMed Scopus (118) Google Scholar; El-Houjeiri et al., 2019El-Houjeiri L. Possik E. Vijayaraghavan T. Paquette M. Martina J.A. Kazan J.M. Ma E.H. Jones R. Blanchette P. Puertollano R. Pause A. The transcription factors TFEB and TFE3 link the FLCN-AMPK signaling Axis to innate immune response and pathogen resistance.Cell Rep. 2019; 26: 3613-3628.e6Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), but these targets exhibit peak expression very early in L1 arrest as if these factors help establish the starvation response (Figures S2D and S2E). Like the global dynamics of the starvation response (Figure 1E), these patterns suggest that the transcriptional response to starvation driven by known regulators is largely mounted early. In contrast, regulation accounting for the relative increase in expression observed for hundreds of genes deep in starvation (e.g., clusters 4 and 6) is unknown.Differential regulation of germline and somatic genes deep into developmental arrestThree of our six largest clusters (clusters 2, 4, and 6) are expressed at relatively low levels upon hatching, exhibit peak expression levels beyond 4 days of starvation, and either maintain or increase expression levels up to 12 days of starvation. These clusters are enriched with genes expressed in several tissues related to reproduction, including “germline,” “gonad primordium,” and “reproductive system” (Figure 3A ) (Angeles-Albores et al., 2016Angeles-Albores D. N Lee R.Y. Chan J. Sternberg P.W. Tissue enrichment analysis for C. elegans genomics.BMC Bioinf. 2016; 17: 366Crossref PubMed Scopus (64) Google Scholar), which was surprising given that transcription is repressed in germ cells in the first few hours of L1 arrest (Belew et al., 2021Belew M.D. Chien E. Wong M. Michael W.M. A global chromatin compaction pathway that represses germline gene expression during starvation.J. Cell Biol. 2021; 220 (e202009197)Crossref PubMed Scopus (3) Google Scholar; Fry et al., 2021Fry A.L. Webster A.K. Burnett J. Chitrakar R. Baugh L.R. Hubbard E.J.A. DAF-18/PTEN inhibits germline zygotic gene activation during primordial germ cell quiescence.PLoS Genet. 2021; 17 (e1009650)Crossref PubMed Scopus (4) Google Scholar). We directly assessed if genes typically expressed in the soma or germline exhibit distinct expression patterns. We used published single-cell RNA-seq data from embryos to define gene sets enriched in somatic or PGCs (Packer et al., 2019Packer J.S. Zhu Q. Huynh C. Sivaramakrishnan P. Preston E. Dueck H. Stefanik D. Tan K. Trapnell C. Kim J. et al.A lineage-resolved molecular atlas of C. elegans embryogenesis at single-cell resolution.Science. 2019; 365: eaax1971Crossref PubMed Scopus (139) Google Scholar). PGC-enriched genes are largely expressed at peak levels late in starvation, based on statistical enrichment of clusters 2, 4, and 6 (Figure 3B), and this was robust to defining PGC enrichment with increasing stringency (Figure S3). In contrast, soma-enriched genes are over-represented in clusters with peak expression within the first few hours of starvation (clusters 1, 3, 5, and 7). Collectively, these results suggest that PGC-enriched genes are more likely to increase in relative expression throughout arrest, while soma-enriched genes are more likely to peak early and decrease, though it should be noted that individual genes may deviate from these patterns.Figure 3Somatic and germline genes have different patterns of regulation during starvationShow full caption(A) Tissue enrichments for clusters 2, 4, and 6, which have peak expression late in starvation. Germline-related tissues are highlighted in pink.(B–E) Z scores over time are plotted for all individual genes in the indicated group and clustered dataset. The number of genes is inset on each graph. Genes are color coded (see legend) by cluster if the cluster is enriched in the gene group with all other genes in gray.(B and E) Cluster enrichment color coded if hypergeometric p < 0.01.(C and D) Cluster enrichment color coded if hypergeometric p < 0.05.(F) Venn diagram of germline-enriched genes plotted in (B) and genes with active (hypergeometric p = 1) or docked (hypergeometric p = 2.4 × 10−7) RNA Pol II.(G) Venn diagram of soma-enriched genes plotted in (B) and genes with active (hypergeometric p = 1.0 × 10−7) or docked (hypergeometric p = 0.90) RNA Pol II.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We extended our analysis with a pair of published datasets examining expression in PGCs sorted from L1-stage larvae, including (1) genes differentially expressed between embryonic and fed L1 PGCs and (2) genes differentially expressed between fed and starved L1 PGCs (Lee et al., 2017Lee C.Y.S. Lu T. Seydoux G. Nanos promotes epigenetic reprograming of the germline by down-regulation of the THAP transcription factor LIN-15B.Elife. 2017; 6: e30201Crossref PubMed Scopus (21) Google Scholar). As expected, genes down-regulated in fed L1 compared with embryonic PGCs are over-represented in clusters that decrease in expression within hours of hatching (clusters 1 and 3), though they are also enriched in clusters that increase late (clusters 4 and 6) (Figure 3C). In contrast, the majority of genes up-regulated in fed L1 relative to embryonic PGCs are part of the late clusters 2, 4, and 6, with cluster 2 significantly over-represented (Figure 3C). Genes down-regulated in starved compared with fed PGCs are over-represented among clusters with peak expression within the first few hours of L1 arrest (clusters 1, 5, and 8), though the late-peaking cluster 6 is also enriched (Figure 3D). In contrast, six of the seven genes that are up-regulated in starved L1 PGCs exhibit peak expression late in arrest, including three genes significantly enriched in cluster 2 (Figure 3D). Together, these observations suggest that genes with differential expression in L1 PGCs, due to either developmental regulation or starvation, show similar patterns early in L1 arrest in our whole-animal data. They also further support the conclusion that many germline genes increase in relative expression levels deep into starvation.Distinct temporal patterns of steady-state expression for germline and soma-enriched genes could be driven by active transcription or differences in transcript stability. To gain insight on this distinction, we used previously defined gene sets early in L1 starvation in which RNA Pol II is “active” or “docked” (Maxwell et al., 2014Maxwell C.S. Kruesi W.S. Core L.J. Kurhanewicz N. Waters C.T. Lewarch C.L. Antoshechkin I. Lis J.T. Meyer B.J. Baugh L.R. Pol II docking and pausing at growth and stress genes in C. elegans.Cell Rep. 2014; 6: 455-466Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Active ge" @default.
• W4304196160 created "2022-10-11" @default.
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• W4304196160 date "2022-10-01" @default.
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• W4304196160 title "Alternative somatic and germline gene-regulatory strategies during starvation-induced developmental arrest" @default.
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• W4304196160 doi "https://doi.org/10.1016/j.celrep.2022.111473" @default.
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