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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 Copyright 2003 by the Genetics Society of America 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 transcrip- tion 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 tran- scription 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.
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