Centromeres are specialized replication domains in heterochromatin - PubMed (original) (raw)

Centromeres are specialized replication domains in heterochromatin

K Ahmad et al. J Cell Biol. 2001.

Abstract

The properties that define centromeres in complex eukaryotes are poorly understood because the underlying DNA is normally repetitive and indistinguishable from surrounding noncentromeric sequences. However, centromeric chromatin contains variant H3-like histones that may specify centromeric regions. Nucleosomes are normally assembled during DNA replication; therefore, we examined replication and chromatin assembly at centromeres in Drosophila cells. DNA in pericentric heterochromatin replicates late in S phase, and so centromeres are also thought to replicate late. In contrast to expectation, we show that centromeres replicate as isolated domains early in S phase. These domains do not appear to assemble conventional H3-containing nucleosomes, and deposition of the Cid centromeric H3-like variant proceeds by a replication-independent pathway. We suggest that late-replicating pericentric heterochromatin helps to maintain embedded centromeres by blocking conventional nucleosome assembly early in S phase, thereby allowing the deposition of centromeric histones.

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Figures

Figure 1

Figure 1

Euchromatin and heterochromatin replicate at distinct times in Kc nuclei. Newly replicated DNA was visualized by incorporation of dig-dUTP (A–C) or BrdU (D), and nuclei were counterstained with DAPI. (A and B) Detection with anti–HP1 antibodies stains the chromocenter; the remainder of each nucleus is euchromatin. (A) Euchromatic replication; (B) heterochromatic replication; (C and D) detection of centromeres by anti–Cid antibody. In the merged images, protein localization is in red and sites of replicating DNA are in green. Nuclei show on average 5.6 (±2.1, n = 254) centromeric spots throughout most of the cell cycle. Incorporation of the DNA label reveals that centromeres replicate with euchromatin. Single optical sections are shown (z = 0.2 μm).

Figure 5

Figure 5

Deposition of Cid-GFP before mitosis. Cells transfected with HS-H3GFP or HS-CidGFP were induced and prepared for cytology after various chase times. We examined 50 metaphase figures from each time point and counted the number of labeled mitotic figures. In the merged images, Cid localization is in red and histone-GFP in green. Each image is a projection of multiple sections through the spread. (A) Mitotic chromosomes that show H3GFP labeling first appear 4 h after induction. H3GFP labels pericentric heterochromatin in these chromosomes, as expected for cells that were in late S phase at the time of induction. (B) Metaphase spreads with Cid-GFP at centromeres appear earlier, 2 h after induction, indicating that these cells were in G2 phase when induced.

Figure 2

Figure 2

Prolonged replication of centromeres. Cells were pulse labeled with dig-dUTP followed by a second pulse 3 h later with Bio-dUTP. Anti–Cid antibodies mark the positions of the centromeres. DAPI staining is white. In the merged images, Cid localization is in red, dig-dUTP in green, and Bio-dUTP in blue. Coincidence of dig-dUTP and Cid is yellow, and Bio-dUTP and Cid is magenta in the merged images. (A) An early S phase nucleus in which replication began in euchromatin before beginning in centromeres (incorporation of the second analogue only). (B) Colocalization of both nucleotide analogues at centromeres, indicating that some centromeres replicate for the entire period between the two pulses. (Insets) Two centromeres from this nucleus that display incorporation of both nucleotide analogues. (C) Labeling of euchromatin and centromeres with first pulse, and the chromocenter with the second pulse. (D) Labeling of distinct subsets of heterochromatin in the chromocenter by the two pulses, but not of centromeres. All images are single optical sections. (E) Number of centromeres labeled by nucleotide analogues. A dig-dUTP nucleotide pulse was delivered to cells and followed by a second pulse of Bio-dUTP 1 h later. Nuclei that had incorporated both nucleotide analogues were categorized by the overall replication patterns shown by each pulse (E/E, euchromatic/euchromatic; E/H, euchromatic/heterochromatic; H/H, heterochromatic/heterochromatic labeling). At least 10 nuclei of each combined pattern were examined. The “heterochromatin/euchromatin” pattern was never observed. Centromeres were scored for the incorporation of each nucleotide analogue (− or +), and the numbers of centromeres showing incorporation of either or both analogues are shown.

Figure 3

Figure 3

Localization of histone-GFP fusion proteins. (A and B) Expression of histones H2B-GFP and H3-GFP from the cid promoter, which is active early in S phase. DAPI staining is white. In the merged images, Cid localization is in red and histone-GFP in green. H2B-GFP localizes to euchromatin and to centromeres (A), but H3-GFP protein localizes only to euchromatin (B). For each histone-GFP fusion, we normalized the fluorescence intensity of GFP at centromeres to that in euchromatin in individual nuclei. The ratio of incorporation of H2B-GFP at centromeres relative to euchromatin was 0.016 (SEM = 0.01, n = 23), while for H3-GFP it was 0.0014 (SEM = 0.0004, n = 10). These ratios are significantly different when compared by a Mann-Whitney test (P = 0.0002). (C and D) Expression of H2B-GFP and H3-GFP, respectively, after induction of a heat-shock promoter. Both histone-GFP fusion proteins give similar labeling of heterochromatin in late S-phase cells. Single optical sections are shown.

Figure 4

Figure 4

Cid-GFP is deposited at centromeres by a replication-independent pathway. (A) Cells transfected with HS-H3GFP (open bars) or HS-CidGFP (grey bars) were treated with aphidicolin to block DNA replication and then induced by heat-shock (no replication of DNA was seen in cells treated with aphidicolin, and then pulsed with dig-dUTP, data not shown). The number of cells showing GFP localization in centromeres (of Cid-GFP) or in any part of the nucleus (H3-GFP) was compared with the number observed in induced, untreated cells. Localization of H3-GFP is replication dependent, but that of Cid-GFP is not. For each sample, 100–200 nuclei were examined. (B) Cells transfected with heat-shock promoter-histone-GFP constructs were induced, and then loaded with dig-dUTP to mark replicating DNA. In the merged images, sites of replicating DNA are in red, and protein localization is in green. Nuclei from top to bottom are in early S phase, late S phase, and gap phase. H3GFP fusion protein localizes to replicating regions in S phase cells and does not localize in gap phase cells. Cid-GFP fusion protein localizes to centromeres in all cell stages. The intensities of GFP signals in each image are shown on the same absolute scale. Single optical sections are shown.

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