Please cite this article in press as: Nadal-Ribelles et al., Control of Cdc28 CDK1 by a Stress-Induced lncRNA, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.01.006
Molecular Cell
Article
Control of Cdc28 CDK1 by a Stress-Induced lncRNAMariona Nadal-Ribelles,1,3 Carme Sole,1,3 Zhenyu Xu,2 Lars M. Steinmetz,2 Eulalia de Nadal,1,* and Francesc Posas1,*1Cell Signaling Unit, Departament de Ciencies Experimentals i de la Salut, Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain2EMBL Heidelberg, Meyerhofstrasse 1, 69117 Heidelberg, Germany3These authors contributed equally to this work*Correspondence: [email protected] (E.d.N.), [email protected] (F.P.)
http://dx.doi.org/10.1016/j.molcel.2014.01.006
SUMMARY
Genomic analysis has revealed the existence of alarge number of long noncoding RNAs (lncRNAs)with different functions in a variety of organisms,including yeast. Cells display dramatic changes ofgene expression upon environmental changes. Uponosmostress, hundreds of stress-responsive genesare induced by the stress-activated protein kinase(SAPK) p38/Hog1. Using whole-genome tiling arrays,we found that Hog1 induces a set of lncRNAs uponstress. One of the genes expressing a Hog1-depen-dent lncRNA in antisense orientation is CDC28, thecyclin-dependent kinase 1 (CDK1) that controls thecell cycle in yeast. Cdc28 lncRNAmediates the estab-lishment of gene looping and the relocalization ofHog1 and RSC from the 30 UTR to the +1 nucleosometo induce CDC28 expression. The increase in thelevels of Cdc28 results in cells able to reenter the cellcycle more efficiently after stress. This may representa general mechanism to prime expression of genesneeded after stresses are alleviated.
INTRODUCTION
The existence of long noncoding RNAs (lncRNAs) is widespread
in eukaryotes from yeast to mammals (Guttman and Rinn, 2012;
Jacquier, 2009). Long noncoding transcripts in yeast influence
gene expression, revealing a new layer of transcriptional regula-
tion (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 (TSS) 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; vanWerven et al., 2012; Hainer
et al., 2011; Uhler et al., 2007). Although in a few cases expres-
sion of specific lncRNAs alters normal mRNA biogenesis, the
general biological relevance and functionality of lncRNAs re-
mains elusive. Remarkably, changes in nutrient availability result
in changes in lncRNA expression (Xu et al., 2009, 2011), indi-
cating that environmental insults and signal transduction path-
ways 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 extracel-
lular 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 tar-
geting several core components of the cell cycle machinery. For
instance, Hog1 controls G1/S transition by the downregulation of
cyclin expression and the stabilization of the Sic1 cyclin-depen-
dent kinase inhibitor (CDKi) (Adrover et al., 2011; Escote et al.,
2004). Hog1 also modulates other phases of the cell cycle,
such as S phase (Duch et al., 2013). Cells unable to delay cell
cycle progression upon osmostress display reduced viability
upon those conditions, suggesting the need to delay 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 re-
cruit 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 open
reading frames (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 re-
modeling 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 stabil-
ity (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; Martınez-Montanes et al., 2010; Weake and Workman,
2010).
Here, we show that the Hog1 SAPK also associates with and
controls the induction of a set of lncRNAs in response to osmo-
stress. One of the genes expressing a stress-induced lncRNA in
antisense orientation is CDC28, the cyclin-dependent kinase 1
(CDK1) that controls the cell cycle in yeast. Induction of the
CDC28 lncRNA permits the increase in the levels of Cdc28,
allowing cells more efficient reentry into the cell cycle after
stress. Therefore, Hog1 directly coordinates the regulation of
Molecular Cell 53, 1–13, February 20, 2014 ª2014 Elsevier Inc. 1
Molecular Cell
CDC28 lncRNA Regulates Cell Cycle upon Stress
Please cite this article in press as: Nadal-Ribelles et al., Control of Cdc28 CDK1 by a Stress-Induced lncRNA, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.01.006
transcription and cell cycle progression by controlling expres-
sion of a stress-induced lncRNA in CDC28.
RESULTS
Hog1 Mediates the Expression of a Set of Stress-Inducible lncRNAsMost 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 was 343 at 0.4 M NaCl
(15 min) and 294 at 1.2 M NaCl (100 min) using a stringent
threshold (see Experimental Procedures). 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 that in 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 (Figure 1). Almost a hundred of them
were shared between the two stress conditions (Figure S1
available online). The average length of these stress-induced
lncRNAs is about 843 nt (Figure S1). Expression of 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) (Figure 1B). Some overlapped with previously anno-
tated cryptic unstable transcripts (CUTs) or stable unannotated
transcripts (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 were present only upon stress (Figures 1C
and S1). Thus, Hog1 mediates the expression of a set of
stress-inducible lncRNAs.
Hog1 Associates with the Promoters of Stress-InducedlncRNAs and Stimulates RNA Pol II Recruitment andGene ExpressionHog1 associates with chromatin of stress-responsive genes
upon stress (Alepuz et al., 2001, 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 �63% of Hog1-dependent lncRNA promoters,
whereas it is recruited to <30% of Hog1-independent lncRNAs
(Figure 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 (Figures 2B and S2A). Therefore, Hog1 associates to and
stimulates the recruitment of RNA Pol II at the promoters of
stress-induced lncRNAs.
Once recruited to stress-responsive genes, Hog1 mediates
chromatin remodeling (Mas et al., 2009; Pelet et al., 2011).
Genome-wide micrococcal nuclease (MNase) digestion of chro-
2 Molecular Cell 53, 1–13, February 20, 2014 ª2014 Elsevier Inc.
matin and deep sequencing (MNase-seq) showed that upon
osmostress, Hog1 mediates dramatic change of nucleosome
occupancy (Nadal-Ribelles et al., 2012). We analyzed the chro-
matin 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 (Figure 2C). Although
levels of nucleosome occupancy decreased slightly upon stress
in hog1D 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 (Figure S2B).
Expression from stress-responsive promoters can be quanti-
tatively measured by the fusion to quadruple Venus (qV)
fluorescent protein (Pelet et al., 2011; Regot et al., 2013). To
characterize one of these lncRNA promoters further, we fused
the 30 untranslated region (30 UTR) of CDC28 in both the sense
and the antisense orientation to qV-yellow fluorescent protein
(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 (Figure 2D). Therefore,
the 50 regions of stress-induced lncRNAs behave as bona fide
stress-responsive promoters.
Induction of CDC28 lncRNA Expression Promotes theInduction of CDC28 Gene Expression upon StressTo 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 (Figure 3A). Tiling arrays are not very sensi-
tive 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).
One of the genes with a clear correlation of the sense and
lncRNA expression was CDC28 (Figure 3B). CDC28 encodes
the main CDK 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
(Figure 3B). Systematic insertion analysis at the 30 UTR of
CDC28 showed that insertion of a KanR marker at 180 nt
downstream of the TTS (lncRNAD) abolished expression of
the lncRNA. In this strain, the induction of CDC28 upon osmo-
stress was impaired (Figures 3B and S3A). Notably, the rrp6
mutation did not alter the induction of the CDC28 sense tran-
scription (Figure S3B). Thus, the presence of the stress-induc-
ible CDC28 lncRNA correlates with induction of the CDC28
gene expression.
We then assessed whether it was the expression of the
lncRNA from the 30 UTR or solely the presence of the
lncRNA that induced CDC28 expression. We created a strain
containing a CDC28::GFP that recapitulated CDC28 gene ex-
pression (CDC28::GFP) and then abolished lncRNA expression
A
B
C
Figure 1. Hog1 Controls Transcription of a Set of lncRNAs upon Stress(A) Osmostress induces expression of a class of lncRNAs. Expression data from tiling arrays of several stress-induced lncRNAs for theWatson (W, top) and Crick
(C, bottom) strands. Normalized signal intensities from duplicate hybridizations of wild-type (WT) and hog1D 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) are shown. Genomic coordinates and gene annotation, transcript boundaries (red lines) and
transcription start site (arrows) are depicted.
(B) Hog1 regulates lncRNA transcription upon stress. lncRNAs induced at least 2-fold upon osmostress (0.4 M NaCl, left; 1.2 M NaCl, right) grouped into three
categories according to the dependence of their expression in hog1D: strong (<50% of wild-type), moderate (50%–75%), none (>75%).
(C) Stress-induced lncRNAs are a family of lncRNAs. WT, hog1D, trf4D, rrp6D, and xrn1D mutant strains were subject to osmostress (0.4 M NaCl), and the
indicated lncRNAs were assessed. See also Figure S1.
Molecular Cell
CDC28 lncRNA Regulates Cell Cycle upon Stress
Please cite this article in press as: Nadal-Ribelles et al., Control of Cdc28 CDK1 by a Stress-Induced lncRNA, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.01.006
(CDC28::GFP lncRNAD) (Figure S3C). We then expressed
CDC28 and its terminator region from a plasmid. This permitted
us to distinguish transcription from the plasmid or the endoge-
nous 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
Molecular Cell 53, 1–13, February 20, 2014 ª2014 Elsevier Inc. 3
A B
C
D
Figure 2. Hog1 Binds and Recruits RNA Pol II at
Genes with lncRNA
(A) Hog1 associates with lncRNA promoters. The percent-
age of Hog1 binding at the Hog1-dependent or -indepen-
dent 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)
was determined by ChIP-seq (0.4 M NaCl for 10 min) for
Hog1-dependent lncRNAs in wild-type (WT) and hog1D.
***p < 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 1 kb up and
downstream from the TTS of wild-type and hog1D mutant
strains under basal conditions (dark blue and red) and 0.4 M
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 30 UTR of CDC28 is an osmores-
ponsive promoter. The 30 UTR of CDC28 fused to the qV-
YFP in the antisense orientation (promoter lncRNA) or in
its natural orientation (terminator sense). Fluorescence in-
tensity was measured by flow cytometry.
Molecular Cell
CDC28 lncRNA Regulates Cell Cycle upon Stress
4 Molecular Cell 53, 1–13, February 20, 2014 ª2014 Elsevier Inc.
Please cite this article in press as: Nadal-Ribelles et al., Control of Cdc28 CDK1 by a Stress-Induced lncRNA, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.01.006
B
0
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1 1.4 1.9 2.9 1.6 1 1.1 1 1.2 1.2 1 1 0.8 0.8 0.8
A
1 15 27 35 9 1 1 1 1 1 1 1 1 1 1
Figure 3. CDC28 Induction Correlates with lncRNA Expression
(A) Expression of sense (x axis) versus lncRNA (y axis), in a log2 scale, in
response to stress in Hog1-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, hog1D, and
lncRNAD strains upon osmostress. See Figure S3 for systematic insertion of a
KanR marker at the 30 UTR region of CDC28. Normalized quantification of
CDC28 sense is shown. Quantifications were done by normalizing by the
loading control gene and the expression in the absence of stress.
Molecular Cell
CDC28 lncRNA Regulates Cell Cycle upon Stress
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increase of the CDC28::GFP, thus indicating a cis effect of the
lncRNA (Figure S3C).
Hog1 Associates with the 30 UTR and the +1 NucleosomeRegions in CDC28 to Promote Chromatin RemodelingTo characterize the mechanism by which Hog1 induces sense
and antisense transcription, we monitored Hog1 association in
CDC28. Hog1 associated with the 30 UTR of CDC28 (the pro-
moter region of the CDC28 lncRNA) upon stress. Strikingly,
Hog1 also associated with a region close to the TSS (region +41
to +125) corresponding to the CDC28 +1 nucleosome. By
contrast, in cells unable to induce CDC28 lncRNA, Hog1 associ-
ation with the TSS was abolished completely and only slightly
reduced at the 30 UTR (Figure 4A).
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 30 UTR of CDC28 changed upon osmostress, as expected
for a stress-responsive promoter. Strikingly, the region corre-
sponding to the +1 nucleosome at the 50 region of CDC28 was
also strongly remodeled upon stress (Figure 4B). The eviction of
the +1 nucleosome was observed in neither hog1- nor lncRNA-
deficient cells (Figure 4B). Therefore, Hog1 association and re-
modeling at the +1 nucleosome region of CDC28 occur in
response to osmostress and in the presence of CDC28 lncRNA.
The RSC Chromatin Remodeling Complex MediatesChromatin Remodeling at the CDC28 +1 NucleosomeRegion upon StressHog1 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 nonpermissive temperature was
similar to that of wild-type but reduced in a SAGA mutant (Fig-
ures 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 30 UTR and +1 nucle-
osome regions of CDC28 in response to stress only in the pres-
ence of Hog1 (Figure 4D). Induction of lncRNA and CDC28
required active Hog1, since a catalytically inactive Hog1 was
unable to associate to chromatin and promote RSC association
(Figures S4B–S4D). By contrast, association of Hog1 was not
altered in the rsc9ts mutant under nonpermissive temperature
(Figure S4E), suggesting that Hog1 mediates the recruitment of
RSC at CDC28 to remodel chromatin upon stress. Correspond-
ingly, chromatin remodeling at the +1 nucleosome, assessed by
MNase digestion, was impaired in the rsc9ts mutant under
nonpermissive temperature (Figure 4E). Thus, recruitment of
RSC by Hog1 is essential to mediate chromatin reorganization
at the +1 nucleosome region and CDC28 gene induction.
The Expression of Both the CDC28 lncRNA and Hog1 IsRequired for CDC28 Induction upon StressIn the absence of CDC28 lncRNA expression, the presence of
Hog1 at the 30 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 an inducible GAL1 promoter in the anti-
sense orientation at the endogenous 30 UTR of CDC28
(CDC28::pGAL1). Expression from the GAL1 promoter is driven
by the Gal4-ER-VP16 activator in the presence of b-estradiol
(Louvion et al., 1993). Although the presence of estradiol strongly
induced expression of theCDC28 lncRNA, this was not sufficient
to stimulate sense transcription (Figure 5A). We then monitored
Hog1 recruitment and found that the presence of estradiol did
not mediate Hog1 recruitment in CDC28 (Figure 5B). Corre-
spondingly, 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
(Figure 5C). Thus, the induction of the CDC28 lncRNA alone is
not sufficient to mediate chromatin remodeling and CDC28
gene induction.
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
Molecular Cell 53, 1–13, February 20, 2014 ª2014 Elsevier Inc. 5
MN
ase
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ectio
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1 18 26 33 15 1 29 39 15 16
KANKAN Figure 4. Binding of Hog1 Induces lncRNA and Promotes Chromatin
Remodeling by the RSC Chromatin Remodeler
(A) Hog1 binds at the 30 UTR and at the +1 nucleosome region. Graphical
representation of the CDC28 locus with the insertion of a KanR marker at the
30 UTR region to disrupt expression of lncRNA (lncRNAD). 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), hog1D, and lncRNAD 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 osmo-
stress. 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 the +1 nucleosome region (amplicon D) and 30 UTR(amplicon J) of CDC28 in wild-type and hog1D strains that were (or were not)
subjected to osmostress (0.4 M NaCl, 5 min).
(E) RSC is essential tomediate chromatin reorganization. Remodeling at the +1
nucleosome in a Rsc9ts strain was assayed byMNase digestion after 10 min of
NaCl. Bars represent the average of the stressed (black bars) compared to the
unstressed (white bars) cells ± SD. See also Figure S4.
Molecular Cell
CDC28 lncRNA Regulates Cell Cycle upon Stress
6 Molecular Cell 53, 1–13, February 20, 2014 ª2014 Elsevier Inc.
Please cite this article in press as: Nadal-Ribelles et al., Control of Cdc28 CDK1 by a Stress-Induced lncRNA, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.01.006
induction upon stress (Figure 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 pre-
vented the generation of the lncRNA. Here, the Gal4DBD-Msn2
didnotpromoteCDC28 sense induction (Figure5E).Correspond-
ingly, Hog1 was recruited at the GAL1 promoter in the 30 UTRregion of CDC28 as well as at the +1 region of CDC28 (Fig-
ure 5F), whereas it was not recruited at the +1 region in the IT
construct (Figure 5G). We then monitored chromatin remodeling
at the +1 region. In contrast to Gal4-ER-VP16 activator, expres-
sion of the CDC28 lncRNA from the GAL1 promoter by the
Gal4DBD-Msn2 activator caused remodeling of the +1 nucleo-
some upon stress (Figure 5H). Thus, induction of the CDC28
lncRNA and the recruitment of Hog1 at the +1 region are required
for chromatin remodeling at the 50 region of CDC28 and CDC28
gene expression.
The Establishment of Gene Looping Permits theRecruitment of Hog1 at the +1 Nucleosome Region andInduction of CDC28
The absence of Hog1 recruitment and remodeling at the 50 regionof 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). Expres-
sion 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 (Figure 6A). Similar results were obtained in a
sua7-1 mutant (Singh and Hampsey, 2007) with impaired gene
looping (Figure S5A). Then, we assessed the recruitment of
Ssu72 and found that there was a clear Hog1-dependent in-
crease in Ssu72 binding upon stress at both the 30 UTR and +1
nucleosome regions (Figure S5B). Thus, the enhanced recruit-
ment of Ssu72 in response to stress does not alter CDC28
pGAL1CDC28
Gal4DBD
Msn2 ZFΔ
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lncRNA
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CDC28 sense
lncRNA
ENO1
NaCl (min) 0 5 15 3010
CDC28::IT::pGAL1*
0 5 15 3010Ø Gal4DBD-Msn2
E
1 24 19 13 71 1 1 1 1
1 0.7 1 1.1 0.61 0.8 0.6 0.7 1.1
1 1 1 1 11 1 1 1 1
Gal4DBD
β-estradiolER
VP16AD
DpGAL1CDC28
X
CDC28 sense
wt +Gal4DBD-ER-
VP16CDC28::pGAL1 +
Gal4DBD-ER-VP16
lncRNA
ENO1
β-E0 5 15 30Time (min)
β-E0 5 15 30
β-E+NaCl0 5 15 30
1 1 0.8 1 1 1.1 1 1.1
1 1 1 1 1 4 12 45 1 1 25 40
1 1.1 1 1.2
Figure 5. Antisense Transcription and Hog1 Recruitment Are Required to Induce CDC28 Expression
(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 b-estradiol (black bars) or b-estradiol and NaCl (gray bars).
CDC28 sense and lncRNA transcripts were assessed by northern blot.
(B and C) We measured Hog1 association (5 min) by ChIP (B) and +1 nucleosome eviction (10 min) by MNase (C) in the indicated strains upon induction with
b-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 SD.
Molecular Cell
CDC28 lncRNA Regulates Cell Cycle upon Stress
Molecular Cell 53, 1–13, February 20, 2014 ª2014 Elsevier Inc. 7
Please cite this article in press as: Nadal-Ribelles et al., Control of Cdc28 CDK1 by a Stress-Induced lncRNA, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.01.006
A
+1 nucleosome 3’UTRB
controlNaCl
+1 n
ucle
osom
eev
ictio
n (A
.U.)
GAL1::SSU72YPGal YPD
0
0.5
1.0
1.5D
O1
CDC28
AciI
TO2
F
A B C D E F G H I J0
1
2
3
4
0
1
2
3
4
YPGal YPD
GAL1::SSU72
YPGal YPD
C
Hog
1-H
A bi
ndin
g
Rsc
1-H
A bi
ndin
g
E
GAL1::SSU72 (YPD)
wthog1ΔlncRNAΔ
RN
A Po
l II b
indi
ng
0
0.5
1.0
NaCl
YPGalYPD
- + - + - + - + ND Dwt hog1Δ YPGal YPD
GAL1::SSU72
NaCl
T+O1
T+O2
TEL
sua7-1wt- + - + ND D
lncRNA
ENO1
NaCl (min)
CDC28 sense
YPGal YPDGAL1::SSU72
0 5 10 15 300 5 10 15 30
1 1.7 1.8 3.2 2 1 1.2 1.5 1.4 1
1 22 28 32 15 1 19 21 30 28
YPD0 5 10 15 30
wt
1 1.7 2 2.6 1.4
1 33 39 41 20
+1 nucleosome
GAL1::SSU72
Figure 6. Gene Looping Allows Recruitment
of Hog1 at the +1 Nucleosome Region as
well as Induction of CDC28
(A) CDC28 induction depends on Ssu72. Wild-type
and GAL1::SSU72 strains grown as stated were
subjected to osmostress. CDC28 sense and
lncRNA transcripts were detected.
(B) Hog1 binding at the +1 nucleosome of CDC28
depends on Ssu72. The GAL1::SSU72 strain was
grown as stated, and Hog1 recruitment was
analyzed by ChIP after 5 min of NaCl at the indi-
cated regions (as in Figure 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 depends
on Ssu72. The GAL1::SSU72 strain was grown as
stated, and remodeling at the +1 nucleosome was
assayed by MNase digestion after 10 min of NaCl.
Bars represent the average of the stressed (black
bars) compared to the unstressed (white bars)
cells ± 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 indi-
cated strains.
(F) Hog1 induces physical interaction between
the +1 nucleosome region and 30 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) is shown. Ampli-
fication of tandem primer pairs is shown, and the
TEL region was used as loading control. Non-
digested (ND) and digested (D) chromatin samples
from the treated WT strain were used as an internal
control of 3C specificity. See also Figures S5
and S6.
Molecular Cell
CDC28 lncRNA Regulates Cell Cycle upon Stress
Please cite this article in press as: Nadal-Ribelles et al., Control of Cdc28 CDK1 by a Stress-Induced lncRNA, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.01.006
lncRNA expression, but it is essential for the increase of CDC28
expression.
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 30 UTR to +1 nucleosome
8 Molecular Cell 53, 1–13, February 20, 2014 ª2014 Elsevier Inc.
regions of CDC28. Association of Hog1
at the 30 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 (Figure 6B). Correspond-
ingly, recruitment of Rsc1 was also abol-
ished at the +1 region in the absence of
Ssu72 (Figure 6C), which prevented chro-
matin remodeling at the +1 region upon
stress (Figure 6D). Moreover, the increase
in RNA Pol II association upon stress at
the CDC28 50 region observed in the
wild-type strain was abolished in cells deficient in hog1,
CDC28 lncRNA, and ssu72 (Figure 6E). Thus, gene looping
mediates the recruitment of Hog1 at the 50 region of CDC28 to
induce chromatin remodeling and RNA Pol II recruitment upon
stress.
Molecular Cell
CDC28 lncRNA Regulates Cell Cycle upon Stress
Please cite this article in press as: Nadal-Ribelles et al., Control of Cdc28 CDK1 by a Stress-Induced lncRNA, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.01.006
To further confirm the establishment of gene looping between
the 30 UTR and promoter regions, we applied the 3C assay (see
Experimental Procedures). We found that there was a clear
increase in gene looping formation upon stress between the
30 UTR and the +1 nucleosome regions, as detected by the pres-
ence 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 (Figures 6F and S5C). Notably, 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 (Figures 6F and S5C), but not in a rsc9ts
mutant (Figure S5D). Therefore, gene looping is critical for the
recruitment of Hog1 from 30 UTR to the +1 nucleosome region
of CDC28 to promote chromatin remodeling and induce
CDC28 gene expression.
An lncRNA in MMF1 Induces Hog1 Recruitment andChromatin Remodeling at the +1 Nucleosome RegionTo assess whether genes other than CDC28 displayed a similar
regulatory mechanism, we choseMMF1 because it expresses a
strong, stress-induced lncRNA (Figure 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 dynamic
transcriptome analysis (DTA) experiments (Miller et al., 2011;
Romero-Santacreu et al., 2009). We created a mutant in anti-
sense MMF1 lncRNA expression (Figure S6A). Hog1 was re-
cruited at the 30 UTR and the 50 region, but not in the body of
theMMF1 gene, depending on the presence of the lncRNA (Fig-
ure 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). Notably, 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 ex-
pression as in CDC28.
We then asked whether the 30 UTR region of CDC28 could
confer osmoinduction in a nonosmoresponsive gene. We re-
placed the 30 UTR region of a non-stress responsive gene,
MBA1, with the 30 UTR region of CDC28 (300 bp downstream
of STOP codon). We choseMBA1 because its expression under
normal conditions and the length of the gene are similar to those
of CDC28. Replacement of the MBA1 terminator by CDC28 led
to the transcription of an lncRNA from the 30 UTR in MBA1,
and most important, it conferred Hog1-dependent osmoinduc-
tion (Figure S6E), suggesting that the role of CDC28 30 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 ofCdc28 that Permits Cells to Reenter the Cell Cycle MoreEfficiently in Response to StressWe then asked whether an increase of CDC28 mRNA results
in an increase of Cdc28 protein production upon stress.
Endogenous [35S]methionine Cdc28 protein increased �2-fold
in response to stress in wild-type cells, whereas no increase
was observed in a CDC28 lncRNA-deficient strain (Figure 7A).
Thus, stress-induced Cdc28 lncRNA expression leads to in-
creased levels of Cdc28 kinase.
Hog1 promotes an immediate, but transient, cell cycle delay
that permits 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 reentry 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 Experi-
mental Procedures). Cells deficient in CDC28 lncRNA were
able to arrest and exit the cell cycle from cdc15ts synchronization
as efficiently as wild-type in the absence of stress. In contrast,
compared to the wild-type, cells deficient inCDC28 lncRNA pro-
duction delayed cell cycle reentry by approximately 20 min upon
stress (Figure 7B). Notably, overexpression of CDC28 from a
plasmid can suppress the delay on cell cycle progression
observed upon osmostress inCDC28 lncRNAD cells (Figure S7).
Thus, stress-induced CDC28 lncRNA results in an increase of
Cdc28 that permits cells to reenter the cell cycle more efficiently
in response to stress.
DISCUSSION
ASpecific Set of Hog1-Dependent lncRNAs Is Induced inResponse to OsmostressStress-activated protein kinases regulate gene expression to
maximize cellular adaptation to environmental stresses (de
Nadal et al., 2011; Weake and Workman, 2010). In yeast, activa-
tion 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 dependsmostly on the presence of Hog1. Correspond-
ingly, similar to osmoresponsive genes, Hog1 associates to the
promoters of lncRNAs upon stress and stimulates RNA Pol II
recruitment and chromatin remodeling. In fact, fusing the pro-
moter 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 remark-
able, 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 a
different transcriptional unit recruited to this region that is inde-
pendent of the neighboring genes must exist. Accordingly, a
recent study by chromatin immunoprecipitation-exonuclease
(ChIP-exo) precisely positioned distinct transcriptional machin-
eries 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 lncRNAs are only detectable in strains
with deleted components of the nuclear or cytosolic exosome
Molecular Cell 53, 1–13, February 20, 2014 ª2014 Elsevier Inc. 9
A
B
Cdc
28p
leve
lsFo
ldin
duct
ion
Time (min)
wtlncRNAΔ
0
1
2
0 20 40 60 80 100
2C
wt lncRNAΔ
control NaCl
cdc15ts
control NaCl1C
Tim
e (m
in)
95
85
75
65
55
45
35
0 88
74
52
40
59
41
65
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90
88
88
82
70
48
40
86
76
57
44
46
54
60
80
89
89
89
88
88
78
71
C
Figure 7. Changes of Cdc28 Levels Promote Cell Cycle Reentry
upon Stress
(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
Molecular Cell
CDC28 lncRNA Regulates Cell Cycle upon Stress
10 Molecular Cell 53, 1–13, February 20, 2014 ª2014 Elsevier Inc.
Please cite this article in press as: Nadal-Ribelles et al., Control of Cdc28 CDK1 by a Stress-Induced lncRNA, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.01.006
(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 implica-
tions for proper cellular adaptation.
The CDC28 lncRNA and Hog1 Induce ChromatinRemodeling and CDC28 Expression via Gene LoopingTo 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 in the cases of some genes. Remarkably, there was
a positive correlation between the induction of CDC28 and an
lncRNA in CDC28 expressed in antisense orientation. CDC28
expression was dependent on Hog1, since there was induction
of neither the lncRNA nor the CDC28 sense in hog1 cells. This
posed the question of how the SAPK and the induction of an
lncRNA lead to increased gene expression. In clear contrast to
typical osmoresponsive genes in which the SAPK associates
all along the gene (Proft et al., 2006), Hog1 associated at the 30
region of CDC28, which corresponds to the promoter region of
the CDC28 lncRNA, and at a region surrounding the +1 nucleo-
some ofCDC28. Transcription ofCDC28 is not controlled by any
of the transcription factors targeted by Hog1, thus opening the
possibility that Hog1 uses the 30 UTR region to mediate its asso-
ciation to the +1 nucleosome region to promote gene expres-
sion. This interesting Hog1-binding pattern resembles some of
the features of osmoresponsive genes in which Hog1 recruit-
ment at the ORFs depends on the 30 UTR region (Proft et al.,
2006).
Cells deficient in lncRNA still recruit Hog1 at the 30 UTR, butnot at the +1 nucleosome region in CDC28. Remarkably, these
cells induce neither chromatin remodeling nor CDC28 gene in-
duction. On the other hand, when CDC28 lncRNA was induced
by a heterologous activator that does not promote recruitment
of Hog1 at the 30 UTR, the SAPK did not bind at the +1 nucleo-
some region and cells could induce neither chromatin remodel-
ing 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 30 UTR, together with the expression
of the lncRNA, allows 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 affected not by
conditions. Cell cycle progression was analyzed by fluorescence-activated
cell sorting (FACS), and percentage of cells in G2/M is shown. See also
Figure S7.
(C) Schematic representation of Hog1-mediated gene looping between the
30 UTR and the +1 nucleosome region at the CDC28 locus in response to
osmostress.
Molecular Cell
CDC28 lncRNA Regulates Cell Cycle upon Stress
Please cite this article in press as: Nadal-Ribelles et al., Control of Cdc28 CDK1 by a Stress-Induced lncRNA, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.01.006
depletion of RSC, but by depletion of SAGA. In contrast, RSC
was absolutely necessary for chromatin remodeling at the +1
nucleosome region andCDC28 gene induction. Thus, the target-
ing of RSC by Hog1 at the +1 nucleosome region is required for
gene induction.
Unlike osmoresponsive genes in which Hog1 travels with elon-
gating polymerase (Proft et al., 2006), the Hog1-binding pattern
at the CDC28 locus suggested that Hog1 could reach the 50 endof 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 cells containing the sua7-1 mutation (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
30 UTR to the +1 nucleosome position.
Altogether, these data suggest the following tentative model
for the induction of CDC28 by Hog1 (Figure 7C). In response
to osmostress, Hog1 associates at the 30 UTR region of
CDC28 and induces lncRNA transcription. Once antisense tran-
scription is induced, gene looping is established and Hog1 is
transferred to the +1 nucleosome region in CDC28. The recruit-
ment 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
another example has recently demonstrated that DNA looping
facilitates targeting of chromatin remodeling complexes (Yadon
et al., 2013). Taken together, the regulation of CDC28 transcrip-
tion by the induction of a stress-responsive lncRNA provides a
paradigm by which an lncRNA mediates gene induction
through changes in chromatin architecture.
Induction of the CDC28 lncRNA Controls Cell CycleReentry upon StressThe 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 re-
sulted 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 downre-
gulation 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 pro-
tein 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
reentry delay in CDC28 lncRNAD cells might seem modest
(20 min), this lapse of time has proven to be important to maxi-
mize 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 transcriptionalCDC28mod-
ulation, thus achieving a different temporal outcome.
In summary, we present here a mechanism of Cdc28 regula-
tion 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
an 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
Yeast Strains and Plasmids
Full list and description of strains and plasmids used in this study is included in
the Supplemental Experimental Procedures.
Tiling Array
Wild-type (BY4741) and hogD cells were grown to mid-log phase and sub-
jected (or not) to mild osmostress (0.4 M NaCl for 15 min) or hyper osmostress
(1.2 M NaCl for 100 min). Hybridization of tiling array was performed as
described (David et al., 2006).
Definition of Stress-Induced lncRNAs
Stress-induced lncRNAs were defined as upregulated, with a minimum
change of 2-fold in response to stress (at the indicated osmolarity) in the
wild-type (BY4741) strain. Hog1 dependence was determined by the percent-
age of expression in a hog1Dmutant with respect to the wild-type strain. 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 hog1D mutant S. cerevisiae strains were grown to mid-log
phase and exposed to 5 min of osmostress (0.4 M NaCl) for Hog1 immunopre-
cipitation and 10 min 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 (0.4 M NaCl for 10 min) or treated with
b-estradiol (100 nM, 10 min). 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 (0.4 M NaCl, 5 min) or treated with
b-estradiol (100 nM, 5 min). Real-time PCR of the indicated regions was
performed.
3C Analysis
Cells were grown to mid-log phase before being subjected (or not) to osmo-
stress (0.4 M NaCl, 10 min). 3C analysis was performed as described previ-
ously, with minor modifications indicated in the Supplemental Experimental
Procedures.
Molecular Cell 53, 1–13, February 20, 2014 ª2014 Elsevier Inc. 11
Molecular Cell
CDC28 lncRNA Regulates Cell Cycle upon Stress
Please cite this article in press as: Nadal-Ribelles et al., Control of Cdc28 CDK1 by a Stress-Induced lncRNA, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.01.006
Metabolic Labeling
Briefly, indicated PHO85-TAP strains were grown in YPD and shifted to MET
media for 2 hr before being stressed. A mixture of 35S[methionine] and 0.4 M
NaCl was added simultaneously.
Flow Cytometry
Flow cytometry experiments were performed as described (Pelet et al., 2011).
Cells were stressed for 45 min with 0.4 M NaCl. To study cell cycle progres-
sion, cdc15ts (cdc15-2) cells were synchronized at 37�C (incubated for 2 hr)
and released at 25�C to allow cell cycle progression.
ACCESSION NUMBERS
The 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.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
seven figures, and one table and can be found with this article online at
http://dx.doi.org/10.1016/j.molcel.2014.01.006.
ACKNOWLEDGMENTS
We thank L. Subirana, S. Obejas, and A. Fernandez for technical support; Nuria
Conde for ChIP-seq analysis; and Javier Jimenez and Alba Duch for cell cycle
analysis. We thank Christoph Schuller for helpful discussions throughout the
study and the Gal4DBD-Msn2 plasmid. We thank Dr. S. Buratowski and
Dr. N. Proudfoot for strains. M.N.-R. 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.d.N.), the Fundacion Marcelino Botın (FMB), and the Consolider Ingenio
2010 programme CSD2007-0015 (to F.P.). This work was supported by the
National Institutes of Health and Deutsche Forschungsgemeinschaft
(L.M.S.). F.P. and E.d.N. are recipients of an ICREA Academia award (Genera-
litat de Catalunya). The authors declare no competing financial interest.
Received: July 26, 2013
Revised: October 31, 2013
Accepted: December 31, 2013
Published: February 6, 2014
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