Determination of the core promoter regions of the Saccharomyces cerevisiae RPS3 gene

Biochimica et Biophysica Acta 1789 (2009) 741–750

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a g r m

Determination of the core promoter regions of the Saccharomyces cerevisiae RPS3 gene Yoo Jin Joo a, Jin-ha Kim a, Joung Hee Baek a, Ki Moon Seong a, Jae Yung Lee b, Joon Kim a,⁎ a b

Labotoray of Biochemistry, School of Life Sciences and Biotechnology, and BioInsititute, Korea University, Seoul 136-701, Korea Department of Life Sciences, Mokpo National University, Muan-gun, Jeonnam, 534-729, Korea

a r t i c l e

i n f o

Article history: Received 9 July 2009 Received in revised form 12 October 2009 Accepted 13 October 2009 Available online 22 October 2009 Keywords: Rap1p UASrpg T-rich region Transcription factor Ribosomal protein gene

a b s t r a c t Ribosomal protein genes (RPG), which are scattered throughout the genomes of all eukaryotes, are subjected to coordinated expression. In yeast, the expression of RPGs is highly regulated, mainly at the transcriptional level. Recent research has found that many ribosomal proteins (RPs) function in multiple processes in addition to protein synthesis. Therefore, detailed knowledge of promoter architecture as well as gene regulation is important in understanding the multiple cellular processes mediated by RPGs. In this study, we investigated the functional architecture of the yeast RPS3 promoter and identified many putative ciselements. Using β-galactosidase reporter analysis and EMSA, the core promoter of RPS3 containing UASrpg and T-rich regions was corroborated. Moreover, the promoter occupancy of RPS3 by three transcription factors was confirmed. Taken together, our results further the current understanding of the promoter architecture and trans-elements of the Saccharomyces cerevisiae RPS3 gene. © 2009 Elsevier Inc. All rights reserved.

1. Introduction Yeast can budget its use of energy and resources through numerous finely regulated processes in order to adapt to violent environmental conditions. Ribosome biogenesis as the major consumer of cellular energy and resources is among the most wellcharacterized cellular mechanisms, consuming about 90% of a cell's transcriptional activity and 50% of its resources; moreover, up to ∼30– 40% of the cytoplasmic volume of a cell is occupied by ribosomes [1]. The synthesis of ribosomes involves the transcription of tRNA and rRNA, as well as other components involved in ribosome biogenesis. The expression of RPGs is especially well-studied because it provides an effective base for the understanding of gene regulation and for the building of a gene regulatory network [2]. The yeast ribosome contains 137 genes, 59 of which are duplicated. These genes encode 32 small-subunit and 46 large-subunit proteins, which account for 50% of POL II-mediated transcription [1]. The genes are scattered over the entire genome and possess distinctive promoter that share some functional similarities [3]. It is well known that expression of RPGs is tightly co-regulated at the transcriptional level, with ribosome biosynthesis in yeast being equimolar [4]. Despite many investigations into this biological process, the exact mechanism by which these regulons are controlled is not yet completely understood. In numerous reports, it has been revealed that Rap1p (repressor and activator protein 1) is involved in the transcriptional regulation of RPG family genes. Most RPGs have one or more Rap1p-binding sites called ⁎ Corresponding author. Tel.: +82 2 3290 3442; fax: +82 2 927 9028. E-mail address: [email protected] (J. Kim). 1874-9399/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbagrm.2009.10.002

UASrpg (upstream activating sequence of ribosomal protein gene; ACACCCRYACAYM) in their promoters [5,6]. However, the role of Rap1p as a repressor or activator of RPG transcription is contextdependent [7]. Rap1p can activate the transcription of a large number of heavily transcribed genes, including those encoding glycolytic enzymes, RPs, and several components of the transcriptional machinery [6]. In addition, it is responsible for the silencing of HMR and HML loci, as well as regulation of telomere length [8]. The general role of Rap1p appears to be as a “chromatin opener” that facilitates the binding of transcription activators at binding sites adjacent to the T-rich region [9]. Many researchers are currently focusing on the identification of specific transcriptional regulators of RPGs in yeast. These efforts have revealed several proteins, such as Fhl1p, Ifh1p, Sfp1p, Hmo1p, Crf1p, and Esa1p, which are responsible for the regulation of RPG transcription under specific conditions [10–18]. Many signaling pathways, such as the ‘target of rapamycin’ (TOR), ras–cAMP–protein kinase A (PKA), and secretion pathway, contribute to the regulation of RPG transcription in combination with heat shock and amino acid starvation. Moreover, it has been shown that the yeast TOR pathway is mediated through RAS/cAMP signaling [13]. In a recent report, it was proposed that RPGs are regulated by multiple protein factors and mechanisms, as opposed to a unified mechanism as previously thought [19]. Therefore, knowledge of the promoter structure of each gene is required to fully understand the transcriptional regulation of RPGs. RPS3, an essential single-copy gene, has 66% similarity with mammalian rpS3 protein and functions mostly as a ribosomal component. For translational initiation complexes in yeast, efficient UV cross-linking of Rps3p to mRNA was detected at position +11 [20].

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Moreover, Hrr25-dependent phosphorylation of Rps3p is crucial for maturation of the 40S ribosomal complex [21]. Although Rps3p mainly functions in protein synthesis, it displays other non-ribosomal activities such as AP endonuclease activity in yeast [22], apoptosis, metastasis inhibition, and DNA repair in mammalian cells [23–28]. Rps3p is also known as an essential subunit of NK-κB [29]. The intergenic sequence between RPS3 and its 5′-flanking ORF, YNL179C, contains 5 putative Rap1p-binding sites (UASrpg), and two putative Gcn4p-binding sites (GCRE; Gcn4p responsive element as predicted previously [32] and, as such, is quite different from those of mammalian homologues [30,31]. However, essential cis-elements and functional trans-elements have not yet been discovered. In this study, the intergenic region about 1.2 kb upstream of the translation start site was serially deleted in order to elucidate the promoter structure of RPS3. Using a reporter gene assay, the core promoter region of RPS3 containing the UASrpg and T-rich region was identified. To identify trans-elements, reporter assay, EMSA, and chromatin immunoprecipitation (ChIP) were all performed. These results suggest that transcription of yeast RPS3 is mediated by the UASrpg and T-rich region and depends upon the association of Rap1p, Hmo1p, and Fhl1p with the core promoter region. 2. Materials and methods

and BamHI and then inserted into the KpnI and BamHI sites of pRS316-lacZ. For EMSA, the endogenous RAP1 open reading frame (ORF) was obtained by PCR and inserted into pET15b (Novagen) using NdeI and XhoI, generating N-terminal hexahistidine-tagged Rap1 protein (His6-Rap1). To generate a strain containing N-terminal TAPtagged Rap1p, the endogenous RAP1 promoter region (from − 360 bp to −1 bp) was amplified and ligated into the EcoRI and NcoI sites of pBS1761 (EUROSCRAF), resulting in pBS1761-RAP1pro [36]. 2.3. RNA preparation and Northern blotting Total yeast RNA was obtained by performing general hot phenol/ freeze RNA preparation methods as previously described [37]. Six μg of total RNA were separated on a 1% formaldehyde/agarose gel, transferred to a Nylon membrane (NEN, Gene Screen Plus) and then fixed by UV (1500J) using an UV cross-linker. Further blotting was performed according to the procedures of Boehringer Mannheim (DIG Application Manual for Filter Hybridization, Roche). After incubating the membrane in pre-hybridization solution for 2 hours, hybridization with digoxigenin (DIG)-labeled probes specific to RPS3, LacZ, and ACT1 was performed overnight at 65 °C. Chemiluminescent detection was performed with anti-DIG antibody conjugated to alkaline phosphatase and CDP-Star.

2.1. Strains and culture conditions 2.4. β-Galactosidase assay Yeast strains used in this study are summarized in Table 1. Yeast was grown in proper synthetic complement (SC) media (2% glucose and 0.67% yeast nitrogen base without amino acids (Difco), and proper amino acids) or YPD media (1% yeast extract, 2% pepton, and 2% glucose). For β-galactosidase reporter assay, JS143-7D and YJK101 strains were used [33]. The JS143-7D strain was also used for RNA analysis under various conditions [UV, doxorubicin, rapamycin, 3aminotriazole (3-AT) treatment or high confluency]. BY4741 and its deletion variant strains were used for Northern blotting. For chromatin immunoprecipitation (ChIP), strains harboring tri-HA tagged proteins (Hmo1-HA3, Sfp1-HA3, Fhl1-HA3, and Ifh1-HA3) were generated using PCR-based gene modification methods as previously described [34]. To generate a strain containing TAP (tandem affinity purification)-tagged RAP1, an N-terminal TAP tagging cassette was amplified by PCR using pBS1761-RAP1pro as a template. Transformation was performed using general lithium acetate methods [35]. 2.2. Plasmids and primers Primers used in this study are summarized in Table 2. PCR was performed with a specific primer set to obtain the serially or internally deleted 5′-flanking region of RPS3. For construction of reporter constructs and the pRS316-lacZ vector control, the lacZ gene was introduced between the BamHI and ApaI sites of pRS316. Next, the PCR products of the mutant promoter construct were cut with KpnI Table 1 The yeast strains used in this study. Strain JS143-7D YJK101 YJK109 YJK123 YJK119 YJK113 YJK115 BY4741

Relevant genotype

MATa, leu2-3.112, trp1-Δ1, ura3-52 As JS143-7D except gcn4::LEU2 As JS143-7D except rap1::TAP-RAP1-TRP1 As JS143-7D except hmo1::HMO1-HA3-TRP1 As JS143-7D except sfp1::SFP1-HA3-TRP1 As JS143-7D except fhl1::FHL1-HA3-TRP1 As JS143-7D except ifh1::IFH1-HA3-TRP1 MATa, his3-Δ1, leu2-Δ0, met15-Δ0, ura3Δ0 BY4741Δhmo1 As BY4741 except hmo1::kanMX BY4741Δsfp1 As BY4741 except sfp1::kanMX BY4741Δcrf1 As BY4741 except crf1::kanMX BY4741Δrpd3 As BY4741 except rpd3::kanMX

Source or reference [54] [33] In this study In this study In this study In this study In this study Clontech Clontech Clontech Clontech Clontech

Transformation of the GCN4 isogenic strains, JS143-7D and YJK101, was performed with reporter constructs, and positive transformants were selected on a SC-ura plate. Transformants cultured overnight in SC-ura media were reseeded to fresh media to an OD600 of 0.05, followed by growth to early exponential phase. One milliliter of cells in culture mixture was harvested, and liquid β-galactosidase assays were performed in triplicate as previously described [38] with three independent colonies. 2.5. Electrophoresis gel mobility shift assay (EMSA) N-terminal hexahistidine-tagged Rap1 was overexpressed in Escherichia coli BL21-CodonPlus (DE3)-RIL cells (Stratagene) harboring the pET15b-RAP1 plasmid. After 3 hours of IPTG induction, 50-ml cultures were harvested and resuspended in 1 ml of Ni-NTA lysis buffer (pH 8.0) containing four protease inhibitors (50 mM NaH2PO4, 300 mM NaCl, 10 mM immidazole, 1 mM PMSF, 1 μg/ml pepstatin A, 5 μg/ml leupeptin, and 1 μg/ml aprotinin). Crude protein extracts were prepared by sonication. Affinity purification of recombinant Rap1 protein was performed using Ni-NTA agarose beads (Qiagen). A vector control sample (HIS6) was prepared from cells containing pET15b. Double-stranded DNA probes were generated by annealing with two single-stranded complementary DNAs or by PCR with primers specific to the RPS3 promoter region. 32P-labeled DNA probes were individually incubated in a 15-μl binding mixture of purified His6-Rap1p containing 20 mM Tris–HCl (pH 8.0), 60 mM KCl, 0.1 mM EDTA, 20% glycerol, 0.25 mg/ml BSA, and 0.05 mg/μl poly dI/dC. Binding was performed for 30 min at 30 °C while DNA–protein complexes were separated from free probe by 5% Tris–borate–EDTA/polyacrylamide gel electrophoresis. Gels were run at 100 V for 40 min in 0.5× TBE gel running buffer and dried for autoradiography detection using BAS-2500 (FUJIFLIM). 2.6. Chromatin immunoprecipitation (ChIP) ChIP and quantitative PCR analysis were performed as previously described with the following modifications [17]. Cells containing proteins tagged with TAP or tri-HA epitope were cross-linked with 1% formaldehyde and incubated for 20 min at room temperature. Glycine was added for 5 min to a final concentration of 350 mM. Cells were harvested, centrifuged at 5000 × g for 5 min, and washed with 1 ml of

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Table 2 Oligonucleotides used in this study. Name

Sequence

Purpose

JK0411a,b JK0412a,b JK0338a JK0339a JK0407a,b JK0408a,b JK0980a JK0981a JK0982a JK0983a JK0984a JK0985a JK0986a JK0987a JK0988a JK0989a JK0990a JK0991a JK0646 JK0647 JK0222 JK0223 JK0450 JK0451

5′-TTTCCGTAACATCgtTACCTTTAATGT-3′ 5′-ACAGGAAAGGTAacGATGTTACGGAAA-3′ 5′-CTAATTCCTCATGATTAAATGAGACTGTTTTTTGTTTCCGTAACATCCATACCTTTCCTGTATAATATTC-3′ 5′-GAATATTATACAGGAAAGGTATGGATGTTACGGAAACAAAAAACAGTCTCATTTAATCATGAGGAATTAG-3′ 5′-CTAATTCCTCATGATTAAATGAGACTGTTTTTTGTTTCCGTAACATCgtTACCTTTCCTGTATAATATTC-3′ 5′-GAATATTATACAGGAAAGGTAacGATGTTACGGAAACAAAAAACAGTCTCATTTAATCATGAGGAATTAG-3′ 5′-CGGTAAATTAGTTAATTAATTGCACATCCACACATCTGTGACTCACGTTTTTTTATCAGT-3′ 5′-ACTGATAAAAAAACGTGAGTCACAGATGTGTGGATGTGCAATTAATTAACTAATTTACCG-3′ 5′-TTTTCACGTGCTGCGCTGATGTAAGCAGCA-3′ 5′-TGCTGCTTACATCAGCGCAGCACGTGAAAA-3′ 5′-AAAGGTCCACGTCAGTTCCACACAATAACA-3′ 5′-TGTTATTGTGTGGAACTGACGTGGACCTTT-3′ 5′-TTTCCGTAACATCCATACCTTTCCTGTATA-3′ 5′-TATACAGGAAAGGTATGGATGTTACGGAAA-3′ 5′-AATTTTCCAAATCAATGCAGCTCTTTGAAA-3′ 5′-TTTCAAAGAGCTGCATTGATTTGGAAAATT-3′ 5′-TTTTTGAACATTGTTTTGATAACTGAAAAT-3′ 5′-ATTTTCAGTTATCAAAACAATGTTCAAAAA-3′ 5′-CCATTTTTGTAGTTTGTTTGC-3′ 5′-CGCCGGTAAATTCTCTTGTTT-3′ 5′-CCGAATTCCGAATCTCTATCG-3′ 5′-GGCTCGAGGATCATCGGTCAG-3′ 5′-AAAACACCTACCGATATACACGAGG-3′ 5′-CCTTTATATCTAACCAGCATGG-3′

UASrpg3 mutation UASrpg3 mutation EMSA probe (PRw) EMSA probe (PRw) EMSA probe (PRm) EMSA probe (PRm) EMSA probe (UASWT) EMSA probe (UASWT) EMSA probe (UASrpg1) EMSA probe (UASrpg1) EMSA probe (UASrpg2) EMSA probe (UASrpg2) EMSA probe (UASrpg3) EMSA probe (UASrpg3) EMSA probe (UASrpg4) EMSA probe (UASrpg4) EMSA probe (UASrpg5) EMSA probe (UASrpg5) ChIP (RPS3) ChIP (RPS3) ChIP (LacZ) ChIP (LacZ) ChIP (POL1 CDS) ChIP (POL1 CDS)

a b

Rap1p binding elements are indicated by underlined. Mutant nucleotides were indicated by small letter.

PBS. Cell pellets were resuspended in lysis buffer [50 mM HEPES–HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 25 mM β-glycerophosphate, 25 mM NaF, 1 mM PMSF, 1 μg/ml pepstatin A, 5 μg/ml leupeptin, and 1 μg/ml aprotinin]. The resulting cell lysates were extracted using glass beads, and the protein concentration of each sample was determined. Proteins and immunocomplexes from each lysate were recovered by boiling for 5 min in 2× SDS loading buffer. For immunoprecipitation of tri-HA-tagged protein, 6 mg of total cell lysates was incubated with anti-HA antibody (Sigma) for 2 hours at 4°C with rotation. Immunocomplexes were immobilized with 30 μl (bead volume) of protein-A-agarose (Roche). For immunoprecipitation of TAP-Rap1, 6 mg of total cell lysates was incubated with 40 μl (bead volume) of IgG Sepharose beads (GE Healthcare). Following rotation for 2 hours at 4°C, bead–protein complexes were washed seven times in wash buffer [17]. Cross-linked chromatin from immunoprecipitation and input samples was amplified by quantitative PCR using primers specific to RPS3 and POL1 coding sequences (CDS). PCR was performed using 1/40 of the immunoprecipitated DNA and 1/4000 of the total input chromatin DNA as templates. Similar to a previous study, PCR of the POL1 CDS was used as a negative control [18]. 3. Results 3.1. Determination of the yeast RPS3 core promoter region The gene locus of RPS3, along with its 5′-flanking ORF, YNL179C, is located in chromosome XIV (YNL178W). The intergenic sequence

between the two ORFs of RPS3 is about 1.6 kb in length. To determine the core promoter element of RPS3, a region of the intergenic sequence from –1150 bp to +1 bp was investigated. Sequence analysis identified five putative UASrpgs (upstream activating sequence of ribosomal protein gene promoter; Rap1p binding element). Table 3 compares the sequence identities of the UASrpgs to the consensus binding sequence of Rap1p. Among the five elements, UASrpg3 most closely resembles the consensus binding sequence of Rap1p, with UASrpg1 and UASrpg5 in reverse positions. Moreover, we also found two putative Gcn4p-response elements (GCRE) in the promoter upon which eleven reporter constructs serially deleted from the 5′-end were based upon (Fig. 1A). Twelve plasmids, including serial deletion promoter constructs and vector, were transformed into JS143-7D (GCN4 wild type) and YJK101 (gcn4 null), and promoter activities were examined at early log phase. In the β-galactosidase assay, promoter constructs containing the upstream region past – 279 bp had almost identical transcriptional promoter activity. However, deletion of the regions from –279 bp to –262 bp and from –262 bp to –245 bp dramatically reduced the promoter activity (compare pRS316-279Z, -262Z, and -245Z; Fig. 1A). From these data, we conclude that the region from –279 bp to –245 bp is critical for RPS3 transcription. Part of the T-rich region was previously reported to be in the region from – 279 bp to –262 bp [32], whereas UASrpg3 appears to be located in the region from – 262 bp to –245 bp. The Trich region is necessary not only for transcriptional activation of RPGs by Rap1p or Abf1p but also for heavily transcribed genes like DED1 [39–43]. Results of the reporter assay (Fig. 1A) suggest that the upstream T-rich region and UASrpg3 are jointly responsible for

Table 3 A comparison of five UASrpg sequences in the RPS3 promoter region with the conserved Rap1p-binding element. Consensus Rap1p binding sequence

Putative UASrpg in RPS3 promoter region

a b

Location

1 2 3 4 5

These elements are in an inverse orientation. R: A or G. Y: C or T.

(− 857 to − 870)a (− 495 to− 482) (− 259 to − 246) (− 135 to − 122) (− 32 to − 45)a

1

2

3

4

5

6

7

8

9

10

11

12

13

14

A

C

A

Yb

C

C

Rb

Yb

A

C

A

T

Yb

T

A A A A T

C C C A T

A G A A A

T T T T T

C C C C C

A A C A A

G G A A A

C T T T A

G T A G A

C C C C C

A C C A A

G A T G A

C C T C T

A A T T G

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Fig. 1. Functional analysis of the yeast RPS3 promoter region. (A) Serial deletion of the intergenic region between RPS3 and YNL179C. Ten reporter constructs were generated as described in Section 2. The name of each deletion construct indicates the length of the promoter insert. The first translation start codon (ATG) is designated as + 1. β-Galactosidase assay was performed with RPS3 promoter constructs. Wild type (JS143-7D) and gcn4 deletion (YJK101) strains harboring reporter constructs or vector plasmid (pRS316-LacZ) were grown in SC-ura media to early exponential phase. Cells were harvested and the β-galactosidase activity of each promoter construct was measured. Promoter activity of each sample was normalized to pRS316-1150Z in both strains. Results show the averages of three independent experiments performed in triplicate. Error bars indicate standard deviation of each result. GCRE: Gcn4p-response element; UASrpg: upstream activating sequence of ribosomal protein gene. (B) Raw data from β-galactosidase assay in (A) are summarized in the table. Miller units for the 100% of JS143-7D: 156.17; Miller units for the 100% of YJK101 : 189.78; SD: standard deviation.

promoter activity during RPS3 transcription. Using the GCN4 wild type and deletion strains, it was suggested that Gcn4p does not affect basal RPS3 transcription. Therefore, the core promoter region of RPS3 spans from – 279 bp to – 245 bp and contains T-rich region and UASrpg3 elements. 3.2. UASrpg3 and the T-rich region are responsible for basal and TOR1-dependent transcription To more specifically determine cis-elements, three mutant promoter constructs were generated. The 558RmZ construct contains a point mutation in its UASrpg3, the 558uTΔZ construct contains an internal deletion of the upstream T-rich region, and the 558dTΔZ construct contains an internal deletion of the downstream T-rich region (Fig. 2A, left panel). Subsequent reporter analysis with these mutants and two other serial deletion promoter constructs (558Z and 262Z) found that UASrps3 and adjacent T-rich region were essential for RPS3 transcription (Fig. 2A). The promoter activities of four mutant

constructs (558RmZ, 558uTΔZ, 558dTΔZ, and 262Z) were significantly reduced more than that of wild type (558Z). Interestingly, the promoter activity of 558uTΔZ was decreased more intensively than that of 558dTΔZ, implying that the upstream T-rich region is more important for the full transcription of RPS3. To examine how two cis-elements contribute to the regulation of RPS3 expression, a β-galactosidase reporter assay was performed with three types of promoter constructs (Fig. 2B). The basal reporter activities of pRS316-558RmZ and pRS316-262Z were approximately equal and were diminished to about 20–30% of wild type, pRS316558Z. Promoter activities of these constructs were examined under high cell density and amino acid starvation conditions using βgalactosidase assay. Surprisingly, however, the activities of all promoter constructs remained unchanged under all conditions tested. After cell density was increased, the transcription of cells growing in the stationary phase was diminished by about half compared to cells growing exponentially (Fig. 2B). After 3-AT treatment, reporter activity was decreased in both the wild type and pRS316-558RmZ

Fig. 2. β-Galactosidase assay with mutant RPS3 promoters under stress conditions. (A) Cells containing each indicated promoter construct were cultured to early log phase. Cells were harvested and a Miller unit of each sample was determined as described in Section 2. 558: wild type promoter construct with identical sequence as the RPS3 promoter region from − 558 bp to − 1 bp; 558Rm: same as 558 except containing mutant UASrpg3, AACATCgtTACTT; 558uTΔZ: same as 558 except containing internal deletion from − 279 bp to − 262 bp; 558dTΔZ: same as 558 except containing internal deletion from − 240 bp to − 184 bp; 262: wild type promoter construct with identical sequence as the RPS3 promoter region from − 262 bp to + 1 bp. (B) Cells containing the RPS3 reporter plasmid were cultured overnight in SC media and diluted 1:100 in new SC media. After reseeding, cells were harvested on the indicated day and reporter activities were determined (left panel). The data are presented as the ratio of activity in the stationary phase to activity in the log phase (right panel). (C) Two transformants, 558 and 558Rm, treated with 80 mM 3-AT at early log phase were collected for β-galactosidase assay (left panel). The reporter activities after 3AT treatment were normalized to normal conditions (right panel). All experiments were run in triplicate. Error bars indicate standard deviation of each reporter assay. (D) JS143-7D cells transformed with pRS316-558Z, -558uTΔZ, -558dTΔZ, -558RmZ, and -262Z were cultured to an OD600 of 0.5. After 30 min of treatment with 200 ng/ml rapamycin, cells were harvested and total RNA was extracted and subjected to Northern blotting with probe specific to RPS3, LacZ, and ACT1.

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constructs (Fig. 2C). To identify the cis-element responsible for TOR1dependent transcriptional regulation, Northern blotting was performed with transformants containing five mutant constructs (558,

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558uTΔ, 558dTΔ, 558Rm, and 262). Results from this analysis indicated that UASrpg3 as well as both of the upstream and downstream T-rich regions were responsible for transcriptional

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repression upon rapamycin treatment (Fig. 2D). LacZ mRNA of 588 construct as well as endogenous RPS3 expression was decreased significantly by rapamycin treatment. However, mRNA expression of the three mutant constructs was actually slightly increased by rapamycin. 558uTΔ transcription was not detectable, and this result is consistent with the reporter assay showed in Fig 2A. Taken together, these results show that the UASrpg3 and T-rich region are responsible for the basal and TOR1-dependent transcription of RPS3 and that mutation of these elements does not change the pattern of regulation under amino acid starvation or high cell density conditions. The T-rich region may be necessary for full activation of gene transcription in combination with UASrpg3. 3.3. Rap1p directly binds to the RPS3 promoter region Next, to investigate whether Rap1p binds to the core promoter region, EMSA was performed with recombinant His6-tagged Rap1 protein and 32P-labeled DNA probe containing UASrpg3 (Fig. 3A). As more His6-tagged Rap1 was added, the level of protein–DNA binding in the mixture was increased. Furthermore, the amount of remaining free probe was serially decreased, as was protein–DNA complex formation by the addition of cold probe. To identify the binding region, a gel retardation assay was performed with serially deleted mutant dsDNA probes generated by PCR (Fig. 3B). DNA–protein complexes were detected using the probes Ps294, Ps279, and Ps262 and not Ps245. This result suggests that Rap1 binds to the RPS3 promoter region between − 262 bp and −245 bp. We already mentioned that UASrpg3 is located between − 260 bp and − 247 bp (Table 3). Among five putative Rap1 sites, this binding sequence has the highest similarity in reference to the conserved Rap1p binding site with a difference of only one base pair. Moreover, this binding pattern is consistent with the reporter assay in Fig. 1. Since all promoter constructs capable of binding to Rap1 possess some promoter activity, these results collectively show that Rap1p binds to the RPS3 promoter region for the initiation of transcription. Furthermore, to investigate whether any of the other four elements have an additive effect on transcription by Rap1p binding, EMSA competition analysis was performed. As shown in Fig. 3C, only UASrpg3 cold probe could compete with protein–DNA complex formation. UASrpg3 furthermore decreased Rap1-UASWT binding as well as wild type cold probe (cold probe of UASWT). This EMSA result suggests that UASrpg3 is the only functional Rap1p site in the RPS3 promoter region. Next, probes with a mutated UASrpg3 were constructed in order to confirm sequence-specific binding by EMSA (Fig. 4). Both methods of probe construction, annealing with two complementary, single-stranded DNA fragments (Fig. 4A) or PCR with genomic DNA (Fig. 4B), showed that mutant probes bound with much lower affinity than that of wild type. From these results, we revealed that Rap1p acts through UASrpg3 as a functional trans-element for RPS3 transcription. 3.4. Promoter occupancy of RPS3 by Rap1p and other transcriptional activators Many previous studies have revealed that some transcription factors, such as Hmo1p, Sfp1p, Fhl1p, and Ifh1p, were involved in RPG transcription in S. cerevisiae [11,19,44,45]. However, no cis-element derived from an RPG promoter such as the UASrpg of Rap1p has been identified for these factors. It is noteworthy that all these transcriptional activators and repressors, such as Rpd3p, are recruited to the RP promoter in a Rap1p site-dependent manner [10,12,46]. To examine the promoter occupancy of RPS3 by Rap1p and these factors in vivo, strains carrying each epitope tagged with endogenous proteins (TAPRap1, Hmo1-HA3, Sfp1-HA3, Fhl1-HA3, and Ifh1-HA3) were generated. Subsequent ChIP analysis revealed that Rap1p bound strongly to the RPS3 promoter (Fig. 5A). The promoter occupancy of RPS3 by

Fig. 3. EMSA with His6-tagged Rap1 and the RPS3 promoter region. (A) Doublestranded DNA probe containing the RPS3 promoter sequence (− 301 bp to − 232 bp) was generated by annealing with two single-stranded complementary DNAs. EMSA was performed with increasing amounts of His6-tagged Rap1 purified from E. coli (200 ng to 1200 ng) and an appropriate combination of 32P end-labeled DNA probes (0.025 pmol). Increasing amounts of cold competitor of the same sequence were used as a cold probe (10-folds and 50-folds). The DNA–protein complex and unbound probes are indicated by an arrowhead. (B) EMSA of recombinant His6-tagged Rap1 (1200 ng) was performed with serially deleted, 32P end-labeled probes (0.025 pmol) generated by PCR. Each DNA probe contains the RPS3 promoter region. Ps294: − 294 bp to − 156 bp; Ps279: − 279 bp to − 156 bp; Ps262: − 262 bp to − 156 bp; Ps245: − 245 bp to − 156 bp. (C) EMSA of His6-Rap1 (400 ng) was performed with UASWT probe (hot) and indicated cold probe (20-fold to hot probe). Sequences of probes are summarized in Table 2. The sequence of UASWT was artificially generated according to the conserved Rap1p site, and other UASrpg probes contain the identical RPS3 promoter region. The asterisk indicates a nonspecific band.

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Fig. 4. Specific DNA–protein interaction of UASrpg3 from the RPS3 promoter with His6-tagged Rap1. (A) EMSA was performed with probes (corresponding to nucleotide − 301 bp to − 232 bp) harboring wild type UASrpg3 (PRw; ACATCCATACCTTT) or mutant UASrpg3 (PRm; ACATCgtTACCTTT) which were generated by annealing methods described in Fig. 2A and incubated with purified His6-tagged Rap1 protein (600 ng and 1200 ng). (B) EMSA was performed according to the methods in panel A using probes possessing wild type UASrpg3 (PSRw298: − 298 bp to − 156 bp, ACATCCATACCTTT) and mutant UASrpg3 (PSRm298: − 298 bp to − 156 bp, ACATCgtTACCTTT). Three different amounts of recombinant His6tagged Rap1 protein were used (100 ng, 200 ng, and 300 ng).

Hmo1p and Fhl1p was detected, although their amount of coprecipitated chromatin DNA was lower than that of TAP-Rap1 (Figs. 5B and D). However, we could not detect the promoter occupancy of RPS3 by Sfp1p or Ifh1p (Figs. 5C and E). It is possible that binding of these factors was relatively weak or that these factors are involved in other regulatory mechanisms during RP transcription (see the Section 4). The raw quantitative PCR data show that Rap1p, Hmo1p, and Fhl1p clearly associate with the RPS3 promoter (Fig. 5F). These data are in a good agreement with a previous report showing that Rap1p and other factors are recruited to this region in a Rap1p site-dependent manner for transcriptional activation. 3.5. Transcription of RPS3 changes under various stress conditions Expression of the RPS3 gene is important for the physiology of yeast due to its multiple functions previously mentioned. Another study reported that while ribosome biogenesis genes respond rapidly to changes in the environment, they are unresponsive to long-term changes in growth rate [47]. To verify the expression pattern of RPS3, its levels of transcription were examined under various stress conditions such as DNA damage (UV, doxorubicin), TOR kinase inhibition (rapamycin), high cell density, and amino acid starvation (3-AT). Under conditions of DNA damage, RPS3 transcription did not change (Figs. 6A and B). Recently, it was found that the DNA repair activity of rpS3 in humans is regulated by post-translational modification [27,28]. Therefore, it is speculated that the DNA endonuclease activity of yeast Rps3p is also regulated in a posttranscriptional manner, as in protein modification or translocation. However, transcription was severely decreased by treatment with rapamycin or 3-AT, and also mRNA levels in a high cell density state were much lower than that in a low cell density state (Figs. 6C–E). Accordingly, we concluded that RPS3 transcription was finely regulated under various stress conditions in the same manner as other previous reports. Next, to examine the factor-dependent regulation of transcription, Northern blotting was performed with deletion strains of non-essential transcription factors, such as Hmo1p, Sfp1p, Crf1p, and Rpd3p, which are known to be regulators of RP expression (Fig. 6F). Northern blotting found that Sfp1p is required for full transcription under physiological conditions. The basal level RPS3 transcription was reduced by a factor of 2 compared to wild type. Interestingly, it was also revealed that Hmo1p and Crf1p are involved in Tor1-dependent RP transcription. Transcription of RPS3 in the Δhmo1 and Δcrf1 strains was hyposensitive to rapamycin treatment.

However, regulation of RPS3 mRNA under amino acid starvation conditions was not dependent upon trans-elements. Taken together, we conclude that Sfp1p is required for basal RPS3 transcription and that Hmo1p and Crf1p reduce RPS3 transcription upon inhibition of the TOR signaling pathway. 4. Discussion Rap1p is a multifunctional, sequence-specific DNA-binding protein involved in numerous cellular processes such as transcriptional activation and silencing of HMR and HML loci. Moreover, it is an essential factor in the regulation and maintenance of telomere length [48]. Rap1p activates target gene transcription upon binding to UASrpg sequences (ACACCCATACATTT) but does not activate transcription when binding to telomere-like sequences (ACACCCACACACCC) [49]. Generally, the role of Rap1p as an activator appears to be as a “chromatin opener” [9,50]. Here, the structure of the RPS3 promoter in Saccharomyces cerevisiae was investigated, and it was found that a pivotal Rap1p-binding element and the T-rich region act as cis-elements. It was also revealed that Rap1p, Hmo1p and Fhl1p act as trans-elements by associating with the promoter region. We also presented several lines of evidence that Sfp1p and Crf1p are involved in the transcriptional regulation of RPS3. Detailed reporter assays showed that cis-elements are responsible for basal and TOR1-dependent transcription. Therefore, it was suggested that RPS3 is subject to the same transcriptional regulation mechanism by Rap1p and other transcriptional regulators as other RPGs. Interestingly, however, our results present several lines of evidence that other factors are necessary for the activation of RPG. First, we identified a mutant construct with low promoter activity that bound to Rap1p similar to the wild type promoter (Fig. 3C, Ps262). It has been previously reported that the T-rich region is located in the promoter region of RPS3 from −360 bp to −190 bp [32]. Also noteworthy is that a small part just upstream of UASrpg3 plays a pivotal role in RPS3 transcription. In the past, the T-rich sequence downstream of UASrpg was believed to be important for maximal transcription since it prevents nucleosome assembly on the promoter and makes the region more accessible to transcription machinery [39,42]. However, a previous study of the RPS33 gene suggests that the role of the T-rich region is not determined solely by its high thymidine content. Rather, it seems likely that specific sequences are present within the T-rich element [51]. In RPS3, there are upstream and downstream T-rich regions, and it was confirmed that both

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Fig. 5. ChIP analysis for detection of RPS3 promoter occupancy by trans-elements. (A–E) JS143-7D (untagged wild type), YJK109 (TAP-Rap1), YJK123 (Hmo1-HA3), YJK119 (Sfp1-HA3), YJK113 (Fhl1-HA3), and YJK115 (Ifh1-HA3) were cultured to early log phase and harvested for further ChIP analysis. TAP-Rap1 (A) was immunoprecipitated with IgG Sepharose beads and Hmo1-HA3 (B), Sfp1-HA3 (C), Fhl1-HA3 (D), or Ifh1-HA3 (E). Proteins were immunoprecipitated with HA antibody and protein A agarose beads. Input lysates were analyzed by 10% polyacrylamide gel electrophoresis and detected by Western blotting with TAP and HA antibodies. Pgk1p was used as a loading control. Coprecipitated chromatin DNA recovered from immunoprecipitated samples was amplified using primers specific to the RPS3 promoter region containing the T-rich and UASrpg3 elements (JK0646/JK0647), POL1 CDS (JK0450/JK0451). PCR products were analyzed on 1% agarose gel. Untagged wild type strain (JS143-7D) was used as a negative control for immunoprecipitation. (F) Ratio of coprecipitated chromatin DNA determined by semiquantitative PCR was calculated and represented by a graph. ChIP experiments were executed in triplicate with three independent colonies.

regions are responsible for complete transcriptional initiation of RPS3. However, the functional relevance of the upstream T-rich region appears to be more important than that of the downstream T-rich region. This implies that RPS3 transcription relies on the recruitment of specific factors that depend on the T-rich region, especially the upstream region. A recent study proposed that RPGs are regulated by multiple protein factors and mechanisms, rather than by a unified mechanism as previously thought [19]. Considering these facts, it was demonstrated that the T-rich element does not activate RPGs in a prototypical fashion, but rather may mediate transcription in a context-dependent manner. However, as our EMSA assay was performed in vitro, there is a possibility that the in vivo binding of Rap1p with UASrpg of the RPS3 promoter is decreased upon deletion of the upstream T-rich region. Second, we found that mutation of ciselements alters the sensitivity to rapamycin but not to amino acid starvation or high cell density. There is a possibility that stress-specific response elements exist in the RPG promoter. However, based upon recent massive sequence analyses, no such elements have yet been identified. In our experiments, it was revealed that the binding affinity of Rap1p to the 262 construct is identical to that to the 294 or 279

constructs in vitro (Fig. 3C). Therefore, it has been suggested that most RPG-specific transcriptional regulators or mediators are recruited to the RPS3 promoter cooperatively by Rap1p and in a T-rich regiondependent manner under stress conditions. In fact, it was revealed that many transcription apparatuses such as TAFII and Tbp1p bind directly to Rap1p, resulting in transcriptional regulation [52,53]. Moreover, these associations might be changed under stress conditions in yeast cells in order to accommodate the current RPS3 mRNA level. In the case of RPS3, regulatory factors responsible for RPG transcription under amino acid starvation or high cell density conditions appears to function indirectly and independently on two cis-elements, UASrpg and the T-rich region. Finally, this study showed that transcriptional regulators such as Hmo1p, Sfp1p, Fhl1p, and Crf1p appear to be involved in RPS3 transcription during basal and stress conditions. A recent study classified RPS3 as a gene family with a low level of Hmo1p binding [19], and the ChIP result in that study is consistent with our ChIP data (Fig. 5B). Moreover, it was clearly shown that distribution of Rap1 sites relative to start codons in RPG promoters is deeply related with Hmo1p occupancy [10]. In RPS3, the Rap1p site located from −259 bp to − 246 bp is relatively close to the

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Fig. 6. Detection of RPS3 transcript levels under various stress conditions. (A) Exponentially growing cultures of JS143-7D were spread onto Petri dishes and exposed to 40 J/m2 UVC. Control cultures were not exposed. Cells were re-incubated and harvested at the indicated times. Total RNA samples were prepared by hot phenol RNA extraction, followed by electrophoresis of 10 μg of total RNA on 1% formaldehyde/agarose gel. Northern blotting was performed with probes specific to RPS3 and ACT1. (B, C, and E) Exponentially growing cultures of JS143-7D were exposed to 10 μg/ml doxorubicin, 200 ng/μl rapamycin and 80 mM 3-AT. Samples were harvested at the indicated times and total RNA was extracted from each sample. RNA samples (6 μg) were analyzed on 1% formaldehyde agarose gel, followed by Northern blotting with probes specific to RPS3 and ACT1. (D) JS143-7D cells were grown in SC media to early log phase and then harvested, after which the optical density at 600 nm was checked. Six micrograms of total RNA was analyzed on 1% formaldehyde/ agarose gel and Northern blotting was performed with probes specific to RPS3 and ACT1. (F) Wild type and four deletion strains were cultured to early log phase in YPD. Cells were recultured to YPD containing 200 ng/ml of rapamycin or to SC containing minimal concentrations of proper amino acids (0.1×). After 30 min (for control and rapamycin) or 4 hours (for amino acid starvation), cells were harvested and total RNA was extracted. Equal amounts of total RNA (6 μg) were subjected to Northern blot analysis with probes specific to RPS3 and ACT1. Band intensity was determined using a phosphorimager, and ratios of RPS3 to ACT1 were calculated (lower panel). Blotting was performed three times with independent colonies.

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translation start site. Therefore, it was strongly suggested that Hmo1p weakly binds to the RPS3 promoter region, and that Hmo1pdependent transcriptional regulation is only moderate. According to our Northern analysis in Fig. 6F, Hmo1p is involved in transcriptional repression by rapamycin. In the case of Sfp1p, basal transcription is much lower in the deletion strain than in wild type. Although, we could not detect significant association with the RPS3 promoter region, these ChIP results are also consistent with previous reports [16,19]. Recently, it was discovered that Sfp1 interacts directly with TOR complex 1 (TORC1) in a rapamycin-regulated manner whereupon it functions as a negative regulator [45]. This implies that Sfp1p indirectly regulates RPG transcription through direct binding to TORC1. 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