Centromere identity in Drosophila is not determined in vivo by replication timing - PubMed (original) (raw)

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

B Sullivan et al. J Cell Biol. 2001.

Erratum in

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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Figures

Figure 1.

Figure 1.

Drosophila centromeres are replicated primarily in mid to late S phase in vitro in tissue culture cells. (A) Replication labeling strategies used for unsynchronized Drosophila cultured cells. Kc and S2 cells were continuously incubated with BrdU to traverse S and G2 phases. Metaphase arrest served as an anchor point to determine where in S phase replication labeling occurred based on length of exposure to BrdU. In double labeling experiments of interphase nuclei, IdU (early S) was administered for 2 h followed by a chase period of 6 h and then CldU (late S) for 2 h. (B) Kc cells incubated with BrdU for 9 h incorporated label at regions replicated during the last 3 h of S phase. Anti-CID antibodies (red) marked centromeres. The 4th chromosome (arrow), including the centromere, and the centromeres of the X and 3rd chromosomes (arrows) were stained with BrdU. The pericentric region of the 2nd chromosome (arrowhead) adjacent to CID staining was labeled at this time, although the centromere was not. (C) Double labeling of Kc and S2 interphase nuclei with IdU (blue) and CldU (green). Most centromeres (red) did not localize with IdU or CldU, suggesting mid S replication. Merged projections of single optical sections are shown (z = 0.1 μm). Bar units are microns.

Figure 2.

Figure 2.

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.

Figure 3.

Figure 3.

Molecularly and functionally defined centromeres are replicated in late S phase. Single labeling of larval neuroblasts containing _Dp1187_–derived minichromosomes (Fig. 2 B). Centromeres were identified using CID antibodies (red) and sites of replication by anti-BrdU antibodies (green). Gray-scale images (DAPI) identify the minichromosome. Line plots quantitated pixel intensities for each wavelength along the width of the centromere (line). (A) Dp_γ_238 (1.3 Mb) and endogenous X centromeres replicated within 2 h of mitosis when CID staining (arrow) colocalized with BrdU staining (graph). (B) Dp10B (720 kb) containing 420 kb of CEN DNA and no surrounding heterochromatin was replicated 1–2 h before mitosis. (C) DpJ21A (580 kb) containing only half of the CEN DNA replicated earlier (2–4 h before mitosis) than the larger heterochromatin-containing minichromosomes.

Figure 4.

Figure 4.

Labeling of Drosophila larval neuroblast chromosomes for early S replication with IdU (red) and late S replication with CldU (green) shows that deletion of centric heterochromatin shifts centromere replication to mid S. (A) Dpγ238 (1.3 Mb) was completely labeled with CldU. No IdU staining was ever observed on this minichromosome. (B) DpJ21A contains a partially deleted centromere that is replicated primarily in mid S phase. In most cells, DpJ21A was unlabeled for either IdU or CldU (B), suggesting that this minichromosome, including the centromere, replicates primarily in mid S phase. (C) In 20% of metaphases, DpJ21A stained with anti-CldU (late S), suggesting it occasionally replicated at the mid to late S transition. Gray-scale images are shown of each DAPI-stained minichromosome. Bar units are in μm.

Figure 5.

Figure 5.

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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