Asynchronous replication timing of telomeres at opposite arms of mammalian chromosomes

Abstract

Telomeres are defining structural elements of all linear chromosomes, yet information concerning the timing of their replication in higher eukaryotes is surprisingly limited. We developed an approach that allowed a study of telomere replication patterns of specific mammalian chromosomes. In the Indian muntjac (Muntiacus muntjac), replication timing between respective telomeres of homologous chromosomes was highly coordinated, but no such synchrony was evident for p- and q-arm telomeres of the same chromosome. This finding contrasts with the coordinated timing of both ends of each chromosome in yeast. Also in contrast to yeast, where replication of all telomeres is confined to late S phase, we found specific telomeres in Indian muntjac chromosomes that replicated early in S and other telomeres that replicated later. Finally, replication timing of some but not all telomeres was influenced by telomere length. Knowledge of telomere replication timing represents a first step toward understanding the relationship between telomere replication and telomerase action. The approach, which we call replicative detargeting fluorescence in situ hybridization, is widely applicable to different species and genetic loci.

Fig. 1.

Schema of ReDFISH technique. (a) ReDFISH of a chromosome that has replicated fully in the presence of BrdUrd/dC. Newly synthesized DNA incorporating BrdUrd/dC (horizontal stripes) is removed after nicking the DNA with Hoechst 33258 plus UV and digesting nicked DNA with exonuclease III, leaving only the parental strands. The G-rich telomeric strand is the template for lagging strand synthesis and anneals to a Cy3-conjugated C-rich telomeric probe, whereas the C-rich telomeric strand is the template for leading strand synthesis and anneals to an FITC-conjugated G-rich telomeric probe. This pattern defines which telomeric strands replicated at the time of BrdUrd/BrdC labeling. (b) ReDFISH of a partially replicated chromosome. In this example, only the p-arm telomere of the X chromosome was replicating during the 1-h BrdUrd/dC pulse; the q arm was not replicating. As a consequence, only the parental strands are available for hybridization in the p arm (schema, p arms), whereas both strands of unreplicated q-arm DNA survive digestion and hybridize to both probes (schema, q arms).

Fig. 2.

Telomere replication of male and female Indian muntjac chromosomes after G₁/S synchronization with HU. (a) Representative chromosomes after release from HU with hourly BrdUrd/dC pulses. During the first 1-h pulse, only chromosome 2p replicated; thus, it is the only chromosome to show differential hybridization with the two probes (red for lagging strand template and green for leading strand template). Chromosomes were stained with DAPI. Different patterns of telomere replication timing were observed for each chromosome at various time points after release from HU. Each of the five columns illustrates ReDFISH staining for a single cell. (b) Timing patterns of telomere replication of each chromosome end. Male Indian muntjac cells F4869 (PD 13.8, □) and female Indian muntjac cells F4374 (PD 40, ♦) were growth-arrested in low serum and then synchronized at the G₁/S interface by HU. Telomere replication patterns of a total of 64,044 chromosome ends were analyzed by ReDFISH. Note that telomeres replicate throughout S phase.

Fig. 3.

Telomere replication of female Indian muntjac cells as measured during asynchronous growth. (a) Experimental design in which cells were pulse-labeled with BrdUrd/dC for 1 h to determine the length of G₂ phase by anti-BrdUrd staining. Cells were treated with colcemid for 1 h before metaphase harvest. (b) Replication bands of metaphase spreads representing the labeling periods indicated (given as hours before the onset of G₂ phase). Chromosomes were stained with anti-BrdUrd antibody (green) and counter-stained with DAPI (blue). Cells exhibited characteristic replication bands at each time point. (Lower Right and Lower Center) The absence of BrdUrd-positive metaphases after pulsing during the last 4 h before metaphase defines the G₂ period. The 50% positive time point (vertical arrow) is defined as 1 h before G₂/the last hour of S. (c) Time course of telomere replication in asynchronous cells. The telomere replication patterns representing a total of 52,632 chromosome ends of a female Indian muntjac cell (expressing hTERT) were analyzed. Each point represents the percentage of all replicating events of that chromosome end during the 11 h before the onset of G₂ phase. The data from asynchronous cultures are very similar to that for HU-synchronized cells shown in Fig. 2.

Fig. 4.

The timing of telomere replication between homologous chromosomes and between the p and q arms of the same chromosome. (a) Representative metaphase spread of an Indian muntjac cell (F4374) by ReDFISH. Yellow asterisks indicate homologous X chromosomes. Green arrows mark synchronous telomere replication. Red triangles mark homologous chromosome ends that replicated asynchronously. (b) Representative metaphase spread of Indian muntjac cell (F4374) illustrating replication timing of p versus q arms of the same chromosome. A green asterisk indicates a chromosome in which asynchronous timing of telomere replication for the long arm and the short arm is found. A chromosome with synchronous timing of p and q arms is also shown. (c) Timing of telomere replication between homologous chromosomes. About 70% of the homologous chromosome ends replicated within the same hour-long BrdUrd pulse. A total of 22,310 replicating homologous chromosome ends were analyzed. (d) Timing of p-versus q-arm telomere replication. For ≈90% of all chromosomes, the p- and q-arm telomeres did not replicate at the same time. A total of 50,148 replicating chromosome ends were analyzed.

Fig. 5.

The influence of telomere length on the timing of telomere replication. (a) The distribution of telomere length of individual chromosome ends. Telomere fluorescence signals from metaphase chromosomes of Indian muntjac F4374 and F4869, and their telomerase-expressing derivatives were measured by quantitative FISH. Note the significant lengthening in telomeres associated with hTERT expression. The F4374 cells were analyzed while still expressing telomerase (6); the F4869 cells had been infected with a lox-hTERT retrovirus and were analyzed after Cre-mediated excision of hTERT (50) to ensure that any differences were due to the elongation of telomeres rather than the presence of telomerase. (b) Telomere length and replication timing. The potential influence of telomere length on the timing of replication was examined by comparing the pattern of replication in the parental cells (average of both cell types from Fig. 2; diamonds connected by a line) with that after telomere elongation (individual points for each cell line). Only two of the telomeres (1p and Xp) shared a significant change in replication timing. At least 30,000 telomere ends of each cell line were analyzed.