A Cell Cycle-Regulated GATA Factor Promotes Centromeric Localization of CENP-A in Fission Yeast

A Cell Cycle-Regulated GATA Factor Promotes Centromeric Localization of CENP-A in Fission Yeast

Molecular Cell, Vol. 11, 175–187, January, 2003, Copyright 2003 by Cell Press A Cell Cycle-Regulated GATA Factor Promotes Centromeric Localization o...

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Molecular Cell, Vol. 11, 175–187, January, 2003, Copyright 2003 by Cell Press

A Cell Cycle-Regulated GATA Factor Promotes Centromeric Localization of CENP-A in Fission Yeast Ee Sin Chen,1 Shigeaki Saitoh,3 Mitsuhiro Yanagida,1,2 and Kohta Takahashi3,4,* 1 Department of Biophysics Graduate School of Science 2 Department of Gene Mechanisms Graduate School of Biostudies Kyoto University Kitashirakawa Oiwake-cho, Sakyo-ku Kyoto 606-8502 3 Division of Cell Biology Institute of Life Science Kurume University 4 Time’s Arrow and Biosignaling PRESTO, Japan Science and Technology Corporation Aikawa-machi, Kurume Fukuoka 839-0861 Japan

Summary CENP-A, the centromere-specific histone H3 variant, plays a crucial role in organizing kinetochore chromatin for precise chromosome segregation. We have isolated Ams2, a Daxx-like motif-containing GATA factor, and histone H4, as multicopy suppressors of cnp1-1, an S. pombe CENP-A mutant. While depletion of Ams2 results in the reduction of CENP-A binding to the centromere and chromosome missegregation, increasing its dosage restores association of a CENP-A mutant protein with centromeres. Conversely, overexpression of CENP-A or histone H4 suppresses an ams2 disruptant. The intracellular amount of Ams2 thus affects centromeric nucleosomal constituents. Ams2 is abundant in S phase and associates with chromatin, including the central centromeres through binding to GATAcore sequences. Ams2 is thus a cell cycle-regulated GATA factor that is required for centromere function. Introduction Eukaryotic cells possess multiple chromosomes. Faithful chromosome segregation is therefore essential to prevent aneuploidy, which is associated with the tendency to develop cancer and is the cause of many birth defects. Accurate segregation is ensured by the centromere, a chromosomal locus on which kinetochore, a multiprotein/DNA complex, forms to serve as a spindle attachment site. While the chromosome segregation machinery might be highly conserved, the nucleotide sequences in the centromeric loci bear little resemblance among diverse eukaryotic species. Therefore, apart from DNA sequence recognition, other mechanisms such as chromatin-based epigenetic inheritance have been presumed to contribute more to the determination of centromeric identity (Choo, 2001; Henikoff et al., 2001; Karpen and Allshire, 1997; Willard, 1998). A *Correspondence: [email protected]

candidate determinant is CENP-A, an evolutionarily conserved histone H3 variant (Palmer et al., 1991; Sullivan, 2001). CENP-A is essential for chromosome segregation in all organisms so far investigated, and it has been shown to be specifically localized only at active centromeres (Blower and Karpen, 2001; Buchwitz et al., 1999; Howman et al., 2000; Lo et al., 2001; Oegema et al., 2001; Stoler et al., 1995; Takahashi et al., 2000; Vafa and Sullivan, 1997; Warburton et al., 1997). CENP-A behaves as a nucleosome component (Palmer et al., 1987; Shelby et al., 1997; Yoda et al., 2000). The centromere-specific chromatin structure is disrupted in CENPA-deficient mutants of both budding and fission yeast (Meluh et al., 1998; Takahashi et al., 2000). CENP-Acontaining nucleosomes possibly serve to form the primary kinetochore-building scaffold. In order to understand kinetochore formation, it would thus be informative to learn how CENP-A recognizes the centromere locus in the presence of massive amounts of regular histone H3. The centromere of the fission yeast Schizosaccharomyces pombe is several hundred-fold larger than that of budding yeast. The essential regions for mitotic or meiotic centromere functions have been dissected and assigned to two distinct domains. The outer repetitive regions (otr) corresponding to the pericentromeric heterochromatin are required for the reductional segregation of homologous chromosomes in meiosis I, while central regions (cnt and imr) and a portion of the otr are essential for mitotic chromosome segregation (Baum et al., 1994; Chikashige et al., 1989; Clarke and Baum, 1990; Hahnenberger et al., 1991; Matsumoto et al., 1990; Nakaseko et al., 1986; Niwa et al., 1989; Partridge et al., 2000; Takahashi et al., 1992; Watanabe and Nurse, 1999). Higher eukaryotic centromere organization might share similar structural features (Sullivan et al., 2001). The fission yeast CENP-A homolog, SpCENP-A (the gene name is cnp1⫹), is localized at the essential cnt and imr regions, which form specialized chromatin (Takahashi et al., 2000). Two evolutionarily conserved centromere-specific proteins, Mis6 and Mis12, are known to be colocalized with SpCENP-A at the central regions and show the mutant phenotype indistinguishable from that of SpCENP-A (Goshima et al., 1999; Goshima and Yanagida, 2000; Saitoh et al., 1997; Takahashi et al., 1994, 2000). Mis6 is required for recruiting SpCENP-A to the specialized centromere chromatin. Mis6-dependent incorporation of newly synthesized SpCENP-A to the centromere may be a prerequisite for bioriented connection between sister centromeres (Saitoh et al., 1997; Takahashi et al., 2000). However, Mis6 homologs of budding yeast and chicken are not required for loading CENP-A (Measday et al., 2002; Nishihashi et al., 2002). The common role of Mis6 may reside in the alteration of centromere chromatin, albeit its molecular activity is unknown. Mis6 might be specifically involved in the loading of CENP-A in fission yeast. Mis12 also participates in the formation of specialized chromatin, but in a distinct manner. At least two independent pathways

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Figure 1. The ams2⫹ Gene, a Multicopy Suppressor for the SpCENP-A ts Mutant, Encodes a GATA Factor (A) Suppression of the ts phenotype of the cnp1-1 strain, an SpCENP-A ts mutant, by plasmids pAMS1, 2, 3, and 4 on EMM2 plates. pCNP1 is the plasmid carrying the wild-type SpCENP-A gene (cnp1⫹) under the control of the native promoter. The restrictive and permissive temperatures for mutant strains were 33⬚C and 22⬚C, respectively. (B) Suppression of the ts phenotype and mitotic defects in the cnp1-1 mutant by ectopic overexpression of the ams2⫹ gene. Plasmid pnmt41AMS2 is a multicopy plasmid that carries the ams2⫹ gene under the inducible promoter nmt41. Transformant strains carrying the indicated plasmids were streaked on EMM2 plates in the presence (promoter off) or the absence (promoter on) of thiamine and were incubated at 33⬚C or 22⬚C (upper panel). The frequency (%) of unequal chromosome segregation was scored in binucleate cnp1-1 cells carrying the indicated plasmids, which were cultured in EMM2 at 33⬚C (lower graph). (C) Sequence similarity between Ams2 and GATA factors and between Ams2 and mammalian Daxx protein. Ams2 is schematized in the upper panel, with the other three GATA factors found in the S. pombe genome. The putative zinc finger motif (hatched), the basic amino acid stretches rich in arginine and lysine (black), and the domain homologous to Daxx (gray) are shown. Comparisons of Ams2 with the DNA binding regions of GATA factors and the apoptosis-activating fragments in mammalian Daxx proteins are shown in the lower panels. Only those amino acids similar to Ams2 are highlighted, with identical and similar amino acids shaded in black and gray, respectively. The four typical cysteine residues of the zinc finger are marked by arrowheads. Sp, SchizoSaccharomyces pombe; Sc, Saccharomyces cerevisiae; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Xl, Xenopus laevis; Mm, Mus musculus; Hs, Homo sapien. The accession number references are as follows: SpGaf1, L31601; ScDAL80, M77821; ScASH1, P34233; CeELT-2, U25175(EMBL); DmMta1, AF170345; XlTRPS1, AF346837; HsGATA-1, BC009797; HsGATA-3, X55037; HsDaxx, AF015956; MmDaxx, AF006040.

may contribute to the formation of specialized centromere chromatin in fission yeast (Goshima et al., 1999). We report that a GATA-type zinc finger protein, Ams2, is required for proper chromosome segregation, possibly via regulation of SpCENP-A localization. Ams2 was identified as a multicopy suppressor of a SpCENP-A temperature sensitive (ts) mutant. It is a nuclear protein that is enriched in the central centromeres and at promoter regions of presumed target genes. Ams2 binds directly to the GATA-like DNA sequences in vitro. Overexpression of Ams2 but not Mis6 suppresses the localization defect of a SpCENP-A ts mutant, even in the G2 phase. In contrast, Ams2 null mutant shows reduced SpCENP-A binding to the central regions of centromeres.

Results Multicopy Suppressor Screening for cnp1-1 To identify factors that interact with CENP-A, we screened for multicopy suppressors of the S. pombe CENP-A ts mutant, cnp1-1 (L87Q mutation) (Takahashi et al., 2000). The cnp1-1 mutant fails to form colonies at 30⬚C to 36⬚C. Transformants were obtained by plating at 33⬚C or 36⬚C. All of the transformants obtained at 36⬚C carried plasmids that contained SpCENP-A gene (cnp1⫹), whereas 40% of the transformants obtained at 33⬚C contained multicopy suppressor genes. One hundred and six transformants having plasmids with genes other than SpCENP-A were isolated and analyzed. Subcloning and subsequent restriction analyses indicated

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that the plasmids contained four nonoverlapping transforming genes (Figure 1A). They were designated as ams1⫹-ams4⫹ (cenp-a’s multicopy suppressor). Of these, three contained genomic sequences derived from the regions of the three pairs of the histone H3-H4 gene cassettes present in fission yeast. Subcloning established that the ams1⫹, ams3⫹, and ams4⫹ genes were, respectively, one of the three histone H4 genes, hhf1⫹, hhf2⫹, and hhf3⫹ (Matsumoto and Yanagida, 1985). In contrast, the elevated gene dosage of histone H3 caused a delay in colony formation of cnp1-1 at 33⬚C. Basically identical positive and negative interactions of CENP-A mutations with histone H3 and H4 were reported in budding yeast (Glowczewski et al., 2000; Meluh et al., 1998; Smith et al., 1996).

ams2ⴙ Encodes a Daxx-like Motif Containing GATA Factor To examine whether the ams2⫹ can suppress the cnp1-1 mutant, plasmid pnmt41AMS2 was created, which contained the ams2⫹ coding region placed under the control of the inducible nmt41 promoter. Both ts phenotypes and mitotic missegregation in cnp1-1 were rescued at 33⬚C by pnmt41AMS2 only under the inducible condition (Figure 1B, lower and upper panels). The ams2⫹ gene (SPCC 4F11.01, SPCC290.04) encodes a 697 amino acid protein. The middle of the coding region contains a zinc finger DNA binding motif with the two pairs of cysteine residues, a characteristic feature for the GATA factors (Scazzocchio, 2000) (Figure 1C). GATA factors are ubiquitous transcriptional regulators present in fungi, metazoans, and plants, and are involved in nutrient signal response, mating type switching in yeast, and developmental differentiation and morphogenesis in higher eukaryotes. Two GATA-type zinc finger proteins, Gaf1 and Gaf2, have been reported in S. pombe (Hoe et al., 1996, 1998). Another GATAlike sequence, SPCC1393.08p, is found in the genome sequence. Ams2 is the fourth putative GATA-like sequence, but its sequence features are quite different from those of the other three GATA factors (Figure 1C). While authentic GATA-type zinc fingers have a 17 residue loop with a leucine residue in the seventh position, the loop of Ams2 consists of 20 residues with asparagine in the corresponding position (asterisk). Ams2 is thus not a typical GATA factor. However, GATA factors with similar variations have also been reported (Scazzocchio, 2000). A distinct feature of Ams2 is its Daxx-like sequence. Daxx protein was originally identified as an adaptor protein that linked the Fas death receptor to the ASK1JNK MAP kinase cascade for transmission of apoptotic signals in mammalian cells. The sequence similarity is significant but is confined within an approximately 135 aa fragment implicated in apoptotic activities (amino acids 501 to 625 in mouse Daxx) through both the binding and activation of the JNK kinase kinase ASK1 (Chang et al., 1998). The high homology between Daxx and Ams2 may have functional implications regarding the potential protein-protein interactions between Ams2 and unidentified protein kinases. Three other GATA-like factors in S. pombe do not possess the Daxx-like motif.

Ams2 Overproduction Restores Localization of SpCENP-A ts Protein at the Central Centromere To gain an understanding of the suppression mechanism of cnp1-1 by the high-dosage Ams2, we observed the behavior of SpCENP-A ts protein (SpCENP-Ats) at both permissive (20⬚C) and restrictive temperatures (33⬚C–36⬚C) in a wild-type background. To this end, a wild-type strain bearing the SpCENP-Ats-GFP gene extragenically integrated onto the lys1 locus was constructed. The centromere localization of SpCENP-AtsGFP protein was found to be normal at 20⬚C (Figure 2A). After shifting to 33⬚C, the GFP signal of the mutant protein at the centromere was abolished. The mutant protein level determined by immunoblot using anti-GFP antibodies decreased at 33⬚C (lower panel: the level of Cdc2 is shown as a loading control). Upon returning to 20⬚C, the mislocated SpCENP-Ats-GFP had again accumulated, as observed by the centromeric dot signals, thus indicating that the ability of SpCENP-Ats to localize at the centromere is temperature sensitive. The level of mutant SpCENP-Ats protein was also restored 1 hr after the shift to 20⬚C. The recovery of GFP signals occurred rapidly within 0.5–1 hr after the shift to 20⬚C (the doubling time of the cells under this experimental condition was approximately 10 hr). The recovery of SpCENP-Ats protein to the centromere was thus efficient even after the cell had traversed the S phase (exponentially growing S. pombe cells were mostly in the G2 phase). We then addressed the issue of whether or not the high dosage Ams2 and histone H4 could promote the recovery of SpCENP-Ats protein localization to the centromere at restrictive temperatures. Localization of SpCENP-Ats-GFP in the presence of multicopy plasmids carrying either the ams1⫹ (histone H4) or the ams2⫹ gene was observed at 20⬚C and 33⬚C (Figure 2B). Although the signal of SpCENP-Ats-GFP was dot-like at 20⬚C, it became dispersed at 33⬚C in cells carrying the vector. In contrast, the GFP signal persisted as dots at 33⬚C when multicopy plasmid pAMS2 or pH4 was introduced. However, multicopy plasmids of mis6⫹ failed to recover the dot-like localization of SpCENP-Ats. The time-course behavior of SpCENP-Ats protein during the induced overproduction of Ams2 was then studied. Wild-type cells integrated with SpCENP-Ats-GFP and carrying plasmid with the ams2⫹ gene under the control of the inducible promoter nmt41 were cultured in the absence of thiamine (promoter on). After 10–11 hr, the overproduction of Ams2 took place. Coincident with the appearance of Ams2 protein (Figure 2C), the signal of SpCENP-Ats-GFP was observed as a dot in the nucleus. We then determined whether this localization recovery of SpCENP-Ats protein could occur in G2arrested cells using the cdc25-22 mutant. As shown in Figure 2D (lower panel), the initially dispersed signal of SpCENP-Ats-GFP mutant protein at 33⬚C was found to be restored to centromeres in the G2-arrested cdc2522 cells when Ams2 was overproduced (upper panel). Thus, elevating the dosage of Ams2 restored the centromeric localization of SpCENP-Ats protein, even in late G2-arrested cells. To confirm that SpCENP-Ats protein was correctly accumulated back into the central centromeres by overexpression of Ams2, a chromatin immunoprecipitation (ChIP) experiment was carried out using an SpCENP-

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Figure 2. Centromeric Localization of SpCENP-A ts Protein in Cells Overproducing Ams2 (A) Intracellular localization and the level of SpCENP-Ats mutant protein at 20⬚C or 33⬚C. Wild-type cells expressing the tagged SpCENP-AtsGFP or SpCENP-A-GFP integrated at the lys1 locus were cultured in EMM2 at 20⬚C, shifted to 33⬚C for 5 hr, then returned again to 20⬚C. (Upper panel) Localization of GFP signals. (Lower panel) The protein levels of GFP-tagged wild-type (SpCENP-A-GFP) and ts SpCENP-A (SpCENP-Ats). The localizations of GFP-tagged proteins were presented at the indicated time points. Note that the doubling time of the cells was approximately 3 hr at 33⬚C and 10 hr at 20⬚C in this culture medium. The amount of Cdc2 kinase (antibodies to PSTAIRE) was used as a loading control. Bar, 10 ␮m. (B) Mislocalized SpCENP-Ats-GFP mutant protein was restored to centromeres by multicopy plasmid pAMS2 and pH4 but not by pMIS6 in a wild-type background cultured in EMM2 at 33⬚C. (C and D) Induced overproduction of Ams2 results in rapid allocation of SpCENP-Ats-GFP protein to centromeres in wild-type background (C) or in the G2-arrested cells (D). Wild-type (C) and cdc25-22 mutant (D) cells bearing one additional copy of SpCENP-Ats-GFP gene at the lys1 locus were transformed by a multicopy plasmid carrying the Ams2-HA gene under the control of the inducible nmt41 (REP41) promoter. In

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Ats-HA integrant strain (Figure 2E). Exponentially growing cells were fixed by formaldehyde and immunoprecipitated by anti-HA antibodies under the condition that Ams2 was ectopically expressed. When using the central centromere (cnt and imr) primers, approximately 4- to 6-fold stronger signals were detected in the case of Ams2 overexpression over those of the vector control. The PCR signals were not amplified using the outer centromere (otr) primers or using the pericentromeric lys1 locus primers. The distribution of SpCENP-Ats protein accumulated by Ams2 induction was thus tightly restricted to the central region of centromeres, identical to that of the wild-type protein.

ams2 Null Cells Are Defective in Growth with Frequent Missegregation To study the null phenotype of ams2⫹, heterozygous diploids were created by one-step replacement of the Ams2 coding region with the ura4⫹ sequence. After sporulation of the diploids, tetrads were dissected, and all of the germinated spores were found to be viable at 33⬚C, indicating that the ams2⫹ gene was not essential for viability. However, colony formation of the ⌬ams2 deletion haploid (Ura⫹) was exceedingly slow at all temperatures tested (20⬚C–36⬚C), as seen in Figure 3A (33⬚C; other temperatures, data not shown). ⌬ams2 cells exhibited aberrant mitosis with unequal segregation or lagging condensed chromosomes at 33⬚C (Figure 3Ba– 3Bg). To assess the actual occurrence of chromosome missegregation in ⌬ams2, the fluorescent in situ hybridization (FISH) method was applied using a portion of rDNA as the chromosome III-specific probe (Figure 3C). Under the culture conditions employed, 6% of binucleate ⌬ams2 cells showed missegregation with only one rDNA-FISH signal per cell, whereas wild-type cells always showed two separate FISH signals (missegregation was 0%). This loss rate may be underestimated, as missegregation of regular chromosomes leads to cell death and is consequently undetectable. We therefore monitored the stability of a nonessential linear minichromosome Ch10 (Takahashi et al., 1994) in ⌬ams2 cells and found it to be dramatically unstable, even in the selective culture condition (Figure 3D). This suggests that Ams2 depletion severely reduces chromosome stability. Furthermore, when nitrogen-starved G1-arrested cells were released to the complete medium, the first mitosis was abnormal, showing unequal chromosome

segregation mainly with large and small daughter nuclei (data not shown). Centromere Defects in the ⌬ams2 Mutant The above missegregation phenotype in ⌬ams2 might be due to the disruption of the central centromere-specific chromatin structure. To test this possibility, an MNase digestion experiment was carried out using the nuclear chromatin isolated from ⌬ams2 cells (Figure 3E). Genomic DNAs were isolated after MNase digestion, run in agarose gel, and Southern hybridized with the probes from the central or the outer centromeric regions. Ethidium bromide (EtBr) staining of the isolated DNA fragments revealed regular nucleosome ladders (Figure 3E, EtBr). In the wild-type controls, the Southern hybridization pattern for the central centromere probe (imr) was smeared, whereas in ⌬ams2 cells, the hybridization pattern was ladder-like against a rather smeared background, suggesting that the central centromeric chromatin was partially disrupted (Figure 3E, central cen). To determine whether SpCENP-A is associated with the centromere DNAs in the ⌬ams2 mutant, a ChIP experiment was performed using the chromosomally integrated HA-tagged SpCENP-A at the lys1 locus in ⌬ams2 cells (Figure 3F). In the precipitates from ⌬ams2 by antiHA antibody, the amount of PCR-amplified DNAs using the central centromere primers was reproducibly 2- to 4-fold (cnt) and 4- to 10-fold (imr) smaller than those of the precipitates from the wild-type controls (lanes 1 and 3). A ChIP experiment using anti-human H3 antibodies was also carried out using the same cell extracts. The antigen for this antibody contains an amino acid sequence identical to that of the S. pombe histone H3 C-terminal and also very similar to that of the SpCENP-A C-terminal. This antibody was indeed shown to recognize immunoprecipitated SpCENP-A, as well as histone H3 (data not shown). Thus, the amount of PCR signal amplified using this antibody (␣CENP-A⫹H3) likely represents the mixture of SpCENP-A and histone H3 binding. In ⌬ams2 cells, the total amount of SpCENP-A and histone H3 binding at the central centromeres is increased over that of wild-type cells (lanes 2 and 4), despite the decrease in SpCENP-A binding (lanes 1 and 3). Note that such differences were not observed at the lys1 locus (the lower right). These data suggest that the decrease in SpCENP-A was compensated for, at least partly, by histone H3 within the central centromeres in ⌬ams2 cells. Immunoblotting by anti-HA antibodies

(C), transformant cells were first cultured in EMM2 in the presence of thiamine at 33⬚C (0 hr). Induced Ams2 synthesis occurred 10 hr after the removal of thiamine. Immunoblotting of the extracts was undertaken using antibodies against HA, GFP, or Cdc2 (PSTAIRE). Intracellular localization of SpCENP-Ats-GFP indicated that the GFP signal was localized at centromeres after 10 hr. In (D), transformant cells in the cdc2522 background were cultured in EMM2 after the removal of thiamine at 20⬚C for 15 hr and then shifted to 33⬚C (0 hr). Induced production of Ams2 appeared at 4 hr after shifting to 33⬚C under otherwise identical culture conditions employed. Immunoblot was performed using antibodies against HA or Cdc2 (PSTAIRE). Localization of SpCENP-Ats-GFP at centromeres occurred after the induced synthesis of Ams2. Note that cells had been arrested in the G2 phase at 2 hr after the temperature shift to the restrictive condition (data not shown) and the SpCENP-Ats-GFP signals were dispersed. ⫹T, the promoter is in the repressed condition. (E) Wild-type cells bearing the SpCENP-Ats-HA gene at the lys1 locus were transformed by either pREP41 (lane 2) or pREP41 carrying the ams2⫹ gene (lane 1), and were cultured in EMM2 after the removal of thiamine at 33⬚C for 16 hr. SpCENP-Ats-HA was immunoprecipitated (IP) for the ChIP analysis using antibodies against HA. Lane 3 is the control lane using beads alone. Coprecipitated DNAs were amplified by PCR using four different primers (their locations are shown as vertical lines in the illustration for cen1 in Figure 5). The intensity of each IP signal was divided by that of the corresponding whole-cell extract (WCE) signal after the background titration and shown in the right panel as the relative intensity.

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Figure 3. Chromosome Missegregation and SpCENP-A Mislocalization Phenotypes Produced by ⌬ams2 Disruption (A) Slow colony formation of ⌬ams2 on the YPD plates at 33⬚C for 2, 3, or 4 days. (B) Aberrant mitotic nuclear division of ⌬ams2 cells in the EMM2 liquid cultures at 33⬚C. DNA (blue) and microtubules (red). Bar, 10 ␮m. (C) ⌬ams2 cells in the YPD liquid culture at 33⬚C were treated by the FISH method. In 6% of the cells, the rDNA signals were unequally segregated. Wild-type cells are shown as the control. The percentage of binucleate cells showing unequal or equal distributions of the FISH signals is indicated at right. (D) Colony color assay to monitor the stability of linear minichromosome Ch10 in wild-type and ⌬ams2 cells. Exponentially growing cells carrying Ch10 in EMM2 selective medium were plated on YE rich plates and incubated at 26⬚C. Cells that lost the minichromosome produced a red colony. Pink colonies were derived from cell populations that frequently lost their minichromosomes. (E) Nuclear chromatin prepared from growing wild-type and ⌬ams2 cells were digested with MNase for 0, 1, 2, 4, and 8 min, followed by agarose gel electrophoresis and Southern hybridization using either the central (imr) or the outer (otr) centromeric probes (Takahashi et al., 1992). (F) SpCENP-A-HA expressed in either ⌬ams2 or wild-type cells cultured in YES at 26⬚C was immunoprecipitated (IP) for the ChIP analysis using antibodies against HA (␣HA) and the C-terminal region of human histone H3 (␣CENP-A⫹H3). This antibody recognizes both SpCENPA and histone H3 in fission yeast (data not shown). Coprecipitated DNAs were amplified by PCR using the central centromere (cnt1, imr1) and the arm region (lys1) primers (their locations are shown in Figure 5). Lane 5 is the control lane using beads alone. The relative intensity of PCR products (the right panel) was estimated as described in (E). (G) Localization of SpCENP-A-GFP, Mis6-GFP, or Mis12-GFP in wild-type and ⌬ams2 cells cultured in EMM2 at 26⬚C. The percentage of cells showing proper centromeric (gray), weak centromeric (striped), or dispersed nuclear (white) patterns is shown in the right panel.

revealed that the level of SpCENP-A in ⌬ams2 cells was roughly equal to that of wild-type cells (data not shown). A similar behavior between CENP-A and H3 was observed at Drosophila centromeres (Blower et al., 2002). Consistent with the ChIP results, microscopic observation revealed that the SpCENP-A-GFP signal but not the Mis6-GFP nor Mis12-GFP signal was dramatically reduced in ams2 disruptant cells (Figure 3G). Approximately 77% of the ⌬ams2 cells had dispersed nuclear or weak dot SpCENP-A-GFP signals, though the remaining 23% of these cells revealed relatively strong dot signals similar to those seen in wild-type cells. In contrast, the

Mis6 and Mis12 GFP signals appeared to be generally comparable to those in wild-type cells. Ams2 may specifically affect the nucleosomal composition at the central centromeres, which is balanced between histone H3 and SpCENP-A. Interactions of Ams2 with Histones and Centromere Proteins Consistent with the hypothesis that Ams2 plays a role in the regulation of the central centromeric nucleosomes, the retarded growth of the ⌬ams2 mutant was largely restored by overproduction of either SpCENP-A

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Figure 4. Genetic Interactions between Ams2 and Histones and between Ams2 and Kinetochore Proteins (A) Slow-growing ⌬ams2 cells were partly suppressed by plasmids pCNP1 and pH4 carrying the histone H4 gene at 33⬚C. Quantitative data on the doubling time are represented in the lower panel. (B) Suppression of the ts phenotype of mis12-537 but not that of mis6-302 by the plasmid carrying the ams2⫹ gene on EMM2 plates at 36⬚C. (C) Synthetic lethal phenotype of ⌬ams2 mis6-302 double mutant on YPD plates. (D) Interactions of Ams2 with five other gene products found in this study. The arrows indicate suppression of the phenotypes of gene disruptants or ts mutants, while the T-shaped lines represent toxicity upon overexpression. A line with a double arrowhead represents synthetic lethality.

or histone H4 at all temperatures tested (22⬚C, 26⬚C, 30⬚C, 33⬚C, and 36⬚C; Figure 4A and data not shown). The genetic interactions between Ams2 and SpCENP-A were thus reciprocal, suggesting that their relationship was functionally close. Suppression of ⌬ams2 by a high dose of histone H4 suggested that Ams2 might be implicated in the interaction between SpCENP-A and histone H4. Immunoprecipitation (IP) experiments between Ams2 and SpCENP-A were carried out under several different conditions; however, no interaction was detected (see the supplemental data at http://www. molecule.org/cgi/content/full/11/1/175/DC1). Thus, Ams2 most probably does not physically associate with SpCENP-A, at least in the soluble form. Mis6 had been previously reported to be required for the proper localization of SpCENP-A. To elucidate the functional interaction between Ams2 and Mis6, we carried out IP experiments in order to find possible interactions. However, no physical interaction was detected between Ams2 and Mis6 in these IP experiments (see the supplemental data at http://www.molecule.org/cgi/ content/full/11/1/175/DC1). Ams2 overexpression did not complement mis6 ts, and Mis6 overexpression did not complement the growth retardation phenotype of ⌬ams2 (Figure 4B and data not shown). In ⌬ams2 cells,

the intensity and the localization of Mis6-GFP proteins appeared normal (Figure 3G). These data suggest that Ams2 and Mis6 act independently when determining the proper SpCENP-A localization. A finding that is potentially consistent with this conclusion would be that the ⌬ams2 mis6-302 double mutant shows synthetic lethality at 30⬚C (Figure 4C). It was unexpectedly found that Ams2 overproduction could suppress the ts phenotype of mis12-537 but not that of mis6-302 (Figure 4B). Overexpression of Mis12 did not complement the ⌬ams2 phenotype (data not shown). Since ⌬ams2 was found to be synthetically lethal with mis12-537 mutation at any temperature (data not shown), Ams2 most likely functions upstream of the Mis12 pathway. As summarized in Figure 4D, Ams2 interacts in a similar manner as SpCENP-A with histone genes, but SpCENP-A did not interact with Mis12. Ams2 is thus implicated in nucleosomal function as regards the formation of the central specialized chromatin in centromeres. Localization of Ams2 at Chromatin during Chromosome Duplication To determine intracellular localization, the Ams2-GFP fusion gene was created and integrated into the genome

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Figure 5. Cell Cycle-Dependent Alterations of Ams2 (A) Wild-type cells carrying the integrated Ams2-GFP were cultured in EMM2 at 26⬚C. Cells at different stages of the cell cycle demonstrated striking cell cycle-dependent alterations in localization. Ams2-GFP was enriched in the nucleus during G1/S, S, and early G2. Bar, 10 ␮m. (B) cdc25-22 ts mutant expressing Ams2GFP was employed to examine the changes in the amount and the localization of Ams2 during the synchronous cell cycle progressing from late G2 by the temperature shift from 36⬚C to 26⬚C. The septation index and the amounts of Cig2 (S phase cyclin), Cdc13 (mitotic cyclin), and Cdc2 (PSTAIRE, a loading control) were used as reference markers to monitor cell cycle progression. The panel at the lower right shows the cell cycle-dependent fluctuation of Ams2-GFP signals (the numbers indicate minutes after the temperature shift). Bar, 10 ␮m.

by replacing the authentic ams2⫹ gene. The GFP-tagged construct was fully functional as the integrant was able to grow normally. Microscopic observation demonstrated that Ams2-GFP accumulated on the nuclear chromatin in a cell cycle-dependent manner (Figure 5A). The Ams2-GFP signal was observed as intense punctate spots in the nuclear chromatin from telophase to early G2 phase. Nuclear staining peaked during the S phase (septated cells) and early in the G2 phase (small cells immediately after cell division), whereas accumulation was hardly detected in the mid to the late G2 or early M phase. Next, the amount of Ams2 was estimated by immunoblotting using a synchronized cell culture by the release of arrested cdc25-22 mutant (Figure 5B). The Ams2 protein become apparent at 45–60 min after the release from the G2 block (corresponding to the late-M to G1/S phase), peaked at 75–90 min (G1/S to early G2 phase), then disappeared abruptly at 105–120 min (mid to late G2 phase). The results demonstrate that the synthesis

and/or degradation of Ams2 were regulated in a cell cycle-dependent manner. The level of Ams2 became maximal after the peak of Cig2, the S phase cyclin (Yamano et al., 2000). The timing for Ams2 nuclear localization (panel at the bottom right) was identical to that for Ams2 protein accumulation detected by immunoblotting. Ams2 Binds to GATA-Core DNA Sequence at the Central Centromeres We tested the possibility that Ams2 might reside and act at centromeres. To this end, a ChIP experiment was performed using the PCR primers from three centromeres and noncentromeric regions (Figure 6A). In an exponentially growing asynchronous culture, Ams2 was found to predominantly bind to the probes from the central regions but not to those of the outer centromeres. The noncentromeric probes derived from the 20 kb region on the short arm of chromosome II, the ars1 and lys1 loci were also not amplified (a1, a10, etc.; data

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Figure 6. Localization of Ams2 in the Central Centromere (A) The positions of the 31 primers used for the ChIP analysis are schematically shown in the three centromeres and flanking arm regions. Bold and underlined primers represent those that produced positive results for Ams2-HA ChIP, while dotted underlines represent weak interactions. The distribution and frequency of the putative GATA factor binding consensus (HGATAR) are indicated by black circles over the regions around the cen1 and the lys1 locus. One circle represents the presence of one putative consensus sequence. For the ChIP analysis, immunoprecipitation (IP) was performed using wild-type cells containing the integrated Ams2-HA gene and antibody against HA. Coprecipitated DNA was amplified by PCR using the 43 (21 centromeric and 22 arm) different primers derived from cen1, cen2, cen3, or noncentromeric DNA sequences. (B) Representative ChIP results using the probes from each of the four regions (inner centromere, outer centromere, arm, and promoters) are shown. Approximately the same amounts of the PCR product were obtained from the whole-cell extracts (WCE) of cells with (⫹) and without (⫺) Ams2-HA. Lanes 2, 3, and 4 are the control lanes using either beads alone or the extract without Ams2-HA.

not shown). The T14 signal from the outer centromeric region may have been positive, though not strongly so. As all of the central centromere primers tested (T8, cnt1 from cen1, 22, T12, T10, 24, T11 from cen2, T15, 34, 32 from cen3) produced positive signals, Ams2 is certain to be concentrated in the central centromere regions.

To identify other genomic sites where Ams2 is concentrated, we focused on the sites around GATA-like sequence-containing upstream regions of several genes (cdc18⫹, dis1⫹, cig2⫹, mis6⫹, mis12⫹, suc22⫹, and cdc2⫹) for ChIP experiments (Figure 6B and data not shown). Also analyzed was the upstream of heat shock-

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induced gene (hsp9⫹) lacking an obvious GATA-like sequence. Among them, cdc18 ⫹ upstream primers produced relatively strong ChIP signals but not others. Besides the GATA-core sequence, other factors are likely required for Ams2 binding to the chromatin in vivo. Although its regulatory roles in transcription must be further clarified experimentally, Ams2 may be a GATA factor that remodels both the central centromeres and the promoters of the target genes. The consensus recognition core sequences (HGATAR; H ⫽ T/A/C, R ⫽ A/G) for GATA-type zinc finger proteins (Scazzocchio, 2000) are plentiful in the centromere sequences of S. pombe (Figure 6A). For instance, 63 HGATAR sequences exist in the 15,360 bp long central centromeric DNA of cen1 (2.8 times higher than that theoretically calculated). To determine whether Ams2 directly binds to the centromere DNA, a gel mobility shift assay was carried out using GST-Ams2 (Figure 7). Two hundred base pair central centromeric DNA in the middle portion of imr1 (Imr1 DNA; T8) was synthesized for the assay because a set of primers derived from the region produced relatively high amplification in the ChIP experiments (Figure 6A; T8). The region contains a single GATA-core consensus sequence (AGATAA) (Figure 7A). 32 P-end-labeled double-strand (ds) DNA was incubated with increasing concentrations of GST-Ams2, and the resulting mixtures were run in polyacrylamide gel followed by autoradiography (Figure 7B). A shift in electrophoretic mobility was observed in the case of GSTAms2, but not with GST as the negative control. The band shift was observed when a 2-fold molar concentration of GST-Ams2 relative to dsDNA was added. To determine whether such association depends on the GATA-core sequence, Imr1 was divided into two parts for the assay; namely, it either contained the GATA-core (Imr1-w) or it did not (Imr1-w/o) (Figure 7A). Ams2 caused a shift in the electrophoretic mobility of Imr1-w but not of Imr1-w/o DNA, suggesting that the DNA binding activity of Ams2 was sequence specific (Figure 7B). To clarify the binding consensus, different versions of the 35 bp dsDNA sequence were created for the competition assay. These versions contained the sequence around the GATA site in Imr1 (designated Imr1GATA), and the mutant version obtained by replacing the tetra nucleotides GATA with CCCC, Imr1-CCCC, or a non-GATA sequence in the first 35 bp of Imr1 (Imr1no GATA) (Figure 7A). Binding of Ams2 to Imr1 DNA was disrupted when nonlabeled Imr1 or Imr1-GATA DNA was mixed and coincubated as a template competitor (Figure 7C). Neither Imr1-noGATA nor Imr1-CCCC affected the binding activity of Ams2, even at high concentrations (⫻50) (Figure 7C), suggesting that Ams2 directly binds to the central centromeric DNA via the GATA-core sequence. Discussion In this study, we showed that overproduction of a nuclear factor, Ams2, and histone H4 could suppress the ts mutant phenotype of CENP-A, an evolutionarily conserved histone H3 variant, which is exclusively present in centromeres from yeast to human. CENP-A has attracted considerable interest due to its presumed role

Figure 7. Direct Binding of Ams2 to the GATA-Core DNA Sequences (A) Three probes and three competitor oligonucleotides used are indicated together with their nucleotide length. (B and C) Gel mobility shift assay for the interaction between the central centromeric probe DNAs and the bacterially produced GSTAms2 fusion protein, which had been purified by affinity chromatography. (B) A 200 bp long central centromeric DNA corresponding to a small portion of the imr region in chromosome I, which had a single GATA site, was used as the probe (Imr1). Imr1 was divided into two halves either containing the GATA site (Imr1-w) or not (Imr1w/o). Bacterially expressed GST was used as a negative control for DNA binding. (C) Competition was performed with 35 bp dsDNA containing the GATA site (Imr-GATA), the mutant version of the GATA site made by introduction of CCCC mutations into the GATA site (Imr1-CCCC), and nonrelated sequences (no GATA). The amount of proteins preincubated with DNA was 0-, 2-, 10-, and 50-fold of that of the DNA (0.01 pmole). The amount of unlabeled competitor was 0-, 50-, 100-, and 200-fold of the amount of the probes used.

in determining centromere identity. The mechanism by which this centromeric histone is loaded to centromeres thus emerged as an important issue for understanding both kinetochore chromatin formation and function. In this report, we show that the centromere chromatin disrupted in SpCENP-A mutant cells was recovered by increasing the intracellular amount of Ams2. SpCENP-A appeared to be incorporated into the impaired kinetochore chromatin in mutant cells, at the binding sites unoccupied by any histone octamers or by replacing falsely bound octamers containing regular histone H3, when the level of Ams2 was augmented. The relationship between Ams2 and SpCENP-A is expected to be very close, as their genetic interaction is bilateral; the phenotypes of ams2 disruptant and ts cnp1-1 were suppressed

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by an increased amount of SpCENP-A and Ams2, respectively. Ams2 is one of the four GATA-type transcriptional regulators present in S. pombe. The C2C2-type zinc fingers commonly present in the GATA factors recognize a GATA-core DNA consensus in the promoter regions of the target genes. We showed that Ams2 binds to the GATA-core sequence present in the central centromere regions. We restricted the present work report to the study of centromeric function of Ams2, as we consider that the potential transcriptional role of Ams2 must be investigated together with those of the three other GATA factors. The central regions of S. pombe centromeres are thought to be transcriptionally inactive. However, although it is not known whether they are transcribed or not, the imr regions do host numerous tRNA genes (Kuhn et al., 1991; Takahashi et al., 1991). Recently, certain small nuclear RNA transcripts have been found to match sequences of the outer repetitive regions (Reinhart and Bartel, 2002; Volpe et al., 2002). It is potentially of great interest that transcription might be related to centromere function. The erythroid GATA-type transcription factor GATA-1 was reported to cause extensive, cooperative breakage of the histone-DNA contacts in an entirely reversible manner, i.e., the nucleosomes are regenerated upon the removal of GATA-1 (Boyes et al., 1998). Like GATA-1, Ams2 might act as a chromatin modulator, and it may utilize its ability as a transcription factor to gain access to the target site in chromatin at the centromeric region, resulting in the disruption or displacement of nucleosomes around the binding regions. This remodeling step of the centromeric chromatin may be a prerequisite for functional bioriented kinetochore formation. Alternatively, though not a mutually exclusive possibility, Ams2 might promote the incorporation of SpCENP-A-containing nucleosomes; for example, Ams2 may enhance the formation of SpCENP-A-histone H4 tetramers. The results obtained in this study also do not exclude the possibility that the effect of Ams2 on SpCENP-A is indirect, i.e., through the transcriptional regulations of other factors. Another mammalian GATA factor, GATA-4, represents a new class of developmentally regulated transcription factors referred to as pioneer or decisive factors, which are capable of opening compacted chromatin and, in doing so, helping other factors bind to DNA (Cirillo et al., 2002; Lomvardas and Thanos, 2002). As decisive factors are expressed in a highly restricted manner, the gate would open only in the right tissues and at the right time. Similarly, as the decisive factor for subsequent SpCENP-A deposition, the cell cycle-regulated GATA factor Ams2 may also be involved in making the specialized centromeric chromatin at the appropriate time and place, namely, during S phase and at the central centromeres. As a dual-functional GATA factor, Ams2 could coordinate the proper deposition of SpCENP-A into centromeric nucleosomes with functionally related gene transcription during the S phase. Ams2 and Mis6 are both required for and likely play distinct roles in the proper localization of SpCENP-A in fission yeast. The efficient selection mechanism(s) preferentially depositing SpCENP-A in the presence of H3 may require both Ams2-dependent chromatin re-

modeling and Mis6-dependent capturing and/or incorporation of SpCENP-A. In this paper, we demonstrated that SpCENP-A could be incorporated into centromeric nucleosomes very rapidly (Figure 2A), even after the passage of the S phase (Figure 2D). This finding is consistent with the observation that centromeric histones from a variety of organisms localize to centromeres in an S phase-independent manner (Shelby et al., 2000; Ahmad and Henikof, 2002). The integrity of centromeric chromatin could be corrected, with Ams2 enabling the incorporation of centromeric nucleosomes throughout the cell cycle, by actively remodeling nucleosomes. Sequence-specific DNA binding factors such as Ams2 may be involved in the de novo formation of the kinetochore chromatin structure. When centromeres are inactivated, it is known that the neocentromere can form at regions where latent centromeric sequences exist (Choo, 2001). The transcription factors might be implicated in the generation of centromeres during the evolution of eukaryotes. Experimental Procedures Strains and Media S. pombe haploid strains cnp1-1, mis6-302, and mis12-537 were previously described (Goshima et al., 1999; Saitoh et al., 1997; Takahashi et al., 2000). The complete YPD, YE, YES, and minimal EMM2 media were used (Moreno et al., 1991). Antibodies ␣-SpCENP-A polyclonal antibody was made by immunizing rabbits with GST-tagged SpCENP-A. ␣-GFP, ␣-Myc (AB-1), and ␣-HA (12CA5) monoclonal antibodies were purchased from Roche, Oncogene, and Boehringer, respectively. When 12CA5 was used for IP, polyclonal rabbit ␣-HA antibody (Clontech) was used for immunoblot. For (SpCENP-A⫹histone H3)-IP, a rabbit polyclonal antibody against the C-terminal region of human histone H3 (A. Verreault, personal communication) was used. Cell Synchronization, Immunofluorescence Microscopy, and FISH The cdc25-22 block and release experiment was performed as previously described (Takahashi et al., 2000). DAPI (4⬘6-diamidino-2phenylindole) was used to stain the DNA. Cells were fixed with paraformaldehyde, and microtubules were stained with TAT1 antibody (Goshima et al., 1999). The FISH method was applied using rDNA probe YIp10.4 (Takahashi et al., 1994). Gene Cloning An S. pombe genomic DNA library was used for the transformation of cnp1-1. Plasmids were recovered from the Ts⫹Leu⫹ transformants obtained, and the inserted genomic DNA was sequenced. Of 216,000 colonies screened, all of the 98 suppressers recovered at 36⬚C were the cnp1⫹ gene. Therefore, an additional 400,000 colonies were screened at 33⬚C. One hundred and six suppressing sequences, which excluded the cnp1⫹ gene, were obtained and classified into four distinct groups. The ams2⫹ gene was contained in S. pombe cosmid clones, c4F11 and c290 (GenBank accession numbers AL117389 and AL035260, respectively). The presence of a 100 nucleotide intron flanked by consensus splice sites was confirmed by the sequencing of the RT PCR (Takara, RNA PCR kit [AMV] ver 2.1) product. The length of the ORF was confirmed by comparison with the size of the HA-tagged protein expressed from the native or from the nmt promoter (Maundrell, 1990). Gene Integration, Disruption, and Tagging One-step homologous recombination (Rothstein, 1983) was employed for gene integration and disruption. A DNA fragment containing the flanking sequences of the 1 kb long 5⬘ and 3⬘ ends of the ams2⫹ gene ligated to ura4⫹, which replaced the amino acid

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number 8 to 692 of Ams2, was constructed with PCR and used for the disruption of the ams2⫹ gene in the 5A/1D diploid (h⫹/h⫺ leu1/ leu1 ura4/ura4 his2/⫹ ade6-M210/ade6-M216). Gene disruptions were verified using Southern hybridization. GFP and 3⫻ HA tagging were performed at the carboxyl terminus of the ams2⫹, as previously described (Goshima et al., 1999). The GFP-tagged mutant cnp1-1 and wild-type cnp1⫹ genes were integrated onto the lys1⫹ locus in the wild-type background. MNase Digestion, ChIP, and Gel Mobility Shift Assay The MNase digestion was carried out using three centromeric probes described previously (Takahashi et al., 2000). The ChIP method was also performed as previously described (Saitoh et al., 1997; Takahashi et al., 2000). The PCR conditions used for Rad21 (Tomonaga et al., 2000) were adapted with 32 amplification cycles. Information about the PCR primers and amplification conditions is available upon request. For the gel mobility shift assay, 5⬘ endlabeled DNA was mixed with the indicated amounts of bacterially purified GST-Ams2 or GST proteins in binding buffer (20 mM TrisCl [pH 7.5], 50 mM NaCl, 10% glycerol, 1 mM DTT, 1mM EDTA, 100 mg/ml BSA). After incubation at room temperature for 30 min, samples were applied to 4% or 8% nondenaturing polyacrylamide gel in TBE that had been prerun for 1 hr at 4⬚C. Electrophoresis was performed at 4⬚C at 30 V/cm2 . The gel was then dried and exposed for autoradiography. The linear dsDNAs used (Imr1, Imr1-w, and Imr1-w/o) were created by PCR and gel purified. dsDNAs of Imr1GATA, its mutated version (Imr1-CCCC), and no GATA were made for the competition assay by annealing two complementary synthesized oligonucleotides of 35 nt each under buffer conditions of 10 mM Tris-Cl (pH 7.5), 50 mM NaCl, 10 mM MgCl2, and 0.5 mM DTT. Acknowledgments We would like to thank Dr. Alain Verreault (Cancer Research UK, London Research Institute, Clare Hall Laboratories) for providing the anti-histone H3 antibody, and Drs. Chikashi Shimoda and Taro Nakamura (Osaka City University) for the S. pombe genomic library. This work was supported by the CREST research project of the Japan Science Technology Corporation (to M.Y.), the COE Scientific Research Grant (to M.Y.), the Grant-in-Aid for Scientific Research (B) and Scientific Research on Priority Areas “Genome Biology,” “Mechanisms of Oncogenesis and Anti-Oncogenesis,” and “Cell Cycle Control” from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to K.T.). E.S.C. was a recipient of the Pre-Doctoral Foreign Student Fellowship of the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Received: May 7, 2002 Revised: October 22, 2002

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