The histone deacetylase Rpd3/Sin3/Ume6 complex represses an … · 2015-03-26 · We report that the promoters of VTH1 and VTH2 appear to drive bi-directional expression of mitotic

FEBS Letters xxx (2015) xxx–xxx

journal homepage: www.FEBSLetters .org

The histone deacetylase Rpd3/Sin3/Ume6 complex repressesan acetate-inducible isoform of VTH2 in fermenting budding yeast cells

http://dx.doi.org/10.1016/j.febslet.2015.02.0220014-5793/� 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

⇑ Corresponding author at: Inserm U1085, Université de Rennes 1, 263, ave duGénéral 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 Biotech-

nology, 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.

Please cite this article in press as: Stuparevic, I., et al. The histone deacetylase Rpd3/Sin3/Ume6 complex represses an acetate-inducible isoform of Vfermenting budding yeast cells. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.02.022

Igor Stuparevic a,1,2, Emmanuelle Becker a,2, Michael J. Law b,2, Michael Primig a,⇑,2

a Inserm U1085 IRSET, Université de Rennes 1, 35042 Rennes, Franceb Rowan University, School of Osteopathic Medicine, Stratford, NJ 08084, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 19 November 2014Revised 30 January 2015Accepted 12 February 2015Available online xxxx

Edited by Francesc Posas

Keywords:RPD3UME6VTH1VTH250-UTRIsoform

The tripartite Rpd3/Sin3/Ume6 complex represses meiotic isoforms during mitosis. We asked if italso controls starvation-induced isoforms. We report that VTH1/VTH2 encode acetate-inducible iso-forms with extended 50-regions overlapping antisense long non-coding RNAs. Rpd3 and Ume6repress 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 locusto study mechanisms implicating promoter directionality, lncRNA transcription and post-transcrip-tional control of gene expression via 50-UTRs. Since mammalian genes encode transcript isoformsand Rpd3 is conserved, our findings are relevant for gene expression in higher eukaryotes.� 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 vari-able isoforms often include 50- and 30-untranslated regions(UTRs) that change in length when cells respond to environmentalcues [1–7]. The transcriptional mechanisms controlling this phe-nomenon are poorly understood in spite of the fact that UTRs areimportant for mRNA turnover, transcript localization and mRNAtranslation [8,9]. Two well-studied mechanisms act via upstreamopen reading frames (uORFs), which inhibit the translation ofdownstream 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 exori-bonuclease important for normal mitotic growth and efficientmeiosis [7,12–14]. In its absence, numerous lncRNAs such as cryp-tic unstable transcripts (CUTs) and some stable unannotated tran-scripts (SUTs) accumulate in mitosis [15,16]. Many lncRNAs are

expressed via bi-directional promoters that mediate transcriptionof divergent pairs of transcripts [15]; reviewed in [17]. The func-tions of most lncRNAs are not known, but some regulate theexpression of protein-coding genes via promoter interference orthrough a mechanism of antagonistic sense/antisense transcription[18,19]; for review, see [20].

The conserved histone deacetylase (HDAC) Rpd3, the co-repres-sor Sin3, and the upstream regulatory site 1 (URS1) binding proteinUme6 form a complex, which prevents the expression of earlymeiosis-specific transcripts and transcript isoforms during vegeta-tive growth [21–24]; [25]. Its activity is abolished when cellsswitch from fermentation to respiration and sporulation, becauseUme6 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 weredetermined using RNA profiling [28].

VTH1 and VTH2 encode non-essential homologs of the vacuolarprotein sorting receptor PEP1 [29]. Over-expression of VTH2 wasshown to suppress the sorting phenotype of soluble hydrolasesto the yeast vacuole in pep1 mutant cells [30]. Expression profilingstudies showed that both paralogs are constitutively transcribed ingrowing and starving cells and during sporulation [31,32], but cellslacking either Vth1 or Vth2 do not have a meiotic phenotype or agermination defect [33,34].

TH2 in

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2 I. Stuparevic et al. / FEBS Letters xxx (2015) xxx–xxx

We report that the promoters of VTH1 and VTH2 appear to drivebi-directional expression of mitotic isoforms and divergent puta-tive long non-coding RNAs (lncRNAs). In addition, they mediateacetate-induced expression long isoforms with extended 50-UTRs,which partially overlap these antisense lncRNAs. Since VTH2 com-plemented the phenotype of pep1/vps10 mutant cells, we focussedon this gene for further studies [30]. We find that Rpd3 and Ume6repress the 50-extended isoform of VTH2 during vegetative growthin rich medium. Moreover, we observe an antagonistic pattern ofVth2 protein concentration and the expression level of the longisoform in fermenting, respiring and sporulating wild-type cellsand, consistently, in fermenting ume6 mutant cells.

These results show that VTH2 is a useful model locus to studycomplex patterns of mRNA/lncRNA expression and post-transcrip-tional regulation of protein stability. Furthermore, they implicatethe histone deacetylase Rpd3/Sin3/Ume6 complex specifically inthe glucose-mediated repression of VTH2’s long isoform. As such,the target gene constitutes a new class distinct from those encod-ing 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/astrain and a sporulation deficient MATa/a control. RT-PCR assaywere carried out with SK1 MATa/a and JHY222 MATa/a strains[12]. The induction of isoforms was monitored in SK1 MATa/aume6 and rpd3, and JHY222 MATa/a ume6 deletion strains(Table 1). Sporulation experiments were done using rich mediawith glucose (YPD) or acetate (YPA) and sporulation medium (SPII).

Table 1Yeast 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)

[12]

MPY392 JHY222 MATa/MATa HAP1/HAP1 MKT1(D30G)/MKT1(D30G) RME1(INS 308A)/RME1(INS 308A)TAO3(E1493Q)/TAO3(E1493Q) rrp6::kanMX4/rrp6::kanMX4

MPY70 SK1 MATa/MATa ho::LYS2/ho::LYS2 ura3/ura3lys2/lys2 leu2::hisG/leu2::hisG arg4-Nsp/arg4-Bglhis4x::LEU2-URA3/his4B::LEU2

[31]

MPY309 SK1 MATa/MATa ho::LYS2/ho::LYS2 ura3/ura3lys2/lys2

[12]

MPY454 W101 MATa ho::lys5 gal2 [50]MPY441 SK1 MATa/MATa ho::hisG/ho::hisG lys2/lys2 ura3/

ura3 leu2::hisG/leu2::hisG arg4-Nsp/arg4-Bglhis4x::LEU2-URA3/his4B::LEU2 trp1::hisG/trp1::hisG rpd3::KanMX4/rpd3::KanMX4

[51]

MPY542 JHY222 MATa/MATa HAP1/HAP1 MKT1(D30G)/MKT1(D30G) RME1(INS 308A)/RME1(INS 308A)TAO3(E1493Q)/TAO3(E1493Q) ume6::KanMX4/ume6::KanMX4

[25]

MPY702 SK1 MATa/MATa ura3/ura3 leu2/leu2 trp1/trp1lys2/lys2 ho::LYS2/ho::LYS2gal80::LEU2/gal80::LEU2 ume6::TRP1/ume6::TRP1

[52]

MPY765 JHY222 MATa/MATa HAP1/HAP1 MKT1(D30G)/MKT1(D30G) RME1(INS 308A)/RME1(INS 308A)TAO3(E1493Q)/TAO3(E1493Q) VTH2/VTH2-HA::kanMX4

This study

MPY770 W303 MATa/MATa ade2/ADE2 can1-100/CAN1CYH2/cyh2 his3-11,15/his3-11,15 LEU1/leu1-cLEU2/leu2-3,112 trp1-1::URA3::trp1-30D/trp1-1ura3-1/ura3-1 VTH2/VTH2-HA:: kanMX4

This study

MPY771 W303 MATa/MATa ade2/ade2 ade6/ADE6 can1-100/can1ADE2:CAN1 his3-11,15/his3-11,15 leu2-3,112/leu2-3,112 trp1-1/trp1-1 ura3-1/ura3-1ume6D1/ume6D1 VTH2/VTH2-HA:: kanMX4

This study

Please cite this article in press as: Stuparevic, I., et al. The histone deacetylase Rfermenting budding yeast cells. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.f

2.2. Tiling array data

The biological methods and analysis approaches used to processand normalise array data and to identify segments (transcripts) aredescribed in [12]. Expression data from wild type versus rrp6mutant 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 promoterregion was assayed in wild-type cells cultured in YPA and SPII 3 has published (including the oligonucleotide primers used to moni-tor the positive control promoter of SPO13) [25]. Oligonucleotidesfor VTH2 are shown in Table 2.

2.4. RT-PCR

RT-PCR primers were designed as published [25]. RT-PCR assayswere done using 2 lg of total RNA, which was reverse transcribedinto cDNA with Reverse Transcriptase (High Capacity cDNAReverse Transcription kit; Life Technologies, USA) and amplifiedusing Taq Polymerase (Qiagen, France) at 60 �C for 26 cycles.DNA samples were analysed on 2% agarose gels and photographedusing an ImageQuant 350 digital Imaging System at the default set-tings (General Electric, USA). The sequences of oligonucleotide pri-mers are shown in Table 3.

2.5. URS1 motif prediction

Ume6 binding sites were screened for within a 2 kb regionupstream of the annotated VTH2 transcription start site asdescribed in the Saccharomyces Genome Database (SGD) [35]using the Match tool provided by the TRANSFAC Professional data-base release 2011.4. [36]. Motifs M01503, M01898 and M02531were used with cut-offs minimizing the false positives (minFP).11 motifs were predicted, and the one with the highest core scoreand matrix score was selected. Logos were generated using the Rpackage seqLogo [37,38].

2.6. uORF detection in the extended 50-leader sequence of VTH2

The DNA sequence corresponding to the extended isoform(chr10:10592–16124) was extracted from SaccharomycesGenome Database (SGD) [39]. This sequence was screened withORF-FINDER and Expasy server in three reading frames with clas-sical genetic code to search for ORFs longer than 100 nucleotides.Three ORFs were identified upstream of the mitotic transcriptionstart site [40,41].

Table 2Q-PCR primer sequences for ChIP.

Gene Forward primer Reverse primer

VTH2 50-ACTCTTCAAGAATTCCCGCCTAT-3 50-GCGGCGCAATCTTTCG-30

Table 3RT-PCR primer sequences.

Gene Forward primer Reverse primer

VTH2 50-AGGCAAAGGGGCCAAATCTT-3 50-TCCTGTTGATGCGAGGAGAC-30

aVTH2 50-AGCTCAGCCACATTGCACTA-30 50-ACGCACCCAATTCTTCGGAT-30

laVTH2 50-AGCTCAGCCACATTGCACTA-30 50-TCCTGTTGATGCGAGGAGAC-30

pd3/Sin3/Ume6 complex represses an acetate-inducible isoform of VTH2 inebslet.2015.02.022


Table 4Vth2-tagging.

Gene Plasmid C-terminal tag Forward primer Reverse primer

VTH2 pYM14 (1,2) HA 50-GTAGTGCTTCTCACGAGTCCGATTTAGCAGCTGCACGCAGCGAAGACAAGCGTACGCTGCAGGTCGAC-30

50-CCCAACATACAATGCGTGAACAATTTCGTTCGTTTAAAAAGACCTACCTAATCGATGAATTCGAGCTCG-30

I. Stuparevic et al. / FEBS Letters xxx (2015) xxx–xxx 3

2.7. Protein tagging

A PCR-based one-step tagging method was used to generate adiploid strain harbouring Vth2 a C-terminal HA tag using cassetteplasmids and oligonucleotides as reported [42,43]. Several colonieswere analysed by PCR using diagnostic primers to identify strainswhere the gene was successfully tagged. Three colonies were sub-sequently 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 proteinextract 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 and0.1% SDS) and methanol (20% vol/vol anode; 5% vol/vol cathode)for 2 h. Tagged Vth2 was detected with a monoclonal anti-HA anti-body (Life Technologies, USA) at 1:1000. The antibody was incubat-ed in hybridization buffer overnight at 4 �C. The signals wererevealed using the ECL-Plus Chemiluminescence kit (GEHealthcare, USA) and the ImageQuant 350 system (GEHealthcare, USA). Band intensities determined using theImageQuant TL 7.0 software set at default parameters. A rabbitpolyclonal anti-Pgk1 antibody (Invitrogen, USA) was used as aloading control.

3. Results

3.1. Bi-directional promoters mediate expression of VTH1 and VTH2mRNAs and SUT-like potential long non-coding RNAs not regulated byRrp6

VTH1 and VTH2 are paralogs that maintained syntenic neigh-bouring loci. The synteny is mirrored by similar expression profilesfor upstream loci (PAU14 and YIL177C for VTH1 versus PAU1 andYJL225C for VTH2) and downstream loci (IMA3 for VTH1 and IMA4for VTH2), during growth and development. VTH1 and VTH2 pro-moters mediate expression of mitotic isoforms and divergent

lncRNAs termed VDT (VTH Divergent Transcript) 1 and 2. No clearchange of VDT1 and VDT2 levels was observed in diploid rrp6mutant 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-indepen-dent stable unannotated transcripts (SUTs) [15].

3.2. VTH1 and VTH2 encode isoforms with extended 50-UTRs that areinduced when cells transit through meiotic M-phase and duringstarvation

When cells progress through meiosis (SPII 4 h, 6 h, 8 h) theyinduce VTH1 and VTH2 isoforms with extended 50-UTRs.Moreover, we find that rrp6 mutant cells, which sporulate veryslowly and inefficiently, also induce the extended isoforms, albeitat low levels (Fig. 1A and B). We conclude that efficient meiosisper se is not essential for the induction of long VTH1 and VTH2


isoforms. We next analysed tiling array data from synchronizedhaploid (MATa) cells undergoing vegetative growth, asynchronous-ly growing and sporulating diploid (MATa/a) cells, and a starvingdiploid (MATa/a) control [12]. This reveals weak expression ofthe long VTH2 isoform in respiring cells (YPA), and confirms itsstrong induction during sporulation and starvation. Note thatVDT2 is constitutively expressed but strongly accumulates insporulating cells (Fig. 2A). We subsequently compared array datato the output of a non-strand-specific RNA-Sequencing experi-ment, and found that signals corresponding to the VTH2 ORFdecrease as wild-type cells exit meiosis (like in the case of tilingarrays), while signals covering the upstream intergenic regionremained constant. This was not the case for starving control cellsthat yielded homogenous signal for the ORF and the upstreamintergenic region in all samples (Fig. 2B). Taken together, theRNA profiling data show VTH1 and VTH2 gene expression to be con-trolled by a constitutive and potentially bi-directional promoterthat, in the presence of acetate as the sole non-fermentable carbonsource, contains a cryptic transcription start site (TSS), whichmediates expression of long isoforms. These extended mRNAsoverlap with as yet unannotated putative antisense SUTs (VDT1and VDT2). These lncRNAs are constitutively expressed in all celltypes, and their concentrations increase in sporulating and starvingdiploid cells (Fig. 2A and C).

3.3. The VTH2 promoter region harbours predicted URS1 elements

The long VTH1 and VTH2 isoforms are induced during sporula-tion and starvation, contrary to classical meiosis-specific genes.Since meiotic genes are expressed in meiosis-deficient MATa/acells at a low level [21,44], and Ume6 controls genes involved instress response [28], we searched for Ume6 binding sites usingthree PWMs (Fig. 3A). Two matches to the URS1 element are locat-ed upstream of the acetate-induced TSS of VTH2’s long isoform(Fig. 3B). Next, we carried out an in vivo Ume6 binding assay andfound that the protein interacts, albeit weakly, with the VTH2 pro-moter in mitotic diploid cells cultured in rich medium (Fig. 3C). TheSPO13 promoter was used as a positive control as published [25].We conclude that Ume6 binds in vivo to the URS1 motif up-streamof the carbon-source dependent VTH2 isoform in the presence ofglucose.

3.4. VTH2 encodes an isoform directly repressed by Ume6 and Rpd3

We next analysed the 50-region of VTH2 by RT-PCR assays ofsamples from wild-type SK1 and JHY222 (Fig. 3D). In SK1, primerslocated in the 50 acetate-inducible UTR (aVTH2) yielded no band infermenting SK1 cells (YPD), a faint band in respiring cells (YPA),strong signals during meiosis (SPII 2–6 h), and weaker signals dur-ing spore formation and ascus maturation (SPII 8–12 h; Fig. 3E).We found a similar pattern in the wild-type JHY222 strain, whichsporulates less efficiently than SK1 (Fig. 3F). To exclude that a pairof partially overlapping transcripts generates the RNA profiling sig-nals for VTH2 transcripts we carried out an RT-PCR assay with aprimer pair located at the extreme 50- and 30-regions of the longisoform (laVTH2). This experiment reiterated the profile obtainedwith primers located inside the extended UTR (compare Fig. 3Eand F). Primers located within the open reading frame (VTH2)



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 regioncovering 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 bluerectangles 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 theright. (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 signalsobserved 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 andJHY222. 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 predictedURS1 motifs bound by Ume6 in vivo are present right upstream ofthe acetate-induced TSS, we verified if the long isoform of VTH2 isde-repressed in mutant strains during mitotic growth in YPD. BothSK1 mutant ume6 and rpd3 cells and JHY2223 mutant ume6 cellsstrongly accumulate the 50-extended VTH2 transcript duringvegetative growth in rich media with glucose or acetate.Consistently, we find that the isoform does not peak in sporulationmedium in both mutant strains (Fig. 3E and F). Since Ume6physically interacts with the co-repressor Sin3, the corollary is thatthe HDAC complex Ume6/Sin3/Rpd3 represses the acetate-in-ducible isoform of VTH2 in the presence of glucose.

3.5. VDT2 is constitutively expressed and not regulated by Ume6 andRpd3

To validate and extend RNA profiling data we performed RT-PCRassays using primers covering VDT2. The oligonucleotide corre-sponding to the Watson DNA strand was located upstream of the


long VTH2 isoform to distinguish VDT2 from the long isoform ofVTH2 (Fig. 3G). As expected, we found homogenous signals indiploid wild-type strains and rpd3 or ume6 mutant cells from bothSK1 and JHY22 backgrounds. ACT1 was the loading control (Fig. 3Hand I). These data are in keeping with the notion that VTH2 andVDT2 are expressed from what appears to be a constitutive bi-di-rectional 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 andsporulating cells

We next sought to determine the Vth2 protein levels duringmitotic growth and meiotic development. To this end, we taggedthe protein with a C-terminal HA epitope and confirmed that thestrain showed no mitotic phenotype. First, we RT-PCR assayedthe acetate-induced isoform (aVTH2HA) in the tagged strain andfound expression in all samples except YPD (Fig. 4A). Then, we car-ried out a Western blotting analysis and observed a band of theexpected molecular weight (Vth2HA) in extracts from fermentingcells (YPD) but not respiring cells (YPA) and sporulating cells(SPII 2–10 h). Pgk1 yields homogenous signals in all samples



SK1MATa/α

SK1MATα/α

W101

MATa

SK1MATα/α

W101

MATa

VDT2

C

SK1 MATa/α

SK1 MATα/α

VTH2>

YPD

YPA

SPII 4h

SPII 6h

SPII 8h

YPD

YPA

SPII 4h

SPII 6h

SPII 8h

VTH

D

VDT

A

SK1MATa/α

B

YP

DS

PII

SP

IIY

PD

SP

IIS

PII

004

005

006

007

008

009

010 VDT2 5'UTR VTH2 VTH2

log-

trans

form

ed s

igna

ls

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 indicatedto 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 isshown. Blue and red rectangles represent VTH2 and VDT2, respectively. A green rectangle represents a segment corresponding to the extended 50-UTR. Grey rectanglesrepresent 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 isindicated. 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) andthe 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) andsporulating (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 thesporulation-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 onDNA 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 aputative 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-transcrip-tional level when cells switch from fermentation to respiration andsporulation.

3.7. The Vth2 concentration decreases strongly in fermenting ume6mutant cells

We finally asked if there was a negative correlation between theVth2 protein level and the de-repression of the long VTH2 isoformin fermenting ume6 mutant cells. We tagged Vth2 in a strain lack-ing UME6 and verified the expression of VTH2HA and its long iso-form by RT-PCR (Fig. 4B). An RT-PCR assay revealed that ume6mutant cells, but not the wild-type control strain, expressed thelong isoform of VTH2 in YPD. Furthermore, we observed that thefermenting ume6 strain contained around 6-fold less Vth2 proteinthan the corresponding wild-type cells (Fig. 4B). This experiment


confirms that the levels of Vth2 protein and VTH2 isoform arenegatively correlated.

3.8. The extended VTH2 50-UTR contains uORFs

It was proposed that certain types of uORFs present in 50-UTRsinhibit the translation of downstream ORFs expressed in sporulat-ing cells [45]. We therefore examined the extended 50-UTR of VTH2and detected indeed two uORFs encoding peptides of 44 and 51amino acids, respectively, which are not in frame with VTH2, andone uORF encoding a peptide of 64 amino acids, which is in framewith VTH2 (Fig. 4C). This finding lends support to the notion thatthe 50-extended VTH2 isoform is not translated efficiently [45].Taken together, our data suggest that Vth2 is down-regulatedpost-transcriptionally when acetate is the sole non-fermentablecarbon-source.



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) retrievedfrom 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 URS1motifs (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 genomecoordinates 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) Aschematic 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 thecoordinates 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 diploidMATa/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 sporulationmedium (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 theprimers 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 likein 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 infermenting budding yeast cells. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.02.022


YPD

YPA

4 6 8 10

SPII

aVTH2HA

VTH2HA

Vth2HA

Pgk1

JHY222 MATa/α VTH2-HA

WT

ume6

YPD

W303 MATa/αVTH2-HA

Vth2HA

Pgk1

aVTH2HA

VTH2HA

A B

Samples

Ra�o

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s

mRN

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CVTH2 (11475 –16124)5 UTRchr10(+)

acetate TSS (10592)

VTH2 (11475 –16124)uORFs (+1)

VTH2 (11475 –16124)uORFs (+2)

1_MQNSWLSALKTMLQPFASIIYIYIYIYISRPLSFFCQLGRNAGSRDLKKAS*_51

1_MSIIIFRGTGKPYCLERISGKRVCRVFTSFIHILLSKLYLDPES*_44

1_MVDQEANIFEPKSPNLKECKIRGSVLSRQCCNPLRQLYIYIYIYIYPVRFLFFVSWVATQGLET*_64

In frame coding sequence Transcribed region VTH2 coding sequence

Fig. 4. Vth2 protein levels during fermentation, respiration and sporulation. (A) AnRT-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 mediawith glucose or acetate (YPD, YPA), or sporulation medium (SPII) at bi-hourly timepoints 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) orsporulation medium (SPII) at the given time points (x-axis). Pgk1 was employed as aloading control. The strain background is given. Error bars represent the standarddeviation. (B) A Western blot analysis of triplicate samples expressing tagged Vth2in 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 representedas bar diagrams. The standard deviation is given. (C) A schematic shows the UTR inblue and the ORF in grey together with two out-of-frame uORFs (+1 frame) given inred. An in-frame uORF (+2 frame) is shown in red together with the ORF. Thepeptide sequences are given using the single letter amino acid code. An asteriskrepresents the stop codons. The number of amino acids is shown. A black linerepresents DNA.

VTH2

VTH2

URS1

URS1

Ferm

enta

tion

Res

pira

tion

&

star

vatio

n

VTH2URS1

Early

mei

osis

Ume6

Rpd3 Sin3

Rpd3 Sin3

Rpd3 Sin3

VDT2

VDT2

VDT2

uORF uORF

uORF uORF

uORF uORF

Fig. 5. A model for VTH2 regulation. VTH2 and VDT2 are shown as blue and greyrectangles. Blue boxes and arrows represent TSSs. Red rectangles mark URS1 motifs.HDAC subunits are shown in red. Transcripts are given as waved lines. Thin bluelines 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/acells in sporulation medium (respiration & starvation), and (iii) the onset ofsporulation in MATa/a cells (early meiosis) are shown.


4. Discussion

4.1. What controls Vth2 protein levels during growth, starvation andspore development?

The functional significance of the VTH RNA/protein patternsobserved during growth, starvation and development could bethe consequence at least three distinct mechanisms that are notmutually exclusive: (i) Vth2 could become unstable in the presenceof acetate because it is targeted by a protease like in the case ofUme6 [26,46]; (ii) uORFs present in the extended 50-UTR mightinhibit protein translation [45]; (iii) Vth2 protein levels could bedown-regulated because VTH2 mRNA and its antisense VDT2 formdouble-stranded RNA molecules that sequester mRNAs in thenucleus, or that prevent ribosome subunits from associating withthe mRNAs. Extensive experimental work will be required to con-clusively determine the mechanism(s) behind the expression pat-terns observed.

4.2. An epigenetic mechanism regulates VTH2 gene expression

VTH1 and VTH2 are not included in a meiotic isoform expressionprogram, which we have recently discovered using tiling array data


and RNA-Seq data [12,25], because their 50-extended isoforms areexpressed in starving cells and therefore are not strictly meiosis-specific. As such, VTH1 and VTH2 constitute a novel class of genesencoding long isoforms, which accumulate during sporulationand starvation. That is, their transcriptional activation or thestabilization of the mRNAs they encode (or both) does not requiremeiosis and spore formation per se to occur. This distinguishesthe loci from early meiotic genes (like SPO13) and early meioticisoforms (such as CFT2 and RTT10), which are detected only inmeiotic cells. Our findings that the VTH2 promoter containspredicted URS1 motifs and that Rpd3 and Ume6 are needed torepress the long VTH2 (and perhaps VTH1) isoform in the presenceof glucose are not unexpected, given that the HDAC complex isknown to orchestrate metabolic functions with meiotic processes[28] (Fig. 5).

Why are VTH1 and VTH2 detectable in starving cells? It is con-ceivable that their upstream regions contain stress-response ele-ments that are absent in meiosis-specific promoters. An obviousexplanation is the rather low in vivo binding activity of Ume6 tothe VTH2 promoter, which may lead to the gene’s de-repressionin pre-sporulation medium (YPA) and in starving MATa/a controlcells. We can only speculate why Ume6 binding to the URS1 motifsin the VTH2 promoter is weak, but perhaps the answer lies in theprecise base composition of the core motif’s surroundingsequences, that may allow only for inefficient Ume6/Sin3/Rpd3complex formation [31]. Another explanation is that VTH mRNAsare more stable than early meiotic transcripts, which were shownto have extremely short half-lives [47,48]. The results presentedhere indicate that the Rpd3/Sin3/Ume6 complex does not onlyrepress meiosis-specific isoforms but also other classes of tran-scripts 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-re-sponse and filamentous growth, for example.




VTH1 and VTH2 are not essential for growth and developmentunder laboratory conditions in the strain backgrounds investigated[29,30,33,34]. This fact notwithstanding, we propose that VTH2 is auseful model locus to study promoter directionality, the functionalrelationship between partially overlapping pairs of mRNAs andlncRNAs that are in sense/antisense orientation, and mechanismscontrolling post-translational protein stability when cells adapttheir metabolism to environmental cues. Our data implicate theconserved HDAC Rpd3 and its DNA binding subunit Ume6 in theglucose-mediated repression of VTH2’s acetate-inducible long iso-form. Given that Rpd3 is conserved and expressed in many tissues[49], our findings are likely relevant for the regulation of geneexpression during cell growth and cell differentiation in highereukaryotes.

Acknowledgments

We thank Olivier Collin for providing us with access to theGenOuest bioinformatics infrastructure, and Olivier Sallou for sys-tem administration. We thank Y. Liu and A. Lardenois for unpub-lished tiling array data on Rrp6 and K. Waern and M. Snyder forunpublished RNA-Sequencing data. This work was supported bythe Région Bretagne CREATE Grant (R11016NN) awarded toMichael Primig.

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The histone deacetylase Rpd3/Sin3/Ume6 complex represses an … · 2015-03-26 · We report that the promoters of VTH1 and VTH2 appear to drive bi-directional expression of mitotic