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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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HAC-telRNA is a chromatin-associated transcript.A. Immunoblot analyses were performed for the indicated proteins to verify the efficiency of the subcellular fractionation. Cyt, cytoplasmic; NP, nucleoplasmic; Chr, chromatin. One representative result is shown. Similar results were obtained for all proteins samples used for the experiment. B. Subcellular localization of transcripts. RNAs were purified from each subcellular fraction, and the abundance of a test RNA was quantified by RT-real-time PCR. The relative abundance in each fraction is shown. Xp-Yp, Chr Xp-Yp-derived TERRA. Bars indicate s.e.m. from three independent experiments. The relative abundance of HAC-telRNA splice variant 1 (HAC (variant 1)) in the chromatin fraction was significantly larger than that of total HAC-telRNA (HAC) (asterisk, one-tailed Student's t-test). C. Transcript levels of indicated genes in TRF1-knockdown HAC#21-HeLa cells. Total RNAs extracted from cells at 48 hrs post-transfection were examined in triplicate by RT-PCR. A representative result is shown. D. TRF1-knockdown effect. Amounts of RT-PCR products obtained from control knockdown (siCtrl) and TRF1-knockdown (siTRF1) HAC#21-HeLa cells were quantified from SYBR Gold-stained gels. 18S rRNA served as an internal control of three independent knockdown experiments. Bars, s.e.m. from three independent knockdown experiments.
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pone-0088530-g006: HAC-telRNA is a chromatin-associated transcript.A. Immunoblot analyses were performed for the indicated proteins to verify the efficiency of the subcellular fractionation. Cyt, cytoplasmic; NP, nucleoplasmic; Chr, chromatin. One representative result is shown. Similar results were obtained for all proteins samples used for the experiment. B. Subcellular localization of transcripts. RNAs were purified from each subcellular fraction, and the abundance of a test RNA was quantified by RT-real-time PCR. The relative abundance in each fraction is shown. Xp-Yp, Chr Xp-Yp-derived TERRA. Bars indicate s.e.m. from three independent experiments. The relative abundance of HAC-telRNA splice variant 1 (HAC (variant 1)) in the chromatin fraction was significantly larger than that of total HAC-telRNA (HAC) (asterisk, one-tailed Student's t-test). C. Transcript levels of indicated genes in TRF1-knockdown HAC#21-HeLa cells. Total RNAs extracted from cells at 48 hrs post-transfection were examined in triplicate by RT-PCR. A representative result is shown. D. TRF1-knockdown effect. Amounts of RT-PCR products obtained from control knockdown (siCtrl) and TRF1-knockdown (siTRF1) HAC#21-HeLa cells were quantified from SYBR Gold-stained gels. 18S rRNA served as an internal control of three independent knockdown experiments. Bars, s.e.m. from three independent knockdown experiments.

Mentions: To characterize the cellular localization of HAC-telRNA, we fractionated extracts from HAC#21-HeLa cells into cytoplasmic (Cyt), nucleoplasmic (NP), and chromatin-bound (Chr) fractions as previously reported [15]. An immunoblot showed that GAPDH, nuclear hnRNP A1 and chromatin-bound histone H2B were highly enriched in the Cyt and NP fractions, NP fraction and the Chr fraction, respectively, as expected (Fig. 6A). Tubulin was barely detected in the Cyt but was abundant in the NP fraction, probably due to polymerization of tubulin rather than incomplete extraction of the cytoplasm in our experiment. Nucleolin, which is involved in ribosome biogenesis and is typically found in both the nucleolus and the nucleus, was detected in the Chr as well as in the NP fraction. To examine whether RNAs contained in the nucleoplasm or chromatin were separated by the fractionation, test RNA present in each fraction was analyzed by RT-real-time PCR using specific primers. GAPDH transcripts and NEAT1 RNA, which constitutes nuclear bodies called paraspeckles [38], were largely fractionated in the NP and Chr fractions, whereas U1 and XIST RNAs were enriched in the Chr fraction, as expected (Fig. 6B). In contrast, Xp-Yp-derived TERRA was detected in the Chr and NP fractions, as reported previously [15]. We found that HAC-telRNA (detected with primers that recognized both spliced and unspliced HAC-telRNA; PCR in Fig. 4A) was also enriched in the Chr and NP fractions (Fig. 6B), suggesting that HAC-telRNA localizes to chromatin similarly to endogenous TERRA [11]. Interestingly, we observed that spliced HAC-telRNA (variant 1) was enriched in the Chr fraction to a greater extent than the total HAC-telRNA. Taken together, HAC-telRNA is largely bound to chromatin, suggesting that it functions as a non-coding RNA at the telomere.


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)

HAC-telRNA is a chromatin-associated transcript.A. Immunoblot analyses were performed for the indicated proteins to verify the efficiency of the subcellular fractionation. Cyt, cytoplasmic; NP, nucleoplasmic; Chr, chromatin. One representative result is shown. Similar results were obtained for all proteins samples used for the experiment. B. Subcellular localization of transcripts. RNAs were purified from each subcellular fraction, and the abundance of a test RNA was quantified by RT-real-time PCR. The relative abundance in each fraction is shown. Xp-Yp, Chr Xp-Yp-derived TERRA. Bars indicate s.e.m. from three independent experiments. The relative abundance of HAC-telRNA splice variant 1 (HAC (variant 1)) in the chromatin fraction was significantly larger than that of total HAC-telRNA (HAC) (asterisk, one-tailed Student's t-test). C. Transcript levels of indicated genes in TRF1-knockdown HAC#21-HeLa cells. Total RNAs extracted from cells at 48 hrs post-transfection were examined in triplicate by RT-PCR. A representative result is shown. D. TRF1-knockdown effect. Amounts of RT-PCR products obtained from control knockdown (siCtrl) and TRF1-knockdown (siTRF1) HAC#21-HeLa cells were quantified from SYBR Gold-stained gels. 18S rRNA served as an internal control of three independent knockdown experiments. Bars, s.e.m. from three independent knockdown experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0088530-g006: HAC-telRNA is a chromatin-associated transcript.A. Immunoblot analyses were performed for the indicated proteins to verify the efficiency of the subcellular fractionation. Cyt, cytoplasmic; NP, nucleoplasmic; Chr, chromatin. One representative result is shown. Similar results were obtained for all proteins samples used for the experiment. B. Subcellular localization of transcripts. RNAs were purified from each subcellular fraction, and the abundance of a test RNA was quantified by RT-real-time PCR. The relative abundance in each fraction is shown. Xp-Yp, Chr Xp-Yp-derived TERRA. Bars indicate s.e.m. from three independent experiments. The relative abundance of HAC-telRNA splice variant 1 (HAC (variant 1)) in the chromatin fraction was significantly larger than that of total HAC-telRNA (HAC) (asterisk, one-tailed Student's t-test). C. Transcript levels of indicated genes in TRF1-knockdown HAC#21-HeLa cells. Total RNAs extracted from cells at 48 hrs post-transfection were examined in triplicate by RT-PCR. A representative result is shown. D. TRF1-knockdown effect. Amounts of RT-PCR products obtained from control knockdown (siCtrl) and TRF1-knockdown (siTRF1) HAC#21-HeLa cells were quantified from SYBR Gold-stained gels. 18S rRNA served as an internal control of three independent knockdown experiments. Bars, s.e.m. from three independent knockdown experiments.
Mentions: To characterize the cellular localization of HAC-telRNA, we fractionated extracts from HAC#21-HeLa cells into cytoplasmic (Cyt), nucleoplasmic (NP), and chromatin-bound (Chr) fractions as previously reported [15]. An immunoblot showed that GAPDH, nuclear hnRNP A1 and chromatin-bound histone H2B were highly enriched in the Cyt and NP fractions, NP fraction and the Chr fraction, respectively, as expected (Fig. 6A). Tubulin was barely detected in the Cyt but was abundant in the NP fraction, probably due to polymerization of tubulin rather than incomplete extraction of the cytoplasm in our experiment. Nucleolin, which is involved in ribosome biogenesis and is typically found in both the nucleolus and the nucleus, was detected in the Chr as well as in the NP fraction. To examine whether RNAs contained in the nucleoplasm or chromatin were separated by the fractionation, test RNA present in each fraction was analyzed by RT-real-time PCR using specific primers. GAPDH transcripts and NEAT1 RNA, which constitutes nuclear bodies called paraspeckles [38], were largely fractionated in the NP and Chr fractions, whereas U1 and XIST RNAs were enriched in the Chr fraction, as expected (Fig. 6B). In contrast, Xp-Yp-derived TERRA was detected in the Chr and NP fractions, as reported previously [15]. We found that HAC-telRNA (detected with primers that recognized both spliced and unspliced HAC-telRNA; PCR in Fig. 4A) was also enriched in the Chr and NP fractions (Fig. 6B), suggesting that HAC-telRNA localizes to chromatin similarly to endogenous TERRA [11]. Interestingly, we observed that spliced HAC-telRNA (variant 1) was enriched in the Chr fraction to a greater extent than the total HAC-telRNA. Taken together, HAC-telRNA is largely bound to chromatin, suggesting that it functions as a non-coding RNA at the telomere.

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