Limits...
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.

Show MeSH

Related in: MedlinePlus

The specialized chromatin structure and association of Cnp1 at central core region is impaired in mcl1-101 and swi7-H4 mutants.(A) Locations of probes used in Southern hybridization are depicted as horizontal lines. (B) The cells were harvested from cultures incubated at 25°C or 37°C for 6 hrs and permeabilized by enzyme treatment and chromatin was partially digested with MNase. DNA was extracted, separated on agarose gel, and subjected to Southern hybridization using probes shown in (A). Smeared digestion patterns characteristic of central core region (cnt1 and imr1L) were partially replaced with ladder patterns in mcl1-101 and swi7-H4 mutants. Strains were wild-type (JY746), mcl1-101 (NYSPC41), swi7-H4 (TN310) and mis6-302 (NYSPL59). (C) ChIP was performed using the antiserum raised to Cnp1. The cells were incubated at 25°C or 37°C for 6 hrs and then fixed. The ratio of ChIPed DNA to input DNA was calculated as described in Materials and methods and average fold enrichment compared to wild-type strain from three experiments is shown. Localization of Cnp1 was decreased even at permissive temperature in the mcl1-101 mutant, and was further decreased at restrictive temperature. Decreased Cnp1-association was also seen at 37°C in the swi7-H4 mutant. Error bars represent the standard deviation. Strains were same as described in (B).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2376062&req=5

pone-0002221-g004: The specialized chromatin structure and association of Cnp1 at central core region is impaired in mcl1-101 and swi7-H4 mutants.(A) Locations of probes used in Southern hybridization are depicted as horizontal lines. (B) The cells were harvested from cultures incubated at 25°C or 37°C for 6 hrs and permeabilized by enzyme treatment and chromatin was partially digested with MNase. DNA was extracted, separated on agarose gel, and subjected to Southern hybridization using probes shown in (A). Smeared digestion patterns characteristic of central core region (cnt1 and imr1L) were partially replaced with ladder patterns in mcl1-101 and swi7-H4 mutants. Strains were wild-type (JY746), mcl1-101 (NYSPC41), swi7-H4 (TN310) and mis6-302 (NYSPL59). (C) ChIP was performed using the antiserum raised to Cnp1. The cells were incubated at 25°C or 37°C for 6 hrs and then fixed. The ratio of ChIPed DNA to input DNA was calculated as described in Materials and methods and average fold enrichment compared to wild-type strain from three experiments is shown. Localization of Cnp1 was decreased even at permissive temperature in the mcl1-101 mutant, and was further decreased at restrictive temperature. Decreased Cnp1-association was also seen at 37°C in the swi7-H4 mutant. Error bars represent the standard deviation. Strains were same as described in (B).

Mentions: The chromatin structure of the central core domain is known to be unusual since partial micrococcal nuclease (MNase) digestion results in a smeared pattern, rather than the ladder pattern that is indicative of regular nucleosomal packaging [15], [17]. This specialized structure is only associated with a functional centromere context [16]. Importantly, it is reported that integrity of this structure correlates with that of central core silencing [22]. To determine whether Mcl1 and Polα are required for the specialized chromatin structure, chromatin was partially digested with MNase and subjected to Southern hybridization (Figure 4B). In wild-type cells, a typical nucleosomal ladder was observed with a probe corresponding to the heterochromatic domain, outer repeat (otr1L, Figure 4A). In contrast, smeared digestion patterns were detected with probes to central core domain (cnt1 and imr1L, Figure 4A), although wild-type cells shifted to 37°C showed a slightly ladder-like pattern. In mcl1 cells, the smeared patterns of cnt1 and imr1L were partially replaced with a ladder pattern at permissive temperature and the ladder-like pattern of imr1L was significantly enhanced at 37°C compared to wild-type cells. Similar results were obtained in swi7 cells, but only at 37°C. Complete replacement of the smeared pattern was observed in mis6-302 cells at 37°C as reported previously [32]. These data suggest that Mcl1 and Polα are necessary for assembly of the specialized chromatin structure at the central core domain.


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)

The specialized chromatin structure and association of Cnp1 at central core region is impaired in mcl1-101 and swi7-H4 mutants.(A) Locations of probes used in Southern hybridization are depicted as horizontal lines. (B) The cells were harvested from cultures incubated at 25°C or 37°C for 6 hrs and permeabilized by enzyme treatment and chromatin was partially digested with MNase. DNA was extracted, separated on agarose gel, and subjected to Southern hybridization using probes shown in (A). Smeared digestion patterns characteristic of central core region (cnt1 and imr1L) were partially replaced with ladder patterns in mcl1-101 and swi7-H4 mutants. Strains were wild-type (JY746), mcl1-101 (NYSPC41), swi7-H4 (TN310) and mis6-302 (NYSPL59). (C) ChIP was performed using the antiserum raised to Cnp1. The cells were incubated at 25°C or 37°C for 6 hrs and then fixed. The ratio of ChIPed DNA to input DNA was calculated as described in Materials and methods and average fold enrichment compared to wild-type strain from three experiments is shown. Localization of Cnp1 was decreased even at permissive temperature in the mcl1-101 mutant, and was further decreased at restrictive temperature. Decreased Cnp1-association was also seen at 37°C in the swi7-H4 mutant. Error bars represent the standard deviation. Strains were same as described in (B).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2376062&req=5

pone-0002221-g004: The specialized chromatin structure and association of Cnp1 at central core region is impaired in mcl1-101 and swi7-H4 mutants.(A) Locations of probes used in Southern hybridization are depicted as horizontal lines. (B) The cells were harvested from cultures incubated at 25°C or 37°C for 6 hrs and permeabilized by enzyme treatment and chromatin was partially digested with MNase. DNA was extracted, separated on agarose gel, and subjected to Southern hybridization using probes shown in (A). Smeared digestion patterns characteristic of central core region (cnt1 and imr1L) were partially replaced with ladder patterns in mcl1-101 and swi7-H4 mutants. Strains were wild-type (JY746), mcl1-101 (NYSPC41), swi7-H4 (TN310) and mis6-302 (NYSPL59). (C) ChIP was performed using the antiserum raised to Cnp1. The cells were incubated at 25°C or 37°C for 6 hrs and then fixed. The ratio of ChIPed DNA to input DNA was calculated as described in Materials and methods and average fold enrichment compared to wild-type strain from three experiments is shown. Localization of Cnp1 was decreased even at permissive temperature in the mcl1-101 mutant, and was further decreased at restrictive temperature. Decreased Cnp1-association was also seen at 37°C in the swi7-H4 mutant. Error bars represent the standard deviation. Strains were same as described in (B).
Mentions: The chromatin structure of the central core domain is known to be unusual since partial micrococcal nuclease (MNase) digestion results in a smeared pattern, rather than the ladder pattern that is indicative of regular nucleosomal packaging [15], [17]. This specialized structure is only associated with a functional centromere context [16]. Importantly, it is reported that integrity of this structure correlates with that of central core silencing [22]. To determine whether Mcl1 and Polα are required for the specialized chromatin structure, chromatin was partially digested with MNase and subjected to Southern hybridization (Figure 4B). In wild-type cells, a typical nucleosomal ladder was observed with a probe corresponding to the heterochromatic domain, outer repeat (otr1L, Figure 4A). In contrast, smeared digestion patterns were detected with probes to central core domain (cnt1 and imr1L, Figure 4A), although wild-type cells shifted to 37°C showed a slightly ladder-like pattern. In mcl1 cells, the smeared patterns of cnt1 and imr1L were partially replaced with a ladder pattern at permissive temperature and the ladder-like pattern of imr1L was significantly enhanced at 37°C compared to wild-type cells. Similar results were obtained in swi7 cells, but only at 37°C. Complete replacement of the smeared pattern was observed in mis6-302 cells at 37°C as reported previously [32]. These data suggest that Mcl1 and Polα are necessary for assembly of the specialized chromatin structure at the central core domain.

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