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First published online March 19, 2008
doi: 10.1242/10.1242/jcs.024224

Journal of Cell Science 121, 1107-1118 (2008)
Published by The Company of Biologists 2008
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Diminishing HDACs by drugs or mutations promotes normal or abnormal sister chromatid separation by affecting APC/C and adherin

Yuu Kimata1,2, Akihisa Matsuyama3, Koji Nagao1,*, Kanji Furuya1,{ddagger}, Chikashi Obuse1,§, Minoru Yoshida3 and Mitsuhiro Yanagida1,¶

1 CREST Research Program, Japan Science and Technology Corporation, Graduate School of Biostudies, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan
2 Cell Cycle Control Group, Marie Curie Research Institute, The Chart, Oxted, Surrey, RH8 0TL, UK
3 Chemical Genetics Laboratory, RIKEN Institute, Wako, Saitama 351-0198, Japan


Figure 1
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  		Fig. 1. mis4 mutants are sensitive to HDAC inhibition and show synthetic lethality with HDAC mutants. (A) Spot tests of effects of TSA on the growth of a kinetochore mutant mis6-302 and HDAC mutants clr6-1, nmt1-clr6HA, {Delta}clr3, {Delta}hos2 and wild type (WT). These strains were grown at 26°C and spotted at serial cell concentrations on YPD plates lacking or containing TSA (12.5 µg/ml), followed by incubation at the indicated temperatures. (B) Wild type, mis4-242, rad21-K1, psc3-407 and eso1-H17 mutants were spotted on YPD plates lacking or containing TSA (25.0 µg/ml). (C) mis4-242 mutants were cultured in YPD medium containing 12.5 µg/ml TSA at 26°C (upper panel) or in YPD medium at 36°C (lower panel) for 6 hours, then fixed with glutaraldehyde and stained with DAPI. The cells showing unequal chromosome missegregation phenotypes are indicated by arrowheads, and the cells in which chromosomes were condensed because of mitotic arrest due to spindle checkpoint activation by arrows. Scale bar: 10 µm. (D) The indicated strains were grown exponentially and then spotted on YPD plates.

 

Figure 2
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  		Fig. 2. TSA treatment decreases the Mis4 protein level and the chromatin-bound portion of Rad21 in the chromosome arm region. (A) Wild-type and mis4-242 cells were cultured in YPD medium at 26°C and were harvested at the indicated time points after TSA (12.5 µg/ml) was added. Immunoblotting for the cell extracts was carried out for Mis4, Rad21, Cut9, Cdc2 (PSTAIR) and acetylated histone H3 and H4 (AcH3 and AcH4, respectively). (B) Non-tagged wild type (WT No tag), rad21HA integrant strains (WT rad21HA) and cut9-665rad21HA strains were cultured at 26°C, and then incubated at 36°C for 4 hours with or without TSA (12.5 µg/ml). ChIP for Rad21HA was performed using anti-HA antibody followed by PCR with four sets of primers (cnt1 and dg for the central and outer centromere, respectively, and lys1 and c83 for the chromosome arm region). Immunoblotting for the whole cell extracts of the unfixed cells was also carried out using the indicated antibodies (WB). (C) Non-tagged wild-type (No tag) and rad21HA strains were grown exponentially at 26°C and cells were fixed at the indicated time points after TSA (12.5 µg/ml) was added to the cultures. ChIP was carried out as B using seven sets of primers (imr1 for the central centromere on chromosome I, 08c and ARS2004 for the arm).

 

Figure 3
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  		Fig. 3. Histone deacetylase inhibitors suppress temperature sensitivities of S. pombe APC/C mutants. (A) Wild type and APC/C subunit mutants (cut4-533, cut9-665, nuc2-663, cut23-194, cut20-100, apc10-153, apc10-666) were cultured at 26°C and then spotted on YPD plates lacking or containing TSA (12.5 µg/ml). (B) Wild type and cut4-533 mutants were grown at 26°C and then shifted to 36°C (Time=0); at the same time TSA (12.5 µg/ml) was added to the cultures. Cell numbers were counted (left panel) and cytological phenotypes of cut4-533 mutants were observed by staining with DAPI (right panel) at the indicated time points. (C) Effects of various HDAC inhibitors (TSA, CHAP31, FK228 and TPXB) on the growth of wild type and cut4-533 mutants. Cells were cultured exponentially at 26°C, and then the temperature was shifted to 36°C after the cell numbers were adjusted to OD600=0.01. HDAC inhibitors or the same volumes of the buffers (EtOH or DMSO) were added at the indicated concentrations, and cell numbers (OD600) were counted after 24 hour incubation at 36°C. (D) Effects of TSA on ts or null mutants of proteins involved in the APC/C-dependent ubiquitination and proteolysis (ubcP4-140, slp1-362, {Delta}ste9, mts2-1, mts3-1 and {Delta}mad2). The indicated strains were spotted on YPD plates lacking or containing TSA (12.5 µg/ml). Scale bar: 10 µm.

 

Figure 4
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  		Fig. 4. Clr6 HDAC mutations suppress APC/C mutants. (A) clr6 mutations, but not {Delta}clr3 or {Delta}hos2, suppress the ts growth of cut4-533 mutants. The indicated strains were spotted on YPD plates and incubated at the indicated temperatures. In nmt1-clr6HA cells, the expression of HA-tagged clr6+ gene under nmt1 promoter was repressed on YPD plates. (B) The temperature sensitivities of cut9-665, apc10-153 and apc10-666 mutants were suppressed by clr6-1 mutations. The indicated strains were spotted and incubated. (C) The growth and the frequency of mitotic arrest phenotypes of double mutants of cut4-533 and HDAC mutants at the restrictive temperature. The indicated strains were cultured at 26°C and shifted to 36°C. The cell number (left panel) and the frequency of the mitotic delay phenotype (right panel) were measured at the indicated time points.

 

Figure 5
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  		Fig. 5. Inhibition of Clr6 HDAC stimulates the formation of the 20S APC/C complex. (A) Effects of TSA treatment on cellular levels of APC/C subunit proteins. Wild type, cut4-533 and cut9-665 were cultured at 26°C and TSA (12.5 µg/ml) was added into the cultures. Cells were harvested at the indicated time points and the whole cell extracts were examined by imunoblotting to determine the concentrations of APC/C subunit proteins. {alpha}-tubulin was detected by TAT1 antibody as loading control. (B) Wild type and cut4-533 mutants were cultured at 26°C and cells were harvested after 12 hour incubation in the absence or presence of 12.5 µg/ml TSA. The cell extracts were fractionated by 15-40% sucrose gradient centrifugation and APC/C subunits Cut4, Cut9 and Nuc2 were detected by immunoblotting. The fractions corresponding to 4.5S and 16.5-19S were determined using bovine serum alubmin and thyroglobulin A, respectively. (C, left panel) Sucrose gradient centrifugation was performed as in B using the cell extracts of wild-type, cut4-533 and cut4-533clr6-1 cells that were cultured at 26°C or 36°C for 4 hours. A condensin subunit Cnd2 was shown as a marker indicating the fraction corresponding to 13S. (C, right panel) The aliquots of the whole cell extracts of each strain were blotted. (D) Wild-type strains were cultured at 26°C and TSA (12.5 µg/ml) was added. Cells were harvested after 12 hour incubation at 26°C. The cell extract was fractionated and analysed as in B. (E) Wild type (no tag) and apc2HA strain were cultured at 26°C in the absence or presence of TSA (12.5 µg/ml) for 12 hours. Apc2HA protein was immunoprecipitated with anti-HA antibody. Cut4, Cut9 and Nuc2 co-immunoprecipitated with Apc2HA were detected by their specific antibodies.

 

Figure 6
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  		Fig. 6. An APC/C subunit Cut23 is acetylated in vivo. (A) 3HA6His-tagged Cut23 overproduced from pREP1 plasmid in wild type was purified using nickel beads under denaturing conditions and was separated by SDS-PAGE followed by Coomassie Brilliant Blue (CBB) staining. (B) The band of purified Cut23-3HA6His shown in (A) (~64 kDa) was cut out and subjected to survey of the acetylation site using LC-MS/MS after trypsin digestion (Ohta et al., 2002Go). The MS/MS spectra of the tryptic acetylated peptide amino acids 82-95, 123-132, 499-502 of Cut23 obtained by collision-induced dissociation of the [M + 2H]2+ or [M + 3H]3+ precursor ions, m/z 858.55, m/z 606.87, m/z 496.91, m/z 946.02. Each peptide has one possible acetylated lysine residue, K88 in 82-95, K126 in 123-132, K461 in 453-464 or K502 in 499-514. Peptides containing unmodified lysine residues were also detected for each region and these four acetylated peptides were detected in two independent experiments. (C) Localization of the four putative acetylated sites in Cut23 protein. The acetylation sites (Ac) and TPR domains are shown in the diagram. Six destruction box-like sequences (RxxL) in Cut23 are also shown as black bars. (D) Spot tests for indicated strains on YPD plates in the absence or the presence of 12.5 µg/ml TSA.

 

Figure 7
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  		Fig. 7. HDAC inhibition facilitates sister chromatid separation in S. pombe mutants. (A) ts mutants of S. pombe separase Cut1 and securin Cut2 were suppressed by TSA. cut1-206, cut1-T693, cut1-21 and cut2-364 growing exponentially at 26°C were spotted on YPD plates lacking or containing TSA (12.5 µg/ml), and then incubated at 26, 30, 33 and 36°C. (B) Wild type, cut9-665, {Delta}pka1 and cut9-665{Delta}pka1 mutants grown exponentially were diluted and spotted on YPD plates lacking or containing TSA (12.5 µg/m). (C) cut9-665 and cut9-665{Delta}mad2 stains were spotted as B. cut9-665{Delta}mad2 double mutants grew better than the cut9-665 single mutants; however, the temperature sensitivities were further suppressed in the presence of TSA. (D) Results presented in the present study are summarized. Clr6, Clr3 and Hos2 are three HDACs that are sensitive to TSA. Clr6 inhibition facilitates the assembly of APC/C ubiquitin ligase complex in the mutant cells. Inhibition of Clr3 and Hos2 deteriorates chromatin loading of cohesin complex through Mis4 destablisation. In both cases, sister chromatid separation is promoted.

 

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© The Company of Biologists Ltd 2008