Telomere elongation involves intra-molecular DNA replication in cells utilizing alternative lengthening of telomeres

Abstract

Alternative lengthening of telomeres (ALT) is a telomere length maintenance mechanism based on recombination, where telomeres use other telomeric DNA as a template for DNA synthesis. About 10% of all human tumors depend on ALT for their continued growth, and understanding its molecular details is critically important for the development of cancer treatments that target this mechanism. We have previously shown that telomeres of ALT-positive human cells can become lengthened via inter-telomeric copying, i.e. by copying the telomere of another chromosome. The possibility that such telomeres could elongate by using other sources of telomeric DNA as copy templates has not been investigated previously. In this study, we have determined whether a telomere can become lengthened by copying its own sequences, without the need for using another telomere as a copy template. To test this, we transduced an ALT cell line with a telomere-targeting construct and obtained clones with a single tagged telomere. We showed that the telomere tag can be amplified without the involvement of other telomeres, indicating that telomere elongation can also occur by intra-telomeric DNA copying. This is the first direct evidence that the ALT mechanism involves more than one method of telomere elongation.

Figure 1.

Description of the telomere-targeting construct. Graphical representation of the telomere-targeting construct (pHRT) in its linear form (not to scale). The RFP coding sequence is located upstream of a CMV promoter and will not be transcribed in this orientation. These sequences are flanked, respectively, by a splice acceptor and a splice donor site. At the 5′ and 3′ ends of the construct, we introduced 800 bp of telomeric repeats on each side, to promote integration at the telomere of host cells via homologous recombination. Telomeric sequences are shown as (CCCTAA)_n to indicate integration of the construct at the C-rich telomeric DNA strand. If the construct is duplicated, the promoter and the reporter will be in the correct orientation for transcription of mRNA containing the coding sequence for RFP. The splice sites are also in the correct orientation to promote splicing of the telomeric sequences interposed between the two copies of the construct. This will ultimately result in the translation of RFP protein.

Figure 2.

RFP expression in ALT-positive cells transfected with pHRT and pLRH. IIICF/c cells transfected with pLRH (positive control) showed diffuse expression of RFP as early as 2 days after transfection and kept expressing RFP throughout the observation time (left panels). In stable clones of IIICF/c transfected with pHRT, small and discrete clusters of RFP-positive cells were documented by fluorescence microscopy after a variable time of growth in culture, ranging from ∼14 to 25 PDs (right panels). In the right-hand panels, corresponding bright field and red fluorescence images are shown to visualize the clusters of RFP-positive cells among the non-fluorescent cells. Images were taken with a 20× objective.

Figure 3.

FISH analysis in IIICF/c pHRT clones. Metaphase spreads were obtained from IIICF/c pHRT clones and analyzed by FISH. Full metaphases and details of the chromosome tagged are shown. Cells were stained with DAPI to visualize the DNA (in blue) and probed with pHRT plasmid sequences devoid of the telomeric repeats (in green) to visualize the integration site. Early PD clone 3.1 revealed integration of the pHRT construct at the telomere of marker chromosome A (A). In (B), telomere-FISH (red) was combined with pHRT-FISH (green). The co-localization of the red and green signals and the presence of telomeric sequences proximal to the pHRT tag confirm telomeric integration of the pHRT tag. In clone 1.4, integration of pHRT was observed at the telomere of a marker chromosome (C). In clone 3.3, where fluorescence was never observed, integration of pHRT was interstitial within another marker chromosome (D).

Figure 4.

Molecular analysis of pHRT construct integration in IIICF/c pHRT clones 3.3 and 1.4. (A) PCR strategy for testing pHRT construct copy number in the IIICF/c pHRT clones. The forward and reverse PCR primers are oriented so that amplification only occurs if there is more than one copy of the construct. Size of PCR products will depend on the intervening telomere tract length (distances between each primer and the start of the telomeric repeat sequences are indicated). As positive and negative controls, we used primers amplifying a 1960 and a 1509 bp tract of DNA within the construct, respectively, the positive control overlapping with the Southern probe. (B and C) Clones 3.3 and 1.4 were analyzed by PCR–Southern blotting, with the probe depicted in (A). The test reaction primer pairs are indicated above each lane. The presence of bands of correct size in the control lanes of the PCR gels confirmed that these clones contained integrated pHRT DNA. In clone 3.3, the Southern blot probed for pHRT shows no discrete bands in the test lanes, indicating the presence of a single copy of the construct. In clone 1.4, Southern blotting analysis of the three test PCR reactions shows the presence of major amplicons of 2600, 2800 and 3000 bp. These amplicons contain pHRT (middle panel) as well as telomeric (right panel) sequences. This is consistent with amplification having occurred across two copies of the pHRT construct separated by ∼1500 bp of telomeric DNA.

Figure 5.

Molecular analysis of pHRT construct integration in IIICF/c pHRT clone 3.1. (A) Cells from IIICF/c pHRT clone 3.1 were enriched for HcRed expression by FACS. Southern blotting analysis of the three test PCR reactions shows the presence of three products for each amplification reaction. These amplicons contain sequences of the pHRT construct as well as telomeric sequences. The size of each amplicon is consistent with three separate two-copy-sets of the pHRT construct separated by 200, 700 and 1800 bp of telomeric DNA. (B) DNA from HcRed-positive IIICF/c pHRT clone 3.1 cells was serially diluted from 20 ng to 20 pg, the latter estimated as a single-genome-equivalent amount of DNA for these cells. The DNA was then PCR-amplified, as depicted in Figure 4, blotted and probed with pHRT sequences (F1–R3 amplification reactions were blotted on a separate membrane). The lower panel represents a longer exposure of the upper panel, to better visualize the amplicons obtained at 20 pg amounts of DNA (analyzed in triplicate for each set of primers). The two main amplicons, sized ∼1300 and 1800 bp in reaction F1–R1, are amplified separately when the DNA is diluted to single-genome equivalents, suggesting that they are located within separate telomeres.

Figure 6.

(A) Analysis of HT1080 clone 22 and 23 cells by FISH and PCR. FISH analysis performed on metaphase spreads obtained from HT1080 clone 22 cells (telomerase-positive) indicated that integration had occurred at the telomere of the q arm of a large chromosome. (B) PCR and Southern blotting were carried out on DNA pooled from clones 22 and 23, as described earlier. The internal primer pair used as a positive control produces an amplicon of correct size that contains construct-specific sequences, confirming its integration in the genome.

Figure 7.

Potential mechanism generating intra-telomeric duplication events. Graphical representation of three proposed mechanisms for duplication of a telomeric tag. (A) Loop-mediated DNA copying. The tagged telomere folds back and forms a loop structure consisting mostly of construct DNA. If the loop configuration primes new DNA synthesis, this results in duplication of the telomere tag. (B) Unequal t-SCE. Telomeric sequences distal to the construct DNA of one sister chromatid anneal to the complementary strand proximal to the construct DNA in the other sister chromatid. If this is followed by an unequal exchange, one sister chromatid will gain an additional copy of the construct, whereas the other will lose the construct sequences (and the resulting daughter cell will be lost from the population. (C) Sister chromatid copying. Only one sister chromatid undergoes rapid shortening of the telomeric DNA distal to the construct and becomes highly recombinogenic, whereas the other sister chromatid maintains a relatively long telomere. The short ‘distal’ telomere anneals to the ‘proximal’ telomeric DNA of its sister chromatid and this is followed by new DNA synthesis. This results in one sister chromatid gaining an additional copy of construct, whereas the other sister chromatid maintains a single copy. Dotted lines: newly synthesized DNA.