title: A human artificial chromosome recapitulates the metabolism of native telomeres in mammalian cells.
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.
authors.affiliation: Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, Japan.
authors.forename: Michihito
authors.initials: M
authors.lastname: Wakai
authors.affiliation: Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Yonago, Tottori, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Abe
authors.affiliation: Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Yonago, Tottori, Japan ; Chromosome Engineering Research Center, Tottori University, Tottori, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kazuki
authors.affiliation: Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Yonago, Tottori, Japan ; Chromosome Engineering Research Center, Tottori University, Tottori, Japan.
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Oshimura
authors.affiliation: Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, Japan.
authors.forename: Fuyuki
authors.initials: F
authors.lastname: Ishikawa
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry; D001483:Base Sequence; D002453:Cell Cycle; D002460:Cell Line; D002843:Chromatin / Q000737:chemistry; D022222:Chromosomes, Artificial, Human / Q000235:genetics*; D002877:Chromosomes, Human; D017931:DNA Primers / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D011061:Poly A / Q000737:chemistry; D012313:RNA / Q000235:genetics; D012319:RNA Polymerase II / Q000737:chemistry; D034741:RNA, Small Interfering / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA / Q000379:methods*; D016615:Telomere / Q000648:ultrastructure*; D014158:Transcription, Genetic
chemical_list: D000906:Antibodies; D002843:Chromatin; D017931:DNA Primers; D034741:RNA, Small Interfering; D011061:Poly A; D012313:RNA; D012319:RNA Polymerase II