Variant repeats are interspersed throughout the telomeres and recruit nuclear receptors in ALT cells

Abstract

Telomeres in cells that use the recombination-mediated alternative lengthening of telomeres (ALT) pathway elicit a DNA damage response that is partly independent of telomere length. We therefore investigated whether ALT telomeres contain structural abnormalities that contribute to ALT activity. Here we used next generation sequencing to analyze the DNA content of ALT telomeres. We discovered that variant repeats were interspersed throughout the telomeres of ALT cells. We found that the C-type (TCAGGG) variant repeat predominated and created a high-affinity binding site for the nuclear receptors COUP-TF2 and TR4. Nuclear receptors were directly recruited to telomeres and ALT-associated characteristics were induced after incorporation of the C-type variant repeat by a mutant telomerase. We propose that the presence of variant repeats throughout ALT telomeres results from recombination-mediated telomere replication and spreading of variant repeats from the proximal regions of the telomeres and that the consequent binding of nuclear receptors alters the architecture of telomeres to facilitate further recombination.

Figure 1.

Variant repeats are interspersed throughout the telomeres of ALT cells. (A) FISH performed on interphase nuclei using both a Texas red–conjugated (TTAGGG)₃ telomeric probe (red) and Alexa 488–OO-(TCAGGG)₃ or (TGAGGG)₃ variant probe (green) counterstained with DAPI (blue). (B) Representative metaphase spreads of the WI38-VA13/2RA ALT cell line processed for FISH as described in A showing localization of the C- and G-type variant repeat at individual telomeres. Magnified examples are shown in the bottom right. (C) Percentage of each telomeric repeat type generated from deep sequencing analysis of WI38-VA13/2RA and HeLa cells. Variant repeats were counted within reads containing more than six nonconsecutive TBAGGG telomeric repeats. (D) Examples of various telomeric 75-nucleotide reads from sequencing data of WI38-VA13/2RA cells. (E) Distribution of the C- and G-type variant repeats along the length of the telomere in the WI38-VA13/2RA cell line, visualized on chromatin fibers. Bars, 5 µm.

Figure 2.

Nuclear receptors are detected at ALT telomeres. (A) Indirect immunofluorescence staining of IIICF/c nuclei at interphase. COUP-TF2 or TR4 (red) and PML (purple) coupled with telomere FISH (green) and DAPI (blue) counterstaining shows that nuclear receptors are present within APBs (arrowheads). (B) Representative metaphase spreads of the GM847 ALT cell line stained with COUP-TF2 or TR4 (red) and TRF2 (green) immunofluorescence and DAPI (blue) show localization of nuclear receptors to telomeres. Magnified examples are shown in the bottom right. (C) Distribution of TR4 (red) among TRF2 (green) along the length of the telomere in the WI38-VA13/2RA cell line visualized at chromatin fibers. Bars, 5 µm.

Figure 3.

Binding of TR4 and TRF2 to variant telomeric repeat sequences. (A) EMSA titration experiments conducted using double-stranded γ³²P-radiolabeled C- and G-type variant and canonical telomeric repeat probes incubated with purified human TR4. A binding curve generated from two independent titration experiments is also shown (average ± range). (B) EMSA titration experiments and binding curve for human TRF2. (C) Estimation of dissociation constants (K_d) obtained from titration displayed in A and B. The K_d was calculated by quantifying the disappearance of the band corresponding to free DNA and determining the concentration at which 50% of the probe is bound by protein.

Figure 4.

Nuclear receptor depletion results in suppression of ALT phenotypic characteristics. (A) Western blot analysis of whole-cell extracts (10⁵ cell equivalents) from WI38-VA13/2RA and IIICF/c cells 72 h after transfection with siRNA against COUP-TF2 and TR4 shows knockdown of proteins. (B) Quantitation of APBs 72 h after transfection of WI38-VA13/2RA and IIICF/c cells with siRNA against COUP-TF2 and TR4 via indirect immunofluorescence against the PML protein and telomere FISH (mean ± SD; n = 3 independent experiments, quantifying 50 nuclei per replicate). (C) Quantitation of C-circle levels 72 h after transfection of WI38-VA13/2RA and IIICF/c cells (mean ± SD; n = 3). (D) Quantitation of T-SCEs observed via CO-FISH after knockdown of nuclear receptor expression in WI38-VA13/2RA cells (mean ± SD; n = 3, quantifying >2,000 chromosome ends or ∼20 metaphases per replicate). Quantitation of T-SCEs was restricted to chromosome ends with clearly distinguishable sister telomeres.

Figure 5.

Nuclear receptor recruitment upon variant telomeric repeat incorporation. (A) FISH performed on interphase nuclei using both a Texas red–conjugated (TTAGGG)₃ telomeric probe (red) and Alexa 488–OO-(TCAGGG)₃ or (TGAGGG)₃ variant probe (green) counterstained with DAPI (blue). (B) Quantitation of the percentage of nuclear receptor-positive chromosome ends stained with either COUP-TF2 or TR4 on one or both chromatids (mean ± SD; n = 3, quantifying >2,000 chromosome ends or ∼20 metaphases per replicate). (C) Representative metaphase spreads of the HT1080 hTR C-type cell line stained with COUP-TF2 or TR4 (red) and TRF2 (green) immunofluorescence and DAPI (blue). Magnified examples are shown on the bottom right. Bars, 5 µm.

Figure 6.

Incorporation of variant repeats into the telomeres induces ALT-associated characteristics. (A) Quantitation of meta-TIF analysis showing the percentage of chromosome ends stained with γ-H2AX on one or both chromatids in HT1080 cells expressing variant hTR compared with wild-type (mean ± SD; n = 3, quantifying >2,000 chromosome ends or ∼20 metaphases per replicate). (B) Quantitation of C-circle levels in HT1080 cells expressing wild-type and variant hTR in comparison to the IIICF/c ALT cell line (mean ± SD; n = 3) plotted on a logarithmic scale. (C) Representative metaphase spreads of the HT1080 hTR C- and G-type cells processed for neo^R-FISH showing no copying of the neo^R tag at early and late pds. Magnified examples are shown in the bottom right. Bars, 5 µm.

Figure 7.

Model of alterations in telomere architecture during ALT activation. Dispersal of variant sequences among distal telomeric repeats leads to changes in telomere structure such as removal of shelterin, recruitment of nuclear receptors, and elicitation of a DNA damage response that facilitates telomere elongation via HR. Nuclear receptors may also recruit chromatin remodeling complexes capable of altering the heterochromatic state of ALT telomeres. Although this schematic shows only COUP-TF2 and TR4, additional nuclear receptors are likely recruited to the telomeres as either homodimers or heterodimers or even monomers.