Báo cáo khoa học: Sin3 is involved in cell size control at Start in Saccharomyces cerevisiae Octavian Stephan and Christian Koch ppt

Initiation of G1⁄ S-specific transcription of CLN1, CLN2 and PCL1 in a sin3D strain occurs at a reduced cell size compared with a wild-type strain.. In addition, inactivation of the trans

Saccharomyces cerevisiae

Octavian Stephan and Christian Koch

Department of Biology, Friedrich-Alexander-University Erlangen-Nu¨rnberg, Germany

Introduction

Most eucaryotic cells regulate their commitment to cell

division at the G1 to S phase transition In the

bud-ding yeast Saccharomyces cerevisiae, the events in late

G1leading to S-phase entry are collectively referred to

as ‘Start’ [1–3] During the G1phase, yeast cells

moni-tor their size and ensure that they have reached a

suffi-ciently large size for entry into the mitotic cell cycle

One of the earliest events occurring as cells pass

through Start is the transcriptional activation of a

large set of G1⁄ S-specific genes including the G1cyclins

CLN1 and CLN2 and S-phase regulators [4–6] Cln1

and Cln2 with their associated cyclin-dependent kinase

Cdc28 (CDK1) activate the subsequent steps, leading

to the accumulation of Clb5⁄ 6–CDK1 activity, DNA

synthesis, budding and spindle pole body duplication

The periodic expression of G1⁄ S-specific RNAs

depends on the two transcription factor complexes

SBF (Swi4⁄ Swi6) and MBF (Mbp1 ⁄ Swi6) which share

the common subunit Swi6 but contain different

DNA-binding proteins [7–9] Swi4 recognizes short cis-acting sequences called Swi4⁄ 6 cell-cycle box (SCB) elements originally identified in the HO promoter, whereas Mbp1 binds to MluI cell-cycle box (MCB) elements found in many S-phase genes, including cyclins CLB5 and CLB6 [7,10–12] Genes regulated by SBF include the G1 cyclins CLN1, CLN2 and PCL1 [13,14] The timing of CLN1 and CLN2 transcription is of particu-lar importance for the control of cell size because their ectopic expression leads to early entry into the S phase [3,15] Inactivation of SWI4 causes a defect in Start-specific transcription resulting in abnormally large cells with problems in morphogenesis [13,14,16]

Different cyclins are responsible for regulating

G1⁄ S-specific transcription Whereas repression in G2

is caused by Clb1–4⁄ CDK1 activity and leads to the dissociation of Swi4⁄ Swi6 (SBF) from the promoter, activation in late G1 requires Cln3⁄ CDK1 activity [3,15,17–19]

Keywords

G1cyclins; histone deacetylase; Rpd3; Swi4;

Swi6

Correspondence

C Koch, Department of Biology, Chair for

Biochemistry, Friedrich-Alexander-University

Erlangen-Nu¨rnberg, Staudtstr 5, 91058

Erlangen, Germany

Fax: +49 9131 8528254

Tel: +49 9131 8528257

E-mail: ckoch@biologie.uni-erlangen.de

(Received 21 February 2009, revised 7 May

2009, accepted 13 May 2009)

doi:10.1111/j.1742-4658.2009.07095.x

Saccharomyces cerevisiae cells control their cell size at a point in late G1 called Start Here, we describe a negative role for the Sin3⁄ Rpd3 histone deacetylase complex in the regulation of cell size at Start Initiation of

G1⁄ S-specific transcription of CLN1, CLN2 and PCL1 in a sin3D strain occurs at a reduced cell size compared with a wild-type strain In addition, inactivation of the transcriptional regulator SIN3 partially suppressed a cln3D mutant, causing sin3Dcln3D double mutants to start the cell cycle at wild-type size Chromatin immunoprecipitation results demonstrate that Sin3 and Rpd3 are recruited to promoters of SBF (Swi4⁄ Swi6)-regulated genes, and reveal that binding of Sin3 to SBF-specific promoters is cell-cycle regulated We observe that transcriptional repression of SBF-depen-dent genes in early G1 coincides with the recruitment of Sin3 to specific promoters, whereas binding of Sin3 is abolished from Swi4⁄ Swi6-regulated promoters when transcription is activated at the G1 to S phase transition

We conclude that the Sin3⁄ Rpd3 histone deacetylase complex helps to prevent premature activation of the S phase in daughter cells

Abbreviations

CDK, cyclin-dependent kinase; ChIP, chromatin immunoprecipitation; MCB, MluI cell cycle box; SCB, Swi4 ⁄ 6 cell cycle box.

In early G1, SBF is already bound to the promoter

but does not activate transcription [18,19] This

inac-tivity is largely because of binding of the Whi5

repres-sor to SBF [20,21] Whi5 is thought to be the key

target for the Cln3⁄ CDK Phosphorylation of Whi5

leads to its dissociation from SBF and its subsequent

export from the nucleus [20,21]

This mode of regulation is strikingly similar to the

activation of metazoan E2F transcription factors by

cyclin D⁄ Cdk4, which phosphorylates and thereby

inactivates the Rb repressor before S phase [22]

In yeast, the G1cyclin Cln3 is the key regulator that

integrates signals about cell size and growth rate to

promote cell-cycle progression at Start [1,2,23]

Differ-ences in Cln3 protein levels and stability have a

pro-found influence on cell size at Start Activated alleles of

CLN3 lead to smaller cells, whereas a cln3D mutant,

although viable, enters the S phase at a larger cell size

[2] Consistent with a function as a repressor and

important target for Cln3⁄ CDK activity, inactivation

of WHI5 advances cell-cycle entry and largely bypasses

the requirement for CLN3 [20,21] Studies at the HO

promoter have shown that CDK activation in late G1

is important for polymerase recruitment, whereas

recruitment of Srb⁄ mediator complex by SBF occurs

prior to CDK activation [24,25] A number of

addi-tional regulators were shown to affect the amount and

timing of G1⁄ S-specific transcription These include, in

particular, BCK2, which becomes essential in the

absence of CLN3 [26], CCR4 [27], XBP1 [28], MSA1

[29], NRM1 [30] and STB1 [31] Despite their similar

architecture, SBF and MBF are not identically

regu-lated For example, the corepressor Nrm1 specifically

regulates MBF target genes [30] STB1 was reported to

have different effects on MBF- and SBF-regulated

genes although it binds to the common subunit Swi6

[31] Deletion of STB1 in a cln3D strain caused a delay

in G1⁄ S transcription and the accumulation of large

un-budded G1cells [32] suggesting that Stb1 may act as an

activator Further experiments showed that the

interac-tion of Stb1 with Swi6 is abolished upon

phosphoryla-tion of Stb1 through Cln–Cdc28 kinase complexes

[32,33] Earlier studies suggested that Stb1 may

specifi-cally act on MCB elements [31], whereas recent

chro-matin immunoprecipitation (ChIP) assays provided

evidence that Stb1 is recruited to both SCB and MCB

elements in the G1 phase [33] Stb1 was originally

found to interact with the transcriptional corepressor

Sin3 in a two-hybrid assay [34] Recent analysis of G1

-specific mRNA levels in stb1D and sin3D mutants

sug-gested a role for Sin3 and Stb1 in regulating these genes

[33] Sin3 and its associated histone deacetylase Rpd3

act together in large multiprotein complexes on

tran-scriptional repression of many genes [35–39] Through interaction with DNA-binding proteins, Sin3 recruits the deacetylase Rpd3 to specific promoters In particu-lar, the DNA-binding protein Ume6 was shown to recruit Sin3 and Rpd3 deacetylase activity to genes involved in phospholipid biosynthesis, meiosis and sporulation [37,40–43] Genome-wide acetylation stud-ies [44] and genome-wide binding studstud-ies for Rpd3 [39] showed that genes involved in cell growth and cell-cycle control, including the G1-specific gene PCL1, are tar-geted by the Rpd3 deacetylase

In this study, we uncover a role for Sin3 and its associated histone deacetylase Rpd3 in cell size homeo-stasis at the Start of the cell cycle We find that SIN3 represses SBF-dependent transcription in early G1 and show that Sin3 is bound to promoters in G1 and released around the onset of Start transcription We conclude that Sin3 is important for the correct timing

of SBF-dependent transcription in G1

Results

Sin3 represses SBF-dependent transcription Mutations that accelerate cell division relative to cell growth lead to a reduced cell size at Start [45] The tim-ing of Start is mostly determined by the initiation of

G1⁄ S-specific cyclin transcription Activated alleles of the regulator CLN3 lead to smaller cells, whereas loss of CLN3delays CLN1,2 transcription causing cells to start the cell cycle at a larger size [2] We exploited this pheno-type in a screen for novel dose-dependent regulators of

G1⁄ S-specific transcription We transformed cln3D mutants with a multicopy genomic library derived from YEplac181 and used centrifugal elutriation to identify transformants with a reduced cell size in G1 Not sur-prisingly, we found plasmids encoding the known regu-lators CLN1, CLN2, CLN3 and SWI4 (data not shown) In addition, we identified a plasmid encoding a truncated version of SIN3, lacking the C-terminal part

of the coding region (2l SIN3DC) (Fig 1) that led to a reduction of cell size in cln3D cells (Fig 1A) This was accompanied with increased levels of CLN2 RNA and

an increased budding index (Fig 1B) The change in cell size may therefore be the result of increased G1 cyclin expression This was unexpected because Sin3 has been described as a repressor of transcription [46,47] We therefore tested whether the phenotype could be explained by a dominant-negative effect of the truncated SIN3 allele on the function of wild-type SIN3 gene Sin3D mutants were originally identified because they allow HO expression in the absence of SWI5 [46,47] Using a Swi5-dependent HO-ADE2 reporter gene we

found that swi5 mutants transformed with the SIN3DC

plasmid expressed HO-ADE2, suggesting that the

trun-cated allele has a dominant-negative effect (data not

shown)

To test directly whether SIN3 has an effect on the

regulation of G1⁄ S-specific transcription, we compared

synchronized wild-type and sin3D mutant cells Because

we were interested in the timing of G1cyclin expression,

we analysed small G1cells isolated by centrifugal

elutri-ation The collected G1cells were diluted in fresh media

and followed as they progressed through the cell cycle

(Fig 2) Isolation of small unbudded sin3D cells turned

out to be difficult and yielded populations with a mini-mum content of 8–9% budded cells The elutriated sin3D cells initiated budding at a size of 24–26 fL This was smaller than for the congenic wild-type cells, which initiated budding at 32–35 fL (Fig 2A) It is unlikely that the observed difference is caused by a lack of synchrony in the sin3D culture, because cells from the elutriated sin3D population were, on average, slightly smaller than those from the wild-type population (20.9 fL for sin3D and 21.8 fL for wild-type), although more sin3D cells had already passed the S phase (Fig 2C) To analyse SBF-dependent gene regulation, the mRNA level of G1⁄ S-specific genes was determined (Fig 2B) Transcripts of the SBF-regulated genes CLN2 and PCL1 started accumulating at  23 fL in sin3D cells compared with 30 fL in the wild-type pop-ulation, around the time of bud emergence (Fig 2A,B) FACS analysis showed that sin3D mutant cells also rep-licated their DNA at a smaller cell size (Fig 2C) These observations suggest that Sin3 is involved in repression

of Start-specific transcription in G1 and thereby nega-tively regulates cell-cycle initiation In most instances, Sin3 acts together with the histone deacetylase Rpd3 [48] We therefore analysed gene expression in congenic rpd3D cells Rpd3D cells synchronized by elutriation ini-tiated budding at a size of 24–26 fL, comparable with the sin3D strain (Fig 2A) Transcription of SBF-regu-lated genes in the elutriated cells also started at around the same cell size as observed for the sin3D mutant (Fig 2B) It is therefore likely that Sin3 acts together with Rpd3 in the regulation of G1-specific transcripts Because we observed precocious activation of G1 -spe-cific transcription in elutriated sin3 mutant cells, we expected that asynchronously growing mutant cells would be, on average, smaller than a corresponding wild-type population Interestingly, analysis of mean cell size from asynchronous sin3D cultures showed no reduced average size compared with a wild-type popula-tion (Fig 3A–C) The average cell size of a populapopula-tion, however, also depends on the time spent in G2 Indeed,

we found an increased budding index of 73% in sin3D cultures compared with 48% for wild-type cells We also observed that log phase sin3D cells had a signifi-cantly increased percentage of cells that have entered

S phase and replicated their DNA This may, therefore, explain why cells from the sin3 population are, on aver-age, not smaller than the wild-type population

Inactivation of SIN3 suppresses the CLN3 requirement for Start

When wild-type cells reach a critical cell size, activa-tion of G1-specific transcription by Cln3⁄ Cdk1 is rate

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Fig 1 Multicopy plasmids encoding a trunctated SIN3 allele reduce

the mean cell size of cln3D mutants (A) Wild-type (CY979) and cln3D

cells (CY2028) were transformed with YEplac181 or a YEplac181

derivative (pCK1509) encoding a truncated SIN3 allele (2 lm SIN3DC).

This truncated Sin3 polypeptide lacks amino acids 811–1536

Trans-formants were grown at 30 C to log phase in selective medium

lack-ing leucine The frequency distribution of cell size was measured with

a CASY1 cell counter (Scha¨rfe Systems, Innovatis AG, Reutlingen,

Germany) (B) Budding index was determined by counting 250 cells

and the mean cell size of transformants was measured with a

CASY1 cell counter CLN2 RNA levels were determined by northern

blot analysis and quantified with the BIOCAPT v 12.3 software.

limiting for the further events at Start [3,15] There-fore, inactivation of CLN3 results in a large cell phenotype To test whether Cln3 is involved in releas-ing cells from a Sin3-dependent repression of G1 -spe-cific transcription, we analysed the consequences of deleting sin3 in a cln3 mutant The cell size of sin3Dcln3D double mutants from a logarithmically growing culture was compared with that of single mutants and wild-type cells (Fig 3A–C) The average cell size of cln3D cells was reduced to approximately the size of sin3D in the double mutant, suggesting that sin3 is partly epistatic to cln3 (Fig 3A–C) The critical cell size for the initiation of budding and the activa-tion of G1⁄ S-specific transcription was investigated in small G1 cells elutriated from an asynchronous sin3Dcln3D double-mutant culture (Fig 3D,E) Similar

to the sin3D population (see above), it proved difficult

to isolate small unbudded sin3Dcln3D cells, suggesting partial deregulation of cell-cycle entry As can be seen from the FACS profile and cell size measurements

15 min after putting cells into fresh medium (Fig 3F), inactivation of SIN3 in the cln3D mutant led to a reduction in cell size at birth Moreover, although cln3D mutants started budding at a nearly twice the size of wild-type cells, sin3Dcln3D double-mutant cells initiated budding at around the size of wild-type cells (Fig 3D) and activated G1⁄ S-specific transcription at

a much smaller size than cln3D mutants (Fig 3E) Inactivation of SIN3 in a cln3D deletion mutant also caused the cells to replicate their DNA at a smaller cell size (Fig 3F) Hence, inactivation of SIN3 advances Start in a cln3D mutant These data suggest that Cln3 is also involved in releasing cells from a Sin3 dependent repression

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Fig 2 G1⁄ S-specific gene expression in cells synchronized by cen-trifugal elutriation (A) Wild-type (strain CY4196), sin3D (strain CY5538), cln3D (strain CY5713) and sin3Dcln3D double-mutant cells (strain CY5715) were grown to late log phase in YEPGalmedium at

25 C Cell-cycle times were 240 min (Wt; CY4196), 340 min (sin3D; CY5538) and 350 min (rpd3D; CY4061) Cells were har-vested by centrifugation and loaded into an elutriation chamber Small unbudded cells were isolated by centrifugal elutriation and transferred into fresh medium at 25 C To determine the budding index, 250 cells were counted Cell size during outgrowth was measured with a CASY1 cell counter Displayed are mean vol-umes The peak of the cell size distribution at the time of budding was at 26 fL for wild-type For sin3D and rpd3D the peak values were 19 and 20 fL respectively (B) RNA levels of CLN2, PCL1 and TMP1 in samples taken during outgrowth were determined by northern blot analysis The transcript levels were normalized in comparison to the constitutively expressed CMD1 transcript (C) DNA content was analysed by flow cytometry Log, logarithmic growing cells used for elutriation.

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Fig 3 Inactivation of SIN3 rescues the cell size of cln3D mutants (A) Wild-type (strain BY4742 background derived from S288C), congenic sin3D cells (strain CY5538), cln3D (strain CY5713) and sin3Dcln3D double-mutant cells (strain CY5715) were grown to mid-logarithmic phase

in YEPD medium and analysed by DIC microscopy Budding index as percentage of cells with bud was determined by counting 250 cells and was 48% for wild-type, 73% for sin3D, 43% for cln3D and 76% for the sin3Dcln3D double mutant (B) Cell size analysis of wild-type, sin3D, cln3D and sin3Dcln3D double mutants The frequency distribution of cell diameters from asynchronous growing cultures in YEPD was determined using a CASY1 cell counter (C) Cell size analysis of wild-type, sin3D, cln3D and sin3Dcln3D double mutants Displayed are the mean values from 10 independent experiments and their standard error (D) Wild-type (strain CY4196), cln3D (strain CY5713) and sin3Dcln3D double-mutant cells (strain CY5715) were grown to late log phase in YEPGalmedium at 25 C Cell-cycle times were 240 min (Wt; CY4196),

290 min (cln3D; CY5713) and 300 min (sin3Dcln3D; CY5715) Small unbudded cells were isolated by centrifugal elutriation and transferred into fresh medium at 25 C Cell size was measured with a CASY  1 cell counter Displayed are mean volumes The peak of the cell size dis-tribution at the time of budding was at 26 fL for wild-type, 75 fL for cln3D and 22 fL for cln3Dsin3D (E) RNA levels of CLN2, PCL1 and TMP1 in samples taken during outgrowth were determined by northern blot analysis and the transcript levels were normalized to the consti-tutively expressed CMD1 transcript (F) DNA content of samples was analysed by flow cytometry Log, logarithmic growing cells used for elutriation.

To elucidate if this repression by Sin3 is dependent

on SBF (Swi4⁄ Swi6), the cell size of sin3swi4 double

mutants was determined Cell size analysis of log-phase

cultures from double mutants revealed that sin3D does

not reduce the cell size of swi4D mutants, and did not

advance transcriptional activation of G1⁄ S-specific

genes, but instead increased the average size of swi4D

mutants from 84 to 109 fL (data not shown)

Sin3 is recruited to SBF-specific promoters

The effect of Sin3 on cell size and S-phase entry

sug-gests a role for Sin3 in the timing of CLN1 and CLN2

transcription by SBF If Sin3 were directly involved in

regulating SBF (Swi4⁄ Swi6)-dependent transcription in

late G1, it should be present at the relevant promoters

Sin3 does not directly bind to DNA, but is known to

be recruited to specific promoter regions by other

DNA-binding proteins [40,48] We therefore asked

whether Sin3 is targeted to promoters of G1⁄ S-specific

genes in a Swi4⁄ Swi6-dependent manner For this,

SIN3was replaced by an epitope-tagged version at the

SIN3 locus Binding of epitope-tagged Sin3–myc to

G1⁄ S-specific promoters was assayed by ChIP

experi-ments Coprecipitated promoter DNA fragments

encompassing the SBF-binding sites from the promoter

regions of CLN1 and CLN2 were amplified by

multi-plex PCR along with control fragments from their

coding regions and from a nontranscribed region on

chromosome V As shown in Fig 4, the promoter

elements of CLN1 and CLN2 were significantly

enriched compared with control fragments from the

coding region and the nontranscribed region of

chro-mosome V Immunoprecipitations were performed in

triplicate to control for variations in the efficiency of

immunoprecipiation As an additional control for the

specificity of Sin3 binding, cells not expressing the

epi-tope tag were analysed in parallel Sin3–myc binding

to the promoter sequences of CLN1 and CLN2 was

strongly reduced in swi4 and swi6 null mutants

(Fig 4), which further demonstrated the specificity of

the observed interaction These results suggest that

Sin3 effects G1⁄ S transcription directly, and that Sin3

is recruited to G1 cyclin promoters by SBF or by

factors associated with Swi4 or Swi6

Sin3 recruitment is regulated in a

cell-cycle-dependent manner

To detect whether the recruitment of Sin3 and its

asso-ciated histone deacetylase Rpd3 to G1-specific

promot-ers is regulated during the cell cycle, we tested

promoter occupancy in cells that were arrested at

different stages of the cell cycle We analysed cdc28-13 cell-cycle mutants arrested in G1 at 37C, as well as cells that were arrested in G2 with the microtubule depolymerizing drug nocodazole Cdc28-13 mutants arrest in late G1prior to the activation of G1⁄ S-specific transcription Strong Sin3 binding was detected in such cells at the CLN1, CLN2 and PCL1 promoter regions (Fig 5A,D) The stronger signal in the arrested cultures

is most probably a simple reflection of cell-cycle-depen-dent binding Indeed, Sin3 was not associated with the promoters during G2, as we could not significantly coprecipitate CLN2 or PCL1 promoter elements with

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Fig 4 Sin3 binds to G 1 ⁄ S-specific promoters Chromatin immuno-precipitation assays (ChIP) were performed in triplicate using yeast strains carrying a myc tag at the SIN3 locus (CY5386, swi4D; CY5387, wt; CY4849, wt; CY5469, swi6D) ChIP assays with extracts of a strain lacking the myc tag were used as negative con-trols (CY1617) Crude extracts were prepared from formaldehyde cross-linked cells and chromatin precipitated with 9E11 antibodies Precipitates were analysed by multiplex PCR Primers for an untran-scribed region on chromosome V were used as nonspecific control These control primers were applied in the same PCR together with either primers for the amplification of promoter elements or coding regions of CLN1 and CLN2 PCR results with primers for the coding region of CLN2 are displayed in comparison to the PCR results of promoters or the control Products were analysed on a 2% agarose gel WCE, whole cell extract; No Tag, analysis of strains lacking epitope tagged protein.

Sin3–myc from cells arrested with nocodazole

(Fig 5C,D) To provide further evidence for the

spe-cific binding of Sin3, we analysed the recruitment of

Rpd3, the catalytic component of the Sin3⁄ Rpd3

his-tone deacetylase complex, to G1cyclin promoters in G1

(Fig 5B) Cdc28-13 mutants expressing an

epitope-tagged RPD3–HA6 were synchronized in late G1 by

shifting log-phase cultures to 37C for 3 h until all

cells were arrested as large unbudded cells In ChIP

assays with extracts prepared from arrested Rpd3–HA6

cells we observed recruitment of Rpd3–HA6 to

SBF-dependent promoters, although the signal was weaker

than the signal for Sin3–myc (Fig 5B) The binding of

Sin3 and Rpd3 to promoters of SBF-regulated genes in

G1-arrested cells correlates with the transcriptional

repression of CLN1, CLN2 and PCL1 in G1

To analyse if the release of Sin3 from SBF-regulated genes coincides with transcriptional activation of G1 cyclins, we performed an arrest–release experiment Cdc28-13mutants were shifted to 37C until they were arrested as unbudded cells in G1 The cells were subse-quently released from cell-cycle arrest by shifting the culture to 25 C RNA levels and Sin3 binding were analysed from samples taken every 10 min For the arrested culture, ChIP analysis demonstrated strong binding of Sin3–myc to the CLN1 and CLN2 promoter (Fig 6A; 0 min) When cells were released from the cell-cycle block, they synchronously entered the cell cycle (Fig 6C) A peak of G1⁄ S-specific transcription was observed between 10 and 20 min after release Shortly thereafter, cells entered the S phase (FACS profile in Fig 6D) and started budding (Fig 6C)

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Fig 5 Sin3 and Rpd3 are recruited to CLN1, CLN2 and PCL1 promoters in G 1 Chromatin immunoprecipitation (ChIP) assays were per-formed in triplicate using cdc28-13 yeast strains (CY239, CY5327 and CY5555) (A) Cells expressing Sin3–myc (CY5327) were grown in YEPD at 25 C to a titre of 1 · 10 7 mL)1 The culture was subsequently split in two and either arrested in G1 by shifting to 37 C for

165 min or kept at 25 C for the same time Cells were cross-linked with 1% formaldehyde for 20 min at room temperature Cell extracts were subjected to immunoprecipitation with anti-myc (9E11)-coupled Dynabeads The precipitates were analysed by PCR with primers for the amplification of promoter elements of CLN1, CLN2 and PCL1 and the coding regions of CLN2 Precipitation of DNA fragments from an untranscribed region on chromosome V was analysed as a control PCRs were analysed on 2% gels ChIP, chromatin immunoprecipitations; WCE, whole cell extract; No Tag, analysis of strains lacking epitope tagged protein (B) cdc28-13 cells expressing Rpd3–HA6 (CY5555) were treated and ChIPs performed as described in (A) (C) Binding of Sin3–myc (CY4849, wt) to the promoters of CLN2 and PCL2 in nocodazole arrested wild-type cells was analysed by ChIP Extracts were prepared from cells that were grown in YEPD to an D 600 of 0.4 and subse-quently arrested with nocodazole at 25 C for 2.5 h (D) Precipitated DNA from the ChIP assays shown in (A) and (C) was analysed by real-time PCR on a Mx3000P thermocycler using the brilliant II QPCR kit, as described by the manufacturer (Stratagene, Heidelberg, Germany) Values from the untagged control samples were substracted from the signal of the tagged samples Shown are mean values derived from three independent experiments with standard deviations.

The ChIP signal began to fade 10 min after the cells

were released (Fig 6A,B) The decrease in promoter

occupancy by Sin3 correlated best with the timing of

transcriptional activation (Fig 6B) The timing of Sin3 binding is therefore consistent with a role for Sin3 in repression of CLN transcription in the G1phase

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Fig 6 Dissociation of Sin3 from SBF-dependent promoters correlates with activation of G 1 ⁄ S-specific transcription A cdc28-13 mutant expressing Sin3–myc (CY5327) was grown in YEPD to D600= 0.4 and arrested at 37 C for 180 min Cells were released from the G 1 arrest

by shifting the culture to 25 C Samples were taken at the indicated time points (A–D) (A) Sin3–myc binding was analysed by ChIP as in Fig 5 PCRs were analysed on 2% agarose gels and quantified with the BIOCAPT v 12.3 software (B) Northern blot analysis of RNA levels of CLN2 and PCL1 were analysed in parallel with CMD1 as loading control (C) Quantified data from ChIP, CLN2 RNA levels and budding index The transcript levels of the CLN2 RNA were normalized using the constitutively expressed CMD1 transcript ChIP signals for the CLN2 pro-moter region were normalized to the signals of the chromosome V UTR control [(spec ChIP ⁄ control ChIP) tagged – (spec ChIP ⁄ control ChIP) untagged] (D) DNA content analysis by flow cytometry (E,F) ChIP of CLN2 and PCL1 promoter elements from small elutriated G 1 cells expressing Sin3–myc Cells were grown to D600= 2.3 in YEPGalmedium and small G1cells were isolated by centrifugal elutriation from cultures Unbudded cells were inoculated in fresh medium and incubated at 25 C Binding of Sin3–myc to promoters was analysed at different time points by ChIP (F) PCL1 and CMD1 RNA levels determined by hybridization of northern blots with radioactive labelled DNA fragments (G) DNA content of cells was analysed by flow cytometry at the indicated time points.

Because of their abnormally large size, G1-arrested

cell-cycle mutants may not accurately reproduce the

situation found in small wild-type daughter cells in the

early G1 phase We therefore analysed promoter

occu-pancy of Sin3–myc in elutriated wild-type cells Small

G1 cells were isolated by centrifugal elutriation and

allowed to progress through G1 The presence of Sin3–

myc at the CLN2 and PCL1 promoter was compared

with cell-cycle progression Because many cells were

needed for ChIP assays it was not possible to analyse

more than three time points At each time point,

sam-ples from the culture were analysed by ChIP assay and

the RNA levels of G1⁄ S-specific genes and the DNA

content of cells were determined (Fig 6E–G) The data confirmed that SBF-specific promoters are occupied by Sin3 in the G1phase At 160 min, most cells in the cul-ture had left G1 and exhibited no Sin3 binding to the promoter (Fig 6E) Analysis of G1 cyclin expression showed that binding of Sin3 to the SBF-dependent promoters correlated with repression in G1, whereas disappearance of Sin3 from the promoter elements coincided with induction of Start-specific transcription (Fig 6) To elucidate whether Sin3 leaves the promoter together with Rpd3, we performed an arrest-release experiment with cdc28-13 cells expressing Rpd3–HA

As shown in Fig 7, the binding of Rpd3 is very

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Fig 7 Dissociation of Rpd3 from SBF-dependent promoters correlates with activation of G 1 ⁄ S-specific transcription A cdc28-13 mutant expressing Rpd3–myc (CY5555) was grown in YEPD to a titre of 1 · 10 7 cellsÆmL)1and arrested at 37 C for 180 min Shifting the culture

to 25 C released the cells from the G 1 arrest Samples were taken at the indicated time points (A–D) (A) Rpd3–myc binding was analysed

by ChIP as in Fig 5 PCRs were analysed on 2% agarose gels and quantified with the BIOCAPT v 12.3 software (B) RNA levels of CLN2 and PCL1 were analysed by northern blot in parallel with CMD1 as loading control (C) Quantified data from ChIP, CLN2 RNA levels and budding index as described in the legend to Fig 6 The transcript levels of the CLN2 RNA were normalized using the constitutively expressed CMD1 transcript ChIP signals for the CLN2 promoter region were quantified by normalising band intensities of CLN2 promoter fragments to the signals of the chromosome V UTR control (D) DNA content was analysed by flow cytometry at the indicated time points.

similar to the kinetics of Sin3 binding (Fig 6) although

the signal intensities for Rpd3 are generally weaker

We therefore conclude that Sin3 acts together with

Rpd3 at SBF-dependent promoters (Fig 7)

Discussion

In this study, we provide evidence that SIN3 is

involved in the correct timing of G1 cyclin expression

in Saccharomyces cerevisiae at the G1–S phase

transi-tion We have found that inactivation of SIN3 leads to

an advanced induction of Start-specific transcription in

G1 daughter cells and that budding is initiated at a

smaller cell size Consistent with a direct role for Sin3

in repressing gene expression prior to Start, we find

that Sin3 is present at the promoters of SBF-regulated

genes in G1, but leaves the promoter around the time

cells enter the S phase Furthermore, inactivation of

Sin3 suppresses the phenotype of cln3 mutants,

allow-ing them to activate G1⁄ S-specific transcription at a

smaller cell size (Fig 3) Such a phenotype would be

expected if Cln3 with its associated Cdc28 kinase were

involved in the inactivation or repression of

Sin3⁄ Rpd3-dependent histone deacetylation at the

CLN1,2 promoters These data raise several questions

concerning the regulation of G1cyclin transcription In

particular, whether CLN3 acts directly on Sin3⁄ Rpd3

and how Sin3 is recruited to SBF-regulated genes like

CLN2in the G1phase

How is Sin3 recruited to SBF-regulated

promoters?

Sin3 does not bind DNA directly, but associates with

transcriptional regulators to bring the Rpd3 histone

deacetylase to specific sites in chromatin [40,49] There

are different DNA-binding proteins thought to bind to

Sin3 Besides the well-characterized interaction with

Ume6, these include Ash1, Mcm1 and Ssn6 [35,50,51]

At the HO promoter, Sin3 is thought to be recruited

in part by Ash1 [35,51] Veis et al reported

cell-cycle-dependent binding of Sin3 to the G2⁄ M-specific CLB2

promoter [50] Their data further showed that the

recruitment of Sin3 is dependent upon an interaction

with Fkh2 and Mcm1 The removal of Sin3 and the

deacetylase complex does not require B-type cyclins

but Cdc28⁄ Cln activity [50] Similar to the recruitment

of Sin3 to G2⁄ M-specific promoters by the regulatory

factors Mcm1 and Fkh2, we propose that Sin3 is

recruited to G1⁄ S promoters by SBF The

DNA-bind-ing protein Ume6 was shown to be responsible for

Sin3⁄ Rpd3 recruitment at many other sites, for

exam-ple, at SPO13, INO1, IME2 [40,43,52] We found no

Ume6 consensus sites [48] in the promoter regions of CLN1 and CLN2 Any one of the proteins present at the CLN2 promoter in early G1 could, in principle, be responsible for recruiting Sin3 to the promoter These include Swi4, Swi6, Whi5 and Stb1 [18,19,21,33,35] Although recruitment of Sin3 to the CLN2 promoter strongly depends on Swi4 and Swi6 (Fig 4), we found

no significant effects of whi5 or stb1 mutants on the binding of Sin3 to the CLN2 promoter (data not shown) In addition, deleting WHI5 in a sin3 mutant did not reduce the cell size to the level of whi5D single mutants (data not shown) This makes it unlikely that Sin3 is recruited to the promoter via Whi5 Because the absence of Stb1 was observed to increase cell size

of a cln3D mutant [32], it is not likely to mediate Sin3-dependent repression, although it could be important for releasing from Sin3-dependent repression later in the cell cycle However, the timing of SBF binding [18,19], which arrives at the promoter as cells exit mitosis, would be consistent with a direct role as a Sin3⁄ Rpd3 recruiting factor Earlier ChIP results showed that the histone deacetylase Rpd3 is associated with the promoters of cell-cycle genes regulated by SBF, MBF, Fkh1, Fkh2, Mcm1 and Ndd1, and showed that SBF affected Rpd3 binding to CDC20 and PCL1, suggesting that Rpd3 can be recruited by several different transcription factors [39] This is con-sistent with our observation that both Sin3 and Rpd3 are recruited to G1⁄ S-specific promoters in a cell-cycle-dependent manner A situation in which transcrip-tional activators also directly recruit corepressors is in fact quite common, for example, in the case of E2F transcription factors in metazoans [53]

How is Sin3 removed from the promoter in the

S phase?

The observation that deleting SIN3 partly suppresses the size phenotype of cln3 mutants suggests a possible role for Cln3 in the inactivation or subsequent removal

of Sin3⁄ Rpd3 complexes from the promoter The only well-characterized, and presumably critical substrate for Cln3⁄ CDK1 is the repressor Whi5 [20,21] Removal of Sin3 at the beginning of the S phase is probably not a consequence of Whi5 inactivation caused by phosphorylation by Cln3⁄ CDK1 [20,21], because there is no evidence for a direct interaction between Whi5 and Sin3 or Rpd3

Alternatively, Sin3 may be a direct target for Cln3 kinase Sin3 is a phosphoprotein [54] and was found to coprecipitate with Cln2 in a proteomics study of yeast CDKs [54] The timing of Sin3s removal from the promoter (Fig 6) would also be compatible with a