- By measuring (TTAGGG) n tract lengths from the same large DNA molecules that were optically mapped, we simultaneously analyzed telomere length dynamics and subtelomere-linked structural changes at a large number of specific subtelomeric loci in the ALT-positive cell lines U2OS, SK-MEL-2 and Saos-2.. - The frequency of recombinant subtelomeres of SK-MEL-2 (11%) is about half that of U2OS and Saos-2 (24 and 19% respectively). - Very large linear extrachromosomal telomere repeat (ECTR) DNA molecules were found in all three cell lines. - Conclusion: Identifying individual subtelomeres and characterizing linked telomere (TTAGGG) n tract lengths and structural changes using our new single-molecule methodologies reveals the structural consequences of telomere damage, repair and recombination mechanisms in human ALT cells in unprecedented molecular detail and significant differences in different ALT-positive cell lines.. - Keywords: Genomics, Cancer telomeres, Alternative lengthening of telomeres (ALT), U2OS, SK-MEL-2, Saos-2, Single molecule optical mapping. - Full list of author information is available at the end of the article. - Telomeres are nucleoprotein structures located at the tips of eukaryotic chromosomes that prevent the ends of the linear DNA component of chromosomes from being recognized and processed as double-strand breaks, and which provide a means for the faithful completion of chromosomal DNA replication [1, 2]. - the precise sequence of the telomeric DNA motif is deter- mined by the species-dependent RNA component of the RNP enzyme telomerase. - This is supported by evidence that genes encoding HR proteins are necessary for telomere-length mainten- ance in human ALT cells. - ALT probably includes strand invasion of the template molecule and formation of an HR intermediate structure [8, 17]. - We have utilized our recently-developed single-molecule method that simultan- eously measures individual telomere (TTAGGG) n tract lengths and identifies their physically linked DNA to analyze these structures in the telomerase-negative ALT- positive U2OS human osteosarcoma cancer cell line, SK- MEL-2 melanoma cell line and Saos-2 osteosarcoma cell line. - Our recently developed two-color labeling scheme was performed on U2OS, SK-MEL-2 and Saos-2 genomic. - 5p, and 11p in SK-MEL. - 3q and 20q of SK- MEL-2. - One molecule has a telomere length of 17.3 kb compared to another molecule with a telomere length of 0.15 kb.. - Likewise, differing telomere lengths are also seen in mol- ecules from arm 2p of SK-MEL-2 and arm 3p from Saos-2. - All 12 recombinant molecules of the 5p end and 5 recom- binant molecules of the 11q end have extremely short ITSs of less than 500 bp in length. - The three analyzed examples of recombinant 5q ends have longer ITSs with an average telomere length of 6.6 kb ± 3.6. - The SK-MEL-2 has fewest number of ITS.. - We unexpectedly observed a very high level of signal- free telomere ends in these three cell lines. - A total of 57 out of 781 ends completely lacked detectable (TTAG GG) n end signal detected in the U2OS cell line, 38/818 in SK-MEL-2 and 46/594 in Saos-2. - The signal-free ends are distributed unevenly across the arms analyzed for U2OS and SK-MEL-2. - 6q, 8p, 8q, 11q, and 14q of SK-MEL-2 have 23 out of 38 signal-free ends (Table 2). - End telomere End telomere. - 1c shows several 15q ends of U2OS and 8p of SK- MEL-2 which do not contain detectable (TTAGGG)n.. - note that if we had, it would have impacted this metric signifi- cantly for some telomeres (e.g., the average telomere length for 15q would have decreased to 3.5 kb from 4.7 kb). - Recombinant telomeres and ITSs were seen in three ALT positive cell lines. - SK-MEL-2 has the lowest fraction at only 11%. - 1q, 3q, 9p, 18q, and 21q are shown to have high fractions of recombinant telomeres in SK-MEL-2. - The 21q arm telomeres retained ITS length kb) for recombinant molecules is significantly shorter than the end telomere length kb). - The 21q arm of Saos- 2 is also highly recombined with similar average telo- mere length (1.9 kb ± 0.7) in comparison to the end. - telomere length (1.3 kb ± 0.6). - Figure 2b shows that 9p of U2OS has a defined recombination pat- tern, while 7q of SK-MEL-2 lack defined patterns.).. - Examples of very short ITSs at recombinant telomeres are 1q, 6p, 7p, 11p, and 18q of U2OS, 20q of SK-MEL-2 and 3q of Saos-2, all with multiple detected internal telo- meres averaging between 0.4 kb and about 1.3 kb. - Overall, recombinant molecules of U2OS have the highest fraction of ITS loss (108 molecules with ITS loss compared to 182 molecules with ITS) among these three cell lines.. - On the other hand, SK-MEL-2 and Saos-2 have lower frac- tions of ITS loss (5 vs. - 82 of SK-MEL-2, and 3 vs. - the limited number of mole- cules with end telomeres have very short (TTAGGG) n tracts (0.9 kb ± 1.2), with 4 end molecules lacking any signal. - From our previous single-molecule telomere length analyses of senescing primary IMR90 fibroblasts and cancer cell lines UMUC3 and LNCaP, we found that the distribution of very short single telomeres was biased Table 1 U2OS telomere lengths (Continued). - Table 2 SK-MEL-2 telomere lengths. - We therefore looked for unusual (TTAGGG) n length distributions at these telomeres in the U2OS, SK- MEL-2, and Saos-2 cancer cell lines. - Besides very short telomere ends (1.1 kb average telomere length), Saos-2 8q also has a high fraction of recombinant ends, but Saos-2 8q has fewer end telomere and ITS loss. - SK- MEL-2 8q seems to have a different profile compared to U2OS and Saos-2 8q. - Overall, the end telomere lengths of U2OS, SK-MEL-2, and Saos-2 are highly variable ranging from undetectable to ex- tremely long (Tables 1, 2, and 3) in comparison to UMUC3 and LNCaP which are documented to have relatively uniform and short telomere length distributions [37, 38]. - When look- ing specifically at 8q ends, mean (TTAGGG) n tract lengths are similar in the ALT-positive U2OS, SK-MEL-2 and Saos-2 cancer cell lines and telomerase-positive cancer cell lines. - Few very long telomeres are found at U2OS and SK-MEL-2 8q distinguishing the ALT positive from the telomerase positive. - cell lines at this telomere (Fig. - The aver- age end telomere length of U2OS is at 1.6 kb. - SK-MEL-2 14q also has short average telomere length of 1.7 kb with only 4 molecules having end telomere loss. - Saos-2 has only one end telomere loss at 14q with relatively longer average telomere length of 2.3 kb. - The overall end telo- mere length and heterogeneity is higher than in telomerase-positive cancer cell lines. - At some specific ends lengths appear to be very similar, perhaps implying a level of active cis control of the shortest telomeres in both pre- and post-immortalization cells, irrespective of TMM.. - This punctate labeling feature was not observed on any telomeres from IMR90 fibroblasts or from telomerase-positive cell lines [32]. - The punctate feature of the labeling suggests stretches of nontelomeric DNA sequence and/or variant (TTAGGG) n -like repeat DNA interspersed with pure (TTAGGG) n in these telo- mere tracts, as described previously [20, 39]. - Large lin- ear ECTRs comprised 40% of the total telomere signal in Table 2 SK-MEL-2 telomere lengths (Continued). - the average telomere length for these ECTRs was 11.0 kb with the longest measuring 50.4 kb. - The same patterns of long linear ECTRs are also observed in SK-MEL-2 and Saos-2 cell lines.. - Our general global observation of long end telomere tracts and telomere length heterogeneity was anticipated from prior studies of ALT cells [8]. - Overall, there were a surprising number of signal-free ends (8%) in U2OS cells and SK-MEL-2 cell lines (8. - By contrast, there were no signal-free ends found in telomerase negative senes- cing IMR90 fibroblasts, telomerase-positive cancer cell lines (UMUC3, LNCaP) and lymphoblastoid cell lines [32]. - As with the end-telomere characteris- tics, features of the recombinant telomere molecules (in- cluding the percentage of the specific subtelomere molecules involved, and the relative size and retention of the ITS at the fusion site) varied depending upon the linked subtelomere and the cell line. - Third, very large linear ECTR molecules were present in all three ALT positive cell lines. - Previous karyotypic analysis of ALT cell lines [41, 42]. - In a study which in- cluded U2OS and Saos-2 cell lines, clonal structural chromosomal anomalies in the karyotypes of 9 ALT cell lines was 3.7- fold higher than in telomerase-positive cancer cell lines [42]. - The low resolution of the metaphase mapping precluded distinguishing telo- mere capture events (which would stabilize the arm by Table 3 Saos-2 Telomere lengths (Continued). - N/A no data, not enough molecules to measure the telomere length INP inverted nick pair (INP) sites. - If the entirety of these molecules are derived from a single clonal precursor, then this result indicates ongoing evolu- tion of the junction site (ITS length change, ITS loss, or ITS gain) during U2OS propagation. - Subtelomeres we detected using our optical mapping method in six other cell lines but which were rarely found or not found in the dataset (4q, 8p, 10p, 10q, and XqYq of U2OS. - 5p, and 11p in SK-MEL-2. - Alternatively, some of these missing or rearranged subtelomeres may have suffered homozygous deletion in an early stage of the crisis/immortalization process and were simply missing in all subsequent generations of the cell line.. - The location of the telomere is shown with a red “ T. - a Representative data for three chromosome arms (2q U2OS, 2p SK-MEL-2, 3p Saos-2) that have heterogenous end telomere length. - 16q molecules from SK-MEL-2, one with recombination and one without. - two display end telomere (TTAGGG) n loss. - Likewise, molecules from 8p SK-MEL-2, 1 displays telomere loss. - For example, subtelomere specificity of telomere length regulation and stability may relate in part to TERRA. - TERRA is critical to telomere length regulation and stability, transcribed from subtelo- meric promoters, and differentially expressed at single. - a Chromosome 21q molecules with a defined pattern in the recombination partner. - The top U2OS molecule shows 21q with an end telomere. - Chromosome 7q molecules from SK-MEL-2 without a defined pattern in the recombination partner. - The top U2OS molecule shows a structural variant of the 9p end with an end telomere. - The bottom two U2OS molecules show the same structural variant of the 9p end following recombination resulting in two distinct partner fragments, with retention of ITSs in both cases. - Likewise the top 7q SK-MEL-2 molecule has an end telomere while the bottom two show recombinants with ITS retention. - By measuring (TTAGGG) n tract lengths from the same large DNA molecules that were optically mapped, we simultaneously analyzed telomere length dynamics and subtelomere-linked structural changes at a large number of specific subtelomeric loci in the ALT-positive cell lines U2OS, SK-MEL-2 and Saos-2. - Each single molecule telomere length measurement is represented by a dot and the average telomere length for each chromosome arm is shown as a horizontal line. - The SK-MEL-2 cell line was cultured in Eagle’s Minimum Essential Medium supplemented with 10% FBS. - Telomere length analysis. - The telomere labels were found to be the extra labels at the end of molecules or in the middle of recombinant molecules. - The length of the telomeres was inferred from its fluorescent label intensity following the estab- lished protocols [32].. - Representative examples of raw images of single molecules that were imaged are shown below the reference for each of the cell lines. - The Saos-2 molecules retain their ITS., but SK- MEL-2 has only one ITS. - b Comparison of U2OS, Saos-2 and SK-MEL-2 end telomeres and internal telomere length with UMUC3 and LNCaP end telomere length. - ALT: Alternative Lengthening of Telomeres. - Part of the informatics analysis was run on hardware supported by Drexel ’ s University Research Computing Facility. - The funders provided the financial support to the research, but had no role in the design of the study, analysis, interpretations of data and in writing the manuscript.. - Adapting to life at the end of the line: how Drosophila telomeric retrotransposons cope with their job. - Telomere length measurement-caveats and a critical assessment of the available technologies and tools. - TZAP: a telomere-associated protein involved in telomere length control. - Telomere length and telomerase activity in bladder and prostate cancer cell lines. - Loss of ATRX, genome instability, and an altered DNA damage response are hallmarks of the alternative lengthening of telomeres pathway
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