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. Author manuscript; available in PMC: 2015 Feb 20.
Published in final edited form as: Mol Cell. 2014 Feb 6;53(4):549–561. doi: 10.1016/j.molcel.2014.01.006

Control of Cdc28 CDK1 by a stress-induced lncRNA

Mariona Nadal-Ribelles 1,3, Carme Solé 1,3, Zhenyu Xu 2, Lars M Steinmetz 2, Eulàlia de Nadal 1,4, Francesc Posas 1,4
PMCID: PMC4148690  NIHMSID: NIHMS610260  PMID: 24508389

Summary

Genomic analysis has revealed the existence of a large number of long non-coding RNAs (lncRNAs) with different functions in a variety of organisms, including yeast. Cells display dramatic changes of gene expression upon environmental changes. Upon osmostress, hundreds of stress-responsive genes are induced by the stress-activated protein kinase (SAPK) p38/Hog1. Using whole-genome tiling arrays, we found that Hog1 induces a set of lncRNAs upon stress. One of the genes expressing a Hog1-dependent lncRNAs in antisense orientation is CDC28, the CDK1 kinase that controls the cell cycle in yeast. Cdc28 lncRNA mediates the establishment of gene looping and the relocalization of Hog1 and RSC from the 3′UTR to the +1 nucleosome to induce CDC28 expression. The increase in the levels of Cdc28 results in cells able to re-enter the cell cycle more efficiently after stress. This may represent a general mechanism to prime expression of genes needed after stresses are alleviated.

Introduction

The existence of long non-coding RNAs (lncRNAs) is widespread in eukaryotes from yeast to mammals (Guttman and Rinn, 2012; Jacquier, 2009). Long non-coding transcripts in yeast influence gene expression, revealing a new layer of transcriptional regulation (Wei et al., 2011; Wu et al., 2012). LncRNAs might regulate transcription at multiple levels. Sense-oriented lncRNAs of IMD2 and URA2 alter expression by transcriptional interference and transcription start site selection (Kuehner and Brow, 2008; Thiebaut et al., 2008). Expression of lncRNAs can also trigger changes in chromatin epigenetic state or nucleosome occupancy (Houseley et al., 2008; Kim et al., 2012; Margaritis et al., 2012; Pinskaya et al., 2009; van Werven et al., 2012; Hainer et al., 2011; Uhler et al., 2007). Albeit in a few cases expression of specific lncRNAs alters normal mRNA biogenesis, the general biological relevance and functionality of lncRNAs remains elusive. Remarkably, changes in nutrient availability result in changes in lncRNA expression (Xu et al., 2009; Xu et al., 2011), indicating that environmental insults and signal transduction pathways might affect lncRNA transcription.

Exposure of cells to stress requires immediate and specific cellular responses for proper adaptation (Hohmann et al., 2007). Thus, environmental insults require adaptive responses for maximal cell survival (de Nadal et al., 2011). Stress-activated protein kinases (SAPKs) serve to respond and adapt to extracellular changes. Exposure of yeast to high osmolarity results in activation of the p38-related Hog1 SAPK (Saito and Posas, 2012), which is essential to control cell cycle (Clotet and Posas, 2007; Duch et al., 2013) and gene expression (de Nadal and Posas, 2010).

The Hog1 SAPK acts in multiple stages of the cell cycle by targeting several core components of the cell cycle machinery. For instance, Hog1 controls G1/S transition by the down-regulation of cyclin expression and the stabilization of the Sic1 cyclin dependent kinase inhibitor (CDKi) (Adrover et al., 2011; Escote et al., 2004). Hog1 also modulates other phases of the cell cycle such S-phase (Duch et al., 2013). Cells unable of delaying cell cycle progression upon osmostress display reduced viability upon those conditions, suggesting the need of delaying cell cycle for proper adaptation.

The Hog1 SAPK is a key element for reprogramming gene expression in response to osmostress by acting on hundreds of stress-responsive genes. Hog1 is recruited to chromatin to recruit RNA polymerase II (Alepuz et al., 2003; Nadal-Ribelles et al., 2012) and associated factors (de Nadal et al., 2004; Sole et al., 2011; Zapater et al., 2007). Hog1 is present also at the ORFs of stress-responsive genes (Cook and O'Shea, 2012; Nadal-Ribelles et al., 2012; Pokholok et al., 2006; Proft et al., 2006) where it stimulates strong chromatin remodeling by the interplay of the INO80 and the RSC complexes (Klopf et al., 2009; Mas et al., 2009). Chromatin dynamics set a threshold for gene induction upon Hog1 activation (Pelet et al., 2011). In addition to gene induction, Hog1 controls mRNA stability (Miller et al., 2011; Molin et al., 2009; Romero-Santacreu et al., 2009), export (Regot et al., 2013) and translation (Warringer et al., 2010). Thus, Hog1 plays a key role in the regulation of mRNA biogenesis (de Nadal et al., 2011; de Nadal and Posas, 2010; Martinez-Montanes et al., 2010; Weake and Workman, 2010).

Here, we show that the Hog1 SAPK also associates and controls the induction of a novel set of lncRNAs in response to osmostress. One of the genes expressing a stress-induced lncRNA in antisense orientation is CDC28, the CDK1 kinase that controls the cell cycle in yeast. Induction of the CDC28 lncRNA permits the increase in the levels of Cdc28 allowing cells to re-entry more efficiently cell cycle after stress. Therefore, Hog1 directly coordinates the regulation of transcription and cell cycle progression by controlling expression of a stress-induced lncRNA in CDC28.

Results

Hog1 mediates the expression of a new set of stress-inducible lncRNAs

Most transcriptome studies performed to define stress genes have analyzed coding genes. To cover the expression of the whole-genome upon stress, we monitored transcription using strand-specific tiling arrays (David et al., 2006). The number of coding genes induced upon stress were 343 at 0.4 M NaCl (15 min) and 294 at 1.2 M NaCl (100 minutes) using a stringent threshold (see methods). Expression of 56% and 84% of the stress-induced genes depended on Hog1 at 0.4 M and 1.2 M NaCl, respectively. Overall, the number of stress-responsive genes was similar to previous reports (Capaldi et al., 2008; Gasch et al., 2000; Nadal-Ribelles et al., 2012; Posas et al., 2000).

Remarkably, in addition to coding genes, up to 173 lncRNAs were strongly induced upon treatment with 0.4 M NaCl and up to 216 with 1.2 M NaCl (Fig. 1). Almost a hundred of them were shared between the two stress conditions (Fig. S1). The average length of these stress-induced lncRNAs is about 843 nucleotides (Fig. S1). 50% and 91% of the lncRNAs induced by treatment with 0.4 M and 1.2 M NaCl, respectively, depended on the presence of Hog1 (http://steinmetzlab.embl.de/francescData/arrayProfile/index.html) (Fig. 1B). Some overlapped with previously annotated CUTs or SUTs (Wu et al., 2012; Xu et al., 2009). However, most of them were not expressed in the absence of RRP6, TRF4 or XRN1 and present only upon stress (Fig. 1C and S1). Thus, Hog1 mediates the expression of a new set of stress-inducible lncRNAs.

Figure 1. Hog1 controls transcription of a new set of lncRNAs upon stress.

Figure 1

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(A) Osmostress induces expression of a novel class of lncRNAs. Expression data from tiling arrays of several stress-induced lncRNAs for the Watson (W, top) and Crick (C, bottom) strands. Normalized signal intensities from duplicate hybridizations of wild type (wt) and hog1Δ strains under basal conditions (YPD), treated with 0.4 M NaCl for 15 min or treated with 1.2 M NaCl for 100 min (y axis). Genomic coordinates and gene annotation, transcript boundaries (red lines) and transcription start site (arrows). (B) Hog1 regulates lncRNA transcription upon stress. lncRNAs induced at least twofold upon osmostress (0.4 M NaCl, left; 1.2 M NaCl, right) grouped into three categories according to the dependence of their expression in hog1Δ strong (<50% of wild type), moderate (50 – 75%), none (> 75%). (C) Stress-induced lncRNAs are a novel family of lncRNAs. wt, hog1Δ, trf4Δ, rrp6Δ, and xrn1Δ mutant strains were subject to osmostress (0.4 M NaCl) and the indicated lncRNAs were assessed. See also Figure S1.

Hog1 associates with the promoters of stress-induced lncRNAs, stimulates RNA Pol II recruitment and gene expression

Hog1 associates with chromatin of stress-responsive genes upon stress (Alepuz et al., 2001; Alepuz et al., 2003; Cook and O'Shea, 2012; Pokholok et al., 2006; Proft et al., 2006). Actually, Hog1 is present in at least 80% of the Hog1-induced genes upon stress (Nadal-Ribelles et al., 2012). We also found that Hog1 is present in &sim;63% of Hog1-dependent lncRNAs promoters, whereas it is recruited to <30% of Hog1-independent lncRNAs (Fig. 2A). Genome-wide association of RNA Pol II showed that it strongly associates with stress-responsive loci upon stress (Cook and O'Shea, 2012; Nadal-Ribelles et al., 2012). RNA Pol II was also recruited at the stress-induced lncRNAs (2.3 fold increase) upon stress. In contrast, RNA Pol II was not recruited at the Hog1-dependent lncRNA promoters in a hog1 strain (Fig. 2B and Fig. S2A). Therefore, Hog1 associates to and stimulates the recruitment of RNA Pol II at the promoters of stress-induced lncRNAs.

Figure 2. Hog1 binds and recruits RNA Pol II at genes with lncRNA.

Figure 2

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(A) Hog1 associates with lncRNA promoters. The percentage of Hog1 binding at the Hog1-dependent or independent lncRNAs determined by ChIP-seq (0.4 M NaCl, p <0.05). (B) Hog1 stimulates Pol II recruitment to lncRNA promoters. Distribution of RNA Pol II binding (normalized reads, TRPKs) determined by ChIP-seq (0.4 M NaCl for 10 min) for Hog1-dependent lncRNAs in wild type (wt) and hog1 Δ. *** means p-value <0.001 (t-test). (C) Hog1 mediates changes in chromatin architecture at lncRNA promoters. Distribution of nucleosome hits (RPKMs; Reads per Kilobase per Million) expanding 1Kb up and downstream from transcription termination site (TTS) of wild type and hog1Δ mutant strains under basal (dark blue and red) and 0.4M NaCl (light blue and yellow). Plot represents coverage of reads of approximately 90 genes: Hog1-dependent (upper graph), Hog1-independent (lower graph) and non-stress responsive (see Figure S2). Dotted black line marks TTS. (D) Antisense oriented 3′UTR of CDC28 is an osmoresponsive promoter. The 3′UTR of CDC28 fused to the Quadruple-Venus YFP (qV-YFP) in the antisense orientation (promoter lncRNA) or in its natural orientation (terminator sense). Fluorescence intensity was measured by flow cytometry.

Once recruited to stress-responsive genes, Hog1 mediates chromatin remodeling (Mas et al., 2009; Pelet et al., 2011). Genome-wide MNase (Micrococcal Nuclease) digestion of chromatin and deep sequencing (MNase-Seq) showed that upon osmostress Hog1 mediates dramatic change of nucleosome occupancy (Nadal-Ribelles et al., 2012). We analyzed the chromatin organization at genes that expressed Hog1-induced lncRNAs in antisense orientation and found that regions beyond the transcription termination site (TTS) also suffered strong Hog1-dependent chromatin remodeling (Fig. 2C). Although levels of nucleosome occupancy decreased slightly upon stress in hog1Δ cells, this decrease was clearly more prominent in the wild type strain. In contrast, no changes in chromatin structure were observed in promoters of lncRNAs that do not respond to stress (Fig. S2B).

Expression from stress-responsive promoters can be quantitatively measured by the fusion to quadruple Venus (qV) fluorescent protein (Pelet et al., 2011; Regot et al., 2013). To characterize one of these lncRNAs promoters further, we fused the 3′ untranslated region (3′ UTR) of CDC28 in both the sense and the antisense orientation to qV-YFP and assessed gene expression by flow-cytometry in wild type and hog1 strains. Expression of qV-YFP was induced upon stress depending on the presence of Hog1 and only when placed in the antisense orientation (Fig. 2D). Therefore, the 5′regions of stress-induced lncRNAs behave as bona fide stress-responsive promoters.

Induction of CDC28 lncRNA expression promotes the induction of CDC28 gene expression upon stress

To functionally characterize the role of stress-induced lncRNAs, we asked whether there is a correlation between expression of the sense and antisense induced transcription. We found a correlation for only a few relevant cases (8 out of 91; Hog1-dependent lncRNAs at 0.4 M NaCl) in which an increase of the antisense was associated with an increase in the sense transcript (Fig. 3A). Tiling arrays are not very sensitive for slight increases on transcription, thus we analyzed the expression of the 91 genes with stress-induced lncRNAs from previous run-on assays and found that 41 out of 91 genes displayed a positive correlation (Romero-Santacreu et al., 2009).

Figure 3. CDC28 induction correlates with lncRNA expression.

Figure 3

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(A) Expression of sense (x axis) versus lncRNA (y axis), in a log2 scale, in response to stress in HogΔ1-dependent lncRNAs. Highlighted dots represent genes with positive (red), negative (orange) or no (black) correlation. (B) Hog1 and the CDC28 lncRNA are required for CDC28 expression. CDC28 sense and CDC28 lncRNA transcripts were assessed in wt, hog1Δ and lncRNAΔ strains upon osmostress. See Figure S3 for systematic insertion of a KanR marker at the 3′UTR region of CDC28. Normalized quantification of CDC28 sense is shown. Quantifications were done by normalizing by the loading control gene and then in respect to stressed samples referred to the unstressed point (0′ time point).

One of the genes with a clear correlation of the sense and lncRNA expression was CDC28 (Fig. 3B). CDC28 encodes the main CDK kinase that drives progression of the cell cycle in yeast. We found that upon osmostress, there is an increase in CDC28 expression that was not observed in a hog1 strain (Fig. 3B). Systematic insertion analysis at the 3′UTR of CDC28 showed that insertion of a KanR marker at 180 nucleotides downstream of the transcription termination site (lncRNAΔ) abolished expression of the lncRNA. In this strain, the induction of CDC28 upon osmostress was impaired (Fig. 3B and S3A). Of note, the rrp6 mutation did not alter the induction of the CDC28 sense transcription (Fig. S3B). Thus, the presence of the stress-inducible CDC28 lncRNA correlates with induction of the CDC28 gene expression.

We then assessed whether it was the expression of the lncRNA from the 3′UTR or the solely presence of the lncRNA that induced CDC28 expression. We created a strain containing a CDC28::GFP that recapitulated CDC28 gene expression (CDC28::GFP) and then abolished lncRNA expression (CDC28::GFP lncRNAΔ) (Fig. S3C). We then expressed CDC28 and its terminator region from a plasmid. This permitted to distinguish transcription from the plasmid or the endogenous locus within the same cell. Remarkably, in response to osmostress, we could detect an increase in expression of CDC28 sense from the plasmid but we did not observe an increase of the CDC28::GFP, thus indicating a cis effect of the lncRNA (Fig. S3C).

Hog1 associates with the 3′UTR and the +1 nucleosome regions in CDC28 to promote chromatin remodeling

To characterize the mechanism by which Hog1 induces sense and antisense transcription, we monitored Hog1 association in CDC28. Hog1 associated with the 3′ UTR of CDC28 (the promoter region of the CDC28 lncRNA) upon stress. Strikingly, Hog1 also associated with a region close to the transcription start site (TSS) (region +41 to +125) corresponding to the CDC28 +1 nucleosome. By contrast, in cells unable to induce CDC28 lncRNA, Hog1 association with the TSS was abolished completely and only slightly reduced at the 3′ UTR (Fig. 4A).

Figure 4. Binding of Hog1 induces lncRNA and promote chromatin remodeling by the RSC chromatin remodeler.

Figure 4

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(A) Hog1 binds at the 3′UTR and at the +1 nucleosome region. Graphical representation of the CDC28 locus with the insertion of a KanR marker at the 3′UTR region to disrupt expression of lncRNA (lncRNAΔ). Hog1 association at the CDC28 locus was assessed by ChIP at the indicated regions and strains. (B) Induction of CDC28 lncRNA by Hog1 promotes chromatin remodeling at the +1 nucleosome. Nucleosome positioning was assessed by MNase digestion in wild type (wt), hog1Δ and lncRNAΔ strains under control (black) or osmostress (0.4 M NaCl for 10 min, blue). The normalized nucleosome occupancy of one representative experiment is shown (x axis). (C) CDC28 induction depends on RSC activity. Wild type (wt) and rsc9ts strains were grown at restrictive temperature (37°C) and subjected to osmostress. CDC28 sense and lncRNA transcripts were detected by Northern Blot for the indicated times. (D) Hog1 mediates the recruitment of RSC to CDC28. Rsc1 binding was assessed by ChIP at +1 nucleosome region (amplicon D) and 3'UTR (amplicon J) of CDC28 in a wild-type and hog1Δ strains that were or not subjected to osmostress (0.4M NaCl, 5 min). (E) RSC is essential to mediate chromatin reorganization. Remodeling at the +1 nucleosome in a Rsc9ts strain was assayed by MNase digestion after 10 min of NaCl. Bars represent average of the stressed (black bars) compared to the unstressed (white bars) ± SD. See also Figure S4.

The presence of Hog1 at the region corresponding to the +1 nucleosome around the TSS led us to analyze the chromatin architecture by MNase digestion. We found that the chromatin at the 3′ UTR of CDC28 changed upon osmostress, as expected for a stress-responsive promoter. Strikingly, the region corresponding to the +1 nucleosome at the 5′ region of CDC28 was also strongly remodeled upon stress (Fig. 4B). The eviction of the +1 nucleosome was not observed in hog1 nor lncRNA deficient cells (Fig. 4B). Therefore, Hog1 association and remodeling at the +1 nucleosome region of CDC28 occur in response to osmostress and in the presence of CDC28 lncRNA.

The RSC chromatin remodeling complex mediates chromatin remodeling at the CDC28 +1 nucleosome region upon stress

Hog1 stimulates chromatin remodeling at specific stress-responsive loci by recruiting the RSC complex (Mas et al., 2009). Induction of the CDC28 lncRNA in cells deficient in the RSC complex (rsc9ts) under non-permissive temperature was similar to wild type but reduced in a SAGA mutant (Fig. 4C and S4A). Thus, RSC is not necessary for lncRNA expression. In clear contrast, rsc9ts mutant cells did not induce CDC28 expression upon stress. This suggests a key role of RSC for the increase of CDC28 sense upon stress.

Then, we assessed the recruitment of RSC and Hog1 to various regions of CDC28 before and after the addition of NaCl. We found that RSC associates with 3'UTR and +1 nucleosome regions of CDC28 in response to stress only in the presence of Hog1 (Fig. 4D). Induction of lncRNA and CDC28 required of active Hog1, since a catalytically inactive Hog1 was unable to associate to chromatin and to promote RSC association (Fig. S4B, C and D). By contrast, association of Hog1 was not altered in the rsc9ts mutant under non-permissive temperature (Fig. S4E), suggesting that Hog1 mediates the recruitment of RSC at CDC28 to remodel chromatin upon stress. Correspondingly, chromatin remodeling at the +1 nucleosome, assessed by MNase digestion, was impaired in the rsc9ts mutant under non-permissive temperature (Fig. 4E). Thus, recruitment of RSC by Hog1 is essential to mediate chromatin reorganization at the +1 nucleosome region and CDC28 gene induction.

Both the expression of the CDC28 lncRNA and Hog1 are required for CDC28 induction upon stress

In the absence of CDC28 lncRNA expression, the presence of Hog1 at the 3'UTR of CDC28 is not sufficient to increase CDC28 expression. Then, we asked whether the expression of the CDC28 lncRNA alone was sufficient for CDC28 induction. Thus, we inserted at the endogenous 3′ UTR of CDC28 an inducible GAL1 promoter in the antisense orientation (CDC28::pGAL1). Expression from the GAL1 promoter is driven by the Gal4-ER-VP16 activator in the presence of β-estradiol (Louvion et al., 1993). Albeit the presence of estradiol strongly induced expression of the CDC28 lncRNA, this was not sufficient to stimulate sense transcription (Fig. 5A). We then monitored Hog1 recruitment and found that the presence of estradiol did not mediate Hog1 recruitment in CDC28 (Fig. 5B). Correspondingly, chromatin remodeling at the +1 nucleosome did not occur by the sole induction of the CDC28 lncRNA from the GAL1 promoter in the presence of the Gal4-ER-VP16 activator (Fig. 5C). Thus, the induction of the CDC28 lncRNA alone is not sufficient to mediate chromatin remodeling and CDC28 gene induction.

Figure 5. Antisense transcription and Hog1 recruitment are required to induce CDC28 expression.

Figure 5

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(A) lncRNA alone is not sufficient for CDC28 expression. Graphical representation of the CDC28 locus with inducible lncRNA expression achieved by insertion of the pGAL1 promoter (CDC28::pGAL1). Gal4-ER-VP16 activator induces lncRNA in the presence of β-estradiol (black bars) or β-estradiol and NaCl (grey bars). CDC28 sense and lncRNA transcripts were assessed by Northern Blot. (B) Hog1 association (5 min) by ChIP and (C) +1 nucleosome eviction (10 min) by MNase were measured in the indicated strains upon induction with β-estradiol. (D) Hog1 and lncRNA induction are necessary for CDC28 induction. Graphical representation of the CDC28::pGAL1 locus induced by the Gal4-Msn2DBD. Transcript levels were followed as in A. (E) Graphical representation of the CDC28::pGAL1 strain containing an internal terminator (IT). (F) Hog1 and Rsc1 recruitment (5 min) in cells expressing and empty or Gal4-Msn2DBD. (G) Presence of the lncRNA is required for Hog1 recruitment at the +1 nucleosome. Hog1 recruitment (5 min) of cells in the IT strain. (H) +1 nucleosome eviction (10 min) was assessed in CDC28::pGAL1 cells harboring empty vector (Ø) or Gal4-Msn2DBD upon stress. Normalized quantification of CDC28 is shown. Error bars represent standard deviation (SD).

Then, we assessed whether the recruitment of Hog1 together with the CDC28 lncRNA expression from the GAL1 promoter could induce CDC28 gene expression. Expression from CDC28::pGAL1 was then driven by Gal4DBD-Msn2 activator. Msn2 mediates the recruitment of Hog1 to Msn2 dependent genes (Alepuz et al., 2001). Tethering Msn2 to the Gal4-binding domain stimulated stress-inducible transcription of the lncRNA in the CDC28::pGAL1 strain upon stress and restored CDC28 induction upon stress (Fig. 5D). We then inserted a terminator downstream of the GAL1 promoter (IT) (Kopcewicz et al., 2007; Loya et al., 2012) that permitted transcription initiation but prevented the generation of the lncRNA. Here, the Gal4DBD-Msn2 did not promote CDC28 sense induction (Fig. 5E). Correspondingly, Hog1 was recruited at the GAL1 promoter in the 3′ UTR region of CDC28 as well as at the +1 region of CDC28 (Fig. 5F) whereas it was not recruited at the +1 region in the IT construct (Fig. 5G). We then monitored chromatin remodeling at the +1 region. In contrast to Gal4-ER-VP16 activator, expression of the CDC28 lncRNA from the GAL1 promoter by the Gal4DBD-Msn2 activator caused remodeling of the +1 nucleosome upon stress (Fig 5H). Thus, induction of the CDC28 lncRNA and the recruitment of Hog1 at the +1 region are required for chromatin remodeling at the 5′ region of CDC28 and CDC28 gene expression.

The establishment of gene looping permits the recruitment of Hog1 at the +1 nucleosome region and induction of CDC28

The absence of Hog1 recruitment and remodeling at the 5′ region of CDC28 in cells deficient in lncRNA induction prompted us to assess whether the presence of Hog1 at this region was mediated by gene looping formation (O'Sullivan et al., 2004; Tan-Wong et al., 2012). Gene loop formation depends on the essential protein Ssu72 (Ansari and Hampsey, 2005). Expression of SSU72 under GAL1 is repressed in the presence of glucose (YPD). Cells were grown in galactose, shifted to glucose and subjected to osmostress. Depletion of Ssu72 did not alter induction of the CDC28 lncRNA but prevented induction of the CDC28 (Fig. 6A). Similar results were obtained in a sua7-1 mutant (Singh and Hampsey, 2007) with impaired gene looping (Fig. S5A). Then, we assessed the recruitment of Ssu72 and found that there was a clear Hog1-dependent increase in Ssu72 binding upon stress at both, the 3′ UTR and +1 nucleosome regions (Fig. S5B). Thus, the enhanced recruitment of Ssu72 in response to stress does not alter CDC28 lncRNA expression but it is essential for the increase of CDC28 expression.

Figure 6. Gene looping allows recruitment of Hog1 at the +1 nucleosome region and induction of CDC28.

Figure 6

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(A) CDC28 induction depends on Ssu72. Wild type and GAL1::SSU72 strain grown as stated was subjected to osmostress. CDC28 sense and lncRNA transcripts were detected. (B) Hog1 binding at the +1 nucleosome of CDC28 depends on Ssu72. GAL1::SSU72 strain grown as stated and Hog1 recruitment was analyzed by ChIP after 5 min of NaCl at the indicated regions (as in Fig. 4A). (C) Binding of RSC at the +1 nucleosome requires gene looping. Recruitment of Rsc1 was assessed by ChIP at the indicated regions. (D) Chromatin remodeling at the +1 region depend on Ssu72. GAL1::SSU72 strain grown as stated and remodeling at the +1 nucleosome was assayed by MNase digestion after 10 min of NaCl. Bars represent average of the stressed (black bars) compared to the unstressed (white bars) ± SD. (E) Recruitment of RNA Pol II requires gene looping. Binding of RNA Pol II at the +1 nucleosome was assessed by ChIP. Levels in the wild type upon stress were used as a reference for the other indicated strains. (F) Hog1 induces physical interaction between +1 nucleosome region and 3′UTR of CDC28 in response to stress. CDC28 locus is depicted along with the positions of AciI cleavage sites (red lines) and primer position in 3C analysis (arrows). 3C analysis at the indicated strains and conditions in the presence (+) or absence (-) of osmostress (0.4 M NaCl, 10 min). Amplification of tandem primer pairs is shown and TEL region was used as loading control. ND (non-digested) and D (digested) chromatin samples from treated wt strain were used as internal control of 3C specificity. See also Figures S5 and S6.

The lack of CDC28 induction in the absence of Ssu72 and in the sua7-1 mutant suggested that gene looping might mediate the transfer of activities from the 3′UTR to +1 nucleosome regions of CDC28. Association of Hog1 at the 3′UTR region of CDC28 upon stress was not altered by the absence of Ssu72. In clear contrast, the absence of Ssu72 (YPD) completely abrogated the association of Hog1 at the +1 nucleosome region of CDC28 (Fig. 6B). Correspondingly, recruitment of Rsc1 was also abolished at the +1 region in the absence of Ssu72 (Fig. 6C). Correspondingly, chromatin remodeling at the +1 region did not occur upon stress in cells depleted for Ssu72 (YPD) (Fig 6D). Moreover, the increase in RNA Pol II association upon stress at the CDC28 5′ region observed in the wild type strain was abolished in cells deficient in hog1, CDC28 lncRNA and ssu72 (Fig. 6E). Thus, gene looping mediates the recruitment of Hog1 at the 5′ region of CDC28 to induce chromatin remodeling and RNA Pol II recruitment upon stress.

To further confirm that the establishment of gene looping between the 3′UTR and promoter regions, we applied the 3C assay (see methods). We found that there was a clear increase in gene looping formation upon stress between the 3′UTR and the +1 nucleosome regions as detected by the presence of O1-T PCR products. The O1-T PCR product was ligation dependent (D) and it was not detected when an alternative region (O2) was assessed (Fig. 6F and S5C). Of note, the increase in gene looping formation upon stress was dependent on Hog1 and abolished in the absence of SSU72 (YPD) and sua7-1 mutant cells (Fig. 6F and S5C) but not in a rsc9ts mutant (Fig. S5D). Therefore, gene looping is critical for the recruitment of Hog1 from 3′UTR to the +1 nucleosome region of CDC28 to promote chromatin remodeling and induce CDC28 gene expression.

A lncRNA in MMF1 induces Hog1 recruitment and chromatin remodeling at the +1 nucleosome region

To assess whether genes other than CDC28 displayed a similar regulatory mechanism, we chose MMF1 because expresses a strong stress-induced lncRNA (Fig. S6A) and, albeit not seen in the tiling arrays due to insufficient sensitivity, it was reported to be induced upon stress by Hog1 in run-on and DTA experiments (Miller et al., 2011; Romero-Santacreu et al., 2009). We created a mutant in antisense MMF1 lncRNA expression (Fig. S6A). Hog1 was recruited at the 3′UTR and the 5′ regions but not in the body of the MMF1 gene depending on the presence of the lncRNA (Fig. S6B). Nucleosome eviction occurred in response to stress in a Hog1 and lncRNA dependent manner (Figure S6C). Then, we performed 3C experiments in wild type, hog1 and SSU72 shut-off system (pGAL1::SSU72). Of note, in the absence of stress, we could already detect gene looping between the P and T regions. But most remarkably, the wild type strain showed an increase of P-T association in response to stress which was fully dependent on the presence of Hog1 and gene looping (Figure S6D). Thus, albeit some particularities, MMF1 seems to stimulate chromatin remodeling via Hog1 and lncRNA expression as in CDC28.

We then asked whether the 3′UTR region of CDC28 could confer osmo-induction in a non-osmoresponsive gene. We replaced the 3′UTR region of a non-stress responsive gene, MBA1, with the 3′UTR region of CDC28 (300 bp downstream of STOP codon). We chose MBA1 because its expression under normal conditions and the length of the gene are similar to CDC28. Replacement of the MBA1 terminator by CDC28 led to the transcription of a lncRNA from the 3′UTR in MBA1 and, most important, it conferred Hog1-dependent osmo-induction (Fig. S6E), suggesting that the role of CDC28 3′UTR is to confer stress-inducible regulation of gene expression from the terminator region of the gene.

Stress-induced CDC28 lncRNA results in an increase of Cdc28 that permits cells to re-enter the cell cycle more efficiently in response to stress

We then asked whether an increase of CDC28 mRNA results in an increase of Cdc28 protein production upon stress. Endogenous [35S]methionine Cdc28 protein increased ∼twofold in response to stress in wild type cells, whereas no increase was observed in a CDC28 lncRNA deficient strain (Fig. 7A). Thus, stress-induced Cdc28 lncRNA expression leads to increased levels of Cdc28 kinase.

Figure 7. Changes of Cdc28 levels promote cell cycle re-entry upon stress.

Figure 7

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(A) Cdc28 protein levels increase upon stress. 35S-labeled Cdc28 was immunoprecipitated for the indicated lengths of time and strains. Cdc28 levels were normalized against total protein. (B) Increased Cdc28 restores cell cycle progression. The indicated cdc15ts strains were synchronized at anaphase and released under control or stress conditions. Cell-cycle progression was analyzed by fluorescence-activated cell sorter (FACS) and percentage of cells in G2/M is shown. See also Figure S7. (C) Schematic representation of Hog1-mediated gene looping between the 3′UTR and the +1 nucleosome region at the CDC28 locus in response to osmostress.

Hog1 promotes an immediate but transient cell cycle delay that permit stress-adaptation (Clotet and Posas, 2007; Duch et al., 2012). The increase in Cdc28 levels occurred when cells were already recovering from the initial arrest caused by stress. Thus, we hypothesized that this increase of Cdc28 can serve to accelerate cell cycle re-entry after stress. To test this hypothesis, we assessed the exit from the arrest caused by osmostress in a phase of cell cycle with high Cdc28 by synchronizing cells using a temperature sensitive allele of cdc15 (cdc15ts) (see Methods). Cells deficient in CDC28 lncRNA were able to arrest and exit cell cycle from cdc15ts synchronization as efficiently as wild type in the absence of stress. In contrast, cells deficient in CDC28 lncRNA production delayed approximately 20 minutes cell cycle re-entry upon stress compared to the wild type (Fig. 7B). Of note, overexpression of CDC28 from a plasmid can suppress the delay on cell cycle progression observed upon osmostress in CDC28 lncRNAΔ cells (Fig. S7). Thus, stress-induced CDC28 lncRNA results in an increase of Cdc28 that permits cells to re-enter the cell cycle more efficiently in response to stress.

Discussion

A specific set of Hog1-dependent lncRNAs is induced in response to osmostress

Stress-activated protein kinases regulate gene expression to maximize cellular adaptation to environmental stresses (de Nadal et al., 2011; Weake and Workman, 2010). In yeast, activation of Hog1 leads to major changes in gene expression. Here, we provide evidence that, in addition to controlling expression of coding genes, Hog1 also induces a dedicated set of stress-responsive lncRNAs. Upon osmostress, about 200 lncRNAs are rapidly induced. The induction of these stress-induced lncRNAs depends mostly on the presence of Hog1. Correspondingly, Hog1 associates to the promoters of lncRNAs upon stress and stimulates RNA Pol II recruitment and chromatin remodeling similar to osmoresponsive genes. In fact, fusing the promoter of one of these lncRNAs (CDC28) to a GFP reporter showed that expression occurred only upon stress, in antisense orientation and depending on Hog1. This observation is remarkable, since most of the described antisense transcripts have been proposed to arise from bidirectional promoters (Tan-Wong et al., 2012; Xu et al., 2009). The fact that this terminator can function as a heterologous promoter suggests that there must exist a different transcriptional unit recruited to this region that is independent of the neighboring genes. Accordingly, a recent study by ChIP-exo precisely positioned distinct transcriptional machineries at bidirectional promoters, supporting the idea of unique transcription units (Rhee and Pugh, 2012).

Most of the stress-induced lncRNAs are transcribed in response to osmostress. Except for SUTs, which are stable transcripts, the rest of lncRNA are only detectable in strains deleted for components of the nuclear o cytosolic exosome (CUTs and XUTs) (Xu et al., 2009), when gene looping is impaired (Ssu72-restricted transcripts) or by deletion of SET3 (Kim et al., 2012; Tan-Wong et al., 2012). Expression analysis of some representative Hog1-dependent lncRNAs showed that they were not expressed under basal conditions in the absence of RRP6, XRN1 or TRF4. Transcription was only induced upon stress, but stability was altered in these mutants. Thus, Hog1 regulates the transcription of a distinctive class of stress-induced lncRNAs whose induction might have relevant implications for proper cellular adaptation.

The CDC28 lncRNA and Hog1 induce chromatin remodeling and CDC28 expression via gene looping

To unravel the biological function of the stress-induced lncRNAs, we investigated whether there was correlation between the expression of sense and lncRNAs. Overall, this was not evident except for some genes. Remarkably, there was a positive correlation between the induction of CDC28 and a lncRNA in CDC28 expressed in antisense orientation. CDC28 expression was dependent on Hog1 since in hog1 cells there was neither induction of the lncRNA nor the CDC28 sense. This posed the question on how the SAPK and the induction of a lncRNA lead to increased gene expression. In clear contrast to typical osmo-responsive genes in which the SAPK associates all along the gene (Proft et al., 2006), Hog1 associated at the 3′region of CDC28, which corresponds to the promoter region of the CDC28 lncRNA, and at a region surrounding the +1 nucleosome of CDC28. Transcription of CDC28 is not controlled by any of the transcription factors targeted by Hog1, thus opening the possibility that Hog1 uses the 3′UTR region to mediate its association to the +1 nucleosome region to promote gene expression. This interesting Hog1-binding pattern resembles some of the features of osmoresponsive genes, in which Hog1 recruitment at the ORFs depends on the 3′UTR region (Proft et al., 2006).

Cells deficient in lncRNA still recruit Hog1 at the 3′UTR but not at the +1 nucleosome region in CDC28. Remarkably, these cells neither induce chromatin remodeling nor CDC28 gene induction. On the other hand, when CDC28 lncRNA was induced by a heterologous activator that does not promote recruitment of Hog1 at the 3′UTR, the SAPK does not bind at the +1 nucleosome region and cells can neither induce chromatin remodeling nor gene expression. Therefore, the combination of the induction of CDC28 lncRNA transcription and the recruitment of Hog1 is necessary for gene induction. Correspondingly, the combination of artificial tethering of Hog1 to a strain containing the GAL1 at the CDC28 3′UTR together with the expression of the lncRNA allow chromatin remodeling and induction of CDC28. RSC mediates chromatin remodeling at specific stress-responsive genes (Mas et al., 2009). Here, we found that expression of the CDC28 lncRNA was not affected by depletion of RSC but SAGA. In contrast, RSC was absolutely necessary for chromatin remodeling at the +1 nucleosome region and CDC28 gene induction. Thus, the targeting of RSC by Hog1 at the +1 nucleosome region is required for gene induction.

Unlike osmoresponsive genes in which Hog1 travels with elongating polymerase (Proft et al., 2006), Hog1-binding pattern at the CDC28 locus suggested that Hog1 could reach 5′ the end of the gene without traveling through the coding region. In yeast, gene looping has been shown to juxtapose promoter-terminator regions during active transcription (O'Sullivan et al., 2004). Indeed, osmostress stimulates Hog1-mediated gene looping in CDC28. Looping can be prevented by impairing expression of SSU72 or in the mutant sua7-1 (Ansari and Hampsey, 2005; Singh and Hampsey, 2007). Depletion of Ssu72 or sua7-1 mutation did not alter induction of CDC28 lncRNA but completely abolished CDC28 gene induction most likely because Hog1 and RSC cannot be transferred from the 3′UTR to the +1 nucleosome position.

All together suggest the following tentative model for the induction of CDC28 by Hog1 (Fig. 7C). In response to osmostress, Hog1 associates at the 3′UTR region of CDC28 and induces lncRNA transcription. Once antisense transcription is induced, gene looping is established and Hog1 is transferred to the +1 nucleosome region in CDC28. The recruitment of Hog1 serves to target the RSC chromatin remodeler which remodels the +1 region, thus, permitting an increase of the transcription of the CDC28 gene. It is worth noting that recently another example has been demonstrated that DNA looping facilitates targeting of chromatin remodeling complexes (Yadon et al., 2013). Taken together, the regulation of CDC28 transcription by the induction of a stress-responsive lncRNA provides a novel paradigm by which a lncRNA mediates gene induction through changes in chromatin architecture.

Induction of the CDC28 lncRNA controls cell cycle re-entry upon stress

The CDC28 gene encodes the main CDK kinase (CDK1) that drives progression of the cell cycle in yeast. Cdc28 is regulated by several mechanisms, including cyclin association and CDK inhibitors (Bloom and Cross, 2007). However, the increase in transcription of CDC28 observed upon stress was unexpected since transcription of CDC28 was assumed to be constant (Spellman et al., 1998). The increase in CDC28 transcription resulted in an increase of de novo synthesis of Cdc28. In response to osmostress, Hog1 mediates a rapid but transient arrest of cell cycle progression to allow adaptation (Clotet and Posas, 2007; Duch et al., 2012; Saito and Posas, 2012). One of the mechanisms under the control of Hog1 consists in the down-regulation of Cdc28 activity, which seems to be contradictory with an increase of Cdc28 protein. Nevertheless, the increase in Cdc28 protein levels occurred when cells started to recover from stress; thus we postulated that this increase in Cdc28 protein levels should have an effect during the recovery phase. Indeed, cells deficient in CDC28 lncRNA arrested similar to wild type upon stress but reentered cell cycle less efficiently, suggesting that the increase in Cdc28 permits a faster recovery of the cell cycle delay caused by stress. Although cell cycle re-entry delay in CDC28 lncRNAA cells might seem modest (20 minutes), this lapse-time has proven to be important to maximize cell survival upon stress (Escote et al., 2004; Duch et al., 2013). Therefore, Hog1 is able to induce a cell cycle delay and promote the recovery by controlling transcriptional CDC28 modulation, thus achieving a different temporal outcome.

In summary, we present here a new mechanism of Cdc28 regulation through a stress-inducible lncRNA production that is able to alter cell cycle progression in response to environmental challenges. Cdc28 regulation provides a mechanism by which a lncRNA together with a SAPK can mediate gene induction through changes of chromatin architecture. Moreover, this study provides insights into how lncRNAs might affect the regulation of gene expression through chromatin changes in eukaryotic cells.

Experimental Procedures

Website

The following link http://steinmetzlab.embl.de/francescData/arrayProfile/index.html directs to an interface to visualize array expression data. Raw array data are available from ArrayExpress under accession number E-MTAB-1686.

Yeast strains and plasmids

Full list and description of strains and plasmids used in this study is enclosed in Supplemental Experimental Procedures.

Tiling array

Wild type (BY4741) and hogΔ cells were grown to mid-log phase and subjected (or not) to mild osmostress (0.4M NaCl for 15 minutes) or hyper osmostress (1.2M NaCl for 100 minutes). Hybridization of tiling array was performed as described (David et al., 2006).

Definition of stress-induced lncRNAs

Stress-induced lncRNAs were defined as up-regulated with a minimum of two fold change in response to stress (at the indicated osmolarity) in the wild type (BY4741) strain. Hog1 dependence was determined by the percentage of expression in a hog1Δ mutant respect to the wild type strain. Supplemental Table S1 provides the entire list of the osmoresponsive lncRNA, their relationship with the sense transcript and HOG1 dependence.

ChIP-seq and Mnase-seq

Wild type and hog1Δ mutant S. cerevisiae strains were grown to mid-log phase and exposed to 5 minutes of osmostress (0.4 M NaCl) for Hog1 immunoprecipitation , 10 minutes for RNA Pol II immunoprecipitation and nucleosome positioning. ChIP and MNase protocols were performed and purified DNA was sequenced as described. Enrichment of Hog1 and RNA Pol II was done by running the Pyicos enrichment protocol comparing untreated to treated samples (Nadal-Ribelles et al., 2012).

MNase nucleosome mapping

Spheroplasts and digestion with MNase were done with the indicated strains subjected (or not) to osmostress (0.4M NaCl for 10 minutes) or treated with β-estradiol (100nM, 10 minutes). For the analysis of CDC28, DNA was used in a real-time PCR.

ChIP assays

Chromatin immunoprecipitation was done as described previously (Zapater et al., 2007). Briefly, indicated yeast cultures were grown to mid-log phase and exposed (or not) to osmotic stress (0.4M NaCl, 5 minutes) or treated with β-estradiol (100nM, 5 minutes). Real-time PCR of the indicated regions was performed.

3C analysis

Cells were grown to mid-log phase before being subjected or not to osmostress (10 min, 0.4 M NaCl). 3C analysis was performed as described previously with minor modifications indicated in Supplementary Experimental Procedures.

Metabolic labeling

Briefly indicated PHO85-TAP strains were grown in YPD and shifted to MET media for 2 hours before being stressed. A mixture of 35S-methionine and 0.4M NaCl was added simultaneously.

Flow cytometry

Flow cytometry experiments were performed as described (Pelet et al., 2011). Cells were stressed for 45 minutes with 0.4M NaCl. To study cell cycle progression, cdc15ts (cdc15-2) cells were synchronized at 37°C (incubated for 2 hours) and released at 25°C to allow cell cycle progression.

Supplementary Material

01

Highlights.

  • -

    A novel family of lncRNAs is induced by the Hog1 SAPK in response to osmostress.

  • -

    The CDC28 gene expresses a stress-responsive lncRNA in antisense orientation.

  • -

    The CDC28 lncRNA and Hog1 induce CDC28 expression via gene looping.

  • -

    Induction of the CDC28 lncRNA controls cell cycle re-entry upon stress.

Acknowledgments

We thank L. Subirana, S. Obejas and A. Fernandez for technical support, Núria Conde for ChIP-seq analysis and Javier Jimenez and Alba Duch for cell cycle analysis. Christoph Schüller for helpful discussions all along the study and the Gal4DBD-Msn2 plasmid. Dr. S. Buratowski and Dr. N. Proudfoot for strains. M.N. is a recipient of a FIS fellowship. This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (BFU2012-33503 and FEDER to F.P., BFU2011-26722 to E.N), the Fundación Marcelino Botín (FMB) and the Consolider Ingenio 2010 programme CSD2007-0015 to F.P.. The National Institutes of Health and Deutsche Forschungsgemeinschaft to L.M.S.. F.P. and E.N. are recipients of an ICREA Acadèmia (Generalitat de Catalunya).

Footnotes

Supplemental Information: Detailed experimental procedures can be found in the Supplemental Experimental Procedures.

The authors declare no competing financial interest.

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