Fission Yeast Tup1-Like Repressors Repress Chromatin Remodeling at the Fbp1؉ Promoter and the Ade6-M26 Recombination Hotspot

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Fission Yeast Tup1-Like Repressors Repress Chromatin Remodeling at the fbp1؉ Promoter and the ade6-M26 Recombination Hotspot

Kouji Hirota,* Charles S. Hoffman,† Takehiko Shibata‡ and Kunihiro Ohta*,‡,§,1 *Genetic Dynamics Research Unit-Laboratory, The Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama 351-0198, Japan, †Biology Department, Boston College, Chestnut Hill, Massachusetts 02467 and ‡Cellular and Molecular Biology Laboratory, The Institute of Physical and Chemical Research (RIKEN)/CREST of Japan Science and Technology Corporation, Wako-shi, Saitama 351-0198, Japan

ABSTRACT Chromatin remodeling plays crucial roles in the regulation of gene expression and recombination. Transcription of the fission yeast fbp1ϩ gene and recombination at the meiotic recombination hotspot ade6-M26 (M26) are both regulated by cAMP responsive element (CRE)-like sequences and the CREB/ ATF-type transcription factor Atf1•Pcr1. The Tup11 and Tup12 proteins, the fission yeast counterparts of the Saccharomyces cerevisiae Tup1 corepressor, are involved in glucose repression of the fbp1ϩ transcription. We have analyzed roles of the Tup1-like corepressors in chromatin regulation around the fbp1ϩ promoter and the M26 hotspot. We found that the chromatin structure around two regulatory elements for fbp1ϩ was remodeled under derepressed conditions in concert with the robust activation of fbp1ϩ transcription. Strains with tup11⌬ tup12⌬ double deletions grown in repressed conditions exhibited the chromatin state associated with wild-type cells grown in derepressed conditions. Interestingly, deletion of rst2ϩ, encoding a transcription factor controlled by the cAMP-dependent kinase, alleviated the tup11⌬ tup12⌬ defects in chromatin regulation but not in transcription repression. The chromatin at the M26 site in mitotic cultures of a tup11⌬ tup12⌬ mutant resembled that of wild-type meiotic cells. These observations suggest that these fission yeast Tup1-like corepressors repress chromatin remodeling at CRE-related sequences and that Rst2 antagonizes this function.

UKARYOTIC chromosomes are packaged into charomyces pombe have shown that a sequence-specific E highly organized and condensed chromatin struc- transcription factor is involved in the activation of mei- tures. Recent studies have revealed that many DNA- otic homologous recombination. The CREB/ATF-type associated processes, such as transcription, replication, transcription factor Atf1•Pcr1 binds to cAMP-responsive repair, and recombination, are finely regulated by chro- element (CRE)-like sequences, including one in the matin structure. These events preferentially occur at fbp1ϩ promoter (Neely and Hoffman 2000). This same accessible chromatin regions that are devoid of posi- heterodimer induces local chromatin remodeling mei- tioned nucleosomes. Modifications of histones and re- otically around a CRE-like sequence in the meiosis-spe- modeling of chromatin structure are induced to form cific recombination hotspot ade6-M26 locus (T. Yamada, such accessible chromatin regions, where DNA-binding K. Mizuno,K.Hirota,N.Kon,W.P.Wahls et al., proteins and protein complexes can be easily recruited unpublished observation). to DNA molecules. The S. cerevisiae Tup1 protein is a global corepressor Transcriptional activators and repressors in eukary- with WD40 repeats that interacts with the Ssn6 protein otes bind to cis-acting regulatory elements, to activate (Varanasi et al. 1996; Redd et al. 1997). The Ssn6-Tup1 or repress transcription by interacting with coactivators complex is involved in the repression of some genes and corepressors, respectively. These complexes regu- regulated by cell type, glucose, oxygen, DNA damages, late the interaction of RNA polymerases and DNA ele- and other cellular stress signals (Roth 1995; Wahi et al. ments within promoters. They are also assumed to alter 1998). This complex regulates expression of numerous chromatin structure around the regulatory elements to genes controlled by a variety of DNA-binding proteins. gain or reduce DNA accessibility to other sequence- Tup1 can bind to histones, histone deacetylases (HDACs), specific transcription factors (Struhl 1995; Ptashne transcriptional regulators, and RNA polymerase II and Gann 1997; Mannervik et al. 1999). Chromatin (Herschbach et al. 1994; Edmondson et al. 1996; Redd structure has been also shown to influence local recom- et al. 1997; Watson et al. 2000; Wu et al. 2001), sug- bination activities. Analyses in the fission yeast Schizosac- gesting potential roles to regulate transcription by mod- ulating chromatin structure and stability of transcription machinery. In fact, the Ssn6-Tup1 complex has 1Corresponding author: Genetic Dynamics Research Unit-Laboratory, The Institute of Physical and Chemical Research (RIKEN), Wako-shi, been shown to establish repressive chromatin structures Saitama 351-0198, Japan. E-mail: [email protected] around promoters (Cooper et al. 1994; Gavin and Simp-

Genetics 165: 505–515 (October 2003) 506 K. Hirota et al. son 1997; Gavin et al. 2000) and to inhibit the function TABLE 1 of the basal transcription machinery (Redd et al. 1997; S. pombe strains used in this study Lee et al. 2000; Zaman et al. 2001). S. pombe has two partially redundant counterparts of Tup1 (Tup11 and Strain Genotype Tup12), which are involved in transcription repression ϩ ϩ K128 h ade6-M26 leu1-32 of the fbp1 gene encoding the fructose-1,6-bis-phos- Ϫ K131 h ade6-M26 leu1-32 phate (Mukai et al. 1999; Janoo et al. 2001). ϩ ϩ ϩ JK39 h ade6-M26 ura4-D18 tup11::ura4 Transcription of the fbp1 gene is regulated in response tup12::ura4ϩ to glucose concentration in the medium (Vassarotti JK40 hϪ ade6-M26 leu1-32 ura4-D18 tup11::ura4ϩ and Friesen 1985; Hoffman and Winston 1989, 1990, tup12::ura4ϩ Ϫ ϩ 1991). When S. pombe cells sense a high concentration JK42 h ade6-M26 leu1-32 ura4-D18 tup11::ura4 Ϫ ϩ of extracellular glucose, they activate the intracellular JK66 h ade6-M26 leu1-32 ura4-D18 tup12::ura4 JK90 hϩ ade6-M375 ura4-D18 tup11::ura4 cAMP signaling pathway (Maeda et al. 1990; Mochizuki ϩ tup12::ura4 and Yamamoto 1992), leading to the activation of the JK107 hϪ ade6-M26 leu1-32 ura4-D18 tup11::ura4ϩ cAMP-dependent kinase [protein kinase A (PKA)]. The tup12::ura4ϩ rst2::KanR activated PKA signal operates to repress transcription JK108 hϪ ade6-M26 leu1-32 ura4-D18 rst2::KanR ϩ of a certain class of genes such as fbp1 (Hoffman and D20 hϩ/hϪ ade6-M26/ade6-M26 his5-3031/his5ϩ ϩ Winston 1991; Byrne and Hoffman 1993; Jin et al. leu1-32/leu1 1995) by inhibiting the function of the transcriptional activators Rst2 (Kunitomo et al. 2000; Higuchi et al. 2002) and Atf1•Pcr1 (Neely and Hoffman 2000). On recombination hotpot. Thus, we suggest that this class the other hand, glucose starvation stimulates the stress- of corepressors regulates diverse biological processes activated protein kinase (SAPK) pathway, leading to the through a common chromatin-related mechanism con- derepression of the fbp1ϩ transcription (Takeda et al. served between S. cerevisiae and S. pombe. 1995; Kanoh et al. 1996; Stettler et al. 1996). The SAPK signal is mediated by the CREB/ATF-type transcription factor Atf1 (Takeda et al. 1995; Kanoh et al. MATERIALS AND METHODS 1996; Shiozaki and Russell 1996; Wilkinson et al. 1996), a basic leucine-zipper (bZIP) phosphoprotein Fission yeast strains, genetic methods, and media: S. pombe that forms a heterodimer with the Pcr1 bZIP protein strains used in this study are listed in Table 1. General genetic (Watanabe and Yamamoto 1996). Transcriptional con- procedures of S. pombe were carried out as described (Gutz ϩ et al. 1974). Minimal medium (SD; Sherman et al. 1986) was trol of the fbp1 gene requires two upstream cis-acting used for the culture of S. pombe unless otherwise stated. Con- elements called UAS1 and UAS2, which include a CRE- struction of the strains was carried out by mating haploids on like and a stress-response element (STRE)-like DNA sporulation medium (SPA; Gutz et al. 1974) followed by tetrad sequence, respectively (Neely and Hoffman 2000). dissection. Standard rich yeast extract medium (YEL; Gutz Aft1•Pcr1 and Rst2 can bind to UAS1 and UAS2, respec- et al. 1974) was used for culturing cells with glucose at the concentration of 8% (repressing condition), 2% (standard cul- tively (Neely and Hoffman 2000; Higuchi et al. 2002). turing condition), or 0.1% (also containing 3% glycerol; dere- Since Tup11 has been shown to bind histone H3 and pressing condition). Transformation was performed by the H4 (Mukai et al. 1999), Tup11 (possibly Tup12 as well) lithium acetate method as described in Hirota et al. (2001). might repress fbp1ϩ transcription by converting chroma- All strains were grown in 200 ml of YEL in 2-liter flasks at Њ r tin structure to repressive states. 30 . To select Kanamycin-resistance (kan ) colonies, culture suspensions were inoculated on YE plates, incubated for 16 The S. pombe ade6-M26 point mutation (M26) creates hr, and then replica plated onto YE plates containing 100 ␮g/ a meiosis-specific recombination hotspot that requires ml of Geneticin (Sigma, St. Louis). the binding of Atf1•Pcr1 to a CRE-like ATGACGT se- Disruption of the rst2؉ gene: The BglII-SphI fragment (0.3 kb) was eliminated from the cloned rst2ϩ sequence and re- quence around the M26 mutation (Gutz 1971; Schu- r chert et al. 1991; Wahls and Smith 1994; Kon et al. placed by the kan gene prepared from the plasmid pFA6a- KanMX (Ba¨hler et al. 1998). The HindIII fragment carrying 1997; Fox et al. 2000). We previously reported that the rst2::kanr was transformed into the wild-type strain (K131) or chromatin structure is remodeled during meiosis to tup⌬⌬ (a double-deletion mutant of tup11ϩ and tup12ϩ) strain form an accessible DNA region around the M26 se- (JK40). Geneticin-resistant transformants were selected, and the disruption of the rst2ϩ allele was confirmed by PCR reac- quence (Mizuno et al. 1997). In addition, such chromatin ϩ remodeling has been shown to be under the regulation tion using primers for the rst2 region. Northern blot analysis: The probes to detect transcripts of of the PKA and the SAPK pathways (Mizuno et al. 2001). fbp1ϩ and cam1ϩ were prepared from PCR products using a In this study, we have analyzed chromatin structure random-priming kit (Amersham, Piscataway, NJ). The nucleo- ϩ around the fbp1 promoter and the M26 recombination tide sequence of each primer is as described below: hotspot in tup11⌬ and tup12⌬ mutant cells. We demon- fbp1-5Ј, TTGCAGGAACAGCGCCG; strate that S. pombe Tup1-like corepressors Tup11 and fbp1-3Ј, GGGATCGCAAGTGACGG; Tup12 have partially redundant roles to regulate chro- cam1-5Ј, CTACCCGTAACCTTACAG; ϩ matin remodeling in the fbp1 promoter and the M26 cam1-3Ј, TGGAAGAAATGACACGAG. Chromatin Regulation by pombe Tup1-Like Repressors 507

The fbp1ϩ promoter is located 1.5 kbp upstream of the ClaI and separated by electrophoresis in a 1.5% agarose gel fbp1ϩ coding region (Neely and Hoffman 2000). Total RNA (40 cm long) containing TAE buffer. The separated DNA was prepared from S. pombe cells according to the method fragments were alkali transferred to charged Nylon mem- described elsewhere (Elder et al. 1983). For Northern blot branes (Biodyne B membrane, PALL, EA). The probe used analysis, 10 ␮g of total RNA was denatured with formamide, for the indirect end labeling of the fbp1ϩ region was the same separated in 1.5% agarose gels containing formaldehyde (Sam- probe used for Northern analysis for the fbp1ϩ transcription. brook et al. 1989), and blotted to a charged Nylon membrane For the ade6 locus, genomic DNA was digested with XhoI followed (Biodyne B membrane, Pall BioSupport). We repeated experi- by Southern analysis using the probe as described (Mizuno et al. ments at least twice and obtained reproducible results. 1997). Chromatin analysis: Analysis of chromatin structure by indirect end labeling was done according to the method of Mizuno et al. (1997). The DNA samples were analyzed by RESULTS Southern analysis as described below. To analyze chromatin around the fbp1ϩ promoter, genomic DNA was digested with Expression of fbp1؉ is derepressed in a tup11 tup12 double mutant especially in late-log and stationary phases: To examine the relationship between transcription activity and chromatin structure at the fbp1ϩ locus, we ﬁrst performed a Northern analysis on the fbp1ϩ transcription in wild-type (K131) and tup11⌬ tup12⌬ double deletion (tup⌬⌬ JK40) strains under repressed or derepressed conditions. Both strains were cultured to the cell density of midlog phase (referred to as M1 and M2 in Figure 1), late-log phase (L), or prestationary phase (S) in YER containing 8% glucose (repressed condition). The cells at midlog phase were further cultured for 3 hr by transferring the cells into YED containing 0.1% glucose (derepressed condition). In the wild-type cells, a robust activation of fbp1ϩ transcription could be detected by Northern analysis only under derepressed condition. On the other hand, the tup⌬⌬ strain cultured in repressed conditions displayed signiﬁcant activation of fbp1ϩ transcription especially after the late-log phase (Figure 1B, lanes L and S, respectively). This is generally consistent with the previous observation by a ␤-galactosidase reporter assay that the derepression of the fbp1ϩ transcription was observed in tup⌬⌬ cultured in YER (Janoo et al. 2001),

Figure 1.—fbp1ϩ transcription in tup11⌬ tup12⌬ (tup⌬⌬) and tup11⌬ tup12⌬ rst2⌬ (tup⌬⌬ rst2⌬) mutants. (A) The wild- type strain (K131) was cultured in YER (containing 8% glucose), and the cell density was monitored by OD600. Cells were harvested at time points indicated by arrows (M1 and M2, midlog phase; L, late-log phase; S, prestationary phase). (B) Results of Northern analysis. The wild-type and tup⌬⌬ (JK40) cells were cultured in YER and some portions of the cells were transferred into YED (containing 0.1% glucose and 3% glycerol) and cultured for 4 hr (M1 glucose Ϫ). The cells were further cultured up to the cell density of midlog phase I (M1), midlog phase II (M2), late-log phase (L), and prestationary phase (S). The expression of fbp1ϩ was analyzed by Northern blotting. Expression of cam1ϩ (Takeda and Yama- moto 1987) was also analyzed and used as an internal control to normalize the expression levels of fbp1ϩ. The data are averages of two independent experiments. Horizontal bars denote standard deviations. (C) Genetic relationship between tup⌬⌬ and rst2⌬. The wild-type cells, tup⌬⌬, rst2⌬ (JK108), and tup⌬⌬ rst2⌬ (JK107) were cultured in YER to midlog phase (M1) and some of the cells were transferred to YED and cultured for 4 hr (glucose Ϫ). The cells were further cultured up to prestationary phase (glucose ϩ). The fbp1ϩ and cam1ϩ transcripts were measured as described in B. The data are averages of two independent experiments. 508 K. Hirota et al. except that derepression of fbp1ϩ transcription was not conditions at midlog (Figure 2A) and later prestationary detected until late-log phase in this study. Possibly, low (data not shown) stages, the intensity of these bands levels of fbp1ϩ transcription activation could be detected around UAS1 becomes relatively lower, while novel efficiently by the ␤-galactosidase reporter assay, because bands appear within the UAS1 region (Figure 2A, short Northern analysis has a relatively higher threshold for dashed line). Under derepressed conditions, very in- detection. tense bands appear in a region between UAS2 and the .(The transcription activator Rst2 is required for fbp1؉ fbp1ϩ coding sequence (Figure 2A, long dashed line transcription but only in the presence of Tup11 and In the tup⌬⌬ cells under repressed conditions at mid- Tup12 corepressors: The Rst2 transcription activator is log stage, weak bands (corresponding to those observed aC2H2 Zn finger protein that is inactivated through its in transcriptionally active chromatin) within the UAS1 phosphorylation by PKA. On the other hand, Tup11 region are already seen (Figure 2A; M1, glucose ϩ) and and Tup12 have been shown to repress the fbp1ϩ tran- constitutively appear under either repressed or dere- scription in a PKA-independent manner (Janoo et al. pressed conditions. Interestingly, chromatin around the 2001). However, it does not exclude the possibility that fbp1ϩ promoter region under repressed condition at Tup11 and Tup12 negatively regulate the access of Rst2 prestationary stage (Figure 2A; S, glucose ϩ) is totally to its target sequence in the fbp1ϩ promoter. To test this remodeled and very similar to that observed under dere- possibility, we examined fbp1ϩ transcription in tup⌬⌬ pressed conditions (see Figure 2A, lanes M1, glucose Ϫ), cells with or without the rst2 deletion by Northern analy- with several very intense bands detected between UAS2 sis (Figure 1C). In tup⌬⌬ cells at prestationary phase, we and the fbp1ϩ coding region. Figure 2A displays the reproducibly detected robust fbp1ϩ transcription under transition of the MNase-sensitivity patterns around the repressed and derepressed conditions, whereas the rst2 fbp1ϩ region in the tup⌬⌬ strain during midlog (M1, deletion severely inhibited fbp1ϩ transcription under M2), late-log (L), and prestationary phases (S) in the both conditions. More importantly, the tup⌬⌬ rst2⌬ tri- presence of glucose. The band intensity within the UAS1 ple mutant displayed substantial fbp1ϩ transcription un- region first increases until late-log phase, but signifi- der both repressed and derepressed conditions. It cantly decreases in stationary phase. On the other hand, should be noted that the transcript levels in the triple the bands around UAS2 become intense from midlog mutant were slightly lower than those in the tup⌬⌬ mu- to stationary phases, whereas significant changes of the tant. These unexpected results indicate the following: band intensity are not detected within both regions in (1) Rst2 is not essential in the activation of fbp1ϩ tran- the wild-type strain (Figure 2A). scription per se; (2) Rst2 is not involved in Tup11-Tup12- Tup11 and Tup12 act as partially redundant repres- dependent transcription repression, since the repression sors of the fbp1 transcription (Mukai et al. 1999; Janoo can occur in the absence of Rst2; and (3) transcription et al. 2001). We next analyzed effects of single deletions activators other than Rst2 are able to activate fbp1ϩ for tup11ϩ and tup12ϩ genes on chromatin structure transcription when the Tup proteins are absent. in the fbp1ϩ promoter. The wild-type (K131), tup11⌬ Inactivation of Tup11 and Tup12 influences local (JK42), and tup12⌬ (JK66) cells were cultured in YER chromatin structure around the fbp1؉ promoter: Previ- containing glucose and harvested at midlog (M1) and ous reports indicated that Tup11 specifically interacts prestationary (S) phases. Each single deletion exhibited with histones H3 and H4 (Mukai et al. 1999). Therefore, intermediate or partial effects on chromatin structure in it was proposed that Tup11 and Tup12 might affect the fbp1ϩ promoter under repressed conditions (Figure chromatin structure to facilitate access of transcription 2B). The MNase-sensitivity patterns in the single mu- factors to their target DNA site. To test this notion, we tants at prestationary phases (lanes tup11⌬ and tup12⌬, employed an indirect end-labeling analysis using partial S) resembled those at the late-log phase of the tup⌬⌬ digestion of chromatin with MNase. This analysis en- double mutant (Figure 2A, lane tup⌬⌬, L). For example, ables us to map positions of individual nucleosomes the bands around UAS2 in the single mutants were not and nuclease-hypersensitive sites. The strains K131 (wild as intense as those observed in the tup⌬⌬ double mu- type) and JK40 (tup⌬⌬) were cultured in YER (glucose ϩ; tant. Taken together, we concluded that Tup11 and 8% glucose) to the cell density of midlog phase (M1). Tup12 play partially redundant roles to repress chroma- Some of the cells were then transferred to YED (glucose Ϫ; tin remodeling in the fbp1ϩ promoter. 0.1% glucose), and the remaining cells were further Rst2 negatively regulates the Tup11-Tup12 functions cultured up to prestationary phase (S). in chromatin regulation: As mentioned above, Tup11 Figure 2 presents the results of the chromatin analysis and Tup12 repress fbp1ϩ expression in an Rst2-indepen- on the fbp1ϩ promoter region. In the wild-type strain dent manner. To examine the genetic relationship be- (K131), chromatin in the UAS1 regions are protected tween Rst2, Tup11, and Tup12 with respect to chroma- from MNase digestion in the repressed conditions at tin structure, we next investigated MNase sensitivity of both midlog and prestationary stages, although a couple chromatin structure around the fbp1ϩ promoter region of intense bands are observed around UAS1 (Figure 2A, in rst2⌬ (JK108) and tup⌬⌬ rst2⌬ (JK107) mutants. In arrowheads). On the other hand, under derepressed the rst2⌬ and tup⌬⌬ rst2⌬ strains, we could not detect Figure 2.—Chromatin structure around the fbp1ϩ promoter in the tup⌬⌬ strain. (A and B) Culture of the cells and the lane coordinates are as described in Figure 1. Lanes are midlog phase I, M1; midlog phase II, M2; late-log phase, L; and prestationary phase, S. The isolated chromatin from each of the cultures was digested with 0, 20, 30, or 50 units/ml of MNase at 37Њ for 5 min. Purified DNA was digested with ClaI (generating a 3.1-kbp parental fragment) and analyzed by Southern blotting as described in materials and methods. The short- and long-dashed lines indicate the regions with MNase-sensitive sites within UAS1 (the open square indicated by UAS1, Ϫ1566 to Ϫ1573 bp from the first A of the fbp1ϩ open reading frame) and around UAS2 (the open square indicated by UAS2, Ϫ926 to Ϫ938 bp), respectively. The arrowheads show three prominent MNase-sensitive sites surrounding UAS1. The open arrow indicates the fbp1ϩ coding regions. The fbp1ϩ promoter (Ϫ1.9 to Ϫ0.4 kbp) is indicated by a large open box. (B) Effects of single tup11⌬ and tup12⌬ mutations on chromatin structure around the fbp1ϩ promoter. The dashed lines are as described above. We repeated two to three independent experiments and obtained similar results. 510 K. Hirota et al.

Figure 3.—Chromatin structure around the fbp1ϩ promoter in tup⌬⌬, rst2⌬, and tup⌬⌬ rst2⌬ strains. Culture of the cells, chromatin analysis, and the lane coordinates are as described in Figure 2. The dashed lines, the open squares, the large open box, and the open arrow are as described in Figure 2. We repeated two independent experiments and obtained similar results. the weak bands within UAS1, characteristic of transcrip- Tup11 and Tup12 influence chromatin structure tionally active chromatin, observed in wild-type and around the ade6-M26 meiotic recombination hotspot: tup⌬⌬ strains even in derepressed conditions (glucose Ϫ), Since S. cerevisiae Tup1 is a global corepressor involved suggesting that Rst2 plays a crucial role in chromatin in repression of numerous genes, we speculated that modification within UAS1 (Figure 3). Tup11 and Tup12 might affect the chromatin structure The chromatin around UAS2 in rst2⌬ cells is similar elsewhere in the fission yeast genome. The ade6-M26 to that observed in wild-type cells. Interestingly, in the (M26) mutant allele is a well-characterized meiotic re- tup⌬⌬ rst2⌬ triple mutant, transition of chromatin struc- combination hotspot containing a base change that re- ture around UAS2 in response to changes in physiologi- sults in a CRE-like ATGACGT sequence (the base change cal conditions is very similar to that observed in the is underlined; Schuchert et al. 1991). We previously wild-type cells. Thus, the deletion of Rst2 suppresses the demonstrated that the chromatin structure around the tup⌬⌬ effects on the chromatin structure around UAS2. M26 hotspot is remodeled during meiosis (Mizuno et This also means that Rst2 is involved in the chromatin al. 1997). The MNase-sensitivity patterns around the changes around UAS2 in tup⌬⌬ cells of early stationary M26 site indicate that chromatin in the ade6 open read- phase with glucose, but not in the chromatin changes ing frame has phased nucleosomes, which result in peri- bp regular-150ف around the same UAS2 region in glucose-starved tup⌬⌬ odical MNase-cleavage patterns with cells. intervals. In early meiotic prophase, the cleavage pat- On the other hand, it should be noted that fbp1ϩ terns become altered, with an intense band appearing transcription in the tup⌬⌬ rst⌬ triple mutant is signifi- at the M26 mutation site (see Figure 4A). We also re- cantly, but not fully activated at stationary phase in the ported that the binding of Atf1•Pcr1 to the CRE-like presence of glucose (see Figure 1C, lane “tup⌬⌬ rst⌬ ATGACGT sequence is required for the chromatin re- glucose ϩ”). This result indicates that extensive chroma- modeling and that the PKA and mitogen-activated pro- tin remodeling is dispensable for the activation of the tein kinase pathways antagonistically regulate the chro- fbp1ϩ transcription, as reported elsewhere in the case of matin remodeling in response to nitrogen starvation the S. cerevisiae SUC2 promoter (Gavin and Simpson 1997). (Mizuno et al. 2001). Thus, we expected that the chro- Chromatin Regulation by pombe Tup1-Like Repressors 511

Figure 4.—Chromatin structure around the ade6-M26 recombination hotspot in the tup⌬⌬ strain. (A) MNase-sensitivity patterns around ade6- M26 during mitosis and meiosis. The diploid wild- type cells (D20) were cultured in PM ϩ N medium to the density of 1 ϫ 107 cells/ml (mitosis). Then the cells were transferred to PM-N and cultured for 3 hr (meiosis). MNase-digested DNA in chromatin was further cleaved with XhoI and analyzed by Southern blotting with the probe for the sequence adjacent to the XhoI site in the 3Ј region of the ade6 coding sequence. The dashed line indicates the sites of chromatin remodeling observed in meiosis. The open arrow indicates the coding region of the ade6-M26 locus. The arrowhead indicates the position of the M26 mutation. (B) The haploid wild-type (K131), tup⌬⌬ (JK40), and tup⌬⌬ rst2⌬ (JK107) cells were cultured and analyzed as described in Figures 1 and 2. Lanes are midlog phase I, M1; midlog phase II, M2; late-log phase, L; and prestationary phase, S. The arrowhead indicates the position of the M26 mutation. (C) Chromatin structure around ade6-M375 in the tup⌬⌬ strain. The wild-type (K128), ade6- M26 (K128), ade6-M26 tup⌬⌬ (JK39), and ade6- M375 tup⌬⌬ (JK90) strains were cultured in YER to midlog phase (M1) and harvested. We repeated two independent experiments and obtained similar results. The arrowhead indicates the position of the M26 mutation. MNase-sensitivity patterns were analyzed as described in A.

matin remodeling at M26 might be affected by tup11 eling at M26, we analyzed the chromatin structure and tup12 mutations. around M26 in the tup⌬⌬ rst2⌬ triple mutant. The chro- The chromatin structure at M26 in tup11ϩ tup12ϩ matin structure around M26 is constitutively modiﬁed cells was compared to that in tup⌬⌬ cells (Figure 4B) to some extent in the tup⌬⌬ rst2⌬ triple mutant (Figure by indirect end labeling on MNase-treated chromatin. 4B), although the intensity of the band at the M26 We detected chromatin changes at the M26 site in hap- mutation in the triple mutant is signiﬁcantly lower than loid tup⌬⌬ cells that had been cultured in the rich those in the tup⌬⌬ strain. Therefore, in comparison ϩ medium YER to the cell density of midlog to late-log with the case of the fbp1 promoter region, Rst2 is less phases. In the same condition, no chromatin alteration important, but still partly involved in the chromatin could be detected in the wild-type haploid. The control regulation around the M26 recombination hotspot. allele ade6-M375 (M375) has no CRE-like sequence, but has the identical termination codon adjacent to the position of the one created by the M26 mutation (Ponti- DISCUSSION celli et al. 1988; Szankasi et al. 1988). Unlike M26, Transcription of the S. pombe fbp1ϩ gene and meiotic M375 does not show recombination hotspot activity, recombination at ade6-M26 are both regulated by the Atf1•Pcr1 binding, and meiotic chromatin remodeling, Atf1•Pcr1 transcription factor, CRE-like sequences, and thereby serving as an excellent negative control (Fox the SAPK and the PKA pathways (Hoffman and Win- et al. 2000). We observed that chromatin alteration in ston 1990; Byrne and Hoffman 1993; Takeda et al. the ade6 locus observed in the M26 tup⌬⌬ strain is not 1995; Stettler et al. 1996). The S. pombe Tup1-like detected in the M375 tup⌬⌬ strain (Figure 4C), sug- corepressors Tup11 and Tup12 have been shown to gesting that the CRE-like sequence is required for the repress transcription of the fbp1ϩ gene (Mukai et al. mitotic chromatin changes in the tup⌬⌬ strain. From 1999; Janoo et al. 2001). In this study, we demonstrate these data, we conclude that Tup11 and Tup12 are that Tup11 and Tup12 contribute to form repressive involved in the establishment of repressive chromatin chromatin structure in the fbp1ϩ promoter and the mei- structure in the M26 recombination hotspot. otic recombination hotspot M26. We also demonstrated To examine the role of Rst2 in the chromatin remod- that the Rst2 transcription activator may antagonize the 512 K. Hirota et al.

matin alteration, which is characterized by appearance of the intense bands around UAS2. This result leads us to speculate that Tup11 and Tup12 can act to repress the fbp1ϩ transcription by both remodeling-dependent and remodeling-independent mechanisms. The latter repression mechanism may involve the inhibition of the basic transcription factors by Tup11 and Tup12 as reported elsewhere in studies of S. cerevisiae Tup1 (Redd et al. 1997; Lee et al. 2000; Zaman et al. 2001). The presence of multiple states in chromatin structure may reveal discrete mechanistic steps for the derepression of fbp1ϩ transcription. The first step may be the enhanced binding of a sequence-specific transcription activator such as Atf1•Pcr1 to UAS1, which may cause a slight and local increase in MNase sensitivity within UAS1. The second step may be induction of more ex- ϩ Figure 5.—Schematic of chromatin structures associated tended chromatin remodeling in the fbp1 promoter ϩ with fbp1 transcription regulation. Three different possible (possibly up to the UAS2 region), which may be pro- states in chromatin structure are depicted in the schematic. moted by ATP-dependent chromatin remodeling fac- The open arrows indicate the fbp1ϩ locus. The dashed and solid arrows represent partially or fully induced transcripts of tors such as Swi/Snf proteins. Such extensive chromatin the fbp1ϩ gene, respectively. Open and shaded circles show remodeling can create chromosomal regions with high stably positioned (in repressive chromatin) and partially re- DNA accessibility, which is favorable for the loading of modeled (in partially derepressed chromatin) nucleosomes, other basic transcription machinery (see Figure 6). respectively. The hatched and shaded boxes indicate UAS1 Roles of Tup11 and Tup12 in chromatin remodeling: and UAS2. Large and small arrowheads indicate the positions of the prominent and the weak MNase-sensitive sites. Tup11 and Tup12 are the S. pombe homologs of the S. cerevisiae Tup1 global corepressor. Tup1 has been shown to repress chromatin remodeling at SUC2 and STE6 Tup11 and Tup12 function at the fbp1ϩ promoter but promoters (Gavin and Simpson 1997; Ducker and may be less significant at the M26 recombination hot- Simpson 2000). The present results are consistent with spot. These results may provide new insights into the com- the functions of Tup1 to repress chromatin remodeling. plex interactions involving signal transduction pathways, Possibly, Tup11 and Tup12 can compete with functions chromatin remodeling activities, and DNA-binding activi- of Swi/Snf-type ATP-dependent chromatin remodeling ties in fission yeast. factors, as proposed in the S. cerevisiae studies (Gavin Multistage response of chromatin structure in the and Simpson 1997; Gavin et al. 2000; Fleming and fbp1؉ promoter: We were able to detect three different Pennings 2001; Proft and Struhl 2002) (see Figure states of chromatin structure in the wild-type and the 6). Analysis of chromatin in mutants defective for poten- tup⌬⌬ strains under repressed and derepressed condi- tial ATP-dependent chromatin remodeling factors tions (see Figure 5). When the wild-type cells are cul- could further clarify these functional interactions. tured in the presence of glucose (M1-S in Figure 2A), Rst2 may antagonize the chromatin repression by ϩ the fbp1 transcription is strictly repressed, and the Tup1-like repressors: Rst2 is a C2H2 Zn finger transcrip- MNase sensitivity is relatively low except for three in- tion activator that specifically binds to a DNA sequence tense cleavage sites surrounding UAS1 (Figure 5, state in UAS2 of the fbp1ϩ promoter [stress-starvation response 1). In the tup⌬⌬ strain under repressing conditions, element of S. pombe (STREP), CCCCTC; Higuchi et al. weak MNase-sensitive sites appear within UAS1 (Figure 2002]. Most simply, Rst2 is considered as a transcription 5, state 2). Under derepressed conditions, the chroma- activator, independent of Tup proteins. However, pres- tin structure in the wild-type and tup⌬⌬ strains results ent results indicate more complex roles for Rst2 in the in strong cleavage sites around UAS2 (Figure 5, state regulation of transcription and chromatin structure of 3). In late-log phase tup⌬⌬ cells cultured with glucose, the fbp1ϩ promoter. The fbp1ϩ promoter chromatin MNase-sensitivity patterns are intermediate between structure in repressed rst2⌬ cells is very similar to that state 2 and state 3. The activation of fbp1ϩ transcription observed in repressed wild-type cells. Surprisingly, dere- is clearly associated with “state 3 chromatin” in wild-type pressed rst2⌬ cells exhibit the same prominent chroma- and tup⌬⌬ strains, thus representing transcriptionally tin changes around UAS2 as observed in wild-type cells active chromatin. However, it should be noted that par- (Figure 3). Therefore, in contrast to the action of the tial activation of the fbp1ϩ transcription was observed S. cerevisiae transcriptional activator Mcm1 at the STE6 in “state 2 chromatin” in the tup⌬⌬ rst2⌬ mutant at promoter (Gavin et al. 2000), Rst2 does not directly stationary phase cultured with glucose, indicating that mediate or involve chromatin remodeling per se. On the transcription can be activated without the massive chro- other hand, the rst2⌬ suppresses the tup⌬⌬ defects in Chromatin Regulation by pombe Tup1-Like Repressors 513

mutant and the wild-type cells, respectively. This means that Mcm1 is required for both transcription activation and chromatin remodeling. On the other hand, Rst2 is not necessary for both. The role of Rst2 should not be similar to the function of Mat␣2 protein, which cooper- ates with Tup1 to block Mcm1-mediated transcriptional activation and chromatin remodeling activity (Cooper et al. 1994), since the rst2⌬ mutation has little effect on fbp1ϩ transcriptional repression (Figure 1C). All these results lead us to propose that Rst2 antagonizes the ability of Tup11-Tup12 to repress chromatin remodeling in the fbp1ϩ promoter (Figure 5). Rst2 may be classified into a new category of transcription activators that antagonize functions of transcription repressors to inhibit chromatin remodeling. It is possible that Rst2 specifically inhibits the function of Tup11 and Tup12 repressors with respect to chromatin regulation. UAS2 may attract transcriptional regulators other than Rst2: The fbp1ϩ expression levels in the rst2⌬ tup⌬⌬ triple mutant were slightly lower than those in the tup⌬⌬ -reduction), suggesting an Rst2-indepen %30ف) strain dent activation mechanism for the fbp1ϩ transcription. It should be noted that the deletion of cgs1, encoding the regulatory subunit of PKA, in tup⌬⌬ resulted in a more -de %90ف) dramatic reduction in the fbp1ϩ expression Figure 6.—A model for the regulation of expression and ϩ crease; Janoo et al. 2001). Thus, other unknown tran- chromatin structure of the fbp1 promoter. A schematic of scriptional activators that may be regulated by the PKA the chromatin structure and transcription regulation under ϩ repressed (top) and derepressed (bottom) conditions in the pathway could be involved in the activation of fbp1 wild-type cells is shown. Open ovals, rounded rectangles, and transcription. This idea is consistent with the previous circles in the nucleus (shown by big ovals) represent Tup11- observations that at least four factors can bind to UAS2 Tup12, transcription factors, and nucleosomes, respectively. (Neely and Hoffman 2000). In addition, transcriptional Cgs1 and PKA in the cytoplasm are shown in open ovals and repressors such as Scr1, which resembles the S. cerevisiae rectangles. The rounded rectangles represent cAMP and Rst2, as indicated. The open arrows and the horizontal arrows indi- Mig1 repressor that recruits Tup1-Ssn6, may act at UAS2 cate the fbp1ϩ locus and fbp1ϩ transcripts, respectively. The (Neely and Hoffman 2000; Janoo et al. 2001). hatched and shaded boxes represent UAS1 and UAS2, respec- From these results, here we propose a model for the tively. regulation of fbp1ϩ transcription (Figure 6). Under repressive conditions, PKA inhibits the Rst2 function through phosphorylation (Higuchi et al. 2002). In the chromatin structure under repressed prestationary con- absence of active Rst2, Tup11 and Tup12 are recruited ϩ ditions. Chromatin structure of the fbp1 promoter in to UAS2 via Scr1 and prevent chromatin remodeling the tup⌬⌬ rst2⌬ triple mutant under repressive condi- in the fbp1ϩ promoter by inhibiting the function of tions was very similar to that observed in the repressed Atf1•Pcr1 to facilitate chromatin remodeling. In repres- wild-type cells in exponentially growing phases (Figure sive chromatin, interactions of other unknown tran- 3). These data suggest that Rst2 function is to reverse scriptional activators to UAS2 would likely be restricted. the effect of Tup11-12 on chromatin structure. When the cells are shifted into derepression conditions, ϩ Transcription of fbp1 is greatly activated in the tup⌬⌬ Cgs1 inhibits the PKA activity, and then Rst2 becomes rst2⌬ mutant under repressed prestationary conditions activated, possibly by dephosphorylation and transloca- ϩ (Figure 1C), but the chromatin structure of the fbp1 tion into the nucleus, to bind UAS2 and inhibit the promoter still exhibits the “repressed state 1 chromatin” Tup11-Tup12 function that represses chromatin remod- (Figure 3), as mentioned above. Under the same condi- eling. At the same time, possibly through the activation tion, no transcription activation is observed in the wild- by the PKA and SAPK signals, Atf1•Pcr1 is also activated type cells. This is in contrast to the role of the S. cerevisiae and facilitates chromatin remodeling in the fbp1ϩ pro- Mcm1 protein in STE6 transcription activation (Gavin moter. After the chromatin remodeling, unknown tran- et al. 2000). STE6 transcription is derepressed in the tup1⌬ scription activators can easily interact with UAS2, lead- mutant, although the STE6 transcription levels in the ing to transcriptional activation of fbp1ϩ. In this model, tup1⌬ mutant with a mutation of the Mcm1-binding site Rst2 contributes to a more sensitive response of fbp1ϩ are 8- and 16-fold lower than those in the single tup1⌬ transcription to the PKA signals, compared to the regu- 514 K. Hirota et al. lation system solely by Atf1•Pcr1. It would be interesting transcriptional regulators, SSN6 and TUP1, play distinct roles in the establishment of a repressive chromatin structure. Genes Dev. to study the relationship between the Tup11-Tup12 co- 8: 1400–1410. repressors and the PKA-SAPK signaling pathways in fu- Ducker, C. E., and R. T. Simpson, 2000 The organized chromatin ture work. domain of the repressed yeast a cell-specific gene STE6 contains two molecules of the corepressor Tup1p per nucleosome. EMBO Roles of global corepressors in chromatin control of J. 19: 400–409. the M26 meiotic recombination hotspot: The present Edmondson, D. G., M. M. Smith and S. Y. Roth, 1996 Repression results indicate that Tup11-Tup12 play an important domain of the yeast global repressor Tup1 interacts directly with histones H3 and H4. Genes Dev. 10: 1247–1259. role in chromatin regulation at the M26 meiotic recom- Elder, R. T., E. Y. Loh and R. W. Davis, 1983 RNA from the yeast bination hotspots. Establishment of high DNA accessi- transposable element Ty1 has both ends in the direct repeats, a bility through chromatin remodeling has been demon- structure similar to retrovirus RNA. Proc. Natl. Acad. Sci. USA 80: 2432–2436. strated to be important for recombination regulation Fleming, A. B., and S. Pennings, 2001 Antagonistic remodelling by as well as transcription activation (Nicolas 1998; Petes Swi-Snf and Tup1-Ssn6 of an extensive chromatin region forms 2001). 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