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Centromere identity in Drosophila is not determined in vivo by replication timing.

Sullivan B, Karpen G - J. Cell Biol. (2001)

Bottom Line: Minichromosomes with structurally intact centromeres were replicated in late S phase, and those in which centric and surrounding heterochromatin were partially or fully deleted were replicated earlier in mid S phase.We provide the first in vivo evidence that centromeric chromatin is replicated at different times in S phase.These studies indicate that incorporation of CID/CENP-A into newly duplicated centromeres is independent of replication timing and argue against determination of centromere identity by temporal sequestration of centromeric chromatin replication relative to bulk genomic chromatin.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA.

ABSTRACT
Centromeric chromatin is uniquely marked by the centromere-specific histone CENP-A. For assembly of CENP-A into nucleosomes to occur without competition from H3 deposition, it was proposed that centromeres are among the first or last sequences to be replicated. In this study, centromere replication in Drosophila was studied in cell lines and in larval tissues that contain minichromosomes that have structurally defined centromeres. Two different nucleotide incorporation methods were used to evaluate replication timing of chromatin containing CID, a Drosophila homologue of CENP-A. Centromeres in Drosophila cell lines were replicated throughout S phase but primarily in mid S phase. However, endogenous centromeres and X-derived minichromosome centromeres in vivo were replicated asynchronously in mid to late S phase. Minichromosomes with structurally intact centromeres were replicated in late S phase, and those in which centric and surrounding heterochromatin were partially or fully deleted were replicated earlier in mid S phase. We provide the first in vivo evidence that centromeric chromatin is replicated at different times in S phase. These studies indicate that incorporation of CID/CENP-A into newly duplicated centromeres is independent of replication timing and argue against determination of centromere identity by temporal sequestration of centromeric chromatin replication relative to bulk genomic chromatin.

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Late S replication of Drosophila centromeres in vivo. (A) Single and double labeling strategies of larval neuroblasts with BrdU, IdU, and CldU. Colcemid arrest at metaphase allowed determination of the interval of S phase represented by the labeling period. (B) Endogenous centromeres and minichromosome centromeres were studied. (C) Single labeling with BrdU for 2 h showed replication of centric heterochromatin, and CID defined (red) 3rd, 4th, and Y centromeres during very late S phase (30 min before G2 onset). Late S labeling for 3 h (last 1.5 h of S plus 1.5 h of G2) labeled all endogenous centromeres, including the 2nd chromosome, the X, and the X-derived minichromosome (Dp). Bar, 2 μm.
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fig2: Late S replication of Drosophila centromeres in vivo. (A) Single and double labeling strategies of larval neuroblasts with BrdU, IdU, and CldU. Colcemid arrest at metaphase allowed determination of the interval of S phase represented by the labeling period. (B) Endogenous centromeres and minichromosome centromeres were studied. (C) Single labeling with BrdU for 2 h showed replication of centric heterochromatin, and CID defined (red) 3rd, 4th, and Y centromeres during very late S phase (30 min before G2 onset). Late S labeling for 3 h (last 1.5 h of S plus 1.5 h of G2) labeled all endogenous centromeres, including the 2nd chromosome, the X, and the X-derived minichromosome (Dp). Bar, 2 μm.

Mentions: Studies of centromere replication in cultured cells may not reflect the in vivo process, particularly since Drosophila tissue cultured cells used in this and other studies (Ahmad and Henikoff, 2001) are not diploid and may have defects in cell cycle regulation and progression. Therefore, centromere replication was studied in vivo (Fig. 2 A). While studying endogenous centromere replication, we also tested the effects of flanking heterochromatin on centromeric replication timing using the Dp1187 deletion series of structurally distinct minichromosomes with functional kinetochores (Fig. 2 B). Single labeling with BrdU for ≤3 h progressively labeled chromosomal regions that replicated from mid S (3 h before M) to very late S phase (60 min before M) (Fig. 2 B). Centromeres of the 3rd, 4th, and Y chromosomes were replicated very late (60 min before M) (Fig. 2 C). Although CID-associated chromatin of chromosome 2 was not replicated at this time, the surrounding heterochromatin showed BrdU staining. Centromeres of the X and 2nd chromosomes replicated during late S (1.5–2.5 h before M) (Fig. 2 C). After 3 h in BrdU, all Drosophila centromeres were labeled, indicating that in vivo centromere replication occurs primarily in late S phase (Fig. 2 C). Noncentromeric labeling was observed on Drosophila chromosomes in very late S phase, arguing against models proposing that centromeres are the last to replicate in the cell (Csink and Henikoff, 1998).


Centromere identity in Drosophila is not determined in vivo by replication timing.

Sullivan B, Karpen G - J. Cell Biol. (2001)

Late S replication of Drosophila centromeres in vivo. (A) Single and double labeling strategies of larval neuroblasts with BrdU, IdU, and CldU. Colcemid arrest at metaphase allowed determination of the interval of S phase represented by the labeling period. (B) Endogenous centromeres and minichromosome centromeres were studied. (C) Single labeling with BrdU for 2 h showed replication of centric heterochromatin, and CID defined (red) 3rd, 4th, and Y centromeres during very late S phase (30 min before G2 onset). Late S labeling for 3 h (last 1.5 h of S plus 1.5 h of G2) labeled all endogenous centromeres, including the 2nd chromosome, the X, and the X-derived minichromosome (Dp). Bar, 2 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196454&req=5

fig2: Late S replication of Drosophila centromeres in vivo. (A) Single and double labeling strategies of larval neuroblasts with BrdU, IdU, and CldU. Colcemid arrest at metaphase allowed determination of the interval of S phase represented by the labeling period. (B) Endogenous centromeres and minichromosome centromeres were studied. (C) Single labeling with BrdU for 2 h showed replication of centric heterochromatin, and CID defined (red) 3rd, 4th, and Y centromeres during very late S phase (30 min before G2 onset). Late S labeling for 3 h (last 1.5 h of S plus 1.5 h of G2) labeled all endogenous centromeres, including the 2nd chromosome, the X, and the X-derived minichromosome (Dp). Bar, 2 μm.
Mentions: Studies of centromere replication in cultured cells may not reflect the in vivo process, particularly since Drosophila tissue cultured cells used in this and other studies (Ahmad and Henikoff, 2001) are not diploid and may have defects in cell cycle regulation and progression. Therefore, centromere replication was studied in vivo (Fig. 2 A). While studying endogenous centromere replication, we also tested the effects of flanking heterochromatin on centromeric replication timing using the Dp1187 deletion series of structurally distinct minichromosomes with functional kinetochores (Fig. 2 B). Single labeling with BrdU for ≤3 h progressively labeled chromosomal regions that replicated from mid S (3 h before M) to very late S phase (60 min before M) (Fig. 2 B). Centromeres of the 3rd, 4th, and Y chromosomes were replicated very late (60 min before M) (Fig. 2 C). Although CID-associated chromatin of chromosome 2 was not replicated at this time, the surrounding heterochromatin showed BrdU staining. Centromeres of the X and 2nd chromosomes replicated during late S (1.5–2.5 h before M) (Fig. 2 C). After 3 h in BrdU, all Drosophila centromeres were labeled, indicating that in vivo centromere replication occurs primarily in late S phase (Fig. 2 C). Noncentromeric labeling was observed on Drosophila chromosomes in very late S phase, arguing against models proposing that centromeres are the last to replicate in the cell (Csink and Henikoff, 1998).

Bottom Line: Minichromosomes with structurally intact centromeres were replicated in late S phase, and those in which centric and surrounding heterochromatin were partially or fully deleted were replicated earlier in mid S phase.We provide the first in vivo evidence that centromeric chromatin is replicated at different times in S phase.These studies indicate that incorporation of CID/CENP-A into newly duplicated centromeres is independent of replication timing and argue against determination of centromere identity by temporal sequestration of centromeric chromatin replication relative to bulk genomic chromatin.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA.

ABSTRACT
Centromeric chromatin is uniquely marked by the centromere-specific histone CENP-A. For assembly of CENP-A into nucleosomes to occur without competition from H3 deposition, it was proposed that centromeres are among the first or last sequences to be replicated. In this study, centromere replication in Drosophila was studied in cell lines and in larval tissues that contain minichromosomes that have structurally defined centromeres. Two different nucleotide incorporation methods were used to evaluate replication timing of chromatin containing CID, a Drosophila homologue of CENP-A. Centromeres in Drosophila cell lines were replicated throughout S phase but primarily in mid S phase. However, endogenous centromeres and X-derived minichromosome centromeres in vivo were replicated asynchronously in mid to late S phase. Minichromosomes with structurally intact centromeres were replicated in late S phase, and those in which centric and surrounding heterochromatin were partially or fully deleted were replicated earlier in mid S phase. We provide the first in vivo evidence that centromeric chromatin is replicated at different times in S phase. These studies indicate that incorporation of CID/CENP-A into newly duplicated centromeres is independent of replication timing and argue against determination of centromere identity by temporal sequestration of centromeric chromatin replication relative to bulk genomic chromatin.

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