1. A Cell Cycle-Regulated GATA Factor Promotes Centromeric Localization of CENP-A in Fission Yeast 
Ee Sin Chen, Shigeaki Saitoh, Mitsuhiro Yanagida, Kohta Takahashi 
Molecular Cell 
Volume 11, Issue 1, Pages (January 2003)
DOI: /S (03)00011-X

2. 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 pnmt41-AMS2 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, AF
Molecular Cell  , DOI: ( /S (03)00011-X)

3. 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-Ats-GFP 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 μm.
(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 (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 cdc25-22 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.
Molecular Cell  , DOI: ( /S (03)00011-X)

4. Figure 3
Chromosome Missegregation and SpCENP-A Mislocalization Phenotypes Produced by Δams2 Disruption
(A) Slow colony formation of Δams2 on the YPD plates at 33°C for 2, 3, or 4 days.
(B) Aberrant mitotic nuclear division of Δams2 cells in the EMM2 liquid cultures at 33°C. DNA (blue) and microtubules (red). Bar, 10 μm.
(C) Δams2 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 Δams2 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 Δams2 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 Δams2 or wild-type cells cultured in YES at 26°C was immunoprecipitated (IP) for the ChIP analysis using antibodies against HA (αHA) and the C-terminal region of human histone H3 (αCENP-A+H3). This antibody recognizes both SpCENP-A 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 Δams2 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.
Molecular Cell  , DOI: ( /S (03)00011-X)

5. Figure 4
Genetic Interactions between Ams2 and Histones and between Ams2 and Kinetochore Proteins
(A) Slow-growing Δams2 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 mis but not that of mis6-302 by the plasmid carrying the ams2+ gene on EMM2 plates at 36°C.
(C) Synthetic lethal phenotype of Δams2 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.
Molecular Cell  , DOI: ( /S (03)00011-X)

6. 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 μm.
(B) cdc25-22 ts mutant expressing Ams2-GFP 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 μm.
Molecular Cell  , DOI: ( /S (03)00011-X)

7. 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.
Molecular Cell  , DOI: ( /S (03)00011-X)

8. 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 GST-Ams2 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 (Imr1-w/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.
Molecular Cell  , DOI: ( /S (03)00011-X)