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Histone H2B mutations in inner region affect ubiquitination, centromere function, silencing and chromosome segregation

2006; Springer Nature; Volume: 25; Issue: 11 Linguagem: Inglês

10.1038/sj.emboj.7601110

1460-2075

Takeshi Maruyama, Takahiro Nakamura, Takeshi Hayashi, Mitsuhiro Yanagida,

Genetics and Neurodevelopmental Disorders

Article11 May 2006free access Histone H2B mutations in inner region affect ubiquitination, centromere function, silencing and chromosome segregation Takeshi Maruyama Takeshi Maruyama Search for more papers by this author Takahiro Nakamura Takahiro Nakamura Search for more papers by this author Takeshi Hayashi Takeshi Hayashi Search for more papers by this author Mitsuhiro Yanagida Corresponding Author Mitsuhiro Yanagida Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto, Japan Search for more papers by this author Takeshi Maruyama Takeshi Maruyama Search for more papers by this author Takahiro Nakamura Takahiro Nakamura Search for more papers by this author Takeshi Hayashi Takeshi Hayashi Search for more papers by this author Mitsuhiro Yanagida Corresponding Author Mitsuhiro Yanagida Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto, Japan Search for more papers by this author Author Information Takeshi Maruyama, Takahiro Nakamura, Takeshi Hayashi and Mitsuhiro Yanagida 1 1Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto, Japan *Corresponding author. Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan. Tel.: +81 75 753 4205; Fax: +81 75 753 4208; E-mail: [email protected] The EMBO Journal (2006)25:2420-2431https://doi.org/10.1038/sj.emboj.7601110 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The reiterated nature of histone genes has hampered genetic approach to dissect the role of histones in chromatin dynamics. We here report isolation of three temperature-sensitive (ts) Schizosaccharomyces pombe strains, containing amino-acid substitutions in the sole histone H2B gene (htb1+). The mutation sites reside in the highly conserved, non-helical residues of H2B, which are implicated in DNA–protein or protein–protein interactions in the nucleosome. In the allele of htb1-72, the substitution (G52D) occurs at the DNA binding loop L1, causing disruption of the gene silencing in heterochromatic regions and lagging chromosomes in anaphase. In another allele htb1-223 (P102L) locating in the junction between α3 and αC, the mutant residue is in contact with H2A and other histones, leading to structural aberrations in the central centromere chromatin and unequal chromosome segregation in anaphase. The third allele htb1-442 (E34K) near α1 displayed little defect. Evidence is provided that monoubiquitinated H2B is greatly unstable in P102L mutant, possibly owing to proteasome-independent destruction or enhanced deubiquitination. Histone H2B thus plays an important role in centromere/kinetochore formation. Introduction In eukaryotic cells, DNA is packaged repetitively into nucleosomes by means of interactions among four classes of histones, H2A, H2B, H3 and H4 (Richmond et al, 1984; van Holde, 1988; Kornberg and Lorch, 1999; Elgin and Workman, 2002). The crystal structure of the nucleosome core particle consists of approximately 147 bp of DNA organized into a superhelix around an octamer of two of each of these four histones (Luger et al, 1997; Luger, 2003; Richmond and Davey, 2003). Histones are phosphorylated, acetylated, methylated, ubiquitinated and sumoylated (Peterson and Laniel, 2004). Extensive post-translational modifications mainly occur in the tail regions of histones, and affect transcriptional activation, repression, chromatin assembly, heterochromatin gene silencing, centromere chromatin, response to DNA damages, histone deposition, chromosome segregation, mitosis, meiosis and spermatogenesis: histone tails may be viewed as complex protein–protein interaction surface that are regulated by numerous modifications (e.g., Grewal and Elgin, 2002; Fischle et al, 2003a, 2003b; Hayashi et al, 2004; Korber and Horz, 2004; Tagami et al, 2004). Histone tails contribute significantly neither to the structure of individual nucleosomes nor to the stability, but they are thought to strongly affect the folding of nucleosomal arrays into higher-order structure. What then would be the role of inner histone structure in cellular physiological regulations? Does it solely contribute toward the assembly to nucleosomal structure? Post-translational modifications infrequently occur in the inner structure, which is often not exposed to the outer surface of nucleosome. However, K79 in the L1 region between α1 and α2 of H3 (L and α represent, respectively, loop and α-helix; Luger et al, 1997) is methylated by a methylase Dot1 and this methylation has been shown to be implicated in meiotic checkpoint control and telomeric silencing (San-Segundo and Roeder, 2000; Feng et al, 2002; Ng et al, 2002; van Leeuwen et al, 2002). Amino-acid substitution mutants in the inner histone structure have been scarce, as genetic analyses of histone functions in metazoan cells are hampered because of the high copy numbers of the histone genes in the genome. In this study, we characterized the temperature-sensitive (ts) mutants of the fission yeast Schizosaccharomyces pombe histone H2B gene. The mutation sites reside in the highly conserved, non-helical, inner regions of histone H2B. Our results suggested that the inner structure of H2B is essential for proper ubiquitination, centromere function, gene silencing and chromosome segregation. Results Isolation of S. pombe histone H2B mutants To isolate histone H2B mutants, ts mutants were randomly isolated, followed by characterization of cytological mutant phenotypes (Hayashi et al, 2004). Then, mass transformation using plasmid pH2AH2B that carried the paired genes of histone H2A–H2B (Yuasa et al, 2004) was performed by introducing plasmid into ∼500 individual strains. The resulting transformants were screened for their ability to produce colonies at the restrictive temperature (36°C). Three mutant strains (72, 223, 442), whose ts phenotype was fully suppressed by plasmid pH2AH2B at 36°C, were obtained. Subcloning established that the histone H2B gene (designated htb1+: Matsumoto and Yanagida, 1985), but not H2A gene, was capable of suppressing the ts phenotype (Figure 1A). S. pombe has the sole gene of H2B, but the two H2A and three paired H3–H4 genes are present in the genome (Matsumoto and Yanagida, 1985); one may expect that only htb1+ can generate ts mutations. Figure 1.S. pombe H2B mutations reside in the conserved, non-helical regions. (A) Plasmid pH2B suppresses the ts phenotype of htb1-72 and htb1-223. (B) htb1-72 and htb1-223 mutants fail to produce colonies at 36°C on the complete YPD plates. (C) Amino-acid sequences of fission yeast and human histone H2B. Identical and similar amino acids are indicated in black and gray boxes, respectively. Substituted amino acids in the three htb1 mutants are indicated by arrows (see text). The α-helices are labeled as α1, α2, α3 and αC, whereas L1 and L2 represent the DNA binding loops. (D, E) The nucleosome structure (D, front view; E, side view; White et al, 2001) is depicted with the positions of altered amino acids. The atomic coordinates file (ID; 1ID3) was obtained from Brookhaven Protein Databank (PDB) and visualized with VMD (version 1.8.3). The DNA phosphodiester backbone is shown in gray. The octamer histone chains are schematized by the helical rods and the interhelical loops. Histone H2B subunits are indicated in red. The asterisks indicate the position of K that is associated with monoubiquitin. (F) The 3D structure of ubiquitin (PDB ID; 1UBQ) (Vijay-Kumar et al, 1987). Download figure Download PowerPoint To determine whether the ts mutations actually resided in the htb1 locus, an integration vector pYC11 carrying the htb1+ gene and the Saccharomyces cerevisiae LEU2 was chromosomally integrated in the leu1 mutant strain by homologous recombination (because of the low sequence homology between LEU2 and leu1+, homologous integration occurred at the htb1+ locus). The resulting Leu+ integrant strain was crossed with ts mutants, and tetrad dissection was performed. The Ts− and Leu+ phenotypes were tightly linked. With the finding that pH2B, but not pH2A, suppressed the ts phenotype (Figure 1A), we concluded that the ts mutants were derived from the htb1 locus. After several rounds of crossing with the wild-type strain for removing extra silent mutations, if any, the ts phenotypes of three ts strains (htb1-72, -223, -442) at 36°C were characterized. The Ts+ and Ts− phenotypes were segregated 2:2 for the strains 72 and 223, but not for htb1-442. The 442 strain appeared to contain a leaky ts and an additional silent mutation (data not shown). We hence employed mostly the two strains 72 and 223 for subsequent physiological experiments. In Figure 1B, the failure of htb1-72 and htb1-223 to form colonies on the complete YPD plate at 36°C is shown. The strain htb1-72 showed the ts phenotype severer than htb1-223 as it did not form colonies at 33°C. Mutation sites are located in the distinct, but in the conserved and non-helical regions Mutant genes of histone H2B were obtained from the isolated ts strains by the PCR method, and their nucleotide sequences were determined. It was thus established that single-nucleotide substitutions existed for each of the three htb1 mutant strains. For htb1-72, only the 52nd amino-acid residue showed the nucleotide change from GGT to GAT in comparison with the wild-type sequence. For htb1-223, the 102nd residue was altered from CCC to CTC. The resulting amino-acid substitutions in htb1-72 and htb1-223, respectively, were 52Gly to Asp (G52D) and 102Pro to Leu (P102L). The 442 segregant that showed the weak ts phenotype contained a nucleotide change in the 34th residue (from GAA to AAA), causing the change from Glu to Lys (E34K). These three mutations occurred at the highly conserved residues in histone H2B. The amino acids of the mutation sites were conserved between fission yeast (Pombe) and human histone H2B, as shown in Figure 1C. One of them (72) resides in the middle of histone fold, α1–L1–α2–L2–α3, whereas the location of the two others (223 and 442) is not within but nearby the histone fold. Residue G52D (72) is positioned in the loop L1 that exists between α1 and α2 helices (Figure 1C). L1 and L2 are the sites to be bound to DNA. In contrast, the P102L residue (223) locates in the short linker between the two helices, α3 and the αC extension. The long αC extension is unique to H2B, and defines the outer limit of the protein surface of nucleosome core particle. P102L is thus close in contact with H2Aα2 by extensive hydrophobic interaction (Luger et al, 1997). The E34K (442) leaky mutation is in the conserved, amino-terminal tail that is connected to the amino-terminal edge of α1 helix. The physical locations of mutant residues are indicated by the thick arrows in Figure 1D (front view) and Figure 1E (side view) in the nucleosome core structure determined by crystallography (Luger et al, 1997; White et al, 2001; Richmond and Davey, 2003). The thin arrows with asterisk represent the K119 site to bind to monoubiquitin (Figure 1E and F). The ts mutations identified in this study are thus all located in the conserved and non-helical regions. The G52D substitution is in contact with the minor groove of DNA, whereas P102L is in the central region of the histone octamer in the front view (Figure 1D) and in the middle of the outer sides in the side view (Figure 1E). P102L hence interacts with the histone H2A in the same nucleosome, or also with the histones of adjacent nucleosomes (Schalch et al, 2005). The E34K substitution is close in contact with DNA. Missegregation and chromatin shrinkage occur in htb1 mutants We investigated whether these htb1 mutations affected the cell division cycle of S. pombe. Exponentially growing cultures of wild-type, htb1-72 and htb1-223 strains at 26°C in the complete YPD were shifted to 36°C for 12 h. Cells were scored for viability and observed by DAPI staining after glutaraldehyde fixation. Interestingly, htb1-223, but not htb1-72, showed frequent unequal nuclear division after 8 h at 36°C (arrowheads in Figure 2A, lower right panel), the characteristic phenotype seen in the centromere/kinetochore protein mutants (Takahashi et al, 1994, 2000; Saitoh et al, 1997; Goshima et al, 1999; Hayashi et al, 2004). Quantitative data (Figure 2B, red line with rectangles in the middle panel) showed that the frequency of cells with unequal daughter nuclei increased after 4 h at 36°C in coincidence with the gradual decrease of cell viability (bottom panel), and reached 38% of binucleated cells. Typical centromere/kinetochore protein mutants mis6 and mis12 produced ∼80% unequal nuclei in binucleate cells so that the unequal segregation phenotype of htb1-223 was not severe as mis6 and mis12. The unequal nuclear division phenotype was scarce (a few %) in htb1-72. Instead, the nuclear chromatin observed by DAPI staining appeared to be shrunk (12 h; Figure 2A, middle right panel). Cell viability of htb1-72 decreased at the time of nuclear chromatin shrinkage. The gross chromatin organization in htb1-72 appeared to be altered. Additionally, the thick septa indicative of cytokinesis delay were observed in htb1-72. Figure 2.Cellular phenotypes of histone H2B mutants htb1-72 and htb1-223. Asynchronously growing htb1 mutant cells in YPD medium were shifted to 36°C for 0–12 h. (A) Mutant cells cultured at 26°C (left panels) or 36°C for 8 or 12 h (right panels) were stained by DAPI. Upper panels, wild type; middle panels, htb1-72; lower panels, htb1-223. Arrowheads indicate binucleate cells displaying unequal nuclear division. Bar, 10 μm. (B) Top, cell number increase; middle, the frequencies of unequal nuclear division; bottom, cell viability. (C) Unequal chromosome segregation was examined by the cen1(lys1)-GFP method (see text). Three examples of htb1-223 mutant cells cultured at 36°C for 10 h displaying unequal segregation of cen1(lys1)-GFP signals are shown. Bar, 10 μm. Download figure Download PowerPoint To confirm that the unequal nuclear division phenotype of htb1-223 was due to chromosome missegregation, the cen1-GFP method (Nabeshima et al, 1998) was employed. After 10 h at 36°C, 25% of the binucleate htb1-223 cells (50/204) exhibited unequal nuclear division. As shown in Figure 2C, the cen1-GFP signals failed to segregate in these cells. Missegregation of cen1-GFP signals was observed in 22% (11/50) of cells showing unequal nuclear division. We thus concluded that the unequal nuclear division phenotype in htb1-223 mutant was owing to chromosome missegregation. Histone H2B mutants were sensitive to a protein synthesis inhibitor To understand the causes for the histone H2B mutants to produce distinct phenotypes, we examined whether these mutants might be differentially sensitive to drugs and irradiation. Effects of hydroxyurea (DNA replication inhibitor) and UV irradiation were tested, but their sensitivities were similar to that of wild type (data not shown). Cycloheximide (Cyh, a protein synthesis inhibitor), however, showed different sensitivities as shown in Figure 3A. At 30 and 33°C (semi-restrictive temperature), both htb1-72 and htb1-223 were sensitive to Cyh. At 30°C, htb1-223 was more sensitive than htb1-72. Introducing pH2B plasmid into the mutants restored the hypersensitivity (data not shown), indicating that the htb1 mutations caused both ts and Cyh phenotypes. The addition of Cyh thus produced the synthetic lethal effect on htb1 mutations. Figure 3.Cyh sensitivity and turnover of Rhp6-dependent ubiquitination of H2B in htb1 mutants. (A) The htb1 mutants are hypersensitive to Cyh, a protein synthesis inhibitor. Both htb1-72 and htb1-223 were slower in growth than wild type in the presence of Cyh. Cells were serially diluted (1:5) and spotted onto the YPD plates containing Cyh (0, 2 and 5 μg/ml). The highest-density spots contained 5 × 104 cells. (B) The cell number increase (left panel) and the frequencies of unequal nuclear division (right) are shown for wild type (open) and htb1-223 (closed) in the absence (circles) or presence (rectangles, triangles) of Cyh at 33°C. (C) Immunoblot patterns using polyclonal antibodies against histone H2B (anti-H2B) and monoclonal antibodies against HA (anti-HA). Extracts of cells expressing endogenous H2B (Rep1 vector) and ectopically expressed H2B tagged with HA (pRep1-htb1HA) in the presence of thiamine (promoter off) and absence of thiamine (14 h after removal of thiamine; promoter on) were used for immunoblot. The 17 kDa H2B band and the 24–25 kDa uH2B bands (indicated by the arrows) were detected in cells carrying vector. In cells carrying pRep1-htb1HA, additional bands for H2B-3HA (asterisks) were detected by anti-H2B and anti-HA antibodies. (D) Immunoblot was performed for extracts of wild type, Δrhp6 carrying vector or plasmid pRHP6. The presumed monoubiquitinated H2B band was missing in Δrhp6. (E) Wild type, htb1-72 and htb1-223 were cultured in the absence (−) or presence of (+) of Cyh (100 μg/ml) at 36°C for 0–180 min. In the case of +Cyh, strains were first cultured at 36°C for 60 min and then followed by the addition of Cyh. Immunoblot patterns by short and long exposure are shown. The positions for H2B and uH2B are indicated by arrowheads. (F) Wild type, mts2-1, htb1-223 and the double mutant mts2-1 htb1-223 were cultured first at 26°C, and then shifted to 36°C for 8 h. Immunoblot patterns using anti-H2B antibodies indicated that the level of decrease of uH2B was not arrested in the proteasome mutant mts2-1 background. Download figure Download PowerPoint We then tested whether Cyh affected the cellular phenotypes of the htb1-223 mutant at 33°C, the semi-restrictive temperature. To this end, the culture of htb1-223 grown at 26°C was shifted to 33°C for 2 h in the absence of Cyh followed by the addition of Cyh (2 and 5 μg/ml) for the continued culture at 33°C for 12 h (Figure 3B). For control, wild type without or with Cyh (5 μg/ml) was tested. In the absence of Cyh, the frequency of unequal nuclear division was negligible in htb1-223 at 33°C. In the presence of Cyh, however, the cell number increase (left panel) was considerably retarded and the frequency of unequal nuclear division (right panel) strikingly increased after 4 h. These results suggested that the level of functional H2B protein might decrease in the presence of Cyh in htb1-223 mutant at the semi-restrictive temperature (see below). Rhp6-mediated monoubiquitination of H2B The above results suggested that mutant histone H2B might be unstable or faster in turnover than that of wild-type histone H2B. The inhibition of protein synthesis might reduce the level of mutant histone H2B. To test this hypothesis, polyclonal antibodies against a 16 amino-acid peptide of the amino-terminal tail sequence SAAEKKPASKAPAGKA of S. pombe histone H2B were raised. Immunoblot of cell extracts were carried out using affinity-purified anti-H2B antibodies. An intense ∼17 kDa band (corresponding to histone H2B) was seen in wild-type cells carrying vector plasmid pREP1 (Figure 3C, left). A weak ∼25 kDa band (indicated by the arrows) was observed in the long exposed immunoblot patterns. This 25 kDa band corresponds to monoubiquitinated histone H2B (designated uH2B; see below). The assignment of the bands was confirmed using extracts of cells that expressed the HA-tagged histone H2B under the control of the inducible nmt1 promoter (Figure 3C). Antibodies against H2B (left panel) and against HA (right panel) were used to detect HA-tagged H2B that was overproduced in the absence of thiamine (promoter, on) for 14 h after the removal of thiamine. The HA-tagged H2B band (indicated by asterisks) was dramatically increased in the absence of thiamine. Polyclonal antibodies against H2B thus detected both endogenous and ectopically expressed H2B. The 25 kDa upper H2B band was produced in an Rhp6-dependent manner. As shown in Figure 3D, the upper band disappeared in the deletion mutant Δrhp6 (Kitamura et al, 2001). This band was restored in Δrhp6 carrying plasmid pRHP6 (constructed by PCR method in this study). Rhp6 is a homolog of budding yeast Rad6, a ubiquitin conjugating enzyme, which is known to be responsible for monoubiquitination of histone H2B (Robzyk et al, 2000). We hence supposed that the 25 kDa band was an Rhp6-mediated monoubiquitinated band. The band intensity of uH2B in the absence of Cyh was approximately 10% of total cellular histone H2B in wild type. Monoubiquitinated H2B-223 mutant protein is unstable We then tested whether mutant H2B was less stable than wild type in the presence or absence of Cyh. Cyh (100 μg/ml) was added to the cultures of wild type, htb1-72 and htb1-223, 60 min after the shift to 36°C, and cell extracts were prepared. Immunoblot patterns of wild-type and mutant extracts using antibodies against histone H2B are shown in Figure 3E. Although the levels of H2B remained high and did not alter significantly, the uH2B level in htb1-223 mutant was already low before the shift (roughly 50% compared with that in wild-type cells), and decreased after the shift to 36°C in the absence of Cyh. In the presence of Cyh, the level of uH2B-223 rapidly decreased. The levels of wild-type uH2B and other mutant uH2B-72 also decreased in the presence of Cyh. The quantitative densitometric analyses of immunoblot patterns indicated that the half-life of uH2B-223 in the presence of Cyh at 36°C was only 15 min, whereas those in wild type and htb1-72 were 88 and 86 min, respectively. In the absence of Cyh, mutant H2B-223 became unstable by the temperature shift to 36°C, and the apparent half-life of uH2B-223 was 92 min. The level of H2B in wild type and htb1-72 did not alter in the absence of Cyh. We therefore concluded that the half-life of uH2B was specifically short in htb1-223. Instability of uH2B in htb1-223 is not due to 26S proteasome-dependent proteolysis A question was raised whether instability of ubiquitinated H2B was due to proteolysis. Immunoblot of H2B protein was carried out for extracts in a proteasome mutant mts2-1 and the double mutant mts2-1 htb1-223. As shown in Figure 3F, the level of uH2B was not affected at all in the absence of 26S proteasome function for wild-type H2B, but mutant H2B-223 protein destruction seemed to be slightly suppressed so that mutant uH2B might go through the proteasome for degradation. However, the decay in the level of uH2B might be principally owing to deubiquitination rather than 26S-dependent proteolysis. In any case, uH2B became highly dynamic in htb1-223 cells in the presence of Cyh so that chromatin would be strongly altered if uH2B was indispensable. Ubiquitination-deficient H2B does not support cell viability To investigate whether instability of uH2B has any physiological consequence, we constructed a plasmid containing htb1-K119R mutant gene (pH2B-K119R) that had a lysine-to-arginine substitution at the conserved monoubiquitination site K119 of histone H2B to R by site-directed mutagenesis. Plasmid pH2B-K119R tagged with FLAG and under the inducible promoter nmt1 failed to produce the normal band of uH2B (Supplementary Figure 1). We first tested whether high-copy plasmid pH2B-K119R could complement the ts phenotype of htb1 mutants. As shown in Figure 4A, pH2B-K119R suppressed neither the ts phenotype of htb1-72 nor htb1-223. Secondly, we examined whether htb1-223 mutant carrying plasmid pH2B-K119R displayed the missegregation phenotype at 36°C. Although the appearance was delayed, the missegregation phenotype was still observed (closed diamonds in the middle panel in Figure 4B). In addition, the septation index increased, indicating that cytokinesis was delayed, resembling the phenotype of htb1-72. In control htb1-223 cells carrying wild-type plasmid pH2B, no missegregation phenotype was observed. Figure 4.H2B K119R mutant substituted at monoubiquitination site fails to suppress the phenotype of htb1 mutant. (A) Plasmid carrying the htb1-K119R gene failed to suppress the ts phenotype of htb1-72 and htb1-223 mutants. (B) The cultures of htb1-223 mutant carrying vector, pH2B, or pH2B-K119R were asynchronously grown in the EMM2 medium and shifted to 36°C. The cell number increase (left panel), the frequencies of unequal nuclear division phenotype (middle) and the septation index (right) after the shift-up are shown. Download figure Download PowerPoint CENP-A localization was partly diminished in htb1-223 To examine whether Cnp1, the centromere-specific histone H3 variant CENP-A in fission yeast (Takahashi et al, 2000), could be normally localized to the centromere in htb1-223 mutant, the cnp1+ gene was tagged with GFP and integrated onto the chromosome and expressed under the native promoter. Cnp1-GFP signal was observed after methanol fixation. In control htb1+ cells, Cnp1-GFP signals were observed as dots signals in nuclei throughout the cell cycle. After shifting-up to 36°C, the frequencies of cells with dots signals in htb1+ were 83% (0 h), 76% (4 h) and 76% (8 h) (Figure 5A, bottom panel). However, in htb1-223 cells cultured at 36°C for 8 h, the frequencies of Cnp1-GFP signals were significantly diminished from 82% at 26°C (0 h) to 53% at 36°C (6 h). Examples of the dot and dispersed signals of Cnp1-GFP are shown in the upper panels of Figure 5A. Quantitative data are indicated in the lower panel. The blue portions represent the fractions of cells that displayed the dispersed Cnp1-GFP signals. The frequencies of centromeric Cnp1-GFP in htb1-72 cells, however, are similar as those in htb1+. These results suggested that Cnp1 was not properly loaded onto centromere in htb1-223 or that centromeric chromatin architecture was structurally spread whereas Cnp1 binding to centromeres remained. We examined whether Rhp6 has any influence over Cnp1 localization, and found that Cnp1 localization was normal in Δrhp6 deletion mutant cells (data not shown), suggesting that monoubiquitinated H2B did not seem to be implicated in Cnp1 localization. Figure 5.Centromere-specific chromatin structure is partly disrupted in htb1-223 mutant cells. (A) Cnp1-GFP signals (green) were observed in the htb1 mutant background. DAPI was used to stain DNA (purple). Bar, 10 μm. For quantitative measurements, cells were grouped into three classes based on the localization patterns of Cnp1: the dots signals (orange), the dispersed signal (blue) and no detected signal (gray). (B) CHIP experiment was performed using the central centromere probes of cnt1, imr1, the outer heterochromatic repeat dg and pericentric lys1 (lower left). The organization of cen1 is schematized (top). Relative signal intensities were determined and quantitative data are shown (lower right, the value for wild type 0 h is 1.0). The amount of cnt1 and imr1 DNAs co-precipitated with the Cnp1-HA fusion was reduced in htb1-223 mutant cells at 37°C (anti-HA for cnt1 and imr1). (C) Micrococcal nuclease experiments. Nuclear chromatin were prepared from wild-type, htb1-72, htb1-223 and mis6-302 cells that were cultured at 37°C for 7 h, and digested with micrococcal nuclease for 0, 1, 2, 4 and 8 min, followed by agarose gel electrophoresis and Southern hybridization using the three centromeric DNA probes cnt1, imr1 and otr1. The ethidium bromide staining patterns of the gel are also shown. Download figure Download PowerPoint To further substantiate the above localization result, chromatin immunoprecipitation (CHIP) was performed under the htb1 mutant background. The cnp1+ gene was tagged with HA and integrated onto the chromosome under the native promoter (Takahashi et al, 2000). Cnp1-HA was immunoprecipitated after formaldehyde fixation. The levels of PCR signals in the htb1-223 mutant background were estimated, using the central centromere primers (cnt and imr). As shown in Figure 5B, the CHIP patterns (lower left) and their quantitative estimations (lower right) indicated that the levels of the central centromere sequences bound to Cnp1 were reduced after the shift-up to 37°C. Wild-type and mis6-302 are also shown as a control: it is known that Cnp1 centromere localization is greatly reduced in mis6-302 (Takahashi et al, 2000). These results supported the possibility that htb1-223 mutation had an effect on Cnp1 centromere localization, and showed that the degree of association between centromeric DNA and Cnp1 was diminished. The t-test was conducted and the P-values obtained were 0.024 and 0.013 for cnt and imr, respectively. A possibility was raised that, if nucleosome density was altered by the H2B mutation, then the measurements of nucleosome modifications and variants are not informative without correction for nucleosome content. We therefore performed CHIP using anti-H2B antibodies. No significant change in the nucleosome density was found in the centromeric regions (Figure 5B). Partial disruption of specialized centromere chromatin in htb1-223 The above experiments could not establish that centromeric chromatin was disrupted in htb1-223 mutant cells. To examine whether centromeric structure was defective in htb1-223, we performed a micrococcal nuclease digestion experiment. When nuclear chromatin of wild-type S. pombe is digested with micrococcal nuclease, Southern blot using the probes of inner centromere DNAs displays a specialized chromatin with smeared digestion pattern (Polizzi and Clarke, 1991; Takahashi et al, 1992). In cnp1, mis6 and mis12 centromere-defective mutants (Saitoh et al, 1997; Goshi