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Impaired coenzyme A synthesis in fission yeast causes defective mitosis, quiescence-exit failure, histone hypoacetylation and fragile DNA.

Nakamura T, Pluskal T, Nakaseko Y, Yanagida M - Open Biol (2012)

Bottom Line: The mutant becomes auxotrophic to pantothenate at permissive temperature, displaying greatly decreased levels of CoA, acetyl-CoA and histone acetylation.Moreover, ppc1-537 mutant cells failed to restore proliferation from quiescence.Additionally, double-strand break repair is defective in the ppc1-537 mutant, producing fragile broken DNA, probably owing to diminished histone acetylation.

View Article: PubMed Central - PubMed

Affiliation: Okinawa Institute of Science and Technology Graduate University, Tancha 1919-1, Onna, Okinawa 904-0495, Japan.

ABSTRACT
Biosynthesis of coenzyme A (CoA) requires a five-step process using pantothenate and cysteine in the fission yeast Schizosaccharomyces pombe. CoA contains a thiol (SH) group, which reacts with carboxylic acid to form thioesters, giving rise to acyl-activated CoAs such as acetyl-CoA. Acetyl-CoA is essential for energy metabolism and protein acetylation, and, in higher eukaryotes, for the production of neurotransmitters. We isolated a novel S. pombe temperature-sensitive strain ppc1-537 mutated in the catalytic region of phosphopantothenoylcysteine synthetase (designated Ppc1), which is essential for CoA synthesis. The mutant becomes auxotrophic to pantothenate at permissive temperature, displaying greatly decreased levels of CoA, acetyl-CoA and histone acetylation. Moreover, ppc1-537 mutant cells failed to restore proliferation from quiescence. Ppc1 is thus the product of a super-housekeeping gene. The ppc1-537 mutant showed combined synthetic lethal defects with five of six histone deacetylase mutants, whereas sir2 deletion exceptionally rescued the ppc1-537 phenotype. In synchronous cultures, ppc1-537 cells can proceed to the S phase, but lose viability during mitosis failing in sister centromere/kinetochore segregation and nuclear division. Additionally, double-strand break repair is defective in the ppc1-537 mutant, producing fragile broken DNA, probably owing to diminished histone acetylation. The CoA-supported metabolism thus controls the state of chromosome DNA.

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Histone acetylation is diminished in ppc1-537 that strongly interacts with histone acetyltransferase and deacetylase mutants. (a) WT, clr6-1 and ppc1-537 mutant strains were first grown at 26°C, then transferred to 36°C for 0, 4, 8 and 12 h, and harvested. Their extracts were prepared and immunoblotted using antibodies against acetylated histone H3 and H4 as indicated and antibodies against histone H3. Cdc2 antibody PSTAIRE was the loading control. (b) Results of the double mutants between ppc1-537 and acetyltransferase mutants Δhat1 and mst1-L344S, and six deacetylase mutants. Additive defects were observed except for the case of double mutant with Δsir2, which was rescued, and with Δhat1, which showed no effect. (c) The single and double mutants of ppc1-537 and acetyltransferase (hat1, mst1) and deacetylase (hst2, hst4, sir2, clr6, clr3, phd1) mutants. Cells grown exponentially were diluted, spotted on YPD plate (two spots for the double mutant), and then incubated at 26°C, 30°C or 33°C. (d) Protein and (e) mRNA transcript levels of FLAG-tagged Ppc1 in the genetic background of WT, Δhst2, Δhst4 and Δsir2. The level of Ppc1-FLAG chromosomally integrated and expressed under the native promoter was determined by anti-FLAG antibody. (f) The levels of acetylated histone H4 K12 in WT, Δsir2, ppc1-537 and the double mutant were determined by antibody against acetylated H4K12. Anti-tubulin antibody TAT1 was determined as the loading control.
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RSOB120117F6: Histone acetylation is diminished in ppc1-537 that strongly interacts with histone acetyltransferase and deacetylase mutants. (a) WT, clr6-1 and ppc1-537 mutant strains were first grown at 26°C, then transferred to 36°C for 0, 4, 8 and 12 h, and harvested. Their extracts were prepared and immunoblotted using antibodies against acetylated histone H3 and H4 as indicated and antibodies against histone H3. Cdc2 antibody PSTAIRE was the loading control. (b) Results of the double mutants between ppc1-537 and acetyltransferase mutants Δhat1 and mst1-L344S, and six deacetylase mutants. Additive defects were observed except for the case of double mutant with Δsir2, which was rescued, and with Δhat1, which showed no effect. (c) The single and double mutants of ppc1-537 and acetyltransferase (hat1, mst1) and deacetylase (hst2, hst4, sir2, clr6, clr3, phd1) mutants. Cells grown exponentially were diluted, spotted on YPD plate (two spots for the double mutant), and then incubated at 26°C, 30°C or 33°C. (d) Protein and (e) mRNA transcript levels of FLAG-tagged Ppc1 in the genetic background of WT, Δhst2, Δhst4 and Δsir2. The level of Ppc1-FLAG chromosomally integrated and expressed under the native promoter was determined by anti-FLAG antibody. (f) The levels of acetylated histone H4 K12 in WT, Δsir2, ppc1-537 and the double mutant were determined by antibody against acetylated H4K12. Anti-tubulin antibody TAT1 was determined as the loading control.

Mentions: In clr6-1 mutant cells, the level of histone H3 and H4 acetylation moderately increased (figure 6a), as reported [48]. By contrast, the levels of histone acetylation detected by antibodies against acetylated histones H4 AcH4 (at K5, K8, K12 and K16), AcH3 (K9) and AcH3 (K14) [49] were all diminished in ppc1-537 for 0–12 h at 36°C, while the histone H3 protein level did not change. These results were consistent with a notion that histone acetylation was diminished in ppc1-537 owing to the deficiency of CoA and acetyl-CoA.Figure 6.


Impaired coenzyme A synthesis in fission yeast causes defective mitosis, quiescence-exit failure, histone hypoacetylation and fragile DNA.

Nakamura T, Pluskal T, Nakaseko Y, Yanagida M - Open Biol (2012)

Histone acetylation is diminished in ppc1-537 that strongly interacts with histone acetyltransferase and deacetylase mutants. (a) WT, clr6-1 and ppc1-537 mutant strains were first grown at 26°C, then transferred to 36°C for 0, 4, 8 and 12 h, and harvested. Their extracts were prepared and immunoblotted using antibodies against acetylated histone H3 and H4 as indicated and antibodies against histone H3. Cdc2 antibody PSTAIRE was the loading control. (b) Results of the double mutants between ppc1-537 and acetyltransferase mutants Δhat1 and mst1-L344S, and six deacetylase mutants. Additive defects were observed except for the case of double mutant with Δsir2, which was rescued, and with Δhat1, which showed no effect. (c) The single and double mutants of ppc1-537 and acetyltransferase (hat1, mst1) and deacetylase (hst2, hst4, sir2, clr6, clr3, phd1) mutants. Cells grown exponentially were diluted, spotted on YPD plate (two spots for the double mutant), and then incubated at 26°C, 30°C or 33°C. (d) Protein and (e) mRNA transcript levels of FLAG-tagged Ppc1 in the genetic background of WT, Δhst2, Δhst4 and Δsir2. The level of Ppc1-FLAG chromosomally integrated and expressed under the native promoter was determined by anti-FLAG antibody. (f) The levels of acetylated histone H4 K12 in WT, Δsir2, ppc1-537 and the double mutant were determined by antibody against acetylated H4K12. Anti-tubulin antibody TAT1 was determined as the loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3472395&req=5

RSOB120117F6: Histone acetylation is diminished in ppc1-537 that strongly interacts with histone acetyltransferase and deacetylase mutants. (a) WT, clr6-1 and ppc1-537 mutant strains were first grown at 26°C, then transferred to 36°C for 0, 4, 8 and 12 h, and harvested. Their extracts were prepared and immunoblotted using antibodies against acetylated histone H3 and H4 as indicated and antibodies against histone H3. Cdc2 antibody PSTAIRE was the loading control. (b) Results of the double mutants between ppc1-537 and acetyltransferase mutants Δhat1 and mst1-L344S, and six deacetylase mutants. Additive defects were observed except for the case of double mutant with Δsir2, which was rescued, and with Δhat1, which showed no effect. (c) The single and double mutants of ppc1-537 and acetyltransferase (hat1, mst1) and deacetylase (hst2, hst4, sir2, clr6, clr3, phd1) mutants. Cells grown exponentially were diluted, spotted on YPD plate (two spots for the double mutant), and then incubated at 26°C, 30°C or 33°C. (d) Protein and (e) mRNA transcript levels of FLAG-tagged Ppc1 in the genetic background of WT, Δhst2, Δhst4 and Δsir2. The level of Ppc1-FLAG chromosomally integrated and expressed under the native promoter was determined by anti-FLAG antibody. (f) The levels of acetylated histone H4 K12 in WT, Δsir2, ppc1-537 and the double mutant were determined by antibody against acetylated H4K12. Anti-tubulin antibody TAT1 was determined as the loading control.
Mentions: In clr6-1 mutant cells, the level of histone H3 and H4 acetylation moderately increased (figure 6a), as reported [48]. By contrast, the levels of histone acetylation detected by antibodies against acetylated histones H4 AcH4 (at K5, K8, K12 and K16), AcH3 (K9) and AcH3 (K14) [49] were all diminished in ppc1-537 for 0–12 h at 36°C, while the histone H3 protein level did not change. These results were consistent with a notion that histone acetylation was diminished in ppc1-537 owing to the deficiency of CoA and acetyl-CoA.Figure 6.

Bottom Line: The mutant becomes auxotrophic to pantothenate at permissive temperature, displaying greatly decreased levels of CoA, acetyl-CoA and histone acetylation.Moreover, ppc1-537 mutant cells failed to restore proliferation from quiescence.Additionally, double-strand break repair is defective in the ppc1-537 mutant, producing fragile broken DNA, probably owing to diminished histone acetylation.

View Article: PubMed Central - PubMed

Affiliation: Okinawa Institute of Science and Technology Graduate University, Tancha 1919-1, Onna, Okinawa 904-0495, Japan.

ABSTRACT
Biosynthesis of coenzyme A (CoA) requires a five-step process using pantothenate and cysteine in the fission yeast Schizosaccharomyces pombe. CoA contains a thiol (SH) group, which reacts with carboxylic acid to form thioesters, giving rise to acyl-activated CoAs such as acetyl-CoA. Acetyl-CoA is essential for energy metabolism and protein acetylation, and, in higher eukaryotes, for the production of neurotransmitters. We isolated a novel S. pombe temperature-sensitive strain ppc1-537 mutated in the catalytic region of phosphopantothenoylcysteine synthetase (designated Ppc1), which is essential for CoA synthesis. The mutant becomes auxotrophic to pantothenate at permissive temperature, displaying greatly decreased levels of CoA, acetyl-CoA and histone acetylation. Moreover, ppc1-537 mutant cells failed to restore proliferation from quiescence. Ppc1 is thus the product of a super-housekeeping gene. The ppc1-537 mutant showed combined synthetic lethal defects with five of six histone deacetylase mutants, whereas sir2 deletion exceptionally rescued the ppc1-537 phenotype. In synchronous cultures, ppc1-537 cells can proceed to the S phase, but lose viability during mitosis failing in sister centromere/kinetochore segregation and nuclear division. Additionally, double-strand break repair is defective in the ppc1-537 mutant, producing fragile broken DNA, probably owing to diminished histone acetylation. The CoA-supported metabolism thus controls the state of chromosome DNA.

Show MeSH
Related in: MedlinePlus