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

Corresponding author: Kunihiro Ohta, The Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama 351-0198, Japan., kohta{at}postman.riken.go.jp (E-mail)

Communicating editor: P. RUSSELL

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Chromatin remodeling plays crucial roles in the regulation of gene expression and recombination. Transcription of the fission yeast fbp1⁺ 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 fbp1⁺ transcription. We have analyzed roles of the Tup1-like corepressors in chromatin regulation around the fbp1⁺ promoter and the M26 hotspot. We found that the chromatin structure around two regulatory elements for fbp1⁺ was remodeled under derepressed conditions in concert with the robust activation of fbp1⁺ 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 rst2⁺, 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.

EUKARYOTIC chromosomes are packaged into highly organized and condensed chromatin structures. Recent studies have revealed that many DNA-associated processes, such as transcription, replication, repair, and recombination, are finely regulated by chromatin structure. These events preferentially occur at accessible chromatin regions that are devoid of positioned nucleosomes. Modifications of histones and remodeling of chromatin structure are induced to form such accessible chromatin regions, where DNA-binding proteins and protein complexes can be easily recruited to DNA molecules.

Transcriptional activators and repressors in eukaryotes bind to cis-acting regulatory elements, to activate or repress transcription by interacting with coactivators and corepressors, respectively. These complexes regulate the interaction of RNA polymerases and DNA elements within promoters. They are also assumed to alter chromatin structure around the regulatory elements to gain or reduce DNA accessibility to other sequence-specific transcription factors (STRUHL 1995 ; PTASHNE and GANN 1997 ; MANNERVIK et al. 1999 ). Chromatin structure has been also shown to influence local recombination activities. Analyses in the fission yeast Schizosaccharomyces pombe have shown that a sequence-specific transcription factor is involved in the activation of meiotic homologous recombination. The CREB/ATF-type transcription factor Atf1•Pcr1 binds to cAMP-responsive element (CRE)-like sequences, including one in the fbp1⁺ promoter (NEELY and HOFFMAN 2000 ). This same heterodimer induces local chromatin remodeling meiotically around a CRE-like sequence in the meiosis-specific recombination hotspot ade6-M26 locus (T. YAMADA, K. MIZUNO, K. HIROTA, N. KON, W. P. WAHLS et al., unpublished observation).

The S. cerevisiae Tup1 protein is a global corepressor with WD40 repeats that interacts with the Ssn6 protein (VARANASI et al. 1996 ; REDD et al. 1997 ). The Ssn6-Tup1 complex is involved in the repression of some genes regulated by cell type, glucose, oxygen, DNA damages, and other cellular stress signals (ROTH 1995 ; WAHI et al. 1998 ). This complex regulates expression of numerous genes controlled by a variety of DNA-binding proteins. Tup1 can bind to histones, histone deacetylases (HDACs), transcriptional regulators, and RNA polymerase II (HERSCHBACH et al. 1994 ; EDMONDSON et al. 1996 ; REDD et al. 1997 ; WATSON et al. 2000 ; WU et al. 2001 ), suggesting potential roles to regulate transcription by modulating chromatin structure and stability of transcription machinery. In fact, the Ssn6-Tup1 complex has been shown to establish repressive chromatin structures around promoters (COOPER et al. 1994 ; GAVIN and SIMPSON 1997 ; GAVIN et al. 2000 ) and to inhibit the function of the basal transcription machinery (REDD et al. 1997 ; LEE et al. 2000 ; ZAMAN et al. 2001 ). S. pombe has two partially redundant counterparts of Tup1 (Tup11 and Tup12), which are involved in transcription repression of the fbp1⁺ gene encoding the fructose-1,6-bis-phosphate (MUKAI et al. 1999 ; JANOO et al. 2001 ).

Transcription of the fbp1⁺ gene is regulated in response to glucose concentration in the medium (VASSAROTTI and FRIESEN 1985 ; HOFFMAN and WINSTON 1989 , HOFFMAN and WINSTON 1990 , HOFFMAN and WINSTON 1991 ). When S. pombe cells sense a high concentration of extracellular glucose, they activate the intracellular cAMP signaling pathway (MAEDA et al. 1990 ; MOCHIZUKI and YAMAMOTO 1992 ), leading to the activation of the cAMP-dependent kinase [protein kinase A (PKA)]. The activated PKA signal operates to repress transcription of a certain class of genes such as fbp1⁺ (HOFFMAN and WINSTON 1991 ; BYRNE and HOFFMAN 1993 ; JIN et al. 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 the other hand, glucose starvation stimulates the stress-activated protein kinase (SAPK) pathway, leading to the derepression of the fbp1⁺ transcription (TAKEDA et al. 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. 1996 ; SHIOZAKI and RUSSELL 1996 ; WILKINSON et al. 1996 ), a basic leucine-zipper (bZIP) phosphoprotein that forms a heterodimer with the Pcr1 bZIP protein (WATANABE and YAMAMOTO 1996 ). Transcriptional control of the fbp1⁺ gene requires two upstream cis-acting elements called UAS1 and UAS2, which include a CRE-like and a stress-response element (STRE)-like DNA sequence, respectively (NEELY and HOFFMAN 2000 ). Aft1•Pcr1 and Rst2 can bind to UAS1 and UAS2, respectively (NEELY and HOFFMAN 2000 ; HIGUCHI et al. 2002 ). Since Tup11 has been shown to bind histone H3 and H4 (MUKAI et al. 1999 ), Tup11 (possibly Tup12 as well) might repress fbp1⁺ transcription by converting chromatin structure to repressive states.

The S. pombe ade6-M26 point mutation (M26) creates a meiosis-specific recombination hotspot that requires the binding of Atf1•Pcr1 to a CRE-like ATGACGT sequence around the M26 mutation (GUTZ 1971 ; SCHUCHERT et al. 1991 ; WAHLS and SMITH 1994 ; KON et al. 1997 ; FOX et al. 2000 ). We previously reported that the chromatin structure is remodeled during meiosis to form an accessible DNA region around the M26 sequence (MIZUNO et al. 1997 ). In addition, such chromatin remodeling has been shown to be under the regulation of the PKA and the SAPK pathways (MIZUNO et al. 2001 ).

In this study, we have analyzed chromatin structure around the fbp1⁺ promoter and the M26 recombination hotspot in tup11 and tup12 mutant cells. We demonstrate that S. pombe Tup1-like corepressors Tup11 and Tup12 have partially redundant roles to regulate chromatin remodeling in the fbp1⁺ promoter and the M26 recombination hotpot. Thus, we suggest that this class of corepressors regulates diverse biological processes through a common chromatin-related mechanism conserved between S. cerevisiae and S. pombe.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Fission yeast strains, genetic methods, and media:
S. pombe strains used in this study are listed in Table 1. General genetic procedures of S. pombe were carried out as described (GUTZ et al. 1974 ). Minimal medium (SD; SHERMAN et al. 1986 ) was used for the culture of S. pombe unless otherwise stated. Construction of the strains was carried out by mating haploids on sporulation medium (SPA; GUTZ et al. 1974 ) followed by tetrad dissection. Standard rich yeast extract medium (YEL; GUTZ et al. 1974 ) was used for culturing cells with glucose at the concentration of 8% (repressing condition), 2% (standard culturing condition), or 0.1% (also containing 3% glycerol; derepressing condition). Transformation was performed by the lithium acetate method as described in HIROTA et al. 2001 . All strains were grown in 200 ml of YEL in 2-liter flasks at 30°. To select Kanamycin-resistance (kan^r) colonies, culture suspensions were inoculated on YE plates, incubated for 16 hr, and then replica plated onto YE plates containing 100 µg/ml of Geneticin (Sigma, St. Louis).

Disruption of the rst2⁺ gene:
The BglII-SphI fragment (0.3 kb) was eliminated from the cloned rst2⁺ sequence and replaced by the kan^r gene prepared from the plasmid pFA6aKanMX (BAHLER et al. 1998 ). The HindIII fragment carrying rst2::kan^r was transformed into the wild-type strain (K131) or tup (a double-deletion mutant of tup11⁺ and tup12⁺) strain (JK40). Geneticin-resistant transformants were selected, and the disruption of the rst2⁺ allele was confirmed by PCR reaction using primers for the rst2⁺ region.

Northern blot analysis:
The probes to detect transcripts of fbp1⁺ and cam1⁺ were prepared from PCR products using a random-priming kit (Amersham, Piscataway, NJ). The nucleotide sequence of each primer is as described below:

• fbp1-5', TTGCAGGAACAGCGCCG;

• fbp1-3', GGGATCGCAAGTGACGG;

• cam1-5', CTACCCGTAACCTTACAG;

• cam1-3', TGGAAGAAATGACACGAG.

The fbp1⁺ promoter is located 1.5 kbp upstream of the fbp1⁺ coding region (NEELY and HOFFMAN 2000 ). Total RNA was prepared from S. pombe cells according to the method described elsewhere (ELDER et al. 1983 ). For Northern blot analysis, 10 µg of total RNA was denatured with formamide, separated in 1.5% agarose gels containing formaldehyde (SAMBROOK et al. 1989 ), and blotted to a charged Nylon membrane (Biodyne B membrane, Pall BioSupport). We repeated experiments at least twice and obtained reproducible results.

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 Southern analysis as described below. To analyze chromatin around the fbp1⁺ promoter, genomic DNA was digested with ClaI and separated by electrophoresis in a 1.5% agarose gel (40 cm long) containing TAE buffer. The separated DNA fragments were alkali transferred to charged Nylon membranes (Biodyne B membrane, PALL, EA). The probe used for the indirect end labeling of the fbp1⁺ region was the same probe used for Northern analysis for the fbp1⁺ transcription. For the ade6 locus, genomic DNA was digested with XhoI followed by Southern analysis using the probe as described (MIZUNO et al. 1997 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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 fbp1⁺ locus, we first performed a Northern analysis on the fbp1⁺ 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 Fig 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).

View larger version (28K): In this window In a new window Download PPT slide   Figure 1. fbp1+ 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 -). 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 fbp1+ was analyzed by Northern blotting. Expression of cam1+ (TAKEDA and YAMAMOTO 1987 ) was also analyzed and used as an internal control to normalize the expression levels of fbp1+. 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 -). The cells were further cultured up to prestationary phase (glucose +). The fbp1+ and cam1+ transcripts were measured as described in B. The data are averages of two independent experiments.

In the wild-type cells, a robust activation of fbp1⁺ 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 fbp1⁺ transcription especially after the late-log phase (Fig 1B, lanes L and S, respectively). This is generally consistent with the previous observation by a ß-galactosidase reporter assay that the derepression of the fbp1⁺ transcription was observed in tup cultured in YER (JANOO et al. 2001 ), except that derepression of fbp1⁺ transcription was not detected until late-log phase in this study. Possibly, low levels of fbp1⁺ transcription activation could be detected efficiently by the ß-galactosidase reporter assay, because Northern analysis has a relatively higher threshold for detection.

The transcription activator Rst2 is required for fbp1⁺ transcription but only in the presence of Tup11 and Tup12 corepressors:
The Rst2 transcription activator is a C₂H₂ Zn finger protein that is inactivated through its phosphorylation by PKA. On the other hand, Tup11 and Tup12 have been shown to repress the fbp1⁺ transcription in a PKA-independent manner (JANOO et al. 2001 ). However, it does not exclude the possibility that Tup11 and Tup12 negatively regulate the access of Rst2 to its target sequence in the fbp1⁺ promoter. To test this possibility, we examined fbp1⁺ transcription in tup cells with or without the rst2 deletion by Northern analysis (Fig 1C). In tup cells at prestationary phase, we reproducibly detected robust fbp1⁺ transcription under repressed and derepressed conditions, whereas the rst2 deletion severely inhibited fbp1⁺ transcription under both conditions. More importantly, the tup rst2 triple mutant displayed substantial fbp1⁺ transcription under both repressed and derepressed conditions. It should be noted that the transcript levels in the triple mutant were slightly lower than those in the tup mutant. These unexpected results indicate the following: (1) Rst2 is not essential in the activation of fbp1⁺ transcription per se; (2) Rst2 is not involved in Tup11-Tup12-dependent transcription repression, since the repression can occur in the absence of Rst2; and (3) transcription activators other than Rst2 are able to activate fbp1⁺ transcription when the Tup proteins are absent.

Inactivation of Tup11 and Tup12 influences local chromatin structure around the fbp1⁺ promoter:
Previous reports indicated that Tup11 specifically interacts with histones H3 and H4 (MUKAI et al. 1999 ). Therefore, it was proposed that Tup11 and Tup12 might affect chromatin structure to facilitate access of transcription factors to their target DNA site. To test this notion, we employed an indirect end-labeling analysis using partial digestion of chromatin with MNase. This analysis enables us to map positions of individual nucleosomes and nuclease-hypersensitive sites. The strains K131 (wild type) and JK40 (tup) were cultured in YER (glucose +; 8% glucose) to the cell density of midlog phase (M1). Some of the cells were then transferred to YED (glucose -; 0.1% glucose), and the remaining cells were further cultured up to prestationary phase (S).

Fig 2 presents the results of the chromatin analysis on the fbp1⁺ promoter region. In the wild-type strain (K131), chromatin in the UAS1 regions are protected from MNase digestion in the repressed conditions at both midlog and prestationary stages, although a couple of intense bands are observed around UAS1 (Fig 2A, arrowheads). On the other hand, under derepressed conditions at midlog (Fig 2A) and later prestationary (data not shown) stages, the intensity of these bands around UAS1 becomes relatively lower, while novel bands appear within the UAS1 region (Fig 2A, short dashed line). Under derepressed conditions, very intense bands appear in a region between UAS2 and the fbp1⁺ coding sequence (Fig 2A, long dashed line).

View larger version (52K): In this window In a new window Download PPT slide   Figure 2. Chromatin structure around the fbp1+ promoter in the tup strain. (A and B) Culture of the cells and the lane coordinates are as described in Fig 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 37° 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, -1566 to -1573 bp from the first A of the fbp1+ open reading frame) and around UAS2 (the open square indicated by UAS2, -926 to -938 bp), respectively. The arrowheads show three prominent MNase-sensitive sites surrounding UAS1. The open arrow indicates the fbp1+ coding regions. The fbp1+ promoter (-1.9 to -0.4 kbp) is indicated by a large open box. (B) Effects of single tup11 and tup12 mutations on chromatin structure around the fbp1+ promoter. The dashed lines are as described above. We repeated two to three independent experiments and obtained similar results.

In the tup cells under repressed conditions at midlog stage, weak bands (corresponding to those observed in transcriptionally active chromatin) within the UAS1 region are already seen (Fig 2A; M1, glucose +) and constitutively appear under either repressed or derepressed conditions. Interestingly, chromatin around the fbp1⁺ promoter region under repressed condition at prestationary stage (Fig 2A; S, glucose +) is totally remodeled and very similar to that observed under derepressed conditions (see Fig 2A, lanes M1, glucose -), with several very intense bands detected between UAS2 and the fbp1⁺ coding region. Fig 2A displays the transition of the MNase-sensitivity patterns around the fbp1⁺ region in the tup strain during midlog (M1, M2), late-log (L), and prestationary phases (S) in the presence of glucose. The band intensity within the UAS1 region first increases until late-log phase, but significantly decreases in stationary phase. On the other hand, the bands around UAS2 become intense from midlog to stationary phases, whereas significant changes of the band intensity are not detected within both regions in the wild-type strain (Fig 2A).

Tup11 and Tup12 act as partially redundant repressors of the fbp1 transcription (MUKAI et al. 1999 ; JANOO et al. 2001 ). We next analyzed effects of single deletions for tup11⁺ and tup12⁺ genes on chromatin structure in the fbp1⁺ promoter. The wild-type (K131), tup11 (JK42), and tup12 (JK66) cells were cultured in YER containing glucose and harvested at midlog (M1) and prestationary (S) phases. Each single deletion exhibited intermediate or partial effects on chromatin structure in the fbp1⁺ promoter under repressed conditions (Fig 2B). The MNase-sensitivity patterns in the single mutants at prestationary phases (lanes tup11 and tup12, S) resembled those at the late-log phase of the tup double mutant (Fig 2A, lane tup, L). For example, the bands around UAS2 in the single mutants were not as intense as those observed in the tup double mutant. Taken together, we concluded that Tup11 and Tup12 play partially redundant roles to repress chromatin remodeling in the fbp1⁺ promoter.

Rst2 negatively regulates the Tup11-Tup12 functions in chromatin regulation:
As mentioned above, Tup11 and Tup12 repress fbp1⁺ expression in an Rst2-independent manner. To examine the genetic relationship between Rst2, Tup11, and Tup12 with respect to chromatin structure, we next investigated MNase sensitivity of chromatin structure around the fbp1⁺ promoter region in rst2 (JK108) and tup rst2 (JK107) mutants. In the rst2 and tup rst2 strains, we could not detect the weak bands within UAS1, characteristic of transcriptionally active chromatin, observed in wild-type and tup strains even in derepressed conditions (glucose -), suggesting that Rst2 plays a crucial role in chromatin modification within UAS1 (Fig 3).

View larger version (70K): In this window In a new window Download PPT slide   Figure 3. Chromatin structure around the fbp1+ promoter in tup, rst2, and tup rst2 strains. Culture of the cells, chromatin analysis, and the lane coordinates are as described in Fig 2. The dashed lines, the open squares, the large open box, and the open arrow are as described in Fig 2. We repeated two independent experiments and obtained similar results.

The chromatin around UAS2 in rst2 cells is similar to that observed in wild-type cells. Interestingly, in the tup rst2 triple mutant, transition of chromatin structure around UAS2 in response to changes in physiological conditions is very similar to that observed in the wild-type cells. Thus, the deletion of Rst2 suppresses the tup effects on the chromatin structure around UAS2. This also means that Rst2 is involved in the chromatin changes around UAS2 in tup cells of early stationary phase with glucose, but not in the chromatin changes around the same UAS2 region in glucose-starved tup cells.

On the other hand, it should be noted that fbp1⁺ transcription in the tup rst triple mutant is significantly, but not fully activated at stationary phase in the presence of glucose (see Fig 1C, lane "tup rst glucose +"). This result indicates that extensive chromatin remodeling is dispensable for the activation of the fbp1⁺ transcription, as reported elsewhere in the case of the S. cerevisiae SUC2 promoter (GAVIN and SIMPSON 1997 ).

Tup11 and Tup12 influence chromatin structure around the ade6-M26 meiotic recombination hotspot:
Since S. cerevisiae Tup1 is a global corepressor involved in repression of numerous genes, we speculated that Tup11 and Tup12 might affect the chromatin structure elsewhere in the fission yeast genome. The ade6-M26 (M26) mutant allele is a well-characterized meiotic recombination hotspot containing a base change that results in a CRE-like ATGACGT sequence (the base change is underlined; SCHUCHERT et al. 1991 ). We previously demonstrated that the chromatin structure around the M26 hotspot is remodeled during meiosis (MIZUNO et al. 1997 ). The MNase-sensitivity patterns around the M26 site indicate that chromatin in the ade6 open reading frame has phased nucleosomes, which result in periodical MNase-cleavage patterns with 150-bp regular intervals. In early meiotic prophase, the cleavage patterns become altered, with an intense band appearing at the M26 mutation site (see Fig 4A). We also reported that the binding of Atf1•Pcr1 to the CRE-like ATGACGT sequence is required for the chromatin remodeling and that the PKA and mitogen-activated protein kinase pathways antagonistically regulate the chromatin remodeling in response to nitrogen starvation (MIZUNO et al. 2001 ). Thus, we expected that the chromatin remodeling at M26 might be affected by tup11 and tup12 mutations.

View larger version (87K): In this window In a new window Download PPT slide   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 + N medium to the density of 1 x 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 3' 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 Fig 1 and Fig 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.

The chromatin structure at M26 in tup11⁺ tup12⁺ cells was compared to that in tup cells (Fig 4B) by indirect end labeling on MNase-treated chromatin. We detected chromatin changes at the M26 site in haploid tup cells that had been cultured in the rich medium YER to the cell density of midlog to late-log phases. In the same condition, no chromatin alteration could be detected in the wild-type haploid. The control 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 (PONTICELLI et al. 1988 ; SZANKASI et al. 1988 ). Unlike M26, M375 does not show recombination hotspot activity, Atf1•Pcr1 binding, and meiotic chromatin remodeling, thereby serving as an excellent negative control (FOX et al. 2000 ). We observed that chromatin alteration in the ade6 locus observed in the M26 tup strain is not detected in the M375 tup strain (Fig 4C), suggesting that the CRE-like sequence is required for the mitotic chromatin changes in the tup strain. From these data, we conclude that Tup11 and Tup12 are involved in the establishment of repressive chromatin structure in the M26 recombination hotspot.

To examine the role of Rst2 in the chromatin remodeling at M26, we analyzed the chromatin structure around M26 in the tup rst2 triple mutant. The chromatin structure around M26 is constitutively modified to some extent in the tup rst2 triple mutant (Fig 4B), although the intensity of the band at the M26 mutation in the triple mutant is significantly lower than those in the tup strain. Therefore, in comparison with the case of the fbp1⁺ promoter region, Rst2 is less important, but still partly involved in the chromatin regulation around the M26 recombination hotspot.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Transcription of the S. pombe fbp1⁺ gene and meiotic recombination at ade6-M26 are both regulated by the Atf1•Pcr1 transcription factor, CRE-like sequences, and the SAPK and the PKA pathways (HOFFMAN and WINSTON 1990 ; BYRNE and HOFFMAN 1993 ; TAKEDA et al. 1995 ; STETTLER et al. 1996 ). The S. pombe Tup1-like corepressors Tup11 and Tup12 have been shown to repress transcription of the fbp1⁺ gene (MUKAI et al. 1999 ; JANOO et al. 2001 ). In this study, we demonstrate that Tup11 and Tup12 contribute to form repressive chromatin structure in the fbp1⁺ promoter and the meiotic recombination hotspot M26. We also demonstrated that the Rst2 transcription activator may antagonize the Tup11 and Tup12 function at the fbp1⁺ promoter but may be less significant at the M26 recombination hotspot. These results may provide new insights into the complex interactions involving signal transduction pathways, chromatin remodeling activities, and DNA-binding activities in fission yeast.

Multistage response of chromatin structure in the fbp1⁺ promoter:
We were able to detect three different states of chromatin structure in the wild-type and the tup strains under repressed and derepressed conditions (see Fig 5). When the wild-type cells are cultured in the presence of glucose (M1-S in Fig 2A), the fbp1⁺ transcription is strictly repressed, and the MNase sensitivity is relatively low except for three intense cleavage sites surrounding UAS1 (Fig 5, state 1). In the tup strain under repressing conditions, weak MNase-sensitive sites appear within UAS1 (Fig 5, state 2). Under derepressed conditions, the chromatin structure in the wild-type and tup strains results in strong cleavage sites around UAS2 (Fig 5, state 3). In late-log phase tup cells cultured with glucose, MNase-sensitivity patterns are intermediate between state 2 and state 3. The activation of fbp1⁺ transcription is clearly associated with "state 3 chromatin" in wild-type and tup strains, thus representing transcriptionally active chromatin. However, it should be noted that partial activation of the fbp1⁺ transcription was observed in "state 2 chromatin" in the tup rst2 mutant at stationary phase cultured with glucose, indicating that transcription can be activated without the massive chromatin 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 fbp1⁺ 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 ).

View larger version (31K): In this window In a new window Download PPT slide   Figure 5. Schematic of chromatin structures associated with fbp1+ transcription regulation. Three different possible states in chromatin structure are depicted in the schematic. The open arrows indicate the fbp1+ locus. The dashed and solid arrows represent partially or fully induced transcripts of the fbp1+ gene, respectively. Open and shaded circles show stably positioned (in repressive chromatin) and partially remodeled (in partially derepressed chromatin) nucleosomes, respectively. The hatched and shaded boxes indicate UAS1 and UAS2. Large and small arrowheads indicate the positions of the prominent and the weak MNase-sensitive sites.

The presence of multiple states in chromatin structure may reveal discrete mechanistic steps for the derepression of fbp1⁺ 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 extended chromatin remodeling in the fbp1⁺ promoter (possibly up to the UAS2 region), which may be promoted by ATP-dependent chromatin remodeling factors such as Swi/Snf proteins. Such extensive chromatin remodeling can create chromosomal regions with high DNA accessibility, which is favorable for the loading of other basic transcription machinery (see Fig 6).

View larger version (29K): In this window In a new window Download PPT slide   Figure 6. A model for the regulation of expression and chromatin structure of the fbp1+ promoter. A schematic of the chromatin structure and transcription regulation under repressed (top) and derepressed (bottom) conditions in the wild-type cells is shown. Open ovals, rounded rectangles, and circles in the nucleus (shown by big ovals) represent Tup11-Tup12, transcription factors, and nucleosomes, respectively. Cgs1 and PKA in the cytoplasm are shown in open ovals and rectangles. The rounded rectangles represent cAMP and Rst2, as indicated. The open arrows and the horizontal arrows indicate the fbp1+ locus and fbp1+ transcripts, respectively. The hatched and shaded boxes represent UAS1 and UAS2, respectively.

Roles of Tup11 and Tup12 in chromatin remodeling:
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 promoters (GAVIN and SIMPSON 1997 ; DUCKER and SIMPSON 2000 ). The present results are consistent with the functions of Tup1 to repress chromatin remodeling. Possibly, Tup11 and Tup12 can compete with functions of Swi/Snf-type ATP-dependent chromatin remodeling factors, as proposed in the S. cerevisiae studies (GAVIN and SIMPSON 1997 ; GAVIN et al. 2000 ; FLEMING and PENNINGS 2001 ; PROFT and STRUHL 2002 ) (see Fig 6). Analysis of chromatin in mutants defective for potential ATP-dependent chromatin remodeling factors could further clarify these functional interactions.

Rst2 may antagonize the chromatin repression by Tup1-like repressors:
Rst2 is a C₂H₂ Zn finger transcription activator that specifically binds to a DNA sequence in UAS2 of the fbp1⁺ promoter [stress-starvation response element of S. pombe (STREP), CCCCTC; HIGUCHI et al. 2002]. Most simply, Rst2 is considered as a transcription activator, independent of Tup proteins. However, present results indicate more complex roles for Rst2 in the regulation of transcription and chromatin structure of the fbp1⁺ promoter. The fbp1⁺ promoter chromatin structure in repressed rst2 cells is very similar to that observed in repressed wild-type cells. Surprisingly, derepressed rst2 cells exhibit the same prominent chromatin changes around UAS2 as observed in wild-type cells (Fig 3). Therefore, in contrast to the action of the S. cerevisiae transcriptional activator Mcm1 at the STE6 promoter (GAVIN et al. 2000 ), Rst2 does not directly mediate or involve chromatin remodeling per se. On the other hand, the rst2 suppresses the tup defects in chromatin structure under repressed prestationary conditions. Chromatin structure of the fbp1⁺ promoter in the tup rst2 triple mutant under repressive conditions was very similar to that observed in the repressed wild-type cells in exponentially growing phases (Fig 3). These data suggest that Rst2 function is to reverse the effect of Tup11-12 on chromatin structure.

Transcription of fbp1⁺ is greatly activated in the tup rst2 mutant under repressed prestationary conditions (Fig 1C), but the chromatin structure of the fbp1⁺ promoter still exhibits the "repressed state 1 chromatin" (Fig 3), as mentioned above. Under the same condition, no transcription activation is observed in the wild-type cells. This is in contrast to the role of the S. cerevisiae Mcm1 protein in STE6 transcription activation (GAVIN et al. 2000 ). STE6 transcription is derepressed in the tup1 mutant, although the STE6 transcription levels in the tup1 mutant with a mutation of the Mcm1-binding site are 8- and 16-fold lower than those in the single tup1 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 Mat2 protein, which cooperates with Tup1 to block Mcm1-mediated transcriptional activation and chromatin remodeling activity (COOPER et al. 1994 ), since the rst2 mutation has little effect on fbp1⁺ transcriptional repression (Fig 1C).

All these results lead us to propose that Rst2 antagonizes the ability of Tup11-Tup12 to repress chromatin remodeling in the fbp1⁺ promoter (Fig 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 fbp1⁺ expression levels in the rst2 tup triple mutant were slightly lower than those in the tup strain (30% reduction), suggesting an Rst2-independent activation mechanism for the fbp1⁺ transcription. It should be noted that the deletion of cgs1, encoding the regulatory subunit of PKA, in tup resulted in a more dramatic reduction in the fbp1⁺ expression (90% decrease; JANOO et al. 2001 ). Thus, other unknown transcriptional activators that may be regulated by the PKA pathway could be involved in the activation of fbp1⁺ transcription. This idea is consistent with the previous observations that at least four factors can bind to UAS2 (NEELY and HOFFMAN 2000 ). In addition, transcriptional repressors such as Scr1, which resembles the S. cerevisiae Mig1 repressor that recruits Tup1-Ssn6, may act at UAS2 (NEELY and HOFFMAN 2000 ; JANOO et al. 2001 ).

From these results, here we propose a model for the regulation of fbp1⁺ transcription (Fig 6). Under repressive conditions, PKA inhibits the Rst2 function through phosphorylation (HIGUCHI et al. 2002 ). In the absence of active Rst2, Tup11 and Tup12 are recruited to UAS2 via Scr1 and prevent chromatin remodeling in the fbp1⁺ promoter by inhibiting the function of Atf1•Pcr1 to facilitate chromatin remodeling. In repressive chromatin, interactions of other unknown transcriptional activators to UAS2 would likely be restricted. When the cells are shifted into derepression conditions, Cgs1 inhibits the PKA activity, and then Rst2 becomes activated, possibly by dephosphorylation and translocation into the nucleus, to bind UAS2 and inhibit the Tup11-Tup12 function that represses chromatin remodeling. At the same time, possibly through the activation by the PKA and SAPK signals, Atf1•Pcr1 is also activated and facilitates chromatin remodeling in the fbp1⁺ promoter. After the chromatin remodeling, unknown transcription activators can easily interact with UAS2, leading to transcriptional activation of fbp1⁺. In this model, Rst2 contributes to a more sensitive response of fbp1⁺ transcription to the PKA signals, compared to the regulation system solely by Atf1•Pcr1. It would be interesting to study the relationship between the Tup11-Tup12 corepressors and the PKA-SAPK signaling pathways in future work.

Roles of global corepressors in chromatin control of the M26 meiotic recombination hotspot:
The present results indicate that Tup11-Tup12 play an important role in chromatin regulation at the M26 meiotic recombination hotspots. Establishment of high DNA accessibility through chromatin remodeling has been demonstrated to be important for recombination regulation as well as transcription activation (NICOLAS 1998 ; PETES 2001 ). Therefore, Tup1-like corepressors are proposed to establish repressive chromatin around the M26 recombination hotspots to prevent activation of recombination during exponential growing stages. This idea may at least partly account for the reason that the M26 hotspot is activated only during meiosis, while meiotic activation of M26 needs meiosis-specific expression of some recombination genes. Analysis of the M26 recombination activity in tup diploids would be interesting. However, we have been unable to examine M26 recombination hotspot activity in mitotic tup diploids, since the tup mutants were extremely unstable in diploid state.

We found that Rst2 is only partially involved in chromatin regulation in the M26 recombination hotspot, while Rst2 is required for the full level of chromatin remodeling at M26. The partial involvement of Rst2 in chromatin remodeling at M26 may be due to the lack of potential strong binding sites for Rst2 in the ade6-M26 locus. It is possible that Rst2 may interact with the Tup1-like corepressors even in the absence of a consensus target DNA sequence, although we cannot exclude a possibility that weak cryptic Rst2 binding sites are present in the ade6-M26 locus. It would be interesting in the future to investigate more detailed molecular mechanisms of the Tup11 and Tup12 corepressors to repress chromatin remodeling under various physiological conditions.

	ACKNOWLEDGMENTS

We thank H. Murakami, T. Yamada, and H. Seo for critically reading the manuscript. This work was supported by grants from the Human Frontier Science Program; the "Bioarchitect Research Program" of the Institute of Physical and Chemical Research; Core Research for Evolutional Science and Technology program of Japan Science and Technology Corporation; the Ministry of Education, Science, Culture, and Sports, Japan; and by a research grant from the National Institutes of Health (GM46226 to C.S.H.).

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