Misregulation of cell cycle-dependent methylation of budding yeast CENP-A contributes to chromosomal instability

Abstract

Centromere (CEN) identity is specified epigenetically by specialized nucleosomes containing evolutionarily conserved CEN-specific histone H3 variant CENP-A (Cse4 in Saccharomyces cerevisiae, CENP-A in humans), which is essential for faithful chromosome segregation. However, the epigenetic mechanisms that regulate Cse4 function have not been fully defined. In this study, we show that cell cycle-dependent methylation of Cse4-R37 regulates kinetochore function and high-fidelity chromosome segregation. We generated a custom antibody that specifically recognizes methylated Cse4-R37 and showed that methylation of Cse4 is cell cycle regulated with maximum levels of methylated Cse4-R37 and its enrichment at the CEN chromatin occur in the mitotic cells. Methyl-mimic cse4-R37F mutant exhibits synthetic lethality with kinetochore mutants, reduced levels of CEN-associated kinetochore proteins and chromosome instability (CIN), suggesting that mimicking the methylation of Cse4-R37 throughout the cell cycle is detrimental to faithful chromosome segregation. Our results showed that SPOUT methyltransferase Upa1 contributes to methylation of Cse4-R37 and overexpression of UPA1 leads to CIN phenotype. In summary, our studies have defined a role for cell cycle-regulated methylation of Cse4 in high-fidelity chromosome segregation and highlight an important role of epigenetic modifications such as methylation of kinetochore proteins in preventing CIN, an important hallmark of human cancers.

FIGURE 1:

Methylation of Cse4-R37 is cell cycle regulated. (A) Specificity of ^MeCse4-R37 antibodies to methylated Cse4. Western blots showing specific reactivity of the ^MeCse4-R37 antibodies. Cse4 was enriched with Ni-NTA agarose from whole cell extracts of logarithmically growing wild-type (YMB7289) and cse4-R37A (YMB7287) strains and analysed by Western blotting using α-HA and α-^MeCse4-R37 antibodies. (B) Cell morphology analysis show synchronization in G1, S-phase and G2/M. Wild-type strain (YMB7289) was grown to early logarithmic phase at 25°C and synchronized in G1 (3-μM α-factor treatment), S-phase (0.2-M HU treatment), and G2/M (20-μg/mL NOC treatment). Cell cycle stages were determined based on nuclear position and cell morphology by microscopic examination of at least 100 cells for each sample. Different stages of the cell cycle: G1, S-phase (S), and G2/M. Average ± standard error (SE) derived from three biological replicates is shown. (C) The levels of methylated Cse4-R37 are higher in G2/M (mitotic) cells. Cse4 was enriched with Ni-NTA agarose using whole cell extracts from wild-type strain (YMB7289) synchronized in G1, S-phase, and G2/M and analysed by Western blotting using α-HA and α-^MeCse4 antibodies. (D) Enrichment of relative methylation of Cse4 in G1, S-phase, and G2/M. Ratio of methylated Cse4-R37 (^MeHA-Cse4) to the total Cse4 (HA-Cse4) was calculated using Image J (Schneider et al., 2012). Three biological replicates were done. Average ± SE is shown. **p value < 0.01, Student’s t test.

FIGURE 2:

Methylated Cse4-R37 is highly enriched and associates with CEN chromatin in mitotic cells. (A) Enrichment of methylated Cse4-R37 increases during the mitosis. Wild-type strain (YMB10574) grown in YPD at 25°C to early logarithmic phase and synchronized in G1 with α-factor, released into pheromone-free medium, and sampled at 20 min time intervals. α-factor was readded at 80 min to block cells in next G1. Cse4 was enriched with Ni-NTA agarose from whole cell extracts and analysed by Western blotting using α-HA and α-^MeCse4-R37 antibodies. Western blots show the levels of Cse4 (HA-Cse4) and methylated Cse4-R37 (^MeHA-Cse4) during the cell cycle. (B) Enrichment of relative methylation of Cse4-R37 during the cell cycle. Ratio of methylated Cse4-R37 (^MeHA-Cse4) to the total Cse4 (HA-Cse4) was determined with Image J (Schneider et al., 2012). Average from three biological replicates ± SE. ***p value < 0.001, Student’s t test. (C) Cell cycle stages were determined based on nuclear position and cell morphology by microscopic examination of at least 100 cells for each sample. Different stages of the cell cycle: G1, S-phase (S), metaphase (Meta), anaphase (Ana) and Telophase (Tel). Average ± SE derived from three biological replicates is shown. (D) Protein levels of Cse4 and cse4-R37A are largely similar in G2/M cells. Wild-type (WT; YMB10574) and cse4-R37A (YMB11651) strains were grown to early logarithmic phase at 25°C and synchronized with nocodazole in G2/M. Western blotting of protein extracts using α-HA and α-Tub2 (loading control) antibodies. (E and F) Methylated Cse4-R37 associates with CEN chromatin in G2/M. ChIP was performed in WT ( YMB10574) and cse4-R37A (YMB11651) strains from (D) using α-HA agarose and α-^MeCse4-R37 antibodies. Enrichment of Cse4 and ^MeCse4-R37 at CENs (CEN1 and CEN3) and a negative control (ACT1) was determined by qPCR and is shown as % input. Average from three biological replicates ± SE. *p value < 0.05, Student’s t test.

FIGURE 3:

Reduced association of methyl mimic cse4-R37F with CEN chromatin and kinetochores. (A) Levels of cse4-R37F are reduced at CEN chromatin. Wild-type (WT, YMB10574), cse4-R37A (YMB11651) and cse4-R37F (YMB11652) strains were grown to early logarithmic phase at 25°C and synchronized with nocodazole in G2/M and ChIP was performed using α-HA agarose antibodies. Enrichment of Cse4 and its mutants at CENs (CEN1 and CEN3) and a negative control (ACT1) was determined by qPCR and is presented as % input. Average from three biological replicates ± SE. *p value < 0.05, Student’s t test. (B) Expression of Cse4, cse4-R37A and cse4-R37F. Western blotting of protein extracts from strains in (A) using α-HA and α-Tub2 (loading control) antibodies. (C) Reduced association of GFP-cse4-R37F at the kinetochore. Representative images showing GFP-Cse4 fluorescence at the kinetochores in wild-type (T785), cse4-R37A (T786) and cse4-R37F (T796) strains. (D) Quantitation of GFP fluorescence intensity from strain imaged in panel (C). GFP-signals were quantified using Image J and analysed in R-Studio. n = the number of cells examined.

FIGURE 4:

Methyl mimic cse4-R37F strain exhibits defects in kinetochore integrity and CIN phenotype. (A) Levels of Mif2 are reduced at CEN chromatin in cse4-R37F strains. ChIP was performed with α-Mif2 antibodies using chromatin from strains in Figure 3A. Enrichment of Mif2 at CENs (CEN1 and CEN3) and a negative control (ACT1) was determined by qPCR and is presented as percent input. Average from three biological replicates ± SE. *p value < 0.05, Student’s t test. (B) Expression of Mif2 in wild-type, cse4-R37A, and cse4-R37F strains. Western blotting of protein extracts from strains in Figure 3A using α-Mif2 and α-Tub2 (loading control) antibodies. (C) Methyl mimic cse4-R37F strain exhibits CIN. Frequency of CF loss in wild-type (WT; YPH1015), cse4-R37A (YMB11649), and cse4-R37F (YMB11650) was determined as described in Materials and Methods. At least 1000 colonies each from three independent transformants were counted. Values are average ± SE. **p value < 0.01, Student’s t test.

FIGURE 5:

Methyl mimic cse4-R37F strains exhibits negative genetic interactions with chromosome segregation mutants. (A) GO analysis of the negative genetic interactors for biological process and cellular component based on SGA of cse4-R37F with nonessential and essential gene mutations. (B) Methyl mimic cse4-R37F strains exhibits negative genetic interactions with ame1-4, ctf19Δ, mcm21Δ, irc15Δ, chl4Δ, and mad1Δ. Analysis of meiotic progeny of tetrad analysis from mating of cse4-R37F with isogenic BY4741 strains carrying mutations in kinetochore components as indicated. The number of expected and observed double mutants observed are shown. (C) Representative image from tetrad dissection showing SL when cse4-R37F was combined with kinetochore mutant ame1-4. The four spores (S1–S4) from individual asci and their genotypes are marked.

FIGURE 6:

Reduced CEN association of kinetochore proteins Ctf19 and Ame1 in methyl mimic cse4-R37F strain. (A) Levels of Ctf19 are reduced at CEN chromatin in cse4-R37F strains. ChIP was performed with α-Ctf19 antibodies using chromatin from same strains as used in Figure 3A. Enrichment of Ctf19 at CENs (CEN1 and CEN3) and a negative control (ACT1) was determined by qPCR and is presented as % input. Average from three biological replicates ± SE. *p value < 0.05, Student’s t test. (B) Levels of Ame1 are reduced at CEN chromatin in cse4-R37F strains. ChIP was performed with α-Myc agarose antibodies using chromatin from wild-type (WT, YMB11828), cse4-R37A (YMB11829), and cse4-R37F (YMB11830) strains grown to early logarithmic phase at 25°C and synchronized with nocodazole in G2/M. Enrichment of Ame1 at CENs (CEN1 and CEN3) and a negative control (ACT1) was determined by qPCR and is presented as % input. Average from three biological replicates ± SE. **p value < 0.01, Student’s t test. (C) Expression of Ctf19 in wild-type, cse4-R37A, and cse4-R37F strains are similar. Western blotting of protein extracts was done using α-Ctf19 and α-Tub2 (loading control) antibodies. (D) Expression of Ame1 in wild-type, cse4-R37A, and cse4-R37F strains are similar. Western blotting of protein extracts was done using α-Myc (Ame1) and α-Tub2 (loading control) antibodies.

FIGURE 7:

SPOUT methyltransferase Upa1 contributes to methylation of Cse4-R37. (A) Reduced levels of methylated Cse4-R37 in upa1∆ strain. Western blot analysis of extracts prepared from logarithmically grown wild-type (WT) strain with vector (YMB11880) and upa1Δ strain with vector (YMB11881) or 2μ UPA1 (YMB11882) were analysed by α-HA (HA-Cse4) and α-^MeCse4-R37 (^MeHA-Cse4) antibodies. (B) Reduced methylation of Cse4-R37 in upa1∆ strain is complemented by plasmid based UPA1. Quantification of relative methylation of Cse4 of strains from (A). Ratio of methylated Cse4 (^MeHA-Cse4) to the total Cse4 (HA-Cse4) was calculated using Image J. Three biological replicates were done. Average ± SE is shown. **p value < 0.01, ns = statistically not significant, Student’s t test. (C) Increased methylation of Cse4-R37 in mitotic cells of UPA1 overexpressing strain. Western blot analysis of Ni-NTA agarose immunoprecipitated extracts from wild-type (WT) strain with vector (YMB11878) or GAL1-6HIS-HA-UPA1 (YMB11879) strain grown in SC-Ura with galactose + raffinose (2% each) at 25°C for 6 h, synchronized in G2/M with nocodazole and analysed using α-HA (HA-Cse4 or HA-Upa1) and α-^MeCse4-R37 (^MeHA-Cse4) antibodies. (D) Quantification of relative methylation of Cse4 of strains from (C). Ratio of methylated Cse4-R37 (^MeHA-Cse4) to the total Cse4 (HA-Cse4) was calculated using Image J. Three biological replicates were done. Average ± SE is shown. *p value < 0.05, Student’s t test. (E) Overexpression of UPA1 causes errors in chromosome segregation. Frequency of CF loss in wild-type with vector (YMB12214) or GAL1-6HIS-HA-UPA1 (YMB12215) and cse4-R37A strain with vector (YMB12217) or GAL1-6HIS-HA-UPA1 (YMB12218) was determined as described in Materials and Methods. At least 1000 colonies each from three independent transformants were counted. Average ± SE is shown. **p value < 0.01, Student’s t test.

FIGURE 8:

Cell cycle-regulated methylation of Cse4-R37 prevents CIN. In wild-type (Cse4) strains, cell cycle-regulated Cse4 methylation modulates proper kinetochore structure and function leading to faithful chromosome segregation, whereas in methyl mimic cse4-R37F strains exhibit defects in kinetochore structure and function leading to CIN. The model was created with BioRender.com.