Resolution of Sister Telomere Association Is Required for Progression Through Mitosis

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Science  02 Apr 2004:
Vol. 304, Issue 5667, pp. 97-100
DOI: 10.1126/science.1094754


Cohesins keep sister chromatids associated from the time of their replication in S phase until the onset of anaphase. In vertebrate cells, two distinct pathways dissociate cohesins, one acts on chromosome arms and the other on centromeres. Here, we describe a third pathway that acts on telomeres. Knockdown of tankyrase 1, a telomeric poly(ADP-ribose) polymerase caused mitotic arrest. Chromosomes aligned normally on the metaphase plate but were unable to segregate. Sister chromatids separated at centromeres and arms but remained associated at telomeres, apparently through proteinaceous bridges. Thus, telomeres may require a unique tankyrase 1–dependent mechanism for sister chromatid resolution before anaphase.

Telomere length is regulated by the TTAGGG repeat–binding protein TRF1 (1) and its associated factor tankyrase 1 (2), a member of the poly(ADP-ribose) polymerase (PARP) enzyme family (3). Tankyrase 1 localizes to human telomeres (4), as well other subcellular sites (5, 6). Tankyrase 1 contains five discrete TRF1-binding sites (7) and controls TRF1 binding to telomeric DNA through ADP-ribosylation (4). When tankyrase 1 is overexpressed in human cells, TRF1 is released from telomeres and degraded by the proteasome (8). Long-term overexpression of tankyrase 1 induces telomere lengthening in a reaction that depends on its catalytic PARP activity and on telomerase (2, 8, 9).

To inhibit tankyrase 1 expression, we transfected tankyrase 1 small interfering RNA (siRNA) duplexes (10) into asynchronously growing HeLaI.2.11 cells (11). Tankyrase 1 protein was lost at the earliest time point (24 hours) and throughout the time course (Fig. 1A). A similar knockdown was achieved with a different tankyrase 1 siRNA duplex (fig. S1). Cells treated with tankyrase 1 siRNA exhibited a dramatic mitotic arrest, as measured by immunofluorescence (Fig. 1B and fig. S2) and fluorescence-activated cell sorting (FACS) analysis, which indicated a threefold increase in the G2/M population (Fig. 1C). This phenotype was observed with two different tankyrase 1 siRNA duplexes, and it was observed in cells independent of their telomere length or telomerase status (table S1).

Fig. 1.

Knockdown of tankyrase 1 expression results in mitotic arrest. (A) Immunoblot analysis of whole-cell extracts from HeLaI.2.11 cells transfected without (mock) or with (TNKS1) tankyrase 1 siRNA. (B) Histogram showing the percentage of cells in interphase or mitosis after transfection with tankyrase 1 siRNA. About 1000 cells were scored by immunofluorescence for each time point. (C) FACS analysis of HeLaI.2.11 cells 48 hours after transfection with tankyrase 1 siRNA. y axis, cell number. (D) Immunofluorescence analysis of methanol-fixed HeLaI.2.11 cells stained with DAPI (blue) and α-tubulin antibody (green) after treatment with tankyrase 1 siRNA indicates a progression of three classes of abnormal mitotic cells. Scale bar, 2 μm. (E) Tankyrase 1 siRNA cells were cotransfected with siRNA-resistant tankyrase 1 wild-type (WT) or HE/A plasmids. Histogram shows the ratio of normal to abnormal tankyrase 1–expressing mitotic cells scored by immunofluorescence as described in table S2. (F and G) Immunofluorescence of methanol-fixed HeLaI.2.11 cells transfected with tankyrase 1 siRNA showing (F) measurements of the DNA (DAPI) and spindle (α tubulin) or (G) centromere disposition by staining with DAPI (blue), α-tubulin antibody (red), and antibody against centromeres (ACA) (green). Scale bar, 2 μm.

Closer examination of the cells arrested in mitosis indicated a predominance of abnormal mitotic figures, which fell into three distinct classes (Fig. 1D), each peaking in number at a different point in the tankyrase 1 siRNA time course (fig. S3). The earliest phenotype, “fat,” displayed a broad 4′,6′-diamidino-2-phenylindole (DAPI) stain on the metaphase plate (Fig. 1D, b). The second had “misaligned” chromosomes (Fig. 1D, c). And the third and terminal phenotype, “aberrant” displayed a deteriorated spindle and a condensed and disordered DAPI stain (Fig. 1D, d). Aberrant cells did not reenter the cell cycle (fig. S4A) and were not apoptotic (fig. S4B).

To determine whether tankyrase 1 expression could rescue the abnormal mitotic phenotype, we cotransfected cells with tankyrase 1 siRNA and with wild-type (WT) or PARP-dead (HE/A) (9) tankyrase 1 plasmids containing a single-base mismatch to the siRNA oligonucleotide, which rendered them resistant to the siRNA-mediated knockdown (fig. S5). Analysis of the population of mitotic cells expressing tankyrase 1 indicated that wild-type (but not PARP-dead) tankyrase 1 efficiently rescued the abnormal mitotic phenotype (Fig. 1E and table S2).

To gain insight into the mechanism of the arrest induced by tankyrase 1 siRNA treatment, we focused on the earliest phenotypes. Measurements of the “fat” and misaligned “metaphases” indicated that the DAPI staining was much broader and the spindle much shorter than a wild-type metaphase (Fig. 1F and fig. S6) and more resembled an early anaphase figure. Consistent with anaphase entry, centromeres frequently appeared separated (Fig. 1G, b and c), which suggested that chromosomes aligned on the metaphase plate and underwent centromere separation, but then were unable to proceed with anaphase.

Time-lapse analysis of mitosis in a mock-transfected cell and a cell treated with tankyrase 1 siRNA showed that, in both cases, chromosomes congressed normally and by 38 min were aligned on the metaphase plate. However, although the mock-transfected cell continued to progress through mitosis, the tankyrase 1 siRNA cell remained arrested in metaphase (Fig. 2A; Movies S1 and S2). The period of metaphase arrest in tankyrase 1 siRNA cells varied. For example, one cell (Fig. 2B, arrowhead) remained in metaphase from 24 to 188 min (164 min), whereas two other flanking cells (Fig. 2B, arrows) remained arrested in metaphase for the duration of imaging (260 min) (Movie S3).

Fig. 2.

Characterization of the mitotic arrest in tankyrase 1 siRNA cells. (A and B) Time-lapse video live-cell imaging of a HeLa cell line expressing histone H2B tagged with green fluorescent protein (HeLa-H2B-GFP cells) (24) 36 hours after transfection with tankyrase 1 siRNA. (A) Progression of mitosis in a mock-transfected cell versus a tankyrase 1 siRNA cell for 108 min. Scale bar, 10 μm. (B) Larger field which includes the tankyrase 1 siRNA cell shown in (A), (arrowhead) and two flanking cells (arrows) that remain in metaphase for 260 min. Scale bar, 10 μm. (C and D) Immunofluorescence analysis of mitotic cyclins in methanol-fixed HeLaI.2.11 cells stained with DAPI (blue) and cyclin A or B (red) 20 hours after transfection with tankyrase 1 siRNA. Scale bar, 5 μm. (E) Immunoblot analysis of whole-cell extracts from HeLa-SCC1-myc cells 40 hours after treatment with tankyrase 1 siRNA. TNKS1 cells were isolated by mitotic shake-off. Arrow indicates the SCC1myc cleavage product.

Progression through mitosis requires ubiquitin-mediated proteolysis of a number of regulatory proteins by the anaphase-promoting complex (APC) (12). The APC substrates, cyclins A and B, were degraded in fat mitotic figures (Fig. 2, C and D), which indicated that the APC was active. Separation of sister chromatids also depends on the APC, where the final, irreversible step is cleavage of the SCC1 cohesin subunit (13). The SCC1 subunit was cleaved in tankyrase 1 siRNA mitotic cells (Fig. 2E). Thus, the arrest in mitosis was not due to a defect in the APC. Finally, a negative immunostain for the histone γ-H2AX (fig. S7) indicated that the arrest in mitosis was not due to DNA damage.

Cells transfected with tankyrase 1 siRNA entered mitosis and proceeded to metaphase normally; chromosomes aligned on the metaphase plate and sister centromeres separated. However, at that point, the cells were arrested and were delayed or unable to proceed through anaphase. We wondered whether abnormal telomere associations were preventing sister chromatids from separating and moving to the poles. Tankyrase 1 is a positive regulator of telomere length (2). Thus, we asked if there was a gross defect in telomere replication in tankyrase 1 siRNA cells by measuring DNA replication using 5-bromo-2′-deoxyuridine (BrdU) incorporation. Telomeric DNA from cells transfected with tankyrase 1 siRNA sedimented as heavy-light DNA, consistent with at least one round of DNA replication and similar to mock-transfected cells (Fig. 3A). Thus, tankyrase 1 siRNA did not detectably inhibit telomere replication.

Fig. 3.

Telomeres undergo DNA replication and are not covalently fused in tankyrase 1 siRNA cells. (A) Telomeric DNA was isolated from HelaI.2.11 cells after 48 hours of cotreatment with tankyrase 1 siRNA and BrdU, and fractionated on CsCl density gradients. Telomeric DNA was measured by dot-blot analysis using a 32P-labeled telomeric probe. The positions of light-light (LL), heavy-light (HL), and heavy-heavy (HH) DNA are indicated. (B) Southern blot analysis of telomere restriction fragments isolated from HeLaI.2.11 cells after 48 hours of treatment with tankyrase 1 siRNA, and fractionated by pulsed-field gel electrophoresis. The gel was hybridized to a 32P end-labeled (CCCTAA)4 oligonucleotide under native or denatured conditions. (C) Telomeric PNA FISH analysis (FISH using peptide nucleic acid probes) of metaphase spreads of HeLaI.2.11 cells collected 48 hours after transfection with tankyrase 1 siRNA, swollen in hypotonic buffer, and fixed in methanol-acetic acid. Telomeric repeats were detected by using a Cy3-(CCCTAA)3 PNA probe (red). DNA was stained with DAPI (blue). Scale bar, 5 μm.

An alternative explanation for the mitotic arrest phenotype in tankyrase 1 siRNA cells could be covalent ligation of chromosome ends. Inhibition of the telomere repeat–binding protein TRF2 results in loss of the 3′ G-strand telomere tail, ligation of telomere restriction fragments, and end-to-end fusions as observed in metaphase spreads (11). We performed similar analyses to address this question in cells transfected with tankyrase 1 siRNA, and there was no indication of loss of G-strand tails or telomeric fusion products (Fig. 3B).

We further assayed for covalent ligation of telomeres in metaphase spreads using standard procedures, which include hypotonic treatment. Note that this treatment, which was developed to allow visualization of separated chromosome arms, may release chromosomal proteins (14). Typically, in these preparations, sister chromatids remain associated at their centromeres because of tenacious protein-protein interactions (Fig. 3C). Because centromeres were no longer associated in cells transfected with tankyrase 1 siRNA (Fig. 1G and see Fig. 4B, below), chromosomes appeared as separated chromatids, and end-to-end fusions were not observed (Fig. 3C).

Fig. 4.

Sister telomeres (unlike arms and centromeres) remain associated in cells treated with tankyrase 1 siRNA. (A to D) Chromosome-specific FISH analysis of HeLaI.2.11 cells collected by mitotic shake-off 48 hours after treatment with tankyrase 1 siRNA. Cells were fixed directly in methanol-acetic acid without hypotonic swelling and hybridized to chromosome-specific fluorescently labeled probes: telomeres (green), arms (red), and centromeres (red). DNA was stained with DAPI (blue). Scale bar, 2 μm.

Alternatively, sister telomeres could associate through proteinaceous bridges, which may not survive the hypotonic swelling used in metaphase spread preparations. To address this question, we turned to a fluorescent in situ hybridization (FISH) replication timing assay (15), where cells were isolated by mitotic shake-off and fixed directly (without hypotonic treatment) to avoid artifactual separation of sister chromatids. In control mitotic cells, telomeric regions generally appeared as two doublets, which indicated that telomeres had replicated and separated (Fig. 4A; table S3). In contrast, in cells transfected with tankyrase 1 siRNA, two singlets were frequently observed, which indicated that the telomeric regions had not separated (Fig. 4A; table S3). Centromere probes appeared as two doublets in both mock-transfected cells and cells transfected with tankyrase 1 siRNA, but showed greater separation in the latter (Fig. 4B).

In contrast to telomere-specific probes, arm probes showed no difference between cells treated with tankyrase 1 siRNA and mock-transfected cells and generally appeared as two doublets (Fig. 4C; table S3). Thus, unlike telomeres, chromosome arms had separated. Finally, we performed double FISH using arm- and telomere-specific probes from the same chromosome simultaneously. In tankyrase 1 siRNA cells, chromosome arms appeared as two doublets, whereas, in the same cell, telomeres appeared as two singlets (Fig. 4D). Thus, in tankyrase 1 siRNA cells, chromosome arms and centromeres had replicated and separated, but telomeres [although replicated (Fig. 3A)] remained associated.

Knockdown of tankyrase 1 expression blocked cells from completing mitotic anaphase. Although sister chromatids separated at their centromeres and along their arms, they remained associated at their telomeres. Thus, the inability of sister chromatids to segregate to daughter poles appeared to be due to a persistent telomere association. A role for telomeres in sister chromatid separation during mitosis has been suggested in Tetrahymena (16), Schizosaccharomyces pombe (17), and Drosophila (18).

Our results are consistent with the hypothesis that telomeres of sister chromatids normally associate and that resolution of this association is required for progression through anaphase. Our finding that telomere resolution is blocked, whereas centromere and arm resolution appears to occur normally, indicates a unique mechanism for telomeres which is distinct from arms and centromeres. In vertebrates, cohesins are released from chromosomes by two distinct posttranslational mechanisms (13, 19). The bulk of cohesins are released during prophase by a phosphorylation-mediated mechanism (20, 21), whereas the remaining centromeric cohesins are released at metaphase by proteolytic cleavage (22, 23). Our finding that tankyrase 1 PARP activity is required to rescue the abnormal mitotic phenotype implicates a third posttranslational mechanism, poly(ADP-ribosyl)ation, in sister chromatid resolution. Whether telomeres are held together by cohesins or by telomere-specific proteins, such as TRF1 and its associated factors, remains to be determined.

Supporting Online Material

Materials and Methods

Figs. S1 to S7

Tables S1 to S3


Movies S1 to S3

References and Notes

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