A role for histone H4K16 hypoacetylation in Saccharomyces cerevisiae kinetochore function

Abstract

Hypoacetylated H4 is present at regional centromeres; however, its role in kinetochore function is poorly understood. We characterized H4 acetylation at point centromeres in Saccharomyces cerevisiae and determined the consequences of altered H4 acetylation on chromosome segregation. We observed low levels of tetra-acetylated and K16 acetylated histone H4 (H4K16Ac) at centromeres. Low levels of H4K16Ac were also observed at noncentromeric regions associated with Cse4p. Inhibition of histone deacetylases (HDAC) using nicotinamide (NAM) caused lethality in cse4 and hhf1-20 kinetochore mutants and increased centromeric H4K16Ac. Overexpression of Sas2-mediated H4K16 acetylation activity in wild-type cells led to increased rates of chromosome loss and synthetic dosage lethality in kinetochore mutants. Consistent with increased H4K16 acetylation as a cause of the phenotypes, deletion of the H4K16 deacetylase SIR2 or a sir2-H364Y catalytic mutant resulted in higher rates of chromosome loss compared to wild-type cells. Moreover, H4K16Q acetylmimic mutants displayed increased rates of chromosome loss compared to H4K16R nonacetylatable mutants and wild-type cells. Our work shows that hypoacetylated centromeric H4 is conserved across eukaryotic centromeres and hypoacetylation of H4K16 at centromeres plays an important role in accurate chromosome segregation.

Figure 1

Low levels of acetylated H4 are observed at the centromere. Chromatin immunoprecipitation (ChIP) experiments were performed with asynchronously grown wild-type strains expressing Myc-Cse4p (YMB6955). (A) Diagram of centromere (CEN) (C1–4) and pericentromeric regions (L1 and R1) on chromosome III analyzed by ChIP. Solid square represents the CEN III sequence and each vertical line from chromosomal coordinate 113,500 to 115,500 denotes 500-bp increments. Gray arrow represents an uncharacterized open reading frame (YCL001W-B). (B and C) ChIP with antibodies that recognize acetylated isoforms of acetylated histone H4 (tAcH4) to measure the levels of H4 acetylation at centromeric (C1–4) regions or to Myc to measure levels of Myc-Cse4p at centromeric C2 region and pericentromeric (L1 and R1) regions by quantitative PCR. HML serves as a hypoacetylated H4 control (−CTL) region and SNR189 (SNR) (Chr III: 178729–178589) is a hyperacetylated H4 control (+CTL) region. ChIP for total H4 was performed with antibodies to pan H4 (H4P) that recognize modified and unmodified forms. Bar graphs in B show mean fold enrichment for tAcH4 normalized to total H4 from three biological replicates with error bars showing standard error of the mean. Bar graphs in C show mean fold enrichment for Myc-Cse4p normalized to total H4 from two biological replicates.

Figure 2

Kinetochore mutants are sensitized to nicotinamide (NAM). Strains were serially diluted fivefold and spotted onto YPD plates with 0 mM or 30 mM NAM, incubated at the indicated temperatures, and photographed after 3 days. (A) Wild-type CSE4 (RC154) and temperature-sensitive C-terminal cse4 mutants, cse4-1 (RC147) and cse4-111 (MSY1520). (B) Wild-type HHF1 (MSY559) and temperature-sensitive chromosome segregation histone H4 mutant hhf1-20 (MSY554) (top), and strains expressing wild-type histone H4 (YMB8155), nonacetylatable H4K16R (YMB8156), and acetylmimic H4K16Q (YMB8157) (middle and bottom). (C) Temperature-sensitive kinetochore mutants, ndc10-1 (JK421), mif2-2 (6849-10-1), and nonessential kinetochore mutants, mcm21∆ (YPH1715) and ctf19∆ (YPH1713). Note the ndc10-1 mutant is inviable above 30°.

Figure 3

Low levels of H4K16Ac are present at the centromere and at noncentromeric loci enriched for Cse4p. ChIP experiments were performed with asynchronously grown wild-type strains (YMB6955 in B and YMB8077 in C). Immunoprecipitated DNA was subjected to quantitative PCR to determine enrichment for the indicated regions. (A) Diagram of CEN III (C1–4) and pericentromeric regions (L1 and R1) analyzed by ChIP. (B) ChIP experiments with antibodies to acetylated H4K16 (H4K16Ac) and pan H4 to measure levels of H4K16Ac and total H4 at C1–4 and at L1 and R1 or in C at NTS1-1 and NTS1-2 rDNA regions that contain Cse4p. HML (−CTL1) and SPS22 (−CTL2) (Chr III: 42437–42630) serve as hypoacetylated and ARE1 (+CTL) (Chr III: 212230–212450) serves as hyperacetylated H4K16 controls. Bar graphs show mean fold enrichment for H4K16Ac normalized to total H4 from three biological replicates with error bars showing standard error of the mean.

Figure 4

NAM causes an increase in centromeric H4K16Ac in wild-type and cse4-1 strains. ChIP experiments were performed with CSE4 (RC154) and cse4-1 strains (RC147) grown in 0 mM or 30 mM NAM. Immunoprecipitated DNA was subjected to quantitative PCR to determine enrichment for H4K16Ac. (A) Diagram of CEN III and pericentromeric regions. (B) ChIP experiments with antibodies to H4K16Ac and pan H4 to measure levels of H4K16Ac at C1 in CSE4 strains. (C) Same as B except in cse4-1 strains. HML serves as a hypoacetylated H4K16 control region. Bar graphs show mean fold enrichment for H4K16Ac normalized to total H4 from three biological replicates with error bars showing standard error of the mean.

Figure 5

Overexpression of SAS subunits leads to SDL in kinetochore mutants. Strains were transformed with vector (vector) or vector containing the indicated gene under the Gal1 promoter (GAL-). Fivefold serial dilutions of each strain were spotted on SC −Ura plates containing 2% galactose and 2% raffinose, incubated at the indicated temperatures, and photographed after 3–5 days. (A) Wild-type CSE4 (RC154) and C-terminal mutant cse4-1 (RC147). (B) C-terminal mutant cse4-111 (MSY1520). (C) N-terminal mutant cse4-39 (YC190). (D) Wild-type CSE4 (RC154) and C-terminal mutant cse4-1 (RC147). (E) Wild-type HHF1 (MSY559) and temperature-sensitive chromosome segregation mutant hhf1-20 (MSY554) strains. Three or more independent transformants were tested for each experiment.

Figure 6

Catalytic activity of Sas2p contributes to SDL and GAL-SCM3 suppresses the GAL-SAS2 SDL. Strains were transformed with vector (vector) or vector containing the indicated gene under the Gal1 promoter (GAL-). Fivefold serial dilutions of each strain were spotted on SC −Ura plates containing 2% galactose and 2% raffinose, incubated at the indicated temperatures, and photographed after 3–5 days. (A) C-terminal mutants cse4-1 (RC147) and cse4-111 (MSY1520) strains, carrying vector, GAL-SAS2, or the catalytic mutant GAL-sas2m1. (B) C-terminal mutant cse4-1, cse4-111, and histone H4 mutant hhf1-20 (MSY554) strains, carrying vector and GAL-SCM3 or GAL-SAS2 or carrying both GAL-SAS2 and GAL-SCM3. At least three independent transformants were tested for each experiment.

Figure 7

Altered H4K16Ac affects chromosome segregation. Chromosome loss assays were done with wild-type strains carrying a nonessential minichromosome (YPH1015) with vector or SAS2, SAS4, or SAS5 expressed under the GAL1 promoter. Loss of the minichromosome within the first cell division gives rise to colonies that are half red and half white. We measured colonies that were at least half red. (A) Bar graph showing the number of half-sectored colonies per 1000 for each strain on galactose media without (Gal) or with NAM (Gal + 30 mM NAM). (B) Bar graph shows the number of half-sectored colonies per 1000 for SIR2 deletion strain (YMB7911) carrying pRS314 (vector), sir2-H364Y (catalytic mutant), or SIR2. Error bars show standard error of the mean from three biological replicates.

Figure 8

An H4K16Q acetylmimic mutant shows increased chromosome loss. Chromosome loss assays were performed with strains carrying a nonessential minichromosome that expresses wild-type H4 (YMB8155), H4K16R (YMB8156), or H4K16Q (YMB8157). Loss of the minichromosome within the first cell division gives rise to colonies that are half red and half white. We measured colonies that were at least half red. Bar graph shows the number of half-sectored colonies per 1000. Shown are results for three independent experiments and error bars show standard error of the mean.