Replication timing of human telomeres is chromosome arm-specific, influenced by subtelomeric structures and connected to nuclear localization

Abstract

The mechanisms governing telomere replication in humans are still poorly understood. To fill this gap, we investigated the timing of replication of single telomeres in human cells. Using in situ hybridization techniques, we have found that specific telomeres have preferential time windows for replication during the S-phase and that these intervals do not depend upon telomere length and are largely conserved between homologous chromosomes and between individuals, even in the presence of large subtelomeric segmental polymorphisms. Importantly, we show that one copy of the 3.3 kb macrosatellite repeat D4Z4, present in the subtelomeric region of the late replicating 4q35 telomere, is sufficient to confer both a more peripheral localization and a later-replicating property to a de novo formed telomere. Also, the presence of beta-satellite repeats next to a newly created telomere is sufficient to delay its replication timing. Remarkably, several native, non-D4Z4-associated, late-replicating telomeres show a preferential localization toward the nuclear periphery, while several early-replicating telomeres are associated with the inner nuclear volume. We propose that, in humans, chromosome arm-specific subtelomeric sequences may influence both the spatial distribution of telomeres in the nucleus and their replication timing.

Figure 1. Human telomeres replicate within limited and chromosome arm-specific time windows.

(A) The ReDFISH approach is based on the labeling of synchronized cells with BrdU & BrdC in pulses of 1 hour covering the entire S phase. Timing of entry in S-phase after aphidicolin release was ascertained by FACS analysis (not shown). (B) BrdU-C is incorporated in sister chromatid regions that replicate during the pulse. The CO-FISH procedure destroys all base-substituted strands, thus sister telomeres that replicated during the pulse are converted into single strands and therefore recognized exclusively by either C-rich or G-rich specific probes (red and green signals, respectively). Telomeres that are not detargeted by the procedure yield mixed hybridizations (yellow signals). (C) IMR90 metaphase spread after ReDFISH. (D) Overall replication timing of telomeres in IMR90 cells. Diamonds indicate the percentage of detargeted telomeres observed during each pulse of BrdU-C. FACS analysis indicated that the S-phase was finished little after 6 hours (not shown). The vertical line indicates the mean replication timing (mrt) for all telomeres in IMR90 cells (i.e.: 3.12h). (E) Replication timing of individual telomeres in IMR90 cells. The percentage of replicating telomeres for every chromosome arm detected during pulses 1+2 (early S), 3+4 (middle S) and 5+6 (late S) is represented in horizontal bars. The total sum of partial percentages is normalized to 100% with horizontal lines inside bars representing confidence intervals (c.i.) (α = 0.05). For the sake of simplicity, only upper limits for early S and lower limits for late S are represented. Chromosome arms are listed (left) from the lowest to the highest mean replication timing (right). Acrocentric chromosomes are grouped according to the following convention: group D for chromosomes 13, 14 and 15 and group G for chromosomes 21 and 22. (F) Examples of early (19q), middle (6p) and late (4q) replicating telomeres. The vertical black line indicates the mrt for these telomeres, while the vertical grey line indicates the mrt for all telomeres.

Figure 2. No apparent relationship between replication timing of single telomeres and their length.

(A) Example of an IMR90 metaphase treated sequentially for ReDFISH (top) and for subtelomeric FISH (bottom) to distinguish between homologous chromosomes. In IMR90 cells, the subtelomeric probes used (f7501 and ICRF10, revealed in green and red colors, respectively) allow us to distinguish between the homologs of chromosomes 1, 7, 8, 9, and 16. (B) Mean replication timings and relative telomere lengths (measured by Q-FISH) for each allele are plotted side by side. (C) Absence of correlation between mean replication timing and relative telomere lengths.

Figure 3. Telomere elongation by telomerase does not impact on the replication timing of single telomeres.

(A) Expression of telomerase activity increases and homogenizes telomere lengths. Telomere restriction fragment analysis of IMR90 (wt), IMR90+hTERT (T) and IMR90+hTERT+TIN2 (TT) was performed by Southern blotting using RsaI/HinfI-digested DNA and probing with a ³²P labelled telomeric oligonucleotide (CCCTAA4). Mean telomere length was calculated using Telometrics, which showed that telomere length increases in cells bearing telomerase. (B) Overall replication timing of telomeres in IMR90+TT cells. Diamonds indicate the percentage of detargeted telomeres observed during each pulse of BrdU-C. Start of S-phase was ascertained by FACS analysis, which also indicated replication was completed in 5.5 hours (not shown). For comparison, the replication timing of telomeres in the parental IMR90 cells is also shown. Overall telomere replication in IMR90+TT cells appears to occur slightly earlier than in parental cells (mrt = 2.61 indicated by the orange vertical line). (C) Pattern of replication of single telomeres in IMR90+TT cells during the S-phase. The mrt for each telomere is indicated on the right. (D) A significant correlation of telomere-specific mean replication timings exists between IMR90 cells and its immortalized derivative IMR90+TT cell line with longer telomeres, indicating that the relative order of telomere replication was not significantly altered.

Figure 4. The replication timing of single telomeres is conserved among individuals.

(A) The overall telomere replication profile in HCA2+T cells (mean replication timing: 2.45h, green vertical line) is very similar to the one observed in IMR90+TT cells. (B) Pattern of replication of single telomeres in HCA2+T cells during the S-phase. The mrt for each telomere is indicated on the right. (C) A high correlation is observed between telomere-specific mrt of HCA2+T and IMR90+TT cells, indicating that the relative order of telomere replication is conserved. (D) Specifically, no major differences are detected between the profiles of telomere replication of chromosome arms known to carry subtelomeric segmental variations in HCA2+T (H) and IMR90+TT (I).

Figure 5. Telomeres associated with satellite-like subtelomeric repeats are late replicating.

(A) Late telomere replication of the short arms of acrocentric chromosomes in groups D (13, 14, and 15) and G (21 and 22), compared to the respective q arms, in IMR90 cells. Similar replication patterns are detected in all cell lines examined in our study (Figure 3C, Figure 4B, and Figure 7C). (B) Late replication profile of telomeres located on 4q and 10q chromosome ends in IMR90 cells. 4q and 10q are also late replicating in HCA2+T cells (Figure 4B). Colored vertical lines indicate the mean replication score for each telomere. No statistically significant differences are observed between 4q and 10q mrts (significance threshold p<0.0025).

Figure 6. The replication timing of seeded telomeres correlates with their nuclear localization.

(A) Constructs used for random telomere seeding in C33A cells and carrying specific subtelomeric elements events as described in and . T: no subtelomeric sequences; T1X: carries a 3.3kb D4Z4 element ; T8X: carries 8xD4Z4 elements in tandem ; T-Sat: carries a 1.4kb subtelomeric fragment in 4q35, distal to D4Z4 sequences and spanning 4 β-satellite repeats (Boussouar et al., manuscript in preparation); T1X-βSat: carries a D4Z4 element next to the native subtelomeric fragment of 4q spanning 4 β-satellite repeats . (B) Nuclear localization by immuno-3D of randomly seeded telomeres in the C33A cell line. Shown are examples of FISH signals (in red) obtained for control seeded telomeres (T) and telomeres associated with 1 copy (T1X) or multiple copies (T8X) of D4Z4. Lamin B, the reference for the nuclear periphery, is revealed in blue. (C) Constructs carrying only one D4Z4 repeat show a more peripheral localization in immuno-3D. Represented is the mean position of these telomeres (n = 50) in a circle section indicating percentages of volume ratios, calculated as described . The gray shadow indicates the value for mean volume ratio obtained for lamin B. (D) Telomere replication timings of native and seeded telomeres in C33A cells. mrt for each telomere is indicated by colored vertical lines. The grey line indicates the mrt (3.5) for all telomeres in C33T1X -whose overall telomere replication profile is shown on the top panel. The replication profile of telomeres on the short arms of acrocentric chromosomes is also shown (mean replication timing: 4.57). The significance values obtained by comparing mrt for seeded telomeres and the mean replication timing of all native telomeres in the C33A cells are also shown. Only T1X and T1X-βSat (mean replication timings 4.7 and 4.65, respectively) replicate significantly later. (E) Mean volume ratios for nuclear localization (fully colored bars) and mean replication timings (stripy bars) of single telomeres are plotted together. Vertical lines on top of the bars indicate the standard errors for both measurements. The significance values of comparisons taking both sets of data between the control telomeres, on one hand, and the test telomeres, on the other, are given (first, between nuclear localization data; second, between replication timing data). T1X, T-βSat and T1X-βSat replicate significantly later than control telomeres, while only T1X and T1X-βSat are significantly more peripheral than the control. Significance threshold: p<0.003.

Figure 7. Relationship between peripheral nuclear localization and late replication timing of telomeres.

(A) The replication profiles of early replicating telomeres 17q, 12p, 1p, and 5p and late replicating telomeres 12q, 3p, 6q, 4q, and 2p (mean replication timings indicated by colored vertical lines) are represented. The vertical grey lines indicate the median replication score for all telomeres. (B) Examples of nuclear localization analyses by immuno-3D for 12qter (BAC RP11-349K16) and 17qter (BAC RP11-637C24). Hybridizations are revealed in red while lamin B, the reference for nuclear periphery, is revealed in blue. (C) These analyses indicate that the late replicating telomeres 2p, 3p, 4q, 6q, and 12q are more peripheral than the early replicating telomeres 1p, 5p, 12p, and 17q. Represented is the mean position of these telomeres (n = 50) in a circle sector indicating percentages of volume ratios, calculated as described in . The gray shadow indicates the value for volume ratio obtained for lamin B. (D) The mean volume ratios for nuclear localization (fully colored bars) and the mean replication timings (stripy bars) for single telomeres are plotted. Error bars indicate the standard error of the mean. (E) There is a significant correlation between the mean replication timing of single telomeres and their mean volume ratio.