Counteracting Regulation of Chromatin Remodeling at a Fission Yeast cAMP Responsive Element-Related Recombination Hotspot by Stress-Activated Protein Kinase, cAMP-Dependent Kinase and Meiosis Regulators

Corresponding author: Kunihiro Ohta, Genetic Dynamics Research Unit-Laboratory, RIKEN (The Institute of Physical and Chemical Research), Hirosawa 2-1, Wako, Saitama 351-0198, Japan., kohta{at}postman.riken.go.jp (E-mail)

Communicating editor: M. LICHTEN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In fission yeast, an ATF/CREB-family transcription factor Atf1-Pcr1 plays important roles in the activation of early meiotic processes via the stress-activated protein kinase (SAPK) and the cAMP-dependent protein kinase (PKA) pathways. In addition, Atf1-Pcr1 binds to a cAMP responsive element (CRE)-like sequence at the site of the ade6-M26 mutation, which results in local enhancement of meiotic recombination and chromatin remodeling. Here we studied the roles of meiosis-inducing signal transduction pathways in M26 chromatin remodeling. Chromatin analysis revealed that persistent activation of PKA in meiosis inhibited M26 chromatin remodeling, suggesting that the PKA pathway represses M26 chromatin remodeling. The SAPK pathway activated M26 chromatin remodeling, since mutants lacking a component of this pathway, the Wis1 or Spc1/Sty1 kinases, had no M26 chromatin remodeling. M26 chromatin remodeling also required the meiosis regulators Mei2 and Mei3 but not the subsequently acting regulators Sme2 and Mei4, suggesting that induction of M26 chromatin remodeling needs meiosis-inducing signals before premeiotic DNA replication. Similar meiotic chromatin remodeling occurred meiotically around natural M26 heptamer sequences. These results demonstrate the coordinated action of genetic and physiological factors required to remodel chromatin in preparation for high levels of meiotic recombination and eukaryotic cellular differentiation.

DNA accessibility in chromatin plays a pivotal role in eukaryotic gene regulation (WOLFFE 1994 ; TSUKIYAMA and WU 1997 ). Establishment of high DNA accessibility through chromatin remodeling has a crucial role also in the spatial and temporal regulation of recombination initiation (NICOLAS 1998 ; PETES 2001 ), replication (SIMPSON 1990 ), and DNA repair (THOMA 1999 ). For instance, in the budding yeast Saccharomyces cerevisiae, an early event of meiotic recombination, the formation of DNA double strand breaks (DSBs), preferentially occurs at specific chromosomal sites (recombination hotspots) that show high DNA accessibility in chromatin (reviewed in PETES 2001 ). DSB sites are often found in promoter regions on S. cerevisiae chromosomes (BAUDAT and NICOLAS 1997 ), and the high DNA accessibility at DSB sites is at least in part established by certain cis-acting sequences and trans-acting transcription factors (OHTA et al. 1994 ; WU and LICHTEN 1994 ; FAN and PETES 1996 ). However, little is known about intracellular signals that regulate DNA accessibility for recombination activation during meiosis, a highly differentiated stage of fungi and other organisms.

In the fission yeast Schizosaccharomyces pombe, a single G to T nucleotide substitution ade6-M26 activates meiotic homologous recombination at the ade6 locus (called the M26 recombination hotspot; GUTZ 1971 ; SZANKASI et al. 1988 ; FOX and SMITH 1998 ). The substitution creates a heptanucleotide sequence (5'-ATGACGT-3'; underline indicates the M26 mutation), which is essential to recombination hotspot activity and which is closely related to the cAMP response element (CRE) consensus sequence (SCHUCHERT et al. 1991 ; FOX et al. 2000 ; Fig 1A). The earliest inferred event in M26 hotspot activation is the binding to the M26 heptamer sequence of a heterodimeric protein Atf1 (also known as Gad7)-Pcr1 (WAHLS and SMITH 1994 ; KON et al. 1997 ), a member of the activating transcription factor (ATF)/ CRE-binding protein (CREB) family (TAKEDA et al. 1995 ; KANOH et al. 1996 ; SHIOZAKI and RUSSELL 1996 ; WATANABE and YAMAMOTO 1996 ). We previously reported that the M26 mutation causes local remodeling of chromatin structure around the M26 site in meiosis (MIZUNO et al. 1997 ). This is characterized by micrococcal nuclease (MNase) hypersensitivity at M26 and nucleosome disordering in >600 bp at the ade6 locus. The M26 mutation also meiotically enhances MNase hypersensitivity at the ade6 promoter and M26 sites (MIZUNO et al. 1997 ). Since chromatin remodeling requires the M26 heptamer (MIZUNO et al. 1997 ) and both subunits of Atf1-Pcr1 (K. OHTA, K. MIZUNO, N. KON, T. SHIBATA and W. P. WAHLS, unpublished observations), it is likely that Atf1-Pcr1 binding to the heptamer sequence directly mediates M26 chromatin remodeling as well as M26 hotspot activation (KON et al. 1997 ).

View larger version (149K): In this window In a new window Download PPT slide   Figure 1. The PKA pathway negatively regulates M26 chromatin remodeling. (A) DNA sequences of ade6+, ade6-M375 (negative control), and ade6-M26 (recombination hotspot) alleles. White letters indicate G to T transversions that create stop codons near the 5' end of the gene, and the box indicates the heptameric DNA site required for hotspot activity (SCHUCHERT et al. 1991 ). (B) Chromatin structure of ade6-M375 and ade6-M26 in vegetative growth and meiosis. Chromatin was prepared from ade6-M26 (ELD205) and ade6-M375 (ELD203) diploid cells grown in a rich medium, YEL (V), a medium for presporulation conditioning, PM (P), and a sporulation medium, PM-N (S). The isolated chromatin was digested with 20 units/ml of MNase at 37° for 5 min. Purified DNA was XhoI digested and displayed on Southern blots. Probe 1 was used for indirect end labeling. The diagram indicates the ade6 open reading frame (vertical arrows) and the positions of the M375 and M26 mutations (solid and open triangles). Numbers show the positions in nucleotides of the XhoI, BamHI, and HindIII sites and MNase-sensitive sites relative to the first A of the ade6 coding sequence. Open and hatched ovals represent phased and randomly positioned nucleosomes, respectively. Lanes M, -, and N represent DNAsize markers (1953, 1443, and 627 bp), chromatin DNA without MNase digestion, and naked DNA treated with 3 units/ml of MNase, respectively. Note the MNase-hypersensitive site at the M26 site (marked by an open circle) and perturbed positioning of nucleosomes downstream of the M26 site (a dashed line) in sporulation medium (M26, lane S). (C) Suppression of M26 chromatin remodeling by addition of cAMP. ade6-M26 (ELD205) and ade6-M375 (ELD203) diploid cells were cultured in PM medium (lanes 0). The cells were shifted to sporulation medium with or without 10 mM cAMP and cultured for 3 hr (lanes 3). Schematic illustrations for the estimated nucleosome positions are as described in B. (D) M26 chromatin remodeling requires the cgs1+ gene. The ade6-M26 (ELD205), ade6-M375 (ELD203), ade6-M26 cgs1 (KX73), and ade6-M375 cgs1 (KX74) diploid cells were cultured in PM medium to 1 x 107 cells/ml (lanes 0), shifted to sporulation medium, and cultured for 3 hr (lanes 3). Chromatin preparation, partial digestion with MNase, and Southern blot analysis were performed as in Fig 1B. Schematic illustrations for the estimated nucleosome positions are as described in Fig 1B.

Atf1-Pcr1 also plays pivotal roles in S. pombe sexual differentiation and response to environmental stresses (TAKEDA et al. 1995 ; KANOH et al. 1996 ; SHIOZAKI and RUSSELL 1996 ; WATANABE and YAMAMOTO 1996 ; KON et al. 1998 ). A subset of the family of mitogen-activated protein kinases (MAPKs) is designated the stress-activated protein kinase (SAPK) family. These kinases are involved in cellular responses to various environmental stresses. They include S. cerevisiae Hog1, metazoan JNK/SAPK (DAVIS 2000 ), and p38/CSBP/RK (NEBREDA and PORRAS 2000 ). The functions of Atf1-Pcr1 are regulated by the S. pombe SAPK signal transduction pathway (KANOH et al. 1996 ; SHIOZAKI and RUSSELL 1996 ; WILKINSON et al. 1996 ; KON et al. 1998 ). Wis1 and Spc1 (also known as Sty1 or Phh1) are the MAP kinase-kinase and MAP kinase in the S. pombe SAPK cascade, respectively (WILKINSON and MILLAR 1998 ). Wis1 phosphorylates Spc1/Sty1 (MILLAR et al. 1995 ; SHIOZAKI and RUSSELL 1995 ) in response to various environmental stresses such as nitrogen starvation and high osmolarity. Spc1/Sty1 then phosphorylates Atf1 to activate transcription for entry into meiosis and adaptation to environmental stresses (SHIOZAKI and RUSSELL 1995 , SHIOZAKI and RUSSELL 1996 ; DEGOLS et al. 1996 ; STETTLER et al. 1996 ; WILKINSON et al. 1996 ). The phosphorylation of Atf1 by Spc1/Sty1 is assumed to regulate positively the M26 hotspot activity as well as the initiation of meiosis, since disruption of spc1/sty1 or wis1 abolishes the M26 hotspot activity (KON et al. 1998 ; FOX et al. 2000 ).

The intracellular levels of cAMP and the protein kinase A (PKA) pathway also play crucial roles in the regulation of S. pombe meiosis induction (WATANABE et al. 1988 ; MOCHIZUKI and YAMAMOTO 1992 ; YAMAMOTO 1996 ). The intracellular cAMP concentration decreases at the time of meiosis induction (MOCHIZUKI and YAMAMOTO 1992 ). Induction of meiosis is blocked by the addition of cAMP to the sporulation medium (BEACH et al. 1985 ), which increases the intracellular concentration of cAMP, or by disruption of the regulatory subunit (cgs1) of PKA (DEVOTI et al. 1991 ). On the other hand, disruption of the PKA catalytic subunit causes destabilization of the diploid cell cycle, and the cells start meiosis under conditions repressive for wild-type meiosis (MAEDA et al. 1994 ). In addition, the phosphorylation of Atf1 is affected by the disruption of PKA (KANOH et al. 1996 ).

Induction of meiosis in S. pombe involves other signal transduction pathways and regulatory factors. In wild-type cells, meiosis can be initiated only when cells are heterozygous at the mating-type locus (h⁺/h^-; AONO et al. 1994 ). In such cells, starvation activates expression of the mei3 gene (MCLEOD and BEACH 1988 ; VAN HEECKEREN et al. 1998 ). Mei3 inhibits the Pat1 kinase (LI and MCLEOD 1996 ), a negative regulator for meiosis induction (BEACH et al. 1985 ; IINO and YAMAMOTO 1985 ). Inactivation of the Pat1 kinase by Mei3 then results in activation of Mei2, a key positive regulator for meiosis initiation and progression (BEACH et al. 1985 ; SHIMODA et al. 1987 ; WATANABE et al. 1988 , WATANABE et al. 1997 ). Mei2 is phosphorylated and inactivated by Pat1 kinase in rich growth media, but dephosphorylated Mei2 accumulates after Pat1 inactivation. Dephosphorylated (active) Mei2 then generates a signal to start premeiotic DNA replication. Thereafter, to trigger meiosis I, Mei2 forms a complex with MeiRNA encoded by sme2 (WATANABE and YAMAMOTO 1994 ), which facilitates entry of Mei2 into the nucleus and localization of Mei2 to a dot-like intranuclear structure (YAMASHITA et al. 1998 ). These meiosis-inducing signaling pathways in S. pombe offer an excellent model system to study cellular differentiation of eukaryotic cells.

These signaling pathways for meiosis induction are assumed to regulate meiotic recombination (and possibly transcription at certain loci) at the level of chromatin structure, since recombination activation and chromatin remodeling at the CRE-related M26 heptamer are specific to meiotic cells. In addition, some of these signaling pathways regulate the function of Atf1-Pcr1 described above. However, little is known about the relationship between these pathways and the regulation of chromatin remodeling.

We studied the roles of the meiosis-inducing signaling pathways in the remodeling of chromatin at the M26 site and found the following: (1) M26 chromatin remodeling is regulated positively by the SAPK pathway, but negatively by the PKA signaling pathway; (2) M26 chromatin remodeling requires meiosis-inducing signals that are activated by the mating pheromone signaling pathway; (3) M26 chromatin remodeling requires early signals also needed for premeiotic DNA replication but not signals for the later steps; and (4) similar chromatin remodeling can occur at natural CRE sites. It is suggested that regulation of chromatin remodeling by the signal transduction pathways is an important determinant of meiotic recombination activation at CRE-related sequences. In addition, the present findings may provide important insights for understanding regulatory mechanisms of meiotic recombination activation and cellular differentiation at the chromatin level.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Media, sporulation, and strains:
A list of strains is in Table 1. Media and sporulation conditions were as described unless stated otherwise (WATANABE et al. 1988 ; MIZUNO et al. 1997 ). Rich yeast extract medium (MIZUNO et al. 1997 ) was used for the preparation of chromatin from vegetatively growing cells (Fig 1B). For synchronous pat1-114^ts meiosis, strains were cultured at 24° in presporulation medium (PM) and then shifted to 34° as described (IINO and YAMAMOTO 1985 ).

Chromatin analysis:
Analysis of chromatin structure by indirect end labeling was as described (MIZUNO et al. 1997 ). To analyze chromatin around the ctt1 gene, genomic DNA was digested with HindIII for the ctt1 gene and ScaI for the downstream site. Probes used for the indirect end labeling were prepared as follows. For the ade6 locus, plasmid pade6-M26 (SZANKASI et al. 1988 ) was digested with XhoI and the resultant 1.95-kbp fragment was subcloned into the XhoI site of pBluescript (KS+). Probe 1 was prepared from this plasmid (pX2kM26) by digestion with XhoI-EcoRI followed by the purification of the 240-bp fragment in agarose gels. Hybridization probes 71 (294-bp fragment) and 18 (227-bp fragment) were amplified by polymerase chain reaction (PCR), using S. pombe genomic DNA as a template with primer pairs (71F, 5'-CGGCCTCTAAAGGCAATCCG-3'; 71R, 5'-CCTTCAATCTTTGTTTGACG-3') and (18F, 5'-CACTTATATGGGACTTTAGTTCG-3'; 18R, 5'-CTGCTCGGAAATTTCTCTTTC-3'), respectively. The amplified fragments were gel purified before labeling. Nucleosome integrity was analyzed similarly, except that digestion of genomic DNA with XhoI was omitted and a DNA fragment spanning the M26 site or the downstream sites was used as a radioactive probe for Southern hybridization. One microgram of DNA was loaded on each lane for Southern blot analyses. We repeated experiments at least three times and confirmed the reproducibility of all the data unless otherwise stated.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

M26 chromatin remodeling is negatively controlled by the PKA pathway and can be triggered by nutritional starvation:
We previously demonstrated that nucleosomes around the M26 site become rearranged during meiosis (MIZUNO et al. 1997 ). Fig 1B illustrates differences of the nucleosome positioning between ade6-M26 and a negative control ade6-M375, which creates an identical stop codon adjacent to that created by ade6-M26 (Fig 1A). In chromatin from the control (ade6-M375) and from wild-type ade6⁺ (data not shown), we observed an MNase sensitivity pattern characteristic of phased nucleosomes within the coding region of ade6 (periodic appearance of sensitive sites at 150-bp intervals). However, the M26 mutation reduced the nucleosome phasing in premeiotic cells and abolished it during meiosis (MIZUNO et al. 1997 ). In the present study, we define M26 chromatin remodeling as an event with two components: (1) appearance of a MNase-hypersensitive site at the M26 site (Fig 1B, position 170) and (2) perturbation in the positioning of nearby nucleosomes.

To study whether M26 chromatin remodeling occurs constitutively or is temporally regulated, we examined chromatin structure around the M26 site under different physiological conditions (Fig 1B). In diploid cells growing in a rich medium, the pattern of nucleosome positioning at ade6-M26 was indistinguishable from that in wild-type cells and the ade6-M375 mutant (See Fig 1B, lanes V). However, in early stationary phase of a presporulation culture under partially starved condition, the chromatin at ade6-M26 became partially rearranged in diploid cells (See Fig 1B, lanes P). These results suggested that M26 chromatin remodeling might be regulated under physiological conditions.

Starvation reduces the intracellular cAMP level and suppresses the PKA activity (MOCHIZUKI and YAMAMOTO 1992 ; MAEDA et al. 1994 ). Therefore, induction of M26 chromatin remodeling during starvation led us to speculate that the PKA pathway might be involved in regulation of M26 chromatin remodeling. To test this possibility, we examined the effects of adding cAMP to the sporulation medium, which increases the intracellular cAMP concentration, on M26 chromatin remodeling. The addition of cAMP completely inhibited M26 chromatin remodeling as long as 3 hr after transferring cells into the sporulation medium, at which time chromatin is fully remodeled around the M26 site in a normal sporulation medium without cAMP (Fig 1C).

We next examined the role of the PKA pathway in M26 chromatin remodeling. Since diploid cells lacking the catalytic subunit of protein kinase A (Pka1) are very unstable and prone to form haploid cells spontaneously (MAEDA et al. 1994 ), we could not analyze chromatin structure in diploid cells lacking Pka1. Thus, alternatively, we examined the effects of disruption of cgs1⁺, which encodes the regulatory (inhibitory) subunit for PKA. The binding of cAMP to the regulatory subunit causes its dissociation from the catalytic subunit, so that PKA becomes active in the presence of a higher concentration of cAMP, for example, in cells growing vegetatively in a rich medium. On the other hand, when cells face nutritional starvation, the intracellular cAMP concentration decreases and the activity of Pka1 is reduced (MOCHIZUKI and YAMAMOTO 1992 ; MAEDA et al. 1994 ). We found that a cgs1 mutant, in which PKA activity is constitutively high (DEVOTI et al. 1991 ), failed to cause M26 chromatin remodeling even in sporulation medium, an extremely starved condition (Fig 1D). We confirmed that cgs1 mutants (and also other mutants as described below) with ade6+ and ade6-M375 did not undergo such chromatin remodeling (data not shown). These results suggest that the PKA pathway negatively regulates M26 chromatin remodeling.

Positive regulation of M26 chromatin remodeling by the SAPK pathway:
Nutritional starvation activates the S. pombe SAPK cascade (MILLAR et al. 1995 ; SHIOZAKI and RUSSELL 1995 ; STETTLER et al. 1996 ). To study whether M26 chromatin remodeling is regulated by the SAPK pathway, we analyzed the chromatin at ade6-M26 in mutants with deletion of wis1⁺ (MAP kinase-kinase) or spc1⁺ (MAP kinase; Fig 2). Even 3 hr after the cells were transferred to sporulation medium, no M26 chromatin remodeling could be detected in the wis1 and spc1 mutants. These results suggest that the SAPK pathway positively regulates M26 chromatin remodeling during meiosis induction.

View larger version (59K): In this window In a new window Download PPT slide   Figure 2. M26 chromatin remodeling is positively regulated by the SAPK pathway. Chromatin from ade6-M26 (ELD205), ade6-M26 wis1 (KX71), and ade6-M26 spc1 (GP2737) diploid cells was analyzed. Culture conditions, chromatin preparation, partial digestion with MNase, and Southern blot analysis were performed as in Fig 1B. Lane N indicates MNase digestion of naked DNA. Note that the M26 chromatin remodeling was not observed in wis1 or spc1 genes even at 3 hr after a shift into sporulation medium. Schematic illustrations for the estimated nucleosome positions are as described in Fig 1B.

Regulation of M26 chromatin remodeling by the mating pheromone signaling pathway:
In cells heterozygous for the mating-type locus, the mating pheromone signaling pathway is activated by starvation (YAMAMOTO 1996 ). To study the role of the mating pheromone signaling pathway in M26 chromatin remodeling, we first examined chromatin structure in haploid and diploid strains with various genotypes at the mating-type locus. As shown in Fig 3, homozygosity for the mating-type locus prevents remodeling of chromatin around the M26 site. To confirm that heterozygosity for the mating types is required for M26 chromatin remodeling, we analyzed chromatin in special haploid strains expressing both mating-type genes (h⁺ and h^-), in which meiosis can be induced in response to nitrogen starvation (BAHLER et al. 1993 ). Haploid cells with both mating types could undergo M26 chromatin remodeling (Fig 3). Therefore, M26 chromatin remodeling requires heterozygosity at the mating-type locus but seems to operate independently of the ploidy of the cell and the existence of homologous chromosomes.

View larger version (68K): In this window In a new window Download PPT slide   Figure 3. M26 chromatin remodeling requires heterozygosity at the mating-type locus. Chromatin in ade6-M26 haploid cells (K7 and K8) and diploid cells (ELD205, K3, and K4) was analyzed (left). Haploid cells heterozygous for mating type (ade6-M26, K65) were also analyzed (right). As negative and positive controls, the h+/h- ade6-M375 and ade6-M26 diploid cells (ELD203 and ELD205) were used. The cells were cultured in PM medium to 1 x 107 cells/ml. Isolated chromatin was treated with 50 units/ml of MNase. Mating types are indicated by + (h+) and - (h-). Schematic illustrations for the estimated nucleosome positions are as described in Fig 1B.

Heterozygosity at the mating-type locus induces Mei3 during starvation. Activated Mei3 further inactivates Pat1 kinase, which normally suppresses meiosis induction in vegetative growth conditions (BEACH et al. 1985 ; IINO and YAMAMOTO 1985 ; MCLEOD and BEACH 1988 ; LI and MCLEOD 1996 ; WATANABE et al. 1997 ). To confirm that the mating pheromone-signaling pathway is involved in the regulation of the M26 chromatin remodeling, we further analyzed chromatin in the mei3 and pat1-114^ts strains (Fig 4A and Fig B). In a mei3 diploid, M26 chromatin remodeling was barely detectable in sporulation medium. On the other hand, we detected M26 chromatin remodeling in the pat1-114^ts mutant under nonpermissive temperature even in haploid cells. Taking these results together, we conclude that the mating pheromone signaling pathway, via its effects on Mei3 and Pat1 kinases, positively regulates M26 chromatin remodeling.

View larger version (59K): In this window In a new window Download PPT slide   Figure 4. M26 chromatin remodeling in meiosis requires the functions of mei3+ and mei2+, but not sme2+ or mei4+ genes. All schematic illustrations for the estimated nucleosome positions are as described in Fig 1B. Culture, chromatin preparation, partial digestion with MNase, and Southern blot analysis were performed as in Fig 1B, unless otherwise stated. (A) Deletion of the mei3+ gene abolishes M26 chromatin remodeling. Chromatin from ade6- M26 (ELD205) and ade6-M26 mei3 (UX23) diploid cells was analyzed. (B) M26 chromatin remodeling occurs in a pat1-114 mutant at the restrictive temperature. The pat1-114 mutant with ade6-M26 (strain K47, lanes M26) was cultured in presporulation medium (PM) at 24° to 1 x 107 cells/ml (lanes 0) and shifted to 34° for 3 hr to induce meiosis (lanes 3). (C) Mei2 is required for M26 chromatin remodeling. Chromatin from ade6-M26 (ELD205) and ade6-M26 mei2 (UX21) diploid cells was examined. (D) Sme2 is dispensable for M26 chromatin remodeling. Isolated chromatin from ade6-M26 (ELD205) and ade6-M26 sme2 (KX91) diploid cells was digested with 30 units/ml of MNase. (E) Mei4 is dispensable for M26 chromatin remodeling. Chromatin from ade6-M26 (ELD205) and ade6-M26 mei4-B2 (K225) diploid cells was digested with 20 units/ml of MNase.

Regulation by meiosis regulators and the timing of M26 chromatin remodeling:
We next examined the roles of Mei2, Sme2, and Mei4 in M26 chromatin remodeling. The meiosis regulatory gene mei2 is induced by the action of a transcription factor Ste11 (SUGIMOTO et al. 1991 ), and Mei2 is activated by abolition of the Pat1-mediated phosphorylation on Mei2 (WATANABE et al. 1997 ). The activated Mei2 protein allows cells to initiate premeiotic DNA synthesis. Thereafter, RNA encoded by sme2 binds to Mei2 to facilitate its nuclear localization and the formation of the Mei2 dot in the nucleus, which is essential for the progression of S. pombe to meiosis I, although Sme2 RNA itself is dispensable for the function of Mei2 in meiosis I (WATANABE et al. 1997 ; YAMASHITA et al. 1998 ). Mei4 is a transcription factor required for progression of meiosis I and later steps of sporulation (HORIE et al. 1998 ; ABE and SHIMODA 2000 ). Thus, using these mutants, we can estimate genetically the timing of M26 chromatin remodeling.

We found that Mei2 was required for M26 chromatin remodeling, while Sme2 and Mei4 were dispensable (Fig 4, C–E). Therefore, we conclude that M26 chromatin remodeling requires only the early functions of Mei2 and occurs between the first activation stage of Mei2 (before premeiotic DNA replication) and meiosis I.

Chromatin remodeling occurs at natural heptamer sequences:
To study whether the mechanism underlying M26 chromatin remodeling is general or specific to the M26 site, we further tested whether such chromatin remodeling occurs around two natural M26 heptamer sequences present in the S. pombe genome, located 21.5 kbp downstream and 0.43 kbp upstream of the catalase gene ctt1⁺ (Fig 5A). Transcription of ctt1 is activated by Atf1 under stressed conditions (NAKAGAWA et al. 2000 ; NGUYEN et al. 2000 ), suggesting that chromatin remodeling occurs also around these heptamer sequences under nutritional starvation. Thus, we analyzed chromatin structure around these heptamer sequences. Using a probe for the region at the heptamer 21.5 kbp downstream of the ctt1 gene (Fig 5A and Fig B), we reproducibly observed significant induction of MNase sensitivity during meiosis. We also examined chromatin at the ctt1 locus (Fig 5C). The intensity of several bands increased at 3 hr after meiotic induction (compared to those at 0 hr). Those meiotically enhanced MNase-sensitive sites are located mostly in the region upstream of the ctt1 locus, within an 1-kbp region including the M26 heptamer sequence. These results indicate that the naturally existing M26 heptamer sequences can induce local chromatin remodeling during meiosis. This chromatin remodeling also required Spc1/Sty1 and Mei2 (data not shown), suggesting that a common mechanism underlies the chromatin remodeling at natural M26 heptamer sequences and at the M26 hotspot in the ade6 gene.

View larger version (88K): In this window In a new window Download PPT slide   Figure 5. Chromatin remodeling occurs at natural M26 heptamer sequences around the catalase gene ctt1. (A) Schematic representation of two natural M26 heptamer sequences at the ctt1+ gene. The shaded arrow represents the ctt1+ gene; open triangles, natural M26 heptamer sequences; and vertical bars, restriction endonuclease sites. Solid boxes show the hybridization probes 71 (294-bp fragment) and 18 (227-bp fragment). The numbers represent positions from the A residue of the initiation codon of the ctt1+ gene. (B and C) Chromatin remodeling at natural heptamer sequences. Culture conditions and chromatin preparation were as described in Fig 3B. Chromatin of strain ELD205 was partially digested with MNase. The purified DNA was digested with ScaI (B) or HindIII (C). Southern blot hybridization used probes 18 (B) or 71 (C). Horizontal arrows and numbers beside these indicate MNase-sensitive sites and estimated sizes of the MNase-digested fragments, respectively. Vertical solid arrow, the catalase gene ctt1; open arrowheads, the M26 heptamer sequences; solid boxes, probes used for hybridization. The graph in C demonstrates the intensity of each band in the lanes with 20 units of MNase/ ml (0 and 3 hr); similar data were obtained in two independent experiments. Band positions are as indicated in C. Open and solid bars represent the data of premeiotic (0 hr) and meiotic (3 hr) chromatin, respectively. Note that MNase sensitivity in a region distal to the natural M26 heptamers (marked by a dashed line) increased during meiosis.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In this study, we demonstrate that CRE-like sequence-dependent chromatin remodeling during S. pombe meiosis is regulated positively and negatively by the SAPK, PKA, mating pheromone, and meiosis-inducing signaling pathways (Fig 6). It should be emphasized that M26 hotspot activity is also controlled by the SAPK pathway, as well as by Atf1-Pcr1, and is specific to meiosis (KON et al. 1997 ; FOX et al. 2000 ). Thus, there is a close correlation between chromatin remodeling and recombination activity at M26 (see Table 2). The mechanism underlying M26 chromatin remodeling is probably general and not specific to the ade6-M26 recombination hotspot, since similar chromatin remodeling was observed at or near two M26 heptamer sequences that exist naturally in the S. pombe genome (Fig 5).

View larger version (21K): In this window In a new window Download PPT slide   Figure 6. Regulation of the CRE-related chromatin remodeling. In vegetative growth conditions, the PKA pathway inhibits the activity of Atf1-Pcr1 to suppress chromatin remodeling. When cells are shifted to nitrogen starvation, the activity of PKA decreases and in turn the SAPK cascade becomes activated. The activated SAPK facilitates local chromatin remodeling by Atf1-Pcr1. Chromatin remodeling during early meiosis before or during premeiotic DNA replication at the CRE-related sequences requires activation of the mating pheromone signaling pathway and the meiosis regulator Mei2. Sme2 and Mei4, acting later in meiosis, are not required for chromatin remodeling. The inactivated or repressed factors are represented in black boxes. Solid and dashed lines indicate direct and indirect interactions, respectively.

Counteracting regulation of chromatin remodeling by SAPK and PKA pathways:
The requirement for components of the SAPK, mating pheromone, and meiosis-inducing signaling pathways indicates that these pathways positively regulate M26 chromatin remodeling during meiosis. On the other hand, M26 chromatin remodeling was completely abolished when the regulatory (inhibitory) subunit for PKA (Cgs1) was disrupted. In addition, M26 chromatin remodeling was completely blocked by the addition of cAMP to the sporulation medium, which brings about a higher intracellular cAMP concentration and downregulates the PKA activity (BEACH et al. 1985 ; DEVOTI et al. 1991 ; MOCHIZUKI and YAMAMOTO 1992 ; MAEDA et al. 1994 ). These results indicate that the PKA pathway plays an inhibitory role in M26 chromatin remodeling.

Such counteracting regulation of chromatin remodeling at CRE-related sequences might provide a system for sensing the physiological state of cells. The physiological condition might be monitored by the phosphorylation states of the SAPK and PKA phosphorylation sites of Atf1-Pcr1. Indeed, the pattern of phosphorylation on Atf1 is affected by disruption of pka1 (KANOH et al. 1996 ) or spc1/sty1 (KANOH et al. 1996 ; SHIOZAKI and RUSSELL 1996 ; WILKINSON et al. 1996 ), although the critical in vivo phosphorylation sites on Atf1 have not been fully characterized. It would be interesting to examine the effects of altering critical sites phosphorylated by Pka1 and Spc1/Sty1 on M26 chromatin remodeling.

How does phosphorylation by SAPK mediate chromatin remodeling? At least two possible mechanisms are not mutually exclusive. One is that the SAPK-phosphorylated Atf1-Pcr1 has higher affinity to CRE-like sequences. This idea is supported by the observation that Spc1 is required for M26 hotspot activity and efficient binding of Atf1-Pcr1 to the M26 site in vivo (KON et al. 1998 ). In addition, in vitro binding of Atf1-Pcr1 to a CRE-like sequence from the fbp1 promoter is positively and negatively regulated by Spc1 and PKA, respectively (NEELY and HOFFMAN 2000 ). The other possibility is that the SAPK-phosphorylated Atf1-Pcr1 preferentially recruits chromatin remodeling machinery. To test the latter possibility, it would be interesting to study interaction of Atf1-Pcr1 with various components of chromatin remodeling machinery in wild-type and spc1 mutant cells by immunoprecipitation analysis.

CRE-mediated chromatin remodeling in stress response and cellular differentiation:
The regulation of chromatin remodeling by the SAPK and the PKA signaling pathways in S. pombe meiosis gives new insights into chromatin regulation in stress responses and cellular differentiation. The SAPK pathway is well conserved in eukaryotes and has been suggested to play a pivotal role in cellular responses to environmental stimuli such as nitrogen starvation, short wave-length radiation, high temperature, oxidative stress, and high osmolarity (reviewed in DAVIS 2000 ). When S. pombe cells face such stresses, Atf1-Pcr1 is phosphorylated by Spc1/Sty1-SAPK and then activates transcription of loci with CRE-like sequences in the promoter (SHIOZAKI and RUSSELL 1996 ; WILKINSON et al. 1996 ). It would be interesting to study whether environmental stresses other than nitrogen starvation can trigger M26 chromatin remodeling.

The JNK/SAPK pathway and the CREB/ATF factor in multicellular eukaryotes are related to early embryonic development (reviewed in DAVIS 2000 ). The importance of the conserved JNK/SAPK-CREB/ATF system in eukaryotic development leads us to speculate that chromatin regulation mediated by this system plays a general role in cellular differentiation.

Premeiotic DNA replication and M26 chromatin remodeling:
Our previous analysis revealed that M26 chromatin remodeling occurs during premeiotic DNA replication (MIZUNO et al. 1997 ). In the present analysis, we found that M26 chromatin remodeling needs the function of Mei2 that is required for premeiotic DNA replication but not its function required for the later progression assisted by the RNA component Sme2. Therefore, M26 chromatin remodeling may occur between the Mei2-mediated initiation of premeiotic DNA replication and meiosis I. This suggests that M26 chromatin remodeling requires premeiotic DNA replication. However, this may not be the case, since M26 chromatin remodeling can be partially induced under premeiotic conditions where most of the cells are at the G2 stage before premeiotic DNA replication (Fig 1B; MIZUNO et al. 1997 ).

In S. cerevisiae, premeiotic DNA replication is prerequisite to the formation of meiotic DNA DSBs at meiotic recombination hotspots (BORDE et al. 2000 ; SMITH et al. 2001 ). Perhaps cohesin proteins including Rec8 (KLEIN et al. 1999 ; CHA et al. 2000 ) are loaded onto sites for DSB formation as replication forks pass through each potential DSB site. Thereafter, meiotic recombination proteins such as Spo11 (BERGERAT et al. 1997 ; KEENEY et al. 1997 ) and Mre11 are recruited there (FURUSE et al. 1998 ; OHTA et al. 1998 ).

Several meiotic recombination proteins are well conserved in S. pombe and S. cerevisiae. Meiotic recombination in S. pombe requires recombination proteins such as Rec12 (a Spo11 homolog), Rad32 (an Mre11 homolog), and Rec8 (reviewed in DAVIS and SMITH 2001 ). Rec12 and Rec8 are important for the formation of meiotic DSBs in S. pombe meiosis (CERVANTES et al. 2000 ). These and other recombination proteins may be loaded onto S. pombe meiotic chromatin during premeiotic DNA replication to prepare chromosome arms for a high level of meiotic recombination (WATANABE et al. 2001 ). Recruitment of meiotic recombination proteins may be facilitated by the Atf1-Pcr1 binding to CRE-like sequences and the subsequent chromatin remodeling. It would be interesting to study the interaction between CRE-related recombination hotspots and meiotic recombination proteins such as S. pombe Rec12, Rec8, and Rad32.

	ACKNOWLEDGMENTS

We thank Peter Munz for kind initial instructions of S. pombe genetical techniques to K. Mizuno and Wayne P. Wahls for discussions and communication of results. This work was supported by grants from the Human Frontier Science Program; the "Bioarchitect Research Program" of RIKEN; the CREST program of Japan Science and Technology; the Ministry of Education, Science, Culture, and Sports, Japan; the Swiss National Science Foundation; and by research grant GM31693 from the National Institutes of Health (United States).

Manuscript received May 30, 2001; Accepted for publication September 14, 2001.

	LITERATURE CITED

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