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A DNA polymerase alpha accessory protein, Mcl1, is required for propagation of centromere structures in fission yeast.

Natsume T, Tsutsui Y, Sutani T, Dunleavy EM, Pidoux AL, Iwasaki H, Shirahige K, Allshire RC, Yamao F - PLoS ONE (2008)

Bottom Line: The mcl1-101 mutation causes reduced CENP-A(Cnp1) in the central domain and an aberrant increase in histone acetylation in both domains.Mcl1 forms S-phase-specific nuclear foci, which colocalize with those of PCNA and Pol alpha.These results suggest that Mcl1 and Pol alpha are required for propagation of centromere chromatin structures during DNA replication.

View Article: PubMed Central - PubMed

Affiliation: Division of Mutagenesis, National Institute of Genetics, Mishima, Shizuoka, Japan.

ABSTRACT
Specialized chromatin exists at centromeres and must be precisely transmitted during DNA replication. The mechanisms involved in the propagation of these structures remain elusive. Fission yeast centromeres are composed of two chromatin domains: the central CENP-A(Cnp1) kinetochore domain and flanking heterochromatin domains. Here we show that fission yeast Mcl1, a DNA polymerase alpha (Pol alpha) accessory protein, is critical for maintenance of centromeric chromatin. In a screen for mutants that alleviate both central domain and outer repeat silencing, we isolated several cos mutants, of which cos1 is allelic to mcl1. The mcl1-101 mutation causes reduced CENP-A(Cnp1) in the central domain and an aberrant increase in histone acetylation in both domains. These phenotypes are also observed in a mutant of swi7(+), which encodes a catalytic subunit of Pol alpha. Mcl1 forms S-phase-specific nuclear foci, which colocalize with those of PCNA and Pol alpha. These results suggest that Mcl1 and Pol alpha are required for propagation of centromere chromatin structures during DNA replication.

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Acetylation of histone H4 is aberrantly increased in the central kinetochore domain in mcl1 and swi7 mutants.(A) ChIP-on-chip was performed using the antiserum raised to a peptide corresponding to amino acid 2–19 of histone H4 that is acetylated at K5, K8, K12, and K16 (AcH4-KN). The cells were prepared as described in Figure 4C. Values obtained from AcH4-KN in the mcl1 mutant were divided by those in wild-type strain. The orange shading represents the binding ratio of loci that show significant enrichment as described previously [68]. Representative results for subtelomeric and centromeric regions of chromosome I are shown. (B,C) ChIP was performed using AcH4-KN (B) and AcH4-K16 (C), respectively. The cells were prepared as described in Figure 4C. Values obtained in the mutants were normalized to those obtained in wild-type strain. Values are further normalized to euchromatic lys1 locus. Average fold enrichment compared to wild-type are obtained from two repetitions. Strains were wild-type (JY879), mcl1-101 (NYSPC52), swi7-H4 (TN403).
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pone-0002221-g005: Acetylation of histone H4 is aberrantly increased in the central kinetochore domain in mcl1 and swi7 mutants.(A) ChIP-on-chip was performed using the antiserum raised to a peptide corresponding to amino acid 2–19 of histone H4 that is acetylated at K5, K8, K12, and K16 (AcH4-KN). The cells were prepared as described in Figure 4C. Values obtained from AcH4-KN in the mcl1 mutant were divided by those in wild-type strain. The orange shading represents the binding ratio of loci that show significant enrichment as described previously [68]. Representative results for subtelomeric and centromeric regions of chromosome I are shown. (B,C) ChIP was performed using AcH4-KN (B) and AcH4-K16 (C), respectively. The cells were prepared as described in Figure 4C. Values obtained in the mutants were normalized to those obtained in wild-type strain. Values are further normalized to euchromatic lys1 locus. Average fold enrichment compared to wild-type are obtained from two repetitions. Strains were wild-type (JY879), mcl1-101 (NYSPC52), swi7-H4 (TN403).

Mentions: The acetylation of N-terminal tails of histone H3 and H4 is maintained at a lower level in the central core region than in coding regions. Here, mcl1 and, to a lesser extent, swi7 mutants showed sensitivity to the HDAC inhibitor, Trichostatin A (Figure S3), suggesting that Mcl1 and Polα might be required for maintenance of histone hypoacetylation. To examine this possibility, we performed ChIP-on-chip assays using antiserum raised to a peptide corresponding to amino acid 2-19 of histone H4 acetylated at K5, K8, K12, and K16 (H4-KN). We determined the acetylation status of histone H4 across the entire genome (Figure S4A). Acetylation of histone H4 was found at the promoter regions of most genes, whereas H4 was hypoacetylated at centromeric regions. Although acetylation peaks similar to those in wild-type were observed at most genes in the mcl1 mutant, histone H4 acetylation was elevated at the centromere region in mcl1 mutant (Figure S4B). Comparison of H4-KN data obtained from mcl1 and wild-type indicates that acetylation level was increased at centromere and sub-telomeric regions in the mcl1 mutant (Figure 5A).


A DNA polymerase alpha accessory protein, Mcl1, is required for propagation of centromere structures in fission yeast.

Natsume T, Tsutsui Y, Sutani T, Dunleavy EM, Pidoux AL, Iwasaki H, Shirahige K, Allshire RC, Yamao F - PLoS ONE (2008)

Acetylation of histone H4 is aberrantly increased in the central kinetochore domain in mcl1 and swi7 mutants.(A) ChIP-on-chip was performed using the antiserum raised to a peptide corresponding to amino acid 2–19 of histone H4 that is acetylated at K5, K8, K12, and K16 (AcH4-KN). The cells were prepared as described in Figure 4C. Values obtained from AcH4-KN in the mcl1 mutant were divided by those in wild-type strain. The orange shading represents the binding ratio of loci that show significant enrichment as described previously [68]. Representative results for subtelomeric and centromeric regions of chromosome I are shown. (B,C) ChIP was performed using AcH4-KN (B) and AcH4-K16 (C), respectively. The cells were prepared as described in Figure 4C. Values obtained in the mutants were normalized to those obtained in wild-type strain. Values are further normalized to euchromatic lys1 locus. Average fold enrichment compared to wild-type are obtained from two repetitions. Strains were wild-type (JY879), mcl1-101 (NYSPC52), swi7-H4 (TN403).
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Related In: Results  -  Collection

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pone-0002221-g005: Acetylation of histone H4 is aberrantly increased in the central kinetochore domain in mcl1 and swi7 mutants.(A) ChIP-on-chip was performed using the antiserum raised to a peptide corresponding to amino acid 2–19 of histone H4 that is acetylated at K5, K8, K12, and K16 (AcH4-KN). The cells were prepared as described in Figure 4C. Values obtained from AcH4-KN in the mcl1 mutant were divided by those in wild-type strain. The orange shading represents the binding ratio of loci that show significant enrichment as described previously [68]. Representative results for subtelomeric and centromeric regions of chromosome I are shown. (B,C) ChIP was performed using AcH4-KN (B) and AcH4-K16 (C), respectively. The cells were prepared as described in Figure 4C. Values obtained in the mutants were normalized to those obtained in wild-type strain. Values are further normalized to euchromatic lys1 locus. Average fold enrichment compared to wild-type are obtained from two repetitions. Strains were wild-type (JY879), mcl1-101 (NYSPC52), swi7-H4 (TN403).
Mentions: The acetylation of N-terminal tails of histone H3 and H4 is maintained at a lower level in the central core region than in coding regions. Here, mcl1 and, to a lesser extent, swi7 mutants showed sensitivity to the HDAC inhibitor, Trichostatin A (Figure S3), suggesting that Mcl1 and Polα might be required for maintenance of histone hypoacetylation. To examine this possibility, we performed ChIP-on-chip assays using antiserum raised to a peptide corresponding to amino acid 2-19 of histone H4 acetylated at K5, K8, K12, and K16 (H4-KN). We determined the acetylation status of histone H4 across the entire genome (Figure S4A). Acetylation of histone H4 was found at the promoter regions of most genes, whereas H4 was hypoacetylated at centromeric regions. Although acetylation peaks similar to those in wild-type were observed at most genes in the mcl1 mutant, histone H4 acetylation was elevated at the centromere region in mcl1 mutant (Figure S4B). Comparison of H4-KN data obtained from mcl1 and wild-type indicates that acetylation level was increased at centromere and sub-telomeric regions in the mcl1 mutant (Figure 5A).

Bottom Line: The mcl1-101 mutation causes reduced CENP-A(Cnp1) in the central domain and an aberrant increase in histone acetylation in both domains.Mcl1 forms S-phase-specific nuclear foci, which colocalize with those of PCNA and Pol alpha.These results suggest that Mcl1 and Pol alpha are required for propagation of centromere chromatin structures during DNA replication.

View Article: PubMed Central - PubMed

Affiliation: Division of Mutagenesis, National Institute of Genetics, Mishima, Shizuoka, Japan.

ABSTRACT
Specialized chromatin exists at centromeres and must be precisely transmitted during DNA replication. The mechanisms involved in the propagation of these structures remain elusive. Fission yeast centromeres are composed of two chromatin domains: the central CENP-A(Cnp1) kinetochore domain and flanking heterochromatin domains. Here we show that fission yeast Mcl1, a DNA polymerase alpha (Pol alpha) accessory protein, is critical for maintenance of centromeric chromatin. In a screen for mutants that alleviate both central domain and outer repeat silencing, we isolated several cos mutants, of which cos1 is allelic to mcl1. The mcl1-101 mutation causes reduced CENP-A(Cnp1) in the central domain and an aberrant increase in histone acetylation in both domains. These phenotypes are also observed in a mutant of swi7(+), which encodes a catalytic subunit of Pol alpha. Mcl1 forms S-phase-specific nuclear foci, which colocalize with those of PCNA and Pol alpha. These results suggest that Mcl1 and Pol alpha are required for propagation of centromere chromatin structures during DNA replication.

Show MeSH
Related in: MedlinePlus