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. Author manuscript; available in PMC: 2017 Dec 15.
Published in final edited form as: Mol Cell. 2016 Dec 15;64(6):1088–1101. doi: 10.1016/j.molcel.2016.11.020

Survival in quiescence requires the euchromatic deployment of Clr4/SUV39H by Argonaute-associated small RNAs

Richard I Joh 1, Jasbeer S Khanduja 1, Isabel A Calvo 1, Meeta Mistry 2, Christina M Palmieri 1, Andrej J Savol 3, Shannan J Ho Sui 2, Ruslan I Sadreyev 3,4,5, Martin J Aryee 1,5, Mo Motamedi 1,*
PMCID: PMC5180613  NIHMSID: NIHMS832554  PMID: 27984744

Summary

Quiescence (G0) is a ubiquitous stress response through which cells enter reversible dormancy, acquiring distinct properties including reduced metabolism, resistance to stress and long life. G0 entry involves dramatic changes to chromatin and transcription of cells, but the mechanisms coordinating these processes remain poorly understood. Using the fission yeast, here we track G0-associated chromatin and transcriptional changes temporally and show that as cells enter G0, their survival and global gene expression programs become increasingly dependent on Clr4/SUV39H, the sole histone H3 lysine 9 (H3K9) methyltransferase, and RNA interference (RNAi) proteins. Notably, G0 entry results in RNAi-dependent H3K9 methylation of several euchromatic pockets, prior to which Argonaute1-associated small RNAs from these regions emerge. Overall our data reveal a function for constitutive heterochromatin proteins (the establishment of the global G0 transcriptional program) and suggest that stress-induced alterations in Argonaute-associated sRNAs can target the deployment of transcriptional regulatory proteins to specific sequences.

Keywords: Quiescence, G0, H3K9me, Clr4/Suv39H1, RNAi, Argonaute, sRNA, viability, heterochromatin, gene silencing, RNA degradation, nuclear exosome

Graphical abstract

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eTOC Blurb

Joh et al use a time-course quiescence (G0) assay to show that as cells transition to G0, they accumulate Ago1-associated sRNAs from euchromatic genes. These sRNAs recruit the constitutive heterochromatin protein (H3K9 methyltransferase) Clr4 to euchromatic regions, required for the establishment of the global G0 state.

Introduction

Survival in a fluctuating environment is a central feature of life. Evolution has equipped cells with a range of adaptive responses. One is entry into a highly conserved cellular state called quiescence (G0) within which cells resist stress and survive for long periods of time in reversible dormancy (Pardee, 1974; Sang et al., 2008). In organisms ranging from yeast to mammals, G0 entry results in cell cycle arrest, reduction in cell size, metabolism, RNA transcription, protein translation, ribosomal RNA (rRNA) synthesis, and activation of survival, longevity, stress resistance and alternative metabolic pathways (Valcourt et al., 2012). These together establish the G0 program, which imparts distinct cellular and molecular properties to G0 compared to proliferative cells (Coller, 2011). Regulation of G0 is critical for development and tissue maintenance in multicellular organisms and defects to this regulation are linked to several human pathologies (Cheung and Rando, 2013; Sosa et al., 2014). But despite its importance, the mechanisms by which cells transit to and from G0 have remained poorly charted.

Entry into G0 in yeast and mammalian cells involves chromatin compaction and changes to global histone marks. For example, histone hypoacetylation (McKnight et al., 2015; Mews et al., 2014) and redistribution of a constitutive heterochromatic histone mark, histone H4 lysine 20 methylation (H4K20me) (Boonsanay et al., 2016; Evertts et al., 2013), are critical for G0 in yeast and mammalian cells, respectively. Also in quiescent mouse and human cells, the level and distribution of histone H3 lysine 9 methylation (H3K9me), the hallmark of eukaryotic heterochromatin, appears to be different compared to proliferative cells (Grigoryev et al., 2004). G0 entry also requires the establishment of a unique transcriptional program (Coller et al., 2006; Iyer et al., 1999; Marguerat et al., 2012; Slavov and Botstein, 2013), which in part involves the coordinated transcriptional repression of selective groups of genes. This repression may involve the deployment of repressive factors, such as heterochromatin proteins, in G0 specifically.

To model quiescence, we used Schizosaccharomyces pombe, whose G0 state and heterochromatin regulation bear similarities to mammalian cells (Holoch and Moazed, 2015a; Yanagida, 2009). Moreover, depriving (glucose) or starving (nitrogen) S. pombe cells for simple nutrients induces cells to enter G0 uniformly, creating pure, isogenic G0 cell populations (Su et al., 1996; Wei et al., 1993). Also similar to other eukaryotes, H3K9 trimethylation (H3K9me3) demarcates centromeric, telomeric, and ribosomal DNA (rDNA) heterochromatin, catalyzed by the sole H3K9 methyltransferase Clr4, a member of the Clr4-Rik1-Cul4 (CLRC) complex, whose recruitment to centromeres is facilitated by the RNA interference (RNAi) proteins (Martienssen and Moazed, 2015; Volpe et al., 2002). In the fission yeast, the coordinate activities of Argonaute (Ago1), Dicer (Dcr1) and RNA-dependent RNA polymerase (Rdp1) homologs on centromeric (cen) long noncoding RNAs (lncRNAs) create an amplification loop, which generates small interfering RNAs (siRNAs) from cen transcripts (Motamedi et al., 2004). These cen siRNAs are loaded to an Ago1-bearing complex called RNA-induced Transcriptional Silencing (RITS), which also contains of an H3K9mebinding protein (Chp1) and a GW-motif protein (Tas3) (Verdel et al., 2004). RITS recruitment to centromeres requires cen siRNAs (basepairing with cen sequences) and Chp1 binding to H3K9me and is thought to provide a recruitment hub for CLRC, RNAi and other RNA degradation complexes at cen sequences via its physical interactions with these proteins and cen lncRNAs (Martienssen and Moazed, 2015).

In addition to RITS, H3K9me serves as a binding substrate for several other silencing complexes, tethering transcriptional (TGS) and posttranscriptional gene silencing (PTGS) activities to heterochromatin. TGS requires the histone deacetylase Sir2 (Alper et al., 2013; Buscaino et al., 2013; Shankaranarayana et al., 2003), the H3K9me-binding protein Chp2 and the associated Snf2-histone deacetylase (SHREC2) complex (Fischer et al., 2009; Motamedi et al., 2008; Sugiyama et al., 2007; Yamada et al., 2005) which together restrict RNA polymerase II-dependent transcription in heterochromatin. In addition to RNAi, PTGS requires the H3K9me-binding protein Swi6 (Keller et al., 2012; Motamedi et al., 2008), and transcription elongation factor Spt6 (Kiely et al., 2011) which work in parallel with the mRNA quality control factors including the Trf4-Air2-Mtr4 polyadenylation complex TRAMP (Bühler et al., 2007), the conserved exoribonuclease Dhp1 (Chalamcharla et al., 2015), and the multisubunit 3’ to 5’ RNA degradation complex, exosome (Reyes-Turcu et al., 2011; Yamanaka et al., 2013). TRAMP not only prevents the entry of euchromatic transcripts into the RNAi pathway (Buhler et al., 2008), but also interacts with Mlo3, a homolog of Aly/REF, an RNA export factor, and is thought to shunt heterochromatic transcripts for degradation by the RNAi or exosome pathways (Zhang et al., 2011). Together these complexes degrade heterochromatic transcripts in cis relative to their site of synthesis (Bühler et al., 2007; Reyes-Turcu et al., 2011) and with the TGS proteins establish the full repressive state at heterochromatin.

In wild-type (wt) proliferative cells, H3K9me is found primarily at heterochromatic regions, but a few meiotic genes also display this modification (Zofall et al., 2012). H3K9 methylation at these sites is mostly RNAi-independent, but requires the YTH-domain containing protein Mmi1, which binds to and marks meiotic RNAs for degradation (Harigaya et al., 2006). Mmi1 interacts with Red1, a member of the MTREC/NURS complex (Egan et al., 2014; Lee et al., 2013), which facilitates exosome-mediated elimination of these transcripts in proliferative cells. Unlike heterochromatic domains, the transcriptional silencing at these H3K9me islands is largely independent of Clr4, RNAi or TGS factors (Egan et al., 2014) and proceeds via an MTREC/exosome-mediated PTGS mechanism.

Consistent with the distribution of H3K9me in proliferative cells, more than 90% of all Ago1-associated small RNAs (sRNAs) map to heterochromatic regions (Buhler et al., 2008; Halic and Moazed, 2010), demonstrating that heterochromatic transcripts are the primary substrates of RNAi proteins. However, in the absence of TRAMP (Buhler et al., 2008; Reyes-Turcu et al., 2011), exosome, or under certain environmental growth conditions sRNAs and H3K9me mapping to some developmental genes and retrotransposons within the euchromatic regions of the genome (called HOODs) can be detected (Yamanaka et al., 2013). Unlike the meiotic H3K9me islands in wt cells, the transcriptional repression of HOODs requires RNAi-dependent heterochromatin formation. These results reveal that the activities of these RNA processing proteins, which enforce mRNA quality control in the nucleus (Fox and Mosley, 2016), limit the availability of euchromatic transcripts as substrates for RNAi proteins in proliferative cells. Also, overexpression of limiting components of the RNAi machinery (such as Dcr1) leads to emergence of RNAi-dependent H3K9me at euchromatic genes (Yu et al., 2014). Together these studies suggest that the abundance of nuclear exosome activity and paucity of RNAi proteins are critical for limiting RNAi-and H3K9me-dependent silencing to heterochromatic regions in proliferative cells.

Here using a time-course G0 assay, we show that as cells enter G0, their survival and a part of their global G0 transcriptional program become increasingly dependent on Clr4/SUV39H and RNAi proteins. G0 entry results in the accumulation of RNAi-dependent H3K9me at several euchromatic domains, from which G0-specific sRNAs emerge. These genes form distinct euchromatic clusters, are co-regulated by Clr4 and RNAi proteins in G0 and are overrepresented in developmental, cell cycle, and metabolic functions, all of which are important for establishing the G0 state in eukaryotes. The formation of the G0-specific sRNAs is Ago1 dependent, and occurs prior to H3K9me of the corresponding genes. Moreover, they emerge at the same time as the expression of all exosome/TRAMP components is down-regulated, and the expression of all RNAi proteins is up-regulated. Together these data suggest that environmentally-regulated decrease in exosome/TRAMP and increase in RNAi activities permits the targeting of non-heterochromatic transcripts by RNAi factors, producing Ago1-associated sRNAs, which later recruit Clr4-dependent H3K9 methylation to complementary euchromatic regions. These results reveal a new role for Clr4 protein, whose deployment by G0-induced sRNAs to euchromatic regions facilitates the establishment of the global G0 transcriptional program.

Results

RNAi and heterochromatin proteins are required for cell viability in G0

To test the role of heterochromatin proteins in G0, we induced quiescence in prototrophic wild-type (wt) and different mutant S. pombe strains by nitrogen (N) starvation (Su et al., 1996) or glucose (G) deprivation (Pluskal et al., 2011) and assayed cell viability (live cell imaging and plate assays) for 10 days posttreatment. In contrast to the TGS factors (Sir2 and Chp2), we found that the absence of Clr4 or RNAi proteins results in a progressive and dramatic loss of viability independently of the method of G0 induction. These results demonstrate the importance of these proteins for survival within (Figures 1A and 1B) and exit from (Figure S1) G0.

Fig. 1.

Fig. 1

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Survival in G0 requires RNAi and Clr4 proteins and is concomitant with Clr4-dependent transcriptional reprogramming. (A and B) Viability of indicated strains at various time points after (A) nitrogen (N) starvation and (B) glucose deprivation (0.02%). Error bars represent standard deviation (SD) of 3 biological replicates. (C) Heat map (RNA log2 fold change (FC)) depicting the hierarchical clustering of the transcriptome of wt cells at various time points after N starvation relative to proliferative cells (t=0). Consistent with previous reports (Marguerat et al., 2012; Shimanuki et al., 2007), the early (1h, 2h) (N starvation and mating) and late (10h, 24h) (G0) transcriptomes are significantly different from one another. (D) Difference of RNA log2 FC in transcriptomes of clr4Δ relative to wt cells at indicated times following N starvation. Red dots denote transcripts which show a statistically significant change in clr4Δ relative to wt cells (FDR<0.05; 2 biological replicates).

Clr4 is required for the establishment of the global G0 program

Next to test if Clr4 is required for the establishment of the G0 transcriptional program, we performed RNA sequencing (RNA-seq) analyses on samples collected from wt and clr4Δ cells at 0, 1, 2, 4, 10 and 24 hours after N starvation. In the fission yeast, N starvation induces sexual differentiation; however, failing to find a mate of opposite mating type after 8–12 hours, cells reprogram their transcription and enter quiescence uniformly (Yanagida, 2009). Consistent with previous reports (Marguerat et al., 2012; Shimanuki et al., 2007), we found a distinct early N starvation and mating response (e.g. upregulation of mating genes) 1–2 hours post N starvation, followed by a late G0 response (e.g. repression of mating genes) 10 and 24 hours post N starvation in wt cells (Figure 1C). Entry into G0 results in up-regulation of 718 transcripts (G0-activated) and down-regulation of 661 transcripts (G0-repressed) (Table S1). Strikingly, time-course comparisons of the wt versus clr4Δ transcriptomes revealed that as cells enter G0, a part of their transcriptional program becomes increasingly dependent on Clr4 (Figure 1D). This is in contrast to the almost exclusive role of this protein in repressing transcription of heterochromatic genes in proliferative cells (Figure 1D top left panel). Specifically, the expression of 285 euchromatic genes, enriched in reproduction (p<10−49; 6.8-fold enrichment) and carbohydrate metabolism (p<10−4; 3.9-fold enrichment) and transport (p<10−2; 12.7-fold enrichment) functions, is repressed by Clr4 (Clr4-repressed) in G0 (Tables S1 and S2). Together these data reveal that a part of the global G0 transcriptional program is Clr4-regulated, suggesting a direct role for this protein in establishing the G0 state.

G0 entry results in RNAi-dependent accumulation of H3K9me at several euchromatic regions

Clr4/SUV39H is the only H3K9 methyltransferase in the fission yeast and is responsible for its mono (H3K9me1), di (H3K9me2) and tri (H3K9me3) methylation. In proliferative cells, bulk of the H3K9me is found at heterochromatic domains, with some smaller H3K9me peaks at meiotic genes (Zofall et al., 2012). To test if Clr4/SUV39H-dependent H3K9me can be found at a set of euchromatic genes in G0, we performed H3K9me2 chromatin immunoprecipitation (ChIP) experiments in wt and clr4Δ cells at various time points after N starvation. Unexpectedly, we found that as cells enter G0, they accumulate Clr4-dependent H3K9me at a highly transcribed euchromatic gene, act1, which we often use as a negative control in H3K9me2 ChIP assays in proliferative cells (Figure 2A). This G0-mediated H3K9me2 occurs independently of significant changes to H3 levels (Figure S2A). To ask if G0-mediated H3K9 methylation events also occur at other euchromatic regions, we performed deep sequencing of 5 independent H3K9me2 ChIP experiments (ChIP-seq) from wt and clr4Δ cells. Our time-course analyses revealed the emergence of more than 200 H3K9me-enriched euchromatic regions distributed across the three S. pombe chromosomes which show an accumulation of this modification in G0 specifically (Figures 2B – 2D, and Table S3). These regions have an average size of ~4kb and overlap 290 transcripts (H3K9me2-enriched) (Table S1). H3K9me3 ChIP-seq analysis in wt and clr4Δ cells also confirmed the occurrence of trimethylated form of this modification at these peaks in G0 (Figures S2C and S2D). Among these genes (H3K9me-enriched), highly expressed (p<10−57; 5.9-fold enrichment), metabolic (glycolytic, TCA) (p<10−4; 10-fold enrichment), ribosomal (p<10−14; 4.2-fold enrichment), cell cycle (p<10−8; 2.6-fold enrichment), and stress response genes (p<10−6; 2.6-fold enrichment) are overrepresented (Tables S1, S2 and S3). Because the transcriptional regulation of many of these genes is critical for the establishment of G0 state in eukaryotes, these data suggest that H3K9 may play an important role for survival in G0. To test this, we used a strain in which the endogenous copy of histone H3 has a lysine-to-alanine mutation at residue 9 (Mellone et al., 2003). This strain also shows a significant viability defect in G0 compared to the wt control (Figure S2B) suggesting that modification of this residue plays an important role for survival in G0.

Fig. 2.

Fig. 2

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G0 entry results in RNAi-dependent H3K9 methylation of several euchromatic loci. (A) ChIP experiments showing that H3K9me2 accumulates at act1 following N starvation. Fold enrichment of H3K9me at act1 relative to cen1 is depicted. Mean and standard error (SE) of 5 biological replicates are shown. (B and C) H3K9me2 accumulation at several euchromatic regions. Average H3K9me2 enrichment (IP over input) of all ‘H3K9me-enriched’ peaks identified by ChIP-seq in (B) wt and (C) clr4Δ at 0, 1 and 24h after N starvation is shown. Error bars represent 95% bootstrap confidence interval from 1,000 resamplings. (D) The genomic distribution of G0-specific H3K9me2 peaks in wt cells are shown as blue bars. Black boxes depict centromeres (Ch = chromosome). (E) qPCR analyses of H3K9me2 and H3 enrichment at act1 relative to cen1 in G0 (24h) of the indicated strains relative to proliferative (0h) wt cells. Mean and SE of 3 biological replicates are graphed. (F) Average H3K9me2 enrichment of ‘H3K9me-enriched’ peaks in wt and ago1Δ at 0 and 24h after N starvation. Error bars represent 95% bootstrap confidence interval from 1,000 resamplings.

Next, because RNAi facilitates Clr4-mediated H3K9me at centromeres of proliferative cells, we asked whether a similar mechanism also operates at act1 in G0. H3K9me2 ChIP experiments in wt, ago1Δ, rdp1Δ, and dcr1Δ mutant cells revealed that H3K9me2 at act1 in G0 cells is RNAi dependent (Figure 2E). Similarly, two independent H3K9me2 ChIP-seq analyses of wt and ago1Δ cells in proliferation and G0 revealed that H3K9 methylation of these euchromatic pockets in G0 requires Ago1 (Figure 2F). Moreover, these G0-induced euchromatic H3K9me peaks show no significant overlap with the RNAi-independent H3K9me islands reported in proliferative cells (Zofall et al., 2012). These data reveal that G0 entry is concomitant with the accumulation of RNAi-and Clr4-dependent H3K9me at a distinct set of euchromatic genes.

G0-specific Ago1-associated sRNAs emerge from euchromatic genes

The RNAi dependence of H3K9me at act1 and the other euchromatic regions in G0 suggests that Ago1-associated sRNAs may target Clr4 to these regions. The analyses of three independent Ago1 purifications from proliferative (0 hrs) and G0 (24 hrs) wt cells, followed by deep sequencing of the Ago1-associated sRNAs (Ago1-associated sRNA-seq), revealed that G0 cells have a distinct sRNA prolife compared to their proliferative counterparts (Figure 3A and 3B). Importantly, we observed a 10-fold increase in G0-specific sRNAs mapping to coding regions of the genome, including act1 (Figures 3C and S3A). By purifying Ago1 and sequencing the associated sRNAs we were able to identify (see Experimental Procedures, and Figure S3B and S3C) 168 euchromatic genes, distributed across the three S. pombe chromosomes, which show at least a 4-fold enrichment in sRNAs (sRNA-enriched) in G0 (Figure 3D and Table S1). Similar to the Clr4-repressed and H3K9me-enriched genes, this gene set displays an overrepresentation of highly expressed (p<10−9; 3.6-fold enrichment), stress response (p<10−15; 4.6-fold enrichment), ribosomal (p<10−5; 3.9-fold enrichment), and mating response (p<10−2; 2.1-fold enrichment) genes (Table S2).

Fig. 3.

Fig. 3

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G0 entry results in the emergence of Ago1-associated sRNAs from several euchromatic loci. (A and B) Pie charts depicting the distribution of Ago1-associated sRNAs in (A) proliferative (0h) and (B) G0 (24h) cells. These graphs reveal that G0 cells have a distinct sRNA profile and show a five-fold increase in relative abundance of Ago1-associated sRNAs. Here, ‘heterochromatin’ refers to centromeres, telomeres and the mating type locus; rDNA is shown separately. (C) The fraction (%) of uniquely mapped Ago1-associated sRNA reads (mapping quality ≥ 10) in proliferative (0h) and G0 (24h) wt cells. Mean and SE of 3 biological replicates are graphed. (D) The genomic distribution of Ago1-associated sRNA-enriched regions in G0 is shown as red bars. Black boxes represent centromeres (Ch = chromosome). (E and F) Schematic representation of (E) rDNA and (F) telomere 1 (left) and their sRNA and H3K9me2 dynamics in G0. Strand-specific log2 FC of Ago1-associated sRNAs (magenta) and log2 FC of H3K9me2 (orange) in wt G0 (24h) relative to wt proliferative (0h) cells are shown. These data reveal an increase in sRNAs and H3K9me at the rDNA locus and a decrease in H3K9me at telomeres. Note that because the sRNA graphs are strand specific, decrease or no change in sRNA levels will appear as a flat part of the graph.

Interestingly, these data (Figure 3A and 3B) also reveal an increase in the abundance of Ago1-associated sRNAs from rDNA at the expense of a decrease in the relative abundance of sRNAs from other heterochromatic regions. We detected 5-fold increase in both sRNAs and H3K9me at the rDNA locus of G0 cells (Figure 3E), suggesting that Clr4-dependent H3K9me and RNAi proteins play an important role in the regulation of rDNA in G0 (Cam et al., 2005). The increase in sRNA and H3K9me is unique to rDNA and is not observed at other heterochromatic regions (Figure 3F). Together these data demonstrate that G0 cells have a distinct Ago1-associated sRNA profile, with novel species emerging from functionally similar euchromatic genes.

Clr4-repressed, H3K9me- and sRNA-enriched euchromatic genes overlap

Overall, the combined Clr4-repressed, H3K9me- and sRNA-enriched gene set is overrepresented in reproduction, meiotic cell cycle, and several metabolic pathways including glycolysis, TCA, and pyruvate metabolism (Table S2), many of which also play a role in establishing G0 in other eukaryotes (Valcourt et al., 2012). Next, we performed pairwise comparisons between the aforementioned gene sets and found a significant overlap among most gene sets compared to random expectations (p < 10−4; two-tailed binomial test) (Figures 4A and S4A–D). The Clr4-repressed genes display the least amount of overlap with the other gene sets. Gene expression analysis of the Clr4-repressed set revealed several categories exclusive to this group. Some of these categories are regulated by specific transcription factors (Table S2). For example, ste4 and ste11, which are master transcriptional regulator of 62 genes involved in sexual differentiation and cell type specification (Valbuena and Moreno, 2010), is a Clr4-repressed and sRNA-enriched gene (Table S1). Strikingly, more than half (34/62) of the known ste11-regulated genes are Clr4-repressed (p<10−28; 13.5 fold enrichment), but only 3 (pyp2, mei2, and rgs1) are sRNA-or H3K9me-enriched. Other transcription factors, such as Atf21 and Atf31 (p<10−4; 4.4 fold enrichment) and its co-regulated genes (Mata et al., 2007) (p<10−2; 3.8 fold enrichment) also are enriched in the Clr4-repressed gene set exclusively. These data suggest that Clr4 directly represses the expression of key transcription factors and indirectly their target genes.

Fig. 4.

Fig. 4

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Clr4 and RNAi proteins regulate the expression of several euchromatic gene clusters in G0. (A) Fold-enrichment (FE) of overlap among Clr4-repressed, G0-repressed, sRNA-enriched and H3K9me2-enriched genes. The dotted line represents the expected overlap from random sampling of the same number of genes for each gene set (*p < 0.05 and **p < 10−4; two-tailed binomial test). (B) Ranked distances between Clr4-repressed, sRNA- and H3K9me2-enriched gene sets (red line) compared to the distances based on 10,000 random gene samplings (black line). Median distance (black line) and the 95% confidence interval (grey shading) of the 10,000 samplings are depicted. (C) Heat map depicting the spatial distribution of Clr4-repressed, sRNA- and H3K9me2-enriched gene sets. Colors indicate the number of aforementioned genes (0–12) found within a 100kb region.

Clr4-repressed, H3K9me- and sRNA-enriched euchromatic genes form euchromatic clusters

We also noted that the Clr4-repressed, sRNA- and H3K9me2-enriched genes cluster close to one another. To test this statistically, we compared the rank-ordered intergenic distances among the aforementioned genes to those calculated from 10,000 random samplings (Figure 4B). Random sampling was performed using the same number of genes found in our combined genesets (690) and the intergenic distances were measured based on the leftmost coordinates of a gene pair. Our results revealed that these genes are significantly closer to one another than random, forming euchromatic clusters (Figure S4E), which may be co-regulated transcriptionally in G0 (Figure 4C).

RNAi is required for G0-specific H3K9 methylation at euchromatic clusters

To test this model directly, we examined the G0-specific H3K9me and RNA levels of several euchromatic loci, which show an overlap of these gene sets. H3K9me2 ChIP and quantitative reverse-transcriptase PCR (qRT-PCR) revealed that H3K9me2 and Clr4 repression of two such genes, ght3 and cta3, in G0 is RNAi-dependent (Figure 5). Interestingly, we also found that the neighboring shu1 and ibp1 genes are Clr4- and RNAi-repressed and display increased H3K9me in an RNAi-dependent manner (Figures 5C–5F). Similarly, analyses of another euchromatic locus (SPACUNK.4.17) revealed RNAi-dependent H3K9me transcriptional regulation of target and neighboring genes in G0 (Figure S5). Together these results demonstrate that Clr4-dependent H3K9 methylation and transcriptional regulation is RNAi-dependent. Because we do not detect a significant level of Ago1-associated sRNAs from the shu1, ibp1 and mug153 genes, these data also suggest that the sRNA-dependent H3K9me of the target gene (ght3, cta3, SPACUNK.4.17) spreads in cis and affects the transcription of neighboring genes in G0. A similar H3K9me spreading mechanism operates within the mating type region of proliferative cells (Jia et al., 2004). Overall, the functional, genome-wide and statistical analyses presented here suggest a model in which Ago1-associated sRNAs generated specifically in G0 target Clr4 to specific euchromatic regions for the transcriptional regulation of these genes.

Fig. 5.

Fig. 5

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RNAi- and Clr4-dependent H3K9me are required for the transcriptional repression of ght3 and cta3 regions in quiescence. (A and B) Schematic representation of ght3 and cta3 regions and strand-specific fold change (FC) (log2) in RNA and Ago1-associated sRNA and FC (log2) in H3K9me2 levels. The blue, cyan, magenta, and orange graphs represent FC in transcript level in G0 (24h) relative to proliferative (0h) wt cells, FC in transcript level in G0 (24h) clr4Δ relative to G0 (24h) wt cells, FC in Ago1-associated sRNA levels in G0 (24h) relative to proliferative (0h) wt cells, and FC in H3K9me2 in G0 (24h) relative to proliferative (0h) wt cells, respectively. These data are derived from RNA-, sRNA-and H3K9me ChIP-seq experiments. (C and E) qRT-PCR analyses of gene expression and (D and F) qPCR analyses of H3K9me2 ChIP at ght3 and cta3 regions in G0 (24h) of the indicated strains relative to proliferative wt cells. Mean and SE of 3–5 biological replicates are shown.

G0-specific Ago1-associated sRNA emerge prior to H3K9me of euchromatic genes

A prediction of our model is that sRNA biogenesis must precede the Clr4-dependent H3K9 methylation of euchromatic target genes. To chart the emergence of G0-specific sRNAs during the transition to G0, we performed deep sequencing of size-selected (20–30nt) small RNAs (sRNA-seq) from wt, clr4Δ, ago1Δ, and dcr1Δ cells at 0, 1, 2, 4, 10, and 24 hours after N starvation. Consistent with previous reports, we confirmed the requirement of RNAi and Clr4 proteins for the formation of heterochromatic cen sRNAs (Figure S6A) (Halic and Moazed, 2010). Also, we observed that changes in sRNA levels in G0 (in wt and clr4Δ cells) are strong predictors of transcriptional changes in quiescence (Figures S6B and S6C). Comparing the sRNA profiles of clr4Δ, ago1Δ, and dcr1Δ cells revealed a strong overlap among the sRNA-enriched genes in these mutants (Figures 6A and S7A). Each sRNA-enriched gene set also displays a significant overlap with Clr4-repressed genes (Figures S7B and S7C). Together these data suggest that Clr4 and RNAi proteins regulate the expression of an overlapping set of euchromatic genes in G0. Next we examined the dynamics of Ago1-associated ‘sRNA-enriched’ genes (Figure 3 and Table S1) in wt and ago1Δ cells as these cells enter G0. Interestingly, we found that the majority of sRNA alterations occur 1hr post N starvation (r2=0.512 compared to 24h) (Figure 6B), well before the emergence of H3K9me (e.g. 4 hours for act1). Moreover, these changes are Ago1 dependent (Figures 6C and 6D), which together with our ChIP-seq results demonstrate that Ago1-dependent sRNA changes occur earlier than H3K9me as cells transit into G0.

Fig. 6.

Fig. 6

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The emergence of G0-specific Ago1-dependent sRNAs is concomitant with the down-regulation of exosome/TRAMP and up-regulation of RNAi factors. (A) Venn diagram depicting the extensive overlap among the different total sRNA-enriched genes in ago1Δ, clr4Δ, and dcr1Δ strains. (B) Graph depicting the total sRNA dynamics of Ago1-associated sRNA-enriched gene set relative to proliferative (0h) cells. Majority of these changes already occur 1h after N starvation. (C) Graph depicting total sRNA FC (log2) at 1h versus 24h relative to proliferative (0h) wt cells. (D) Graph depicting total sRNA FC (log2) in G0 in wt and ago1Δ cells. Only Ago1-associated sRNA-enriched genes are shown. r is the coefficient of correlation. (E) Relative FC in average expression levels of all exosome, TRAMP and RNAi components in wt cells at the indicated time points after N starvation relative to proliferative wt cells (mean and SD of two biological replicates). (F) Heat map depicting the relative RNA FC of individual RNAi, exosome, and TRAMP components at the indicated time points after N starvation relative to proliferative wt cells. The separate column on the right shows the FC of the same set of genes calculated from a previously published dataset (Marguerat et al., 2012). These data show striking similarities to our transcriptome results.

Down-regulation of exosome and upregulation of RNAi components is concomitant with the emergence of G0-specific Ago1-associated sRNAs

Previous work has shown that in wt proliferative cells, the pervasive RNA degradation by the exosome/TRAMP complexes on euchromatic transcripts restricts Ago1-associated sRNAs and H3K9me regions to heterochromatic domains (Buhler et al., 2008; Chalamcharla et al., 2015; Yamanaka et al., 2013; Zofall et al., 2012). Loss of exosome or TRAMP leads to the emergence of Ago1-associated sRNAs and H3K9me from selective euchromatic loci (called HOODs) in proliferative cells (Yamanaka et al., 2013). Interestingly we find that only Clr4-repressed genes show a statistically significant overlap with HOODs (Figure S7D), suggesting that changes in exosome/TRAMP components may trigger G0-specific sRNA biogenesis of euchromatic genes. Moreover, limited availability of RNAi activity (e.g. Dcr1) prevents RNAi-dependent euchromatic distribution of H3K9me (Yu et al., 2014). Here our data reveal that as cells transition into G0, the level of nearly all components of the exosome/TRAMP complexes and RNAi proteins are down- and up-regulated, respectively (Figures 6E and 6F), at the same time (1hr post N starvation) that the G0-specific Ago1-dependent sRNAs emerge (Figures 6B–6D). These observations together suggest that the concomitant decrease in exosome and increase in RNAi proteins in G0 permits the targeting of non-heterochromatic transcripts by RNAi factors, producing sRNAs which later target Clr4-dependent H3K9 methylation to euchromatic regions (Figure 7).

Fig 7.

Fig 7

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RNAi- and H3K9me-dependent regulation of transcription in quiescence. A decrease in nuclear exosome and cofactors, and an increase in RNAi proteins allow for the generation of Ago1-dependent sRNAs 1h post N starvation. These sRNAs act as specificity factors for the recruitment of CLRC and subsequent H3K9 methylation of complementary euchromatic regions in G0 cells.

Discussion

Using the fission yeast, we show that as cells enter G0, they coopt the existing RNAi-dependent H3K9me system to establish the global G0 transcriptional program. Time-course ChIP-seq and sRNA-seq analyses show the emergence of G0-induced Ago1-associated sRNAs prior to the accumulation of H3K9me at several euchromatic regions. The emergence of G0 sRNAs is concomitant with a decrease and an increase in the level of nearly all components of exosome/TRAMP and RNAi proteins respectively, both of which restrict RNAi activity to heterochromatic transcripts in proliferative cells (Reyes-Turcu et al., 2011; Yamanaka et al., 2013) Together these data suggest that environmentally-induced alterations to Ago-associated sRNA profile can target transcriptional regulatory proteins, including constitutive heterochromatin factors such as Clr4, to specific parts of the genome to establish the G0 state.

RNAi- and H3K9me-dependent regulation of transcription in quiescence

Clr4 belongs to the Suv39H family of methyltransferases whose members are required for heterochromatin formation in eukaryotes. Loss of Clr4/Suv39H results in loss of heterochromatin function, derepression of heterochromatic transcripts, but with little impact on the transcription of euchromatic genes (Figure 1D). The data presented in this paper reveal a physiological condition (G0) within which cells use the constitutive heterochromatin factor, Clr4, to establish their global transcriptional program. This involves RNAi- and Clr4-dependent transcriptional regulation of cell cycle, developmental and several metabolic pathways including glycolysis, TCA, and pyruvate metabolism, all of which are required for establishing the G0 state in eukaryotes (Gray et al., 2004; Valcourt et al., 2012). Indeed mutations in RNAi and Clr4, including the Clr4 histone target H3K9, result in loss of viability of G0 cells (Figure 1A, 1B, and S2B), demonstrating their importance for establishing G0.

In our model (Figure 7), we propose that (1) early transcriptional changes in response to N starvation avail selective euchromatic transcripts for sRNAs biogenesis. This is based on the observation that G0-induced sRNA formation is synchronous with the downregulation and upregulation of all components of the TRAMP/exosome and RNAi complexes, respectively (Figure 6). Three observations in proliferative cells support this mechanism. (i) Cells carrying a mutation in exosome components or under certain environmental conditions form heterochromatin domains (HOODs) at several euchromatic regions whose formation requires MTREC, the associated polyA polymerase (Pla1) and polyA binding protein (Pab2), and the nuclear exoribonuclease Dhp1 (Chalamcharla et al., 2015; Lee et al., 2013; Reyes-Turcu et al., 2011; Yamanaka et al., 2013). These RNA surveillance and processing factors, together with other partners, are thought to survey the transcriptome and ensure mRNA quality in wt cells by degrading antisense, cryptic unstable transcripts (CUTs) and aberrant RNAs in the nucleus (Fox and Mosley, 2016). Our sRNA- or H3K9me-enriched gene sets do not show a significant overlap with HOODs. But the Clr4-repressed gene set shows a small, but significant overlap with HOODs. This overlap though is mostly confined to the meiotic genes (Figure S7D and S7E), suggesting that their repression in G0 uses an MTREC/Clr4-regulated mechanism. (ii) Loss of the TRAMP component Cid14 (Trf5 in S. cerevisiae) results in an increase in sRNAs from euchromatic and rDNA regions (Buhler et al., 2008) similar to the sRNA profile of G0 cells (Figure 3), suggesting that G0 may be physiologically relevant condition in which a decrease in Cid14/TRAMP promotes an increase in RNAi-dependent H3K9me regulation of euchromatin and the rDNA regions. Also, in cid14Δ cells, trace levels of sRNAs are sufficient to induce near wt levels of H3K9me at heterochromatic domains (Buhler et al., 2008). This is similar to G0 in which low levels of antisense sRNAs are sufficient to induce H3K9me at several genes. Long terminal repeats (LTRs) in the fission yeast are another example of silenced loci in proliferative cells from which trace amount of Dcr1-dependent sRNAs are detected, albeit this mechanism is H3K9me independent (Woolcock et al., 2011). (iii) Overexpression of Dcr1, a limiting component of RNAi machinery, results in genome-wide sRNA biogenesis from euchromatic genes, and H3K9me-mediate silencing of a transcript lacking the proper 3’end termination signal (Yu et al., 2014). These together with our sRNA and RNA-seq analyses support the model that in G0 the coordinated decrease in exosome/TRAMP and increase in RNAi proteins may provide several new euchromatic substrates for the RNAi proteins.

(2) We also propose that the G0 sRNAs guide Clr4-mediated H3K9me of complementary genes. In support of this, we find that Ago1 is required for methylation of H3K9me-enriched regions in G0 (Figure 2F) and that the formation of complementary Ago1-dependent G0 sRNAs precedes H3K9me of these regions (Figure 6B–6D). Moreover the total sRNA profiles of clr4Δ, dcr1Δ, and ago1Δ cells show extensive overlap (Figure 6A) suggesting that these proteins are regulating the transcription of an overlapping set of genes in G0. It is important to note that while this paper was under review, another study, using total sRNA-seq analyses, reported that no new loci produced sRNAs in wild-type G0 cells (Roche et al., 2016). Because we performed both Ago1-associated sRNA-seq (Figure 3) and total sRNA-seq (Figure 6) analyses, we were able to determine that the Ago1-associated sRNA-seq (but not total sRNA-seq), analysis provided sufficient resolution for the identification of the Ago1-associated sRNA-enriched gene set in wild-type cells. We also show that the formation of these sRNAs (Figure 6B–D) and H3K9 methylation of several euchromatic genes (Figure 2F, 5, and S5) in G0 is Ago1-dependent. Future experiments will test the requirement of other RNAi factors in the biogenesis of these sRNAs.

(3) Our data also reveal that sRNA-directed H3K9 methylation of a euchromatic gene can spread and affect the transcription of a neighboring gene in G0 (Figure 5 and S5). Indeed spatial analysis of our gene sets reveals several large (20Kb in average) euchromatic gene clusters (Figure 4B, 4C and S4E) within which one (or more) sRNA-mediated nucleation events may be sufficient to induce H3K9me of the local area. How the spreading of H3K9me is regulated in G0 is an interesting question suited for future studies, especially under a physiologically relevant condition such as G0, but we do see a dramatic loss in boundary function at heterochromatic domains in G0 (Figure 3E and 3F). Moreover, the RNAi- and H3K9me-dependent regulation at these genes may still require exosome activity. It is possible that following the downregulation of exosome and emergence of G0-induced sRNAs (1h), restoring exosome activity (10–24h) can help in the repression of H3K9me-marked genes (Figure 6). Future experiments will help decipher the role of exosome in the global regulation of G0 state.

Use of Constitutive heterochromatin for the establishment of G0 state

Entry into G0 is concomitant with global transcriptional repression in eukaryotes (Coller et al., 2006; Iyer et al., 1999; Marguerat et al., 2012; Slavov and Botstein, 2013). Our data reveal that in response to G0, fission yeast cells deploy Clr4 into euchromatic regions to establish the G0 state. Interestingly, mouse muscle stem cells which persist in G0 show a clear enrichment of H3K9me3, and use a constitutive heterochromatin protein, Suv4-20h1, to repress the key muscle transcription factor, MyoD. Loss of Suv4-10h1 results in de-repression of MyoD and loss of the G0 state (Boonsanay et al., 2016). These together suggest that cooption of constitutive heterochromatin factors for the establishment of the G0 state may be conserved from yeast to man.

Reprogramming Ago-associated sRNAs as an adaptive response

We find that in response to environmental stimuli (N starvation), Ago1-associated sRNAs accumulate (1h) and later (10–24h) act as specificity factors for targeting regulatory proteins to different genomic regions. The occurrence of stress-induced Ago1-associated nuclear sRNAs has been documented in other eukaryotes. For example in Neurospora crassa, plants and human cells, DNA double-strand breaks (DSBs) induce the RNAi-dependent formation of a new class of Argonaute-associated nuclear sRNAs (Francia et al., 2012; Gao et al., 2014; Lee et al., 2009). These sRNAs (di/qiRNAs) map to the vicinity of the DSB or other parts of the genome and target Argonaute complexes, through which the recruitment of repair or other regulatory complexes to these regions are thought to take place (Khanduja et al., 2016). Also, chronic exposure to environmental stress or artificially introduced dsRNAs trigger multigenerational inheritance and siRNA- and H3K9me-dependent silencing of the target locus in C. elegans (Buckley et al., 2012; Burton et al., 2011; Gu et al., 2012). In these experiments, siRNA generation also precedes H3K9me in the progeny. Besides stress, developmental programs, such as gametogenesis in flies, use Ago-associated sRNAs (piRNAs) to mediate transposon silencing in germ cells. Even though in part cytoplasmic, the nuclear component of this silencing pathway uses piRNAs to guide Ago-containing silencing complexes to transposons. Also, G0 mammalian cells display a distinct miRNA program (Suh et al., 2012) and in muscle stem cells Dicer expression increases and is required for the maintenance of G0 state (Cheung et al., 2012; Sato et al., 2014). These, together with our discovery of G0-specific sRNAs, suggest that reprograming of sRNAs may be a conserved component of developmental or stress response pathways in eukaryotes, through which regulatory complexes can be targeted to complementary sequences.

Moreover, environmentally regulated changes to RNA quality control activity can alter transcriptomes in response to stress. Several recent studies have shown that RNAi and exosome/TRAMP perform overlapping functions to repress the accumulation of aberrant transcripts in the fission yeast (Buhler et al., 2008; Zhang et al., 2011; Zofall et al., 2009) and plants (Moreno et al., 2013). Based on our results here we speculate that environmentally regulated changes to the RNA quality control activities could help eukaryotes to respond to stress (Roy and Chanfreau, 2014).

Quiescence Induced Transcriptional Silencing (QuieTS)

Our findings reveal three mechanistic themes, which may be conserved in Quiescent-induced Transcriptional Silencing (QuieTS) pathways in eukaryotes. First, we find that G0 entry in the fission yeast results in de novo accumulation of sequence specificity factors (sRNAs) deploying silencing proteins to euchromatic clusters. Similarly in the budding yeast (which lacks RNAi and H3K9me), the nuclear accumulation of two DNA-binding transcription factors (Xbp1 and Stb3) in G0 specifies the recruitment of a class I histone deacetylase (Rpd3) to silence metabolic and translational genes (McKnight et al., 2015). Second, we find that ncRNAs help establish QuieTS. In mice, a G0-induced long ncRNA (lncRNA) located in the rDNA region (called PAPAS) is required for the recruitment of H4K20 histone methyltransferase, Suv4-20h2, and the transcriptional repression of rRNA genes (Bierhoff et al., 2014). Even though these ncRNAs differ in size, their function - to augment rRNA silencing in G0 - is conserved from fission yeast to man (Valcourt et al., 2012). Third, QuieTS in the fission yeast coopts proteins of constitutive heterochromatin to repress several transcription factors (e.g. Ste11). This is also true in mouse muscle stem cells where Suv4-20h1 is required for the maintenance of the G0 state via repression of the key muscle transcription factor, MyoD (Boonsanay et al., 2016). Overall, the discoveries in the fission yeast provides a unifying mechanistic framework for QuieTS pathways in eukaryotes and suggest the existence of a program in which the accumulation of sequence specificity factors (such as sRNAs, lncRNAs, or transcription factors) permit the global deployment of silencing proteins in G0. Considering the importance of quiescence in development and several diseases, future studies may reveal useful targets for their treatment.

Experimental Procedures

Viability assays

Viability was measured by phloxine B staining (Noda, 2008) (Figure 1A and 1B) and counting the number of colonies on YEA plates (Figure S1). See Supplemental Experimental Procedures for details.

RNA-seq

RNA libraries were made from total RNA treated with Ribo-Zero Gold rRNA Removal Kit Yeast (Illumina) to remove rRNAs. See Supplemental Experimental Procedures for details.

ChIP analysis

ChIP was performed on the same number of cells (fixed with 1% formaldehyde) collected at each time point. Samples were analyzed by qPCR or deep sequencing. See Supplemental Experimental Procedures for details.

Ago1-associated sRNA-seq

Ago1-associated sRNAs were isolated and sequenced using the protocol described in Holoch and Moazed (Holoch and Moazed, 2015b). See Supplemental Experimental Procedures for detailed protocol and how sRNA-enriched genes were identified.

sRNA-seq

Small RNA libraries were made from size-selected small RNA (20-30nt by gel extraction). See Supplemental Experimental Procedures for details.

Supplementary Material

1
2
3
4
5

Highlights.

  • G0-specific and RNAi-dependent H3K9me of euchromatic genes

  • Formation of stress-induced Argonautre-associated nuclear sRNAs in G0

  • Use of constitutive heterochromatin to establish global transcription of G0 cells

  • Formation of transcriptionally coregulated euchromatic domains in G0

Acknowledgments

This work was supported by Proton Beam Grant (C06 CA059267), V Scholar and V Scholar Plus Grant to MM, and a Beatriu de Pinos (Generalitat de Catalunya) postdoctoral fellowship to IAC. Bioinformatics analyses by Meeta M and SHS was conducted with the support of Harvard Catalyst (NIH award #UL1 RR 025758). We thank Daniel Holoch and Ruby Yu of the Moazed lab for sRNA purification advice and protocols. We are grateful to Mario Suva, Shawn Gillespie and the Broad Institute for sharing deep-sequencing barcodes and thank Daniel Haber, Johnathan Whetstine and Anders Naar for critical reading and constructive comments on the manuscript.

Footnotes

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Accession numbers

The accession number for the sequencing data reported in this study is NCBI Sequence Read Archive: GSE89151

Supplemental information

Supplemental information includes 7 figures and 5 tables.

Author contributions

MM conceived and designed the study, and with RIJ analyzed and interpreted the results, designed experiments and wrote the manuscript. RIJ performed viability (N starvation), RNA-seq, ChIP-seq, and with CMP performed qRT-PCR and ChIP experiments. JSK performed sRNA- and sRNA-seq purifications and library constructions. IAC performed viability (glucose deprivation) experiments. RNA-, Ago1-associated sRNA- and sRNA-seq bioinformatics and statistical analyses were performed by RIJ, vetted by MJA and RIS. ChIP-seq analyses were done by MM, SHS, with QC and advice from AJS and RIS. Statistical analyses were done by RIJ and reviewed by MJA.

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