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A human artificial chromosome recapitulates the metabolism of native telomeres in mammalian cells.

Wakai M, Abe S, Kazuki Y, Oshimura M, Ishikawa F - PLoS ONE (2014)

Bottom Line: The seeded telomere is associated with telomeric proteins over a length similar to that reported in native telomeres, and is faithfully replicated in mid-S phase in HeLa cells.We found that the seeded telomere on HAC#21 is transcribed from the newly juxtaposed site.These results suggest that transcription into TERRA is locally influenced by the subtelomeric context.

View Article: PubMed Central - PubMed

Affiliation: Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, Japan.

ABSTRACT
Telomeric and subtelomeric regions of human chromosomes largely consist of highly repetitive and redundant DNA sequences, resulting in a paucity of unique DNA sequences specific to individual telomeres. Accordingly, it is difficult to analyze telomere metabolism on a single-telomere basis. To circumvent this problem, we have exploited a human artificial chromosome (HAC#21) derived from human chromosome 21 (hChr21). HAC#21 was generated through truncation of the long arm of native hChr21 by the targeted telomere seeding technique. The newly established telomere of HAC#21 lacks canonical subtelomere structures but possesses unique sequences derived from the target vector backbone and the internal region of hChr21 used for telomere targeting, which enabled us to molecularly characterize the single HAC telomere. We established HeLa and NIH-3T3 sub-lines containing a single copy of HAC#21, where it was robustly maintained. The seeded telomere is associated with telomeric proteins over a length similar to that reported in native telomeres, and is faithfully replicated in mid-S phase in HeLa cells. We found that the seeded telomere on HAC#21 is transcribed from the newly juxtaposed site. The transcript, HAC-telRNA, shares several features with TERRA (telomeric repeat-containing RNA): it is a short-lived RNA polymerase II transcript, rarely contains a poly(A) tail, and associates with chromatin. Interestingly, HAC-telRNA undergoes splicing. These results suggest that transcription into TERRA is locally influenced by the subtelomeric context. Taken together, we have established human and mouse cell lines that will be useful for analyzing the behavior of a uniquely identifiable, functional telomere.

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The seeded subtelomere is replicated in mid-S phase in HeLa cells.A. S-phase progression analyzed by flow-cytometry of G1/S-released cells after thymidine-aphidicolin double-block and propidium iodide staining. B. Histograms indicating the amounts of incorporated BrdU at a test locus during each one-hour pulse in S phase, as determined by real-time PCR quantification of immunoprecipitated genomic DNA by anti-BrdU (shown in arbitrary units). x and z indicate PCR regions in the HAC#21 subtelomere shown in C. Each graph is fitted to a normal distribution (red). C. Replication kinetics represented in the cumulative plots of BrdU-incorporation in B, with fitting curves. The distal subtelomeric DNA in HAC#21 (PCR regions x and z) is replicated with timing between early (ADH5) and late (γ-globin) in S phase.
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pone-0088530-g002: The seeded subtelomere is replicated in mid-S phase in HeLa cells.A. S-phase progression analyzed by flow-cytometry of G1/S-released cells after thymidine-aphidicolin double-block and propidium iodide staining. B. Histograms indicating the amounts of incorporated BrdU at a test locus during each one-hour pulse in S phase, as determined by real-time PCR quantification of immunoprecipitated genomic DNA by anti-BrdU (shown in arbitrary units). x and z indicate PCR regions in the HAC#21 subtelomere shown in C. Each graph is fitted to a normal distribution (red). C. Replication kinetics represented in the cumulative plots of BrdU-incorporation in B, with fitting curves. The distal subtelomeric DNA in HAC#21 (PCR regions x and z) is replicated with timing between early (ADH5) and late (γ-globin) in S phase.

Mentions: To analyze the replication timing of the telomere region in HAC#21, we synchronized HAC#21-HeLa cells at the start of S phase by sequential treatments of cells with thymidine and aphidicolin (Fig. 2A). The synchronous culture was released from the G1-S block by culturing the cells in media without the drug, and split into aliquots. Successive aliquots were labeled with BrdU for 1 hr at consecutive one-hour intervals and chased in the absence of BrdU until 9 hr post-release when most cells exited from S phase. BrdU-labeled nascent DNA was purified by immunoprecipitation with an anti-BrdU antibody, and enrichment of test DNAs was quantified by real-time PCR. All loci analyzed showed a single 1-hr peak interval, during which the BrdU incorporation was greatest (Fig. 2B). It is known that ADH5 and the gamma-globin gene replicate in early and late S phase, respectively [30], [31]. When cumulative BrdU incorporation was correlated with specific test loci in fractions covering the S phase progression, we found that ADH5 and gamma-globin loci replicated at early and late S phase, respectively, as expected (Fig. 2C). To examine the replication kinetics of HAC#21, we chose two primer sets, x and z, which were unique to the HAC#21 subtelomere (Fig. 2C). Regions amplified by primer sets x and z are located 0.1-kb and 3.5-kb proximal to the telomere repeat DNAs of the seeded telomere of HAC#21 (regions x and z, respectively). We found that regions x and z synchronously replicated at mid-S phase (Fig. 2B and C). These results indicate that the telomere-proximal region of the seeded telomere of HAC#21, which is devoid of any endogenous hChr21 subtelomere DNA sequence, synchronously replicates at mid-S phase in HAC#21-HeLa cells.


A human artificial chromosome recapitulates the metabolism of native telomeres in mammalian cells.

Wakai M, Abe S, Kazuki Y, Oshimura M, Ishikawa F - PLoS ONE (2014)

The seeded subtelomere is replicated in mid-S phase in HeLa cells.A. S-phase progression analyzed by flow-cytometry of G1/S-released cells after thymidine-aphidicolin double-block and propidium iodide staining. B. Histograms indicating the amounts of incorporated BrdU at a test locus during each one-hour pulse in S phase, as determined by real-time PCR quantification of immunoprecipitated genomic DNA by anti-BrdU (shown in arbitrary units). x and z indicate PCR regions in the HAC#21 subtelomere shown in C. Each graph is fitted to a normal distribution (red). C. Replication kinetics represented in the cumulative plots of BrdU-incorporation in B, with fitting curves. The distal subtelomeric DNA in HAC#21 (PCR regions x and z) is replicated with timing between early (ADH5) and late (γ-globin) in S phase.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3928237&req=5

pone-0088530-g002: The seeded subtelomere is replicated in mid-S phase in HeLa cells.A. S-phase progression analyzed by flow-cytometry of G1/S-released cells after thymidine-aphidicolin double-block and propidium iodide staining. B. Histograms indicating the amounts of incorporated BrdU at a test locus during each one-hour pulse in S phase, as determined by real-time PCR quantification of immunoprecipitated genomic DNA by anti-BrdU (shown in arbitrary units). x and z indicate PCR regions in the HAC#21 subtelomere shown in C. Each graph is fitted to a normal distribution (red). C. Replication kinetics represented in the cumulative plots of BrdU-incorporation in B, with fitting curves. The distal subtelomeric DNA in HAC#21 (PCR regions x and z) is replicated with timing between early (ADH5) and late (γ-globin) in S phase.
Mentions: To analyze the replication timing of the telomere region in HAC#21, we synchronized HAC#21-HeLa cells at the start of S phase by sequential treatments of cells with thymidine and aphidicolin (Fig. 2A). The synchronous culture was released from the G1-S block by culturing the cells in media without the drug, and split into aliquots. Successive aliquots were labeled with BrdU for 1 hr at consecutive one-hour intervals and chased in the absence of BrdU until 9 hr post-release when most cells exited from S phase. BrdU-labeled nascent DNA was purified by immunoprecipitation with an anti-BrdU antibody, and enrichment of test DNAs was quantified by real-time PCR. All loci analyzed showed a single 1-hr peak interval, during which the BrdU incorporation was greatest (Fig. 2B). It is known that ADH5 and the gamma-globin gene replicate in early and late S phase, respectively [30], [31]. When cumulative BrdU incorporation was correlated with specific test loci in fractions covering the S phase progression, we found that ADH5 and gamma-globin loci replicated at early and late S phase, respectively, as expected (Fig. 2C). To examine the replication kinetics of HAC#21, we chose two primer sets, x and z, which were unique to the HAC#21 subtelomere (Fig. 2C). Regions amplified by primer sets x and z are located 0.1-kb and 3.5-kb proximal to the telomere repeat DNAs of the seeded telomere of HAC#21 (regions x and z, respectively). We found that regions x and z synchronously replicated at mid-S phase (Fig. 2B and C). These results indicate that the telomere-proximal region of the seeded telomere of HAC#21, which is devoid of any endogenous hChr21 subtelomere DNA sequence, synchronously replicates at mid-S phase in HAC#21-HeLa cells.

Bottom Line: The seeded telomere is associated with telomeric proteins over a length similar to that reported in native telomeres, and is faithfully replicated in mid-S phase in HeLa cells.We found that the seeded telomere on HAC#21 is transcribed from the newly juxtaposed site.These results suggest that transcription into TERRA is locally influenced by the subtelomeric context.

View Article: PubMed Central - PubMed

Affiliation: Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, Japan.

ABSTRACT
Telomeric and subtelomeric regions of human chromosomes largely consist of highly repetitive and redundant DNA sequences, resulting in a paucity of unique DNA sequences specific to individual telomeres. Accordingly, it is difficult to analyze telomere metabolism on a single-telomere basis. To circumvent this problem, we have exploited a human artificial chromosome (HAC#21) derived from human chromosome 21 (hChr21). HAC#21 was generated through truncation of the long arm of native hChr21 by the targeted telomere seeding technique. The newly established telomere of HAC#21 lacks canonical subtelomere structures but possesses unique sequences derived from the target vector backbone and the internal region of hChr21 used for telomere targeting, which enabled us to molecularly characterize the single HAC telomere. We established HeLa and NIH-3T3 sub-lines containing a single copy of HAC#21, where it was robustly maintained. The seeded telomere is associated with telomeric proteins over a length similar to that reported in native telomeres, and is faithfully replicated in mid-S phase in HeLa cells. We found that the seeded telomere on HAC#21 is transcribed from the newly juxtaposed site. The transcript, HAC-telRNA, shares several features with TERRA (telomeric repeat-containing RNA): it is a short-lived RNA polymerase II transcript, rarely contains a poly(A) tail, and associates with chromatin. Interestingly, HAC-telRNA undergoes splicing. These results suggest that transcription into TERRA is locally influenced by the subtelomeric context. Taken together, we have established human and mouse cell lines that will be useful for analyzing the behavior of a uniquely identifiable, functional telomere.

Show MeSH
Related in: MedlinePlus