Chromosome instability as a result of double-strand breaks near telomeres in mouse embryonic stem cells - PubMed (original) (raw)

Chromosome instability as a result of double-strand breaks near telomeres in mouse embryonic stem cells

Anthony W I Lo et al. Mol Cell Biol. 2002 Jul.

Free PMC article

Abstract

Telomeres are essential for protecting the ends of chromosomes and preventing chromosome fusion. Telomere loss has been proposed to play an important role in the chromosomal rearrangements associated with tumorigenesis. To determine the relationship between telomere loss and chromosome instability in mammalian cells, we investigated the events resulting from the introduction of a double-strand break near a telomere with I-SceI endonuclease in mouse embryonic stem cells. The inactivation of a selectable marker gene adjacent to a telomere as a result of the I-SceI-induced double-strand break involved either the addition of a telomere at the site of the break or the formation of inverted repeats and large tandem duplications on the end of the chromosome. Nucleotide sequence analysis demonstrated large deletions and little or no complementarity at the recombination sites involved in the formation of the inverted repeats. The formation of inverted repeats was followed by a period of chromosome instability, characterized by amplification of the subtelomeric region, translocation of chromosomal fragments onto the end of the chromosome, and the formation of dicentric chromosomes. Despite this heterogeneity, the rearranged chromosomes eventually acquired telomeres and were stable in most of the cells in the population at the time of analysis. Our observations are consistent with a model in which broken chromosomes that do not regain a telomere undergo sister chromatid fusion involving nonhomologous end joining. Sister chromatid fusion is followed by chromosome instability resulting from breakage-fusion-bridge cycles involving the sister chromatids and rearrangements with other chromosomes. This process results in highly rearranged chromosomes that eventually become stable through the addition of a telomere onto the broken end. We have observed similar events after spontaneous telomere loss in a human tumor cell line, suggesting that chromosome instability resulting from telomere loss plays a role in chromosomal rearrangements associated with tumor cell progression.

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Figures

FIG. 1.

Structure of the linearized pNPT-tel and pNPT2-tel plasmids before and after integration on the end of a chromosome in clones A211 and A405. (A) The pNPT-tel and pNPT2-tel plasmids were linearized with _Not_I prior to transfection so that the telomeric repeats would be in the proper orientation at one end to seed a new telomere. The location of the pSP73 vector (amp/ori), neo gene (neo), HSV-tk gene, and telomeric repeat sequences and the orientation of the neo and HSV-tk genes (arrows) are indicated. The structures of the integrated pNPT-tel plasmid in clone A211 and the pNPT2-tel plasmid in clone A405 are also shown. The location of the _Bam_HI (Bm), _Eco_RI (E), _Not_I (N), _Ssp_I (Ss), and _Xba_I (Xb) restriction sites and the 18-bp recognition site for the I-_Sce_I endonuclease (I-_Sce_I) are shown. The sizes of fragments generated by the various restriction enzymes are indicated for clone A405. (B) Southern blot analysis of genomic DNA isolated from clone A405 digested with _Ssp_I (Ss), _Bam_HI (Bm), _Xba_I (Xb), or _Eco_RI (E) and separated by pulsed-field gel electrophoresis prior to Southern blot anlaysis. Hybridization was performed with the pNTP-Δ plasmid (which is similar to pNPT-tel but lacks telomeric repeat sequences) as a probe. The locations of DNA size markers are shown. Terminal restriction fragments are long and heterogeneous in length due to variations in the lengths of the extensive telomeric repeat sequences found in different cells in the population.

FIG. 2.

FISH analysis of telomeric plasmid integration sites in clones A211 and A405. (i) The location of the integration site (arrow) in A211 and A405 is demonstrated by hybridization with the pNTP-Δ plasmid (green). Chromosomes were counterstained with propidium iodide. (ii) The identity of the chromosome containing the integration sites was determined by hybridization with a chromosome 18-specific painting probe for clone A211 or a chromosome 15-specific painting probe for clone A405 (red). Chromosomes were counterstained with DAPI.

FIG. 3.

Southern blot analysis of genomic DNA from clone A405 and its G418r/Ganr subclones FS-3 and FS-4. Genomic DNA was digested with either _Ssp_I or _Eco_RI and separated by standard agarose gel electrophoresis. Hybridization was performed with the pNTP-Δ plasmid as a probe. The lighter bands that are the same size in all three cell lines are due to cross-hybridization of the probe with the mouse endogenous pgk promoter. The location of DNA size markers and the limit of resolution (LOR) are shown.

FIG. 4.

Cloning and analysis of inverted repeats in the G418r/Ganr subclones isolated from the mouse ES cell clones. (A) The structure of the integrated pNPT-tel plasmid in the parental A211 is compared with the inverted repeats in the FS-1 and FS-2 subclones. Similarly, the structure of the integrated pNPT2-tel plasmid in the parental A405 clone is compared with the inverted repeats in the FS-3 and FS-4 subclones. The fragments cloned by PCR for A211 or by plasmid rescue for A405 are indicated (heavy horizontal lines), as are the sizes of the restriction fragments consistent with Southern blot analysis of genomic DNA (light horizontal lines). The recognition site for I-_Sce_I is indicated, as are some of the restriction enzymes used for mapping and Southern blot analysis, including _Bcl_I (B), _Bam_HI (Bm), _Eco_RI (E), _Sac_I (Sa), and _Ssp_I (Ss). The locations of the adjacent cellular DNA (chromosome 18 for A211 and chromosome 15 for A405), pSP73 vector sequences (amp/ori), neo gene (neo), HSV-tk gene, and telomeric repeat sequences are shown. The location of the oligonucleotide primers (arrows) used to amplify the fragments from subclones FS-1 and FS-2 and the locations of sites of recombination (heavy vertical bars) are shown. (B) Comparison of nucleotide sequences at the sites of recombination in the G418r/Ganr mouse ES cell subclones. The nucleotide sequences at the site of recombination in subclones FS-1 and FS-2 are compared with the sequence of the neo (neo) and ampicillin resistance (amp) genes. The nucleotide sequence at the site of recombination in subclone FS-3 is compared with sequences of the HSV-tk gene (tk) in opposite orientations. Nucleotides that are identical between sequences (asterisks) or show complementarity between the sequences at the site of recombination (boldface) are indicated.

FIG. 4.

FIG. 5.

Chromosome rearrangements in the FS-1 and FS-2 subclones isolated from clone A211. Metaphase spreads from clone A211 (A) and its subclones FS-1 (B) and FS-2 (C) were hybridized with both chromosome 18-specific painting (red) and subtelomeric BAC 31E18 (green) probes (i). An enlarged view of the chromosome 18 homologues (ii) and reverse video of the blue channel showing the DAPI GC banding (iii) are also shown. A different metaphase spread hybridized with both chromosome-18-specific (red) and telomere-specific (green) probes is shown for clone A211 and its subclones, FS-1 and FS-2 (iv). The open arrows point to the normal chromosome-18 homologues, while the closed arrow points to the rearranged chromosome 18.

FIG. 6.

Chromosome rearrangements in the FS-3 subclone isolated from clone A405. Metaphase spreads of the parental clone A405 (A) and subclone FS-3 (B) were hybridized with both chromosome-15-specific painting (red) and subtelomeric BAC 169K7 (green) probes (i). An enlarged view of the chromosome-15 homologues (ii) and reverse video of the blue channel showing the DAPI GC banding (iii) are also shown. A different metaphase spread hybridized with both chromosome 15-specific (red) and telomere-specific (green) probes (iv) is also shown for clone A405 and subclone FS-3. The open arrows point to the normal chromosome 15 homologues, while the closed arrows point to the rearranged chromosome 15. (C) Heterogeneity of the rearranged chromosomes in the different second-generation subclones of FS-3 (i to iv) as indicated by the variable lengths of the translocated fragments from other chromosomes. The junction of the rearranged chromosome (arrow) is indicated by the BAC 169K7 subtelomeric signal (green). Hybridization with the telomere-specific probe (red) is observed at both ends of these rearranged chromosomes, but no telomere-specific hybridization was observed within the rearranged chromosome. The normal (open arrow) and rearranged (closed arrow) homologues from the same metaphase spreads are shown for comparison. (D) Complex rearrangements involving the marker chromosome. A variety of large duplications (i and ii) and dicentric chromosomes (iii to x) are shown. The upper panel shows hybridization with subtelomeric BAC 169K7 (green, arrow in panels i and v) or the PAC 561P12 originally located in the center of chromosome 15 (green, open arrows in panels iii, vii, and ix), while the lower panel shows the corresponding DAPI GC banding by reverse video of the blue channel. Dicentric chromosomes were identified by the presence of pericentric heterochromatin (darkly stained regions) on both ends in the metaphase spreads stained with DAPI.

FIG. 7.

Cloning and analysis of inverted repeats in G418r/Ganr subclones isolated from the human EJ-30 tumor cell line. (A) The structure of the integrated pNCT-tel plasmid in the parental EJ-30 clone is compared with inverted repeats in the G55, G71, G45, and G65 subclones. The fragments cloned by plasmid rescue are indicated (heavy horizontal lines), as are the sizes of the restriction fragments that are consistent with bands observed by Southern blot analysis of genomic DNA (light horizontal lines). The recognition sites are shown for some of the restriction enzymes used for mapping and Southern blot analysis, including _Acc_I (Ac), _Bam_HI (Bm), _Ssp_I (Ss), _Xba_I (Xb), and _Xho_I (Xh). The locations of the adjacent cellular DNA (chromosome 16p), pSP73 vector sequences (amp/ori), neo gene (neo), HSV-tk gene, telomeric repeat sequences, and sites of recombination (black vertical bars) are shown. (B) Nucleotide sequences at the sites of recombination in the G418r/Ganr subclones from EJ-30. The nucleotide sequences at the sites of recombination at either end of the187-bp insert in subclone G71 are compared with human DNA sequence from chromosome 13 (Ch13), a sequence from the HSV-tk gene (tk), and a sequence from the cytomegalovirus (CMV) promoter. The nucleotide sequence at the site of recombination in subclone G45 is compared with the sequence in the HSV-tk gene (tk) and the sequence 4.9 kb from the integration site in chromosome 16 (contig no. NT_000669) in the opposite orientation (Ch16). The nucleotide sequence at the site of recombination in subclone G65 is compared with sequences in the CMV promoter on the HSV-tk gene (CMV) and the sequence 6.3 kb from the integration site in chromosome 16 in the opposite orientation (Ch16). Nucleotides that are identical between sequences (asterisks) or show complementarity between the sequences at the site of recombination (boldface) are indicated.

FIG. 8.

Possible mechanisms for the generation of inverted repeats and rearrangement of the marker chromosome. A DSB in a chromosome before or during DNA replication or DSBs in both sister chromatids after DNA replication results in sister chromatid fusion. Due to the presence of two centromeres, the fused sister chromatids break during anaphase, resulting in the transfer of an inverted repeat to the end of one of the chromosomes. After cell division, the absence of a telomere on the broken chromosome leads to additional fusions, either between sister chromatids or with other chromosomes. (A) Due to the absence of a telomere, after replication the sister chromatids again fuse and break in the next cell cycle, resulting in further amplification of the subtelomeric DNA sequences and a large duplication on the end of the chromosome. The acquisition of a telomere by translocation from another chromosome then stabilizes the broken chromosome. Alternatively, if a new telomere is not acquired, the chromosome can undergo further rounds of sister chromatid fusion or fuse with other chromosomes. (B) The broken chromosome can also fuse with another chromosome. The dicentric chromosome breaks during anaphase when the two centromeres are pulled to opposite daughter cells, resulting in a translocation of the other chromosome to the end of the marker chromosome. The acquisition of a telomere then stabilizes the broken chromosome. Alternatively, if a new telomere is not acquired, the chromosome can undergo sister chromatid fusion or again fuse with another chromosome. The telomeres (░⃞), centromeres (○), and subtelomeric sequences (▸) on the marker chromosome are indicated.

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Chromosome instability as a result of double-strand breaks near telomeres in mouse embryonic stem cells - PubMed (original) (raw)