1. No category

FEBS Letters xxx (2015) xxx–xxx
journal homepage: www.FEBSLetters.org
The histone deacetylase Rpd3/Sin3/Ume6 complex represses
an acetate-inducible isoform of VTH2 in fermenting budding yeast cells
Igor Stuparevic a,1,2, Emmanuelle Becker a,2, Michael J. Law b,2, Michael Primig a,⇑,2
a
b
Inserm U1085 IRSET, Université de Rennes 1, 35042 Rennes, France
Rowan University, School of Osteopathic Medicine, Stratford, NJ 08084, USA
a r t i c l e
i n f o
Article history:
Received 19 November 2014
Revised 30 January 2015
Accepted 12 February 2015
Available online xxxx
Edited by Francesc Posas
Keywords:
RPD3
UME6
VTH1
VTH2
50 -UTR
Isoform
a b s t r a c t
The tripartite Rpd3/Sin3/Ume6 complex represses meiotic isoforms during mitosis. We asked if it
also controls starvation-induced isoforms. We report that VTH1/VTH2 encode acetate-inducible isoforms with extended 50 -regions overlapping antisense long non-coding RNAs. Rpd3 and Ume6
repress the long isoform of VTH2 during fermentation. Cells metabolising glucose contain Vth2,
while the protein is undetectable in acetate and during sporulation. VTH2 is a useful model locus
to study mechanisms implicating promoter directionality, lncRNA transcription and post-transcriptional control of gene expression via 50 -UTRs. Since mammalian genes encode transcript isoforms
and Rpd3 is conserved, our findings are relevant for gene expression in higher eukaryotes.
Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction
Most yeast genes encode more than one transcript; such variable isoforms often include 50 - and 30 -untranslated regions
(UTRs) that change in length when cells respond to environmental
cues [1–7]. The transcriptional mechanisms controlling this phenomenon are poorly understood in spite of the fact that UTRs are
important for mRNA turnover, transcript localization and mRNA
translation [8,9]. Two well-studied mechanisms act via upstream
open reading frames (uORFs), which inhibit the translation of
downstream ORFs, or via the 50 -UTR binding protein Rim4 [10,11].
The yeast transcriptome also includes long non-coding RNAs
(lncRNAs), which are targeted by Rrp6, a conserved 30 –50 exoribonuclease important for normal mitotic growth and efficient
meiosis [7,12–14]. In its absence, numerous lncRNAs such as cryptic unstable transcripts (CUTs) and some stable unannotated transcripts (SUTs) accumulate in mitosis [15,16]. Many lncRNAs are
⇑ Corresponding author at: Inserm U1085, Université de Rennes 1, 263, ave du
Général Leclerc, 35042 Rennes, France. Fax: +33(0)2 23 23 55 50.
E-mail address: [email protected] (M. Primig).
1
Present address: University of Zagreb, Faculty of Food Technology and Biotechnology, 10000 Zagreb, Croatia.
2
IS, ML performed experiments. EB analyzed high-throughput data. MP designed
experiments and wrote the paper. All authors contributed to the manuscript.
expressed via bi-directional promoters that mediate transcription
of divergent pairs of transcripts [15]; reviewed in [17]. The functions of most lncRNAs are not known, but some regulate the
expression of protein-coding genes via promoter interference or
through a mechanism of antagonistic sense/antisense transcription
[18,19]; for review, see [20].
The conserved histone deacetylase (HDAC) Rpd3, the co-repressor Sin3, and the upstream regulatory site 1 (URS1) binding protein
Ume6 form a complex, which prevents the expression of early
meiosis-specific transcripts and transcript isoforms during vegetative growth [21–24]; [25]. Its activity is abolished when cells
switch from fermentation to respiration and sporulation, because
Ume6 is degraded by the Spt-Ada-Gcn5-acetyltransferase (SAGA)
complex, the anaphase promoting complex/cyclosome (APC/C)
and Inducer of Meiosis 1 (Ime1) [26,27]. Ume6 target genes were
determined using RNA profiling [28].
VTH1 and VTH2 encode non-essential homologs of the vacuolar
protein sorting receptor PEP1 [29]. Over-expression of VTH2 was
shown to suppress the sorting phenotype of soluble hydrolases
to the yeast vacuole in pep1 mutant cells [30]. Expression profiling
studies showed that both paralogs are constitutively transcribed in
growing and starving cells and during sporulation [31,32], but cells
lacking either Vth1 or Vth2 do not have a meiotic phenotype or a
germination defect [33,34].
http://dx.doi.org/10.1016/j.febslet.2015.02.022
0014-5793/Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Stuparevic, I., et al. The histone deacetylase Rpd3/Sin3/Ume6 complex represses an acetate-inducible isoform of VTH2 in
fermenting budding yeast cells. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.02.022
2
I. Stuparevic et al. / FEBS Letters xxx (2015) xxx–xxx
We report that the promoters of VTH1 and VTH2 appear to drive
bi-directional expression of mitotic isoforms and divergent putative long non-coding RNAs (lncRNAs). In addition, they mediate
acetate-induced expression long isoforms with extended 50 -UTRs,
which partially overlap these antisense lncRNAs. Since VTH2 complemented the phenotype of pep1/vps10 mutant cells, we focussed
on this gene for further studies [30]. We find that Rpd3 and Ume6
repress the 50 -extended isoform of VTH2 during vegetative growth
in rich medium. Moreover, we observe an antagonistic pattern of
Vth2 protein concentration and the expression level of the long
isoform in fermenting, respiring and sporulating wild-type cells
and, consistently, in fermenting ume6 mutant cells.
These results show that VTH2 is a useful model locus to study
complex patterns of mRNA/lncRNA expression and post-transcriptional regulation of protein stability. Furthermore, they implicate
the histone deacetylase Rpd3/Sin3/Ume6 complex specifically in
the glucose-mediated repression of VTH2’s long isoform. As such,
the target gene constitutes a new class distinct from those encoding early meiosis-specific isoforms [25].
2. Materials and methods
2.1. Yeast strains
The tiling array data were produced with a wild-type SK1 MATa/a
strain and a sporulation deficient MATa/a control. RT-PCR assay
were carried out with SK1 MATa/a and JHY222 MATa/a strains
[12]. The induction of isoforms was monitored in SK1 MATa/a
ume6 and rpd3, and JHY222 MATa/a ume6 deletion strains
(Table 1). Sporulation experiments were done using rich media
with glucose (YPD) or acetate (YPA) and sporulation medium (SPII).
Table 1
Yeast strains.
Strain ID
Background and genotype
References
MPY1
JHY222 MATa/MATa HAP1/HAP1 MKT1(D30G)/
MKT1(D30G) RME1(INS 308A)/RME1(INS 308A)
TAO3(E1493Q)/TAO3(E1493Q)
JHY222 MATa/MATa HAP1/HAP1 MKT1(D30G)/
MKT1(D30G) RME1(INS 308A)/RME1(INS 308A)
TAO3(E1493Q)/TAO3(E1493Q) rrp6::kanMX4/
rrp6::kanMX4
SK1 MATa/MATa ho::LYS2/ho::LYS2 ura3/ura3
lys2/lys2 leu2::hisG/leu2::hisG arg4-Nsp/arg4-Bgl
his4x::LEU2-URA3/his4B::LEU2
SK1 MATa/MATa ho::LYS2/ho::LYS2 ura3/ura3
lys2/lys2
W101 MATa ho::lys5 gal2
SK1 MATa/MATa ho::hisG/ho::hisG lys2/lys2 ura3/
ura3 leu2::hisG/leu2::hisG arg4-Nsp/arg4-Bgl
his4x::LEU2-URA3/his4B::LEU2 trp1::hisG/
trp1::hisG rpd3::KanMX4/rpd3::KanMX4
JHY222 MATa/MATa HAP1/HAP1 MKT1(D30G)/
MKT1(D30G) RME1(INS 308A)/RME1(INS 308A)
TAO3(E1493Q)/TAO3(E1493Q) ume6::KanMX4/
ume6::KanMX4
SK1 MATa/MATa ura3/ura3 leu2/leu2 trp1/trp1
lys2/lys2 ho::LYS2/ho::LYS2
gal80::LEU2/gal80::LEU2 ume6::TRP1/ume6::TRP1
JHY222 MATa/MATa HAP1/HAP1 MKT1(D30G)/
MKT1(D30G) RME1(INS 308A)/RME1(INS 308A)
TAO3(E1493Q)/TAO3(E1493Q) VTH2/VTH2HA::kanMX4
W303 MATa/MATa ade2/ADE2 can1-100/CAN1
CYH2/cyh2 his3-11,15/his3-11,15 LEU1/leu1-c
LEU2/leu2-3,112 trp1-1::URA3::trp1-30 D/trp1-1
ura3-1/ura3-1 VTH2/VTH2-HA:: kanMX4
W303 MATa/MATa ade2/ade2 ade6/ADE6 can1100/can1ADE2:CAN1 his3-11,15/his3-11,15 leu23,112/leu2-3,112 trp1-1/trp1-1 ura3-1/ura3-1
ume6D1/ume6D1 VTH2/VTH2-HA:: kanMX4
[12]
MPY392
MPY70
MPY309
MPY454
MPY441
MPY542
MPY702
MPY765
MPY770
MPY771
2.2. Tiling array data
The biological methods and analysis approaches used to process
and normalise array data and to identify segments (transcripts) are
described in [12]. Expression data from wild type versus rrp6
mutant cells were visualised using the Repro Genomics Viewer
(rgv.genouest.org; Darde et al., in preparation).
2.3. Chromatin immunoprecipitation (ChIP) assay
Ume6 in vivo binding to the URS1 motifs in the VTH2 promoter
region was assayed in wild-type cells cultured in YPA and SPII 3 h
as published (including the oligonucleotide primers used to monitor the positive control promoter of SPO13) [25]. Oligonucleotides
for VTH2 are shown in Table 2.
2.4. RT-PCR
RT-PCR primers were designed as published [25]. RT-PCR assays
were done using 2 lg of total RNA, which was reverse transcribed
into cDNA with Reverse Transcriptase (High Capacity cDNA
Reverse Transcription kit; Life Technologies, USA) and amplified
using Taq Polymerase (Qiagen, France) at 60 °C for 26 cycles.
DNA samples were analysed on 2% agarose gels and photographed
using an ImageQuant 350 digital Imaging System at the default settings (General Electric, USA). The sequences of oligonucleotide primers are shown in Table 3.
2.5. URS1 motif prediction
Ume6 binding sites were screened for within a 2 kb region
upstream of the annotated VTH2 transcription start site as
described in the Saccharomyces Genome Database (SGD) [35]
using the Match tool provided by the TRANSFAC Professional database release 2011.4. [36]. Motifs M01503, M01898 and M02531
were used with cut-offs minimizing the false positives (minFP).
11 motifs were predicted, and the one with the highest core score
and matrix score was selected. Logos were generated using the R
package seqLogo [37,38].
2.6. uORF detection in the extended 50 -leader sequence of VTH2
[31]
[12]
[50]
[51]
The DNA sequence corresponding to the extended isoform
(chr10:10592–16124) was extracted from Saccharomyces
Genome Database (SGD) [39]. This sequence was screened with
ORF-FINDER and Expasy server in three reading frames with classical genetic code to search for ORFs longer than 100 nucleotides.
Three ORFs were identified upstream of the mitotic transcription
start site [40,41].
[25]
[52]
This study
This study
This study
Table 2
Q-PCR primer sequences for ChIP.
Gene
Forward primer
Reverse primer
VTH2
50 -ACTCTTCAAGAATTCCCGCCTAT-3
50 -GCGGCGCAATCTTTCG-30
Table 3
RT-PCR primer sequences.
Gene
Forward primer
Reverse primer
VTH2
aVTH2
laVTH2
50 -AGGCAAAGGGGCCAAATCTT-3
50 -AGCTCAGCCACATTGCACTA-30
50 -AGCTCAGCCACATTGCACTA-30
50 -TCCTGTTGATGCGAGGAGAC-30
50 -ACGCACCCAATTCTTCGGAT-30
50 -TCCTGTTGATGCGAGGAGAC-30
Please cite this article in press as: Stuparevic, I., et al. The histone deacetylase Rpd3/Sin3/Ume6 complex represses an acetate-inducible isoform of VTH2 in
fermenting budding yeast cells. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.02.022
3
I. Stuparevic et al. / FEBS Letters xxx (2015) xxx–xxx
Table 4
Vth2-tagging.
Gene
VTH2
Plasmid
pYM14 (1,2)
C-terminal tag
HA
Forward primer
0
Reverse primer
5 -GTAGTGCTTCTCACGAGTCCGATTTAGCAGCTG
CACGCAGCGAAGACAAGCGTACGCTGCAG
GTCGAC-30
2.7. Protein tagging
A PCR-based one-step tagging method was used to generate a
diploid strain harbouring Vth2 a C-terminal HA tag using cassette
plasmids and oligonucleotides as reported [42,43]. Several colonies
were analysed by PCR using diagnostic primers to identify strains
where the gene was successfully tagged. Three colonies were subsequently validated at the protein level by Western blotting.
Oligonucleotides used are given in Table 4.
2.8. Western blotting
Samples were prepared from fermenting (YPD), respiring (YPA)
and sporulating (SPII) cells as published [12]. 25 lg of total protein
extract was run on a 4–20% gradient gel (BioRad, USA) for 1 h.
Proteins were transferred onto ImmobilonPSQ membranes
(Millipore, France) using an electro-blotter (TE77X; Hoefer, USA)
in modified Towbin buffer (48 mM Tris base, 40 mM glycine and
0.1% SDS) and methanol (20% vol/vol anode; 5% vol/vol cathode)
for 2 h. Tagged Vth2 was detected with a monoclonal anti-HA antibody (Life Technologies, USA) at 1:1000. The antibody was incubated in hybridization buffer overnight at 4 °C. The signals were
revealed using the ECL-Plus Chemiluminescence kit (GE
Healthcare, USA) and the ImageQuant 350 system (GE
Healthcare, USA). Band intensities determined using the
ImageQuant TL 7.0 software set at default parameters. A rabbit
polyclonal anti-Pgk1 antibody (Invitrogen, USA) was used as a
loading control.
3. Results
3.1. Bi-directional promoters mediate expression of VTH1 and VTH2
mRNAs and SUT-like potential long non-coding RNAs not regulated by
Rrp6
VTH1 and VTH2 are paralogs that maintained syntenic neighbouring loci. The synteny is mirrored by similar expression profiles
for upstream loci (PAU14 and YIL177C for VTH1 versus PAU1 and
YJL225C for VTH2) and downstream loci (IMA3 for VTH1 and IMA4
for VTH2), during growth and development. VTH1 and VTH2 promoters mediate expression of mitotic isoforms and divergent
lncRNAs termed VDT (VTH Divergent Transcript) 1 and 2. No clear
change of VDT1 and VDT2 levels was observed in diploid rrp6
mutant cells cultured in YPD, YPA and sporulation medium
(Fig. 1A and B; the complete dataset will be published elsewhere).
The result implies that VDTs fall into the category of Rrp6-independent stable unannotated transcripts (SUTs) [15].
3.2. VTH1 and VTH2 encode isoforms with extended 50 -UTRs that are
induced when cells transit through meiotic M-phase and during
starvation
When cells progress through meiosis (SPII 4 h, 6 h, 8 h) they
induce VTH1 and VTH2 isoforms with extended 50 -UTRs.
Moreover, we find that rrp6 mutant cells, which sporulate very
slowly and inefficiently, also induce the extended isoforms, albeit
at low levels (Fig. 1A and B). We conclude that efficient meiosis
per se is not essential for the induction of long VTH1 and VTH2
50 -CCCAACATACAATGCGTGAACAATTTCGTTCGTTT
AAAAAGACCTACCTAATCGATGAATTCGAGCTCG-30
isoforms. We next analysed tiling array data from synchronized
haploid (MATa) cells undergoing vegetative growth, asynchronously growing and sporulating diploid (MATa/a) cells, and a starving
diploid (MATa/a) control [12]. This reveals weak expression of
the long VTH2 isoform in respiring cells (YPA), and confirms its
strong induction during sporulation and starvation. Note that
VDT2 is constitutively expressed but strongly accumulates in
sporulating cells (Fig. 2A). We subsequently compared array data
to the output of a non-strand-specific RNA-Sequencing experiment, and found that signals corresponding to the VTH2 ORF
decrease as wild-type cells exit meiosis (like in the case of tiling
arrays), while signals covering the upstream intergenic region
remained constant. This was not the case for starving control cells
that yielded homogenous signal for the ORF and the upstream
intergenic region in all samples (Fig. 2B). Taken together, the
RNA profiling data show VTH1 and VTH2 gene expression to be controlled by a constitutive and potentially bi-directional promoter
that, in the presence of acetate as the sole non-fermentable carbon
source, contains a cryptic transcription start site (TSS), which
mediates expression of long isoforms. These extended mRNAs
overlap with as yet unannotated putative antisense SUTs (VDT1
and VDT2). These lncRNAs are constitutively expressed in all cell
types, and their concentrations increase in sporulating and starving
diploid cells (Fig. 2A and C).
3.3. The VTH2 promoter region harbours predicted URS1 elements
The long VTH1 and VTH2 isoforms are induced during sporulation and starvation, contrary to classical meiosis-specific genes.
Since meiotic genes are expressed in meiosis-deficient MATa/a
cells at a low level [21,44], and Ume6 controls genes involved in
stress response [28], we searched for Ume6 binding sites using
three PWMs (Fig. 3A). Two matches to the URS1 element are located upstream of the acetate-induced TSS of VTH2’s long isoform
(Fig. 3B). Next, we carried out an in vivo Ume6 binding assay and
found that the protein interacts, albeit weakly, with the VTH2 promoter in mitotic diploid cells cultured in rich medium (Fig. 3C). The
SPO13 promoter was used as a positive control as published [25].
We conclude that Ume6 binds in vivo to the URS1 motif up-stream
of the carbon-source dependent VTH2 isoform in the presence of
glucose.
3.4. VTH2 encodes an isoform directly repressed by Ume6 and Rpd3
We next analysed the 50 -region of VTH2 by RT-PCR assays of
samples from wild-type SK1 and JHY222 (Fig. 3D). In SK1, primers
located in the 50 acetate-inducible UTR (aVTH2) yielded no band in
fermenting SK1 cells (YPD), a faint band in respiring cells (YPA),
strong signals during meiosis (SPII 2–6 h), and weaker signals during spore formation and ascus maturation (SPII 8–12 h; Fig. 3E).
We found a similar pattern in the wild-type JHY222 strain, which
sporulates less efficiently than SK1 (Fig. 3F). To exclude that a pair
of partially overlapping transcripts generates the RNA profiling signals for VTH2 transcripts we carried out an RT-PCR assay with a
primer pair located at the extreme 50 - and 30 -regions of the long
isoform (laVTH2). This experiment reiterated the profile obtained
with primers located inside the extended UTR (compare Fig. 3E
and F). Primers located within the open reading frame (VTH2)
Please cite this article in press as: Stuparevic, I., et al. The histone deacetylase Rpd3/Sin3/Ume6 complex represses an acetate-inducible isoform of VTH2 in
fermenting budding yeast cells. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.02.022
4
I. Stuparevic et al. / FEBS Letters xxx (2015) xxx–xxx
Fig. 1. VTH1 and VTH2 synteny and gene expression in wild-type versus rrp6 mutant strains. (A) Heatmaps representing yeast Sc_tlg tiling array data are given for a region
covering VTH1 and neighbouring loci. Samples are from cells grown in rich media with glucose or acetate (YPD, YPA) and sporulation medium (SPII 4, 6, 8 h). Light blue
rectangles indicate annotated genes. A black bar represents the extended 50 -UTR. The putative lncRNA VDT1 is represented by a red rectangle. The strains are given to the
right. (B) A similar broad display is given for the VTH2 paralog and its syntenic neighbouring genes. Note that VDT lncRNAs are annotated based on the expression signals
observed with tiling arrays. The 50 - and 30 -ends are determined by the output of the segmentation algorithm shown in Fig. 2 in more detail.
showed identical signals across the entire sample set in SK1 and
JHY222. ACT1 was used as a loading control (Fig. 3E and F).
Given that the VTH2 isoform’s pattern was reminiscent of Rpd3/
Sin3/Ume6-dependent early meiotic genes, and that two predicted
URS1 motifs bound by Ume6 in vivo are present right upstream of
the acetate-induced TSS, we verified if the long isoform of VTH2 is
de-repressed in mutant strains during mitotic growth in YPD. Both
SK1 mutant ume6 and rpd3 cells and JHY2223 mutant ume6 cells
strongly accumulate the 50 -extended VTH2 transcript during
vegetative growth in rich media with glucose or acetate.
Consistently, we find that the isoform does not peak in sporulation
medium in both mutant strains (Fig. 3E and F). Since Ume6
physically interacts with the co-repressor Sin3, the corollary is that
the HDAC complex Ume6/Sin3/Rpd3 represses the acetate-inducible isoform of VTH2 in the presence of glucose.
3.5. VDT2 is constitutively expressed and not regulated by Ume6 and
Rpd3
To validate and extend RNA profiling data we performed RT-PCR
assays using primers covering VDT2. The oligonucleotide corresponding to the Watson DNA strand was located upstream of the
long VTH2 isoform to distinguish VDT2 from the long isoform of
VTH2 (Fig. 3G). As expected, we found homogenous signals in
diploid wild-type strains and rpd3 or ume6 mutant cells from both
SK1 and JHY22 backgrounds. ACT1 was the loading control (Fig. 3H
and I). These data are in keeping with the notion that VTH2 and
VDT2 are expressed from what appears to be a constitutive bi-directional promoter, which is not under the control of the Rpd3/
Sin3/Ume6 repressor complex.
3.6. The Vth2 protein accumulates in fermenting but not respiring and
sporulating cells
We next sought to determine the Vth2 protein levels during
mitotic growth and meiotic development. To this end, we tagged
the protein with a C-terminal HA epitope and confirmed that the
strain showed no mitotic phenotype. First, we RT-PCR assayed
the acetate-induced isoform (aVTH2HA) in the tagged strain and
found expression in all samples except YPD (Fig. 4A). Then, we carried out a Western blotting analysis and observed a band of the
expected molecular weight (Vth2HA) in extracts from fermenting
cells (YPD) but not respiring cells (YPA) and sporulating cells
(SPII 2–10 h). Pgk1 yields homogenous signals in all samples
Please cite this article in press as: Stuparevic, I., et al. The histone deacetylase Rpd3/Sin3/Ume6 complex represses an acetate-inducible isoform of VTH2 in
fermenting budding yeast cells. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.02.022
5
I. Stuparevic et al. / FEBS Letters xxx (2015) xxx–xxx
B
A
010
VDT2
5'UTR VTH2
VTH2
log-transformed signals
W101
MATa
YPD
009
SPII
SK1
MATα/α
SK1
MATa/α
SPII
008
007
006
005
004
VTH2>
C
YPD
SK1 MATa/α
YPA
SPII 4h
SPII 6h
SPII 8h
YPD
SK1 MATα/α
YPA
VDT2
SPII 4h
SPII 6h
SPII 8h
SPII
SK1
MATa/α
D
VTH
SPII
SK1
MATα/α
YPD
VDT
W101
MATa
Fig. 2. VTH2/VDT2 expression in haploid versus diploid cells. (A) A heatmap summarises tiling array expression data for samples from haploid and diploid strains as indicated
to the right. Rich media (YPD, YPA) and sporulation medium (SPII 1–10 h) and time points are indicated at the left. The media are indicated. The chromosome number is
shown. Blue and red rectangles represent VTH2 and VDT2, respectively. A green rectangle represents a segment corresponding to the extended 50 -UTR. Grey rectangles
represent the raw output of the segmentation algorithm. Black lines are DNA. Genome coordinates are given and top (+) and bottom () strands are shown. An ARS element is
indicated. A log-2 colour scale is shown. (B) Bar diagrams show the log2 expression signals in MATa/a cells for VTH2, the extended segment of the long isoform (mUTR) and
the putative antisense lncRNA VDT2 (y-axis) versus the samples in rich media (YPD, YPA) and the twelve hourly time points taken in sporulation medium (SPII, x-axis). (C)
Histograms representing DNA non-strand-specific RNA-Seq data generated with the SK1 strain are shown. RNA was prepared from fermenting (YPD), respiring (YPA) and
sporulating (SPII 4, 6, 8 h) cells as indicated. Mitotic and meiotic samples from the wild-type strain are given in blue and green, respectively. Equivalent samples from the
sporulation-deficient strain are shown in red and orange. Interrupted blue and red lines represent the ORF and the extended 50 -UTR, respectively (the annotation is based on
DNA strand specific tiling arrays). (D) A schematic shows the transcripts emerging from the VTH2 locus. Blue, grey and red rectangles represent the 50 -UTR, the ORF and a
putative SUT (VDT2). Black lines are DNA and arrows are TSSs. Waved lines symbolize transcripts.
(Fig. 4A). We conclude that Vth2 is regulated at the post-transcriptional level when cells switch from fermentation to respiration and
sporulation.
confirms that the levels of Vth2 protein and VTH2 isoform are
negatively correlated.
3.8. The extended VTH2 50 -UTR contains uORFs
3.7. The Vth2 concentration decreases strongly in fermenting ume6
mutant cells
We finally asked if there was a negative correlation between the
Vth2 protein level and the de-repression of the long VTH2 isoform
in fermenting ume6 mutant cells. We tagged Vth2 in a strain lacking UME6 and verified the expression of VTH2HA and its long isoform by RT-PCR (Fig. 4B). An RT-PCR assay revealed that ume6
mutant cells, but not the wild-type control strain, expressed the
long isoform of VTH2 in YPD. Furthermore, we observed that the
fermenting ume6 strain contained around 6-fold less Vth2 protein
than the corresponding wild-type cells (Fig. 4B). This experiment
It was proposed that certain types of uORFs present in 50 -UTRs
inhibit the translation of downstream ORFs expressed in sporulating cells [45]. We therefore examined the extended 50 -UTR of VTH2
and detected indeed two uORFs encoding peptides of 44 and 51
amino acids, respectively, which are not in frame with VTH2, and
one uORF encoding a peptide of 64 amino acids, which is in frame
with VTH2 (Fig. 4C). This finding lends support to the notion that
the 50 -extended VTH2 isoform is not translated efficiently [45].
Taken together, our data suggest that Vth2 is down-regulated
post-transcriptionally when acetate is the sole non-fermentable
carbon-source.
Please cite this article in press as: Stuparevic, I., et al. The histone deacetylase Rpd3/Sin3/Ume6 complex represses an acetate-inducible isoform of VTH2 in
fermenting budding yeast cells. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.02.022
6
I. Stuparevic et al. / FEBS Letters xxx (2015) xxx–xxx
Fig. 3. The expression of VTH2 and VDT2 in rpd3 and ume6 mutants. (A) Logos show the forward and reverse DNA sequences of two URS1 PWMs (M01503, M01898) retrieved
from TRANSFAC. The base positions are indicated (x-axis). The prevalence for each base is given via units (y-axis). (B) A schematic shows the location of the predicted URS1
motifs (green rectangle), the 50 -UTR (light blue) and the gene (light grey). A black line represents DNA. The chromosome number, the DNA strand (+), and the genome
coordinates of the acetate-inducible transcription start site (acetateTSS) and VTH2 are shown. The sequence matches are given in red, the core motifs are indicated in bold. (C)
A bar diagram shows the result of a chromatin immunoprecipitation (IP) experiment as compared to controls lacking the primary antibody (NO AB). Fold-enrichment (y-axis)
is plotted against the sample in rich medium (YPD in blue, SPII 3 h in red, x-axis) for the VTH2 promoter. An error bar is shown. SPO13 was used as a positive control. (D) A
schematic shows the 50 -UTR and the VTH2 ORF. Diagnostic PCR fragments are given as red and black lines. Arrows represent forward and reverse PCR primers for which the
coordinates are given in bp in each strain background. Arrows symbolize primers located within the UTR. (E and F) The output of RT-PCR assays using samples from diploid
MATa/a SK1 and JHY222 wild-type strains (WT) and mutant strains (rpd3, ume6) are given for the isoforms. Cells were cultured in rich media (YPD, YPA) or sporulation
medium (SPII, samples were taken every 2 h from 2 to 12 h). To distinguish the transcripts we used two primer combinations that reveal the short glucose-dependent isoform
(VTH2) or the long acetate-induced isoform (aVTH2) as indicated. ACT1 was used as a loading control. (G) A schematic shows the VTH2 locus. Red arrows represent the
primers used to detect VDT2. The positions as compared to the ORF are given. A black line is the diagnostic PCR fragment. (H and I) The output of an RT-PCR assay is shown like
in panel E.
Please cite this article in press as: Stuparevic, I., et al. The histone deacetylase Rpd3/Sin3/Ume6 complex represses an acetate-inducible isoform of VTH2 in
fermenting budding yeast cells. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.02.022
7
I. Stuparevic et al. / FEBS Letters xxx (2015) xxx–xxx
Vth2HA
Protein
VTH2HA
Vth2HA
Protein
VTH2HA
Pgk1
C
5 UTR
VTH2 (11475 –16124)
uORFs (+1)
VTH2 (11475 –16124)
1_MSIIIFRGTGKPYCLERISGKRVCRVFTSFIHILLSKLYLDPES*_44
1_MQNSWLSALKTMLQPFASIIYIYIYIYISRPLSFFCQLGRNAGSRDLKKAS*_51
uORFs (+2)
VTH2 (11475 –16124)
1_MVDQEANIFEPKSPNLKECKIRGSVLSRQCCNPLRQLYIYIYIYIYPVRFLFFVSWVATQGLET*_64
In frame coding sequence
Transcribed region
URS1
uORF uORF
VTH2
VDT2
Rpd3
Samples
acetate TSS (10592)
chr10(+)
Ume6
Respiration
& starvation
Rao of intensies
Samples
Sin3
Pgk1
W303 MATa/α
VTH2-HA
JHY222 MATa/α VTH2-HA
Rao of intensies
aVTH2HA
mRNA
mRNA
aVTH2HA
Rpd3
Fermentation
10
VTH2 coding sequence
Fig. 4. Vth2 protein levels during fermentation, respiration and sporulation. (A) An
RT-PCR assay (mRNA) and a Western blot (protein) is shown for HA-tagged Vth2
(JHY222 MATa/a Vth2HA) in duplicate samples from cells cultured in rich media
with glucose or acetate (YPD, YPA), or sporulation medium (SPII) at bi-hourly time
points from 2 to 10 h. The short isoform (VTH2HA) and the long isoform (aVTH2HA)
are indicated. A bar diagram displays the intensity ratios of Vth2 over Pgk1 (y-axis)
versus samples from cells in rich medium with glucose (YPD) or acetate (YPA) or
sporulation medium (SPII) at the given time points (x-axis). Pgk1 was employed as a
loading control. The strain background is given. Error bars represent the standard
deviation. (B) A Western blot analysis of triplicate samples expressing tagged Vth2
in a wild-type (WT) strain versus a mutant strain (ume6) cultured in rich medium
(YPD) is presented. The strain background is given. Band intensities are represented
as bar diagrams. The standard deviation is given. (C) A schematic shows the UTR in
blue and the ORF in grey together with two out-of-frame uORFs (+1 frame) given in
red. An in-frame uORF (+2 frame) is shown in red together with the ORF. The
peptide sequences are given using the single letter amino acid code. An asterisk
represents the stop codons. The number of amino acids is shown. A black line
represents DNA.
4. Discussion
4.1. What controls Vth2 protein levels during growth, starvation and
spore development?
The functional significance of the VTH RNA/protein patterns
observed during growth, starvation and development could be
the consequence at least three distinct mechanisms that are not
mutually exclusive: (i) Vth2 could become unstable in the presence
of acetate because it is targeted by a protease like in the case of
Ume6 [26,46]; (ii) uORFs present in the extended 50 -UTR might
inhibit protein translation [45]; (iii) Vth2 protein levels could be
down-regulated because VTH2 mRNA and its antisense VDT2 form
double-stranded RNA molecules that sequester mRNAs in the
nucleus, or that prevent ribosome subunits from associating with
the mRNAs. Extensive experimental work will be required to conclusively determine the mechanism(s) behind the expression patterns observed.
4.2. An epigenetic mechanism regulates VTH2 gene expression
VTH1 and VTH2 are not included in a meiotic isoform expression
program, which we have recently discovered using tiling array data
Sin3
URS1
uORF uORF
VTH2
VDT2
Rpd3
Early meiosis
8
ume6
6
WT
4
YPD
B
SPII
YPA
YPD
A
Sin3
URS1
uORF uORF
VTH2
VDT2
Fig. 5. A model for VTH2 regulation. VTH2 and VDT2 are shown as blue and grey
rectangles. Blue boxes and arrows represent TSSs. Red rectangles mark URS1 motifs.
HDAC subunits are shown in red. Transcripts are given as waved lines. Thin blue
lines are DNA. Three states covering (i) rapid mitotic growth in rich medium
(fermentation), (ii) growth in the presence of acetate and growth arrest of MATa/a
cells in sporulation medium (respiration & starvation), and (iii) the onset of
sporulation in MATa/a cells (early meiosis) are shown.
and RNA-Seq data [12,25], because their 50 -extended isoforms are
expressed in starving cells and therefore are not strictly meiosisspecific. As such, VTH1 and VTH2 constitute a novel class of genes
encoding long isoforms, which accumulate during sporulation
and starvation. That is, their transcriptional activation or the
stabilization of the mRNAs they encode (or both) does not require
meiosis and spore formation per se to occur. This distinguishes
the loci from early meiotic genes (like SPO13) and early meiotic
isoforms (such as CFT2 and RTT10), which are detected only in
meiotic cells. Our findings that the VTH2 promoter contains
predicted URS1 motifs and that Rpd3 and Ume6 are needed to
repress the long VTH2 (and perhaps VTH1) isoform in the presence
of glucose are not unexpected, given that the HDAC complex is
known to orchestrate metabolic functions with meiotic processes
[28] (Fig. 5).
Why are VTH1 and VTH2 detectable in starving cells? It is conceivable that their upstream regions contain stress-response elements that are absent in meiosis-specific promoters. An obvious
explanation is the rather low in vivo binding activity of Ume6 to
the VTH2 promoter, which may lead to the gene’s de-repression
in pre-sporulation medium (YPA) and in starving MATa/a control
cells. We can only speculate why Ume6 binding to the URS1 motifs
in the VTH2 promoter is weak, but perhaps the answer lies in the
precise base composition of the core motif’s surrounding
sequences, that may allow only for inefficient Ume6/Sin3/Rpd3
complex formation [31]. Another explanation is that VTH mRNAs
are more stable than early meiotic transcripts, which were shown
to have extremely short half-lives [47,48]. The results presented
here indicate that the Rpd3/Sin3/Ume6 complex does not only
repress meiosis-specific isoforms but also other classes of transcripts that show a carbon source-dependent pattern of expression.
It is therefore worthwhile to extend future analyses of extended 50 UTRs to samples from cells undergoing various types of stress-response and filamentous growth, for example.
Please cite this article in press as: Stuparevic, I., et al. The histone deacetylase Rpd3/Sin3/Ume6 complex represses an acetate-inducible isoform of VTH2 in
fermenting budding yeast cells. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.02.022
8
I. Stuparevic et al. / FEBS Letters xxx (2015) xxx–xxx
VTH1 and VTH2 are not essential for growth and development
under laboratory conditions in the strain backgrounds investigated
[29,30,33,34]. This fact notwithstanding, we propose that VTH2 is a
useful model locus to study promoter directionality, the functional
relationship between partially overlapping pairs of mRNAs and
lncRNAs that are in sense/antisense orientation, and mechanisms
controlling post-translational protein stability when cells adapt
their metabolism to environmental cues. Our data implicate the
conserved HDAC Rpd3 and its DNA binding subunit Ume6 in the
glucose-mediated repression of VTH2’s acetate-inducible long isoform. Given that Rpd3 is conserved and expressed in many tissues
[49], our findings are likely relevant for the regulation of gene
expression during cell growth and cell differentiation in higher
eukaryotes.
Acknowledgments
We thank Olivier Collin for providing us with access to the
GenOuest bioinformatics infrastructure, and Olivier Sallou for system administration. We thank Y. Liu and A. Lardenois for unpublished tiling array data on Rrp6 and K. Waern and M. Snyder for
unpublished RNA-Sequencing data. This work was supported by
the Région Bretagne CREATE Grant (R11016NN) awarded to
Michael Primig.
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fermenting budding yeast cells. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.02.022