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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.

Show MeSH
Cyclical chromatin assembly model for propagating centromere identity. CENP-A deposition is proposed to determine centromere identity and propagation, but other epigenetic marks may determine these functions. During replication, CENP-A– and H3–containing nucleosomes segregate to daughter chromatids. “Replenishment” occurs via recruitment of CENP-A and H3 to sites already containing the appropriate histone due to H3 and CENP-A chromatin assembly factors or other loading factors. CENP-A and H3 recruitment are unlikely to be simultaneous, since H3 assembly is coupled to replication, and CENP-A assembly is not. Nucleosome and kinetochore assembly transmit centromeric chromatin through mitosis, and the replication/replenishment cycle continues.
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fig5: Cyclical chromatin assembly model for propagating centromere identity. CENP-A deposition is proposed to determine centromere identity and propagation, but other epigenetic marks may determine these functions. During replication, CENP-A– and H3–containing nucleosomes segregate to daughter chromatids. “Replenishment” occurs via recruitment of CENP-A and H3 to sites already containing the appropriate histone due to H3 and CENP-A chromatin assembly factors or other loading factors. CENP-A and H3 recruitment are unlikely to be simultaneous, since H3 assembly is coupled to replication, and CENP-A assembly is not. Nucleosome and kinetochore assembly transmit centromeric chromatin through mitosis, and the replication/replenishment cycle continues.

Mentions: Our results support replication-independent incorporation of CID/CENP-A during centromere assembly. Self-propagation of centromere identity could occur through the action of proteins that incorporate CID/CENP-A into newly replicated regions by recognizing existing CID/CENP-A chromatin (Fig. 5) (Sullivan, 2001). The relative timing of CENP-A protein expression and replication timing in mammals strongly support the idea that centromeres are propagated by recruitment of chromatin assembly or remodeling factors that act after DNA replication (Shelby et al., 2000). Neocentromere formation in Drosophila and humans suggests that these putative CID/CENP-A recruitment factors can assemble centromeric chromatin on normally noncentromeric DNA (Blower and Karpen, 2001; Lo et al., 2001; Maggert and Karpen, 2001). Further studies must identify the proteins and mechanisms responsible for CID/CENP-A recruitment to replicated centromeres in a sequence-independent manner.


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

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

Cyclical chromatin assembly model for propagating centromere identity. CENP-A deposition is proposed to determine centromere identity and propagation, but other epigenetic marks may determine these functions. During replication, CENP-A– and H3–containing nucleosomes segregate to daughter chromatids. “Replenishment” occurs via recruitment of CENP-A and H3 to sites already containing the appropriate histone due to H3 and CENP-A chromatin assembly factors or other loading factors. CENP-A and H3 recruitment are unlikely to be simultaneous, since H3 assembly is coupled to replication, and CENP-A assembly is not. Nucleosome and kinetochore assembly transmit centromeric chromatin through mitosis, and the replication/replenishment cycle continues.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196454&req=5

fig5: Cyclical chromatin assembly model for propagating centromere identity. CENP-A deposition is proposed to determine centromere identity and propagation, but other epigenetic marks may determine these functions. During replication, CENP-A– and H3–containing nucleosomes segregate to daughter chromatids. “Replenishment” occurs via recruitment of CENP-A and H3 to sites already containing the appropriate histone due to H3 and CENP-A chromatin assembly factors or other loading factors. CENP-A and H3 recruitment are unlikely to be simultaneous, since H3 assembly is coupled to replication, and CENP-A assembly is not. Nucleosome and kinetochore assembly transmit centromeric chromatin through mitosis, and the replication/replenishment cycle continues.
Mentions: Our results support replication-independent incorporation of CID/CENP-A during centromere assembly. Self-propagation of centromere identity could occur through the action of proteins that incorporate CID/CENP-A into newly replicated regions by recognizing existing CID/CENP-A chromatin (Fig. 5) (Sullivan, 2001). The relative timing of CENP-A protein expression and replication timing in mammals strongly support the idea that centromeres are propagated by recruitment of chromatin assembly or remodeling factors that act after DNA replication (Shelby et al., 2000). Neocentromere formation in Drosophila and humans suggests that these putative CID/CENP-A recruitment factors can assemble centromeric chromatin on normally noncentromeric DNA (Blower and Karpen, 2001; Lo et al., 2001; Maggert and Karpen, 2001). Further studies must identify the proteins and mechanisms responsible for CID/CENP-A recruitment to replicated centromeres in a sequence-independent manner.

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.

Show MeSH