Probing PML body function in ALT cells reveals spatiotemporal requirements for telomere recombination

Abstract

Promyelocytic leukemia (PML) bodies (also called ND10) are dynamic nuclear structures implicated in a wide variety of cellular processes. ALT-associated PML bodies (APBs) are specialized PML bodies found exclusively in telomerase-negative tumors in which telomeres are maintained by recombination-based alternative (ALT) mechanisms. Although it has been suggested that APBs are directly implicated in telomere metabolism of ALT cells, their precise role and structure have remained elusive. Here we show that PML bodies in ALT cells associate with chromosome ends forming small, spatially well-defined clusters, containing on average 2-5 telomeres. Using an innovative approach that gently enlarges PML bodies in living cells while retaining their overall organization, we show that this physical enlargement of APBs spatially resolves the single telomeres in the cluster, but does not perturb the potential of the APB to recruit chromosome extremities. We show that telomere clustering in PML bodies is cell-cycle regulated and that unique telomeres within a cluster associate with recombination proteins. Enlargement of APBs induced the accumulation of telomere-telomere recombination intermediates visible on metaphase spreads and connecting heterologous chromosomes. The strand composition of these recombination intermediates indicated that this recombination is constrained to a narrow time window in the cell cycle following replication. These data provide strong evidence that PML bodies are not only a marker for ALT cells but play a direct role in telomere recombination, both by bringing together chromosome ends and by promoting telomere-telomere interactions between heterologous chromosomes.

Fig. 1.

Chromosome ends associate with PML bodies to form APBs in VA13 cells. (A) Individual telomeres as detected by anti-TRF2 antibody (green) may appear associated with the surface of naturally large APBs, revealed here by either anti-PML or anti-BLM antibodies (both red). (B) Viral protein ICP0 lacking the ring finger domain fused to CFP (CFP-ICP0*, blue) was used to transiently enlarge the size of APBs (hereafter e-APBs). In e-APBs, telomeric DNA detected by PNA FISH (red) colocalizes with either TRF2 or with TRF1 protein foci (green). (C) Subtelomeric probes ICRFc112-F151 (green) and F7501 (red), cluster in e-APBs containing CFP-ICP0* (blue). These probes detect only a specific subset of chromosome arms (Fig. S3A). Histograms showing the average number of subtelomeric regions detected in 20 metaphase spreads, cell nuclei, and nuclei containing ICP0*. ICP0* associated, subtelomeric signals in proximity of ICP0*; Free, elsewhere in the nucleus. Error bars represent the standard error of the mean (SEM). (D) Histogram showing the average number of TRF2 foci expected and actually detected in interphase nuclei of native and ICP0*-transfected VA13 and HT1080 cells. Error bars, SEM. (E) Coup-TF2 is localized in close proximity of TRF2 clusters in e-APBs. (Scale bars, 2 μm and 1 μm in enlarged images.)

Fig. 2.

Telomere clusters are a common feature of ALT cells. (A) Telomere clusters, detected by colocalization of anti-TRF2 antibodies (green) with CFP-ICP0* (blue), are present in all ALT lines tested (GM847, U2OS, SAOS2), and in VA13 cells constitutively expressing telomerase (VA13+telom.), but are rarely observed in an atypical ALT line without APBs (VA13-C3-cl6). (Scale bars, 2 μm.) (B and C) Quantification of results from (A) based on 20 nuclei corrected for the level of random co-localization of TRF2 with ICP0* as observed in HT1080 cells (2 = 0.65 ± 0.21, 3 = 0.50 ± 0.17, 4 = 0, 5 = 0). Error bars, SEM. (D–G) Telomere clusters are present in G1, S, and G2 phase of the cell cycle but not in M. VA13 cells were transfected with CFP-ICP0* (blue) for 48 h, labeled with BrdU before fixation and stained with anti-TRF2 antibodies (green) and different markers for the cell cycle (red) as follows: G1 cells (D) were identified as negative after a double staining with markers for the S and G2 phases; S phase nuclei (E) were identified with anti-BrdU antibodies; G2 nuclei (F) were identified with anti-CENP-F antibodies and chromosome condensation allowed to distinguish CENP-F positive cells that were in M (G). (H) Quantification of results from (D–G). Error bars, SEM.

Fig. 3.

Recombination protein RAD51 and replication protein A (RPA) preferentially associate with particular telomeres in a cluster. (A and B) IF analyses of VA13 cells fixed 24 h posttransfection with BFP-ICP0* (not represented in the figures) revealing the co-localization of anti-RAD51 (A) or anti-RPA (B) staining (both red) with individual telomeres, as detected by anti-TRF2 antibody (green), in e-APBs. 24% (n = 20) and 35% (n = 20) of RAD51 and RPA nuclear foci, respectively, were found associated with TRF2 in e-APBs. Localization of the same proteins in unperturbed APBs in non-transfected cells is also shown. Scale bars, 2 μm. (C) Models of native PML and native and e-APB structures. Infiltration by ICP0* leads to displacement of proteins from the core of the PML scaffold but preserves the association of telomeres with the outer layer of the body.

Fig. 4.

Enlargement of APBs perturbs the resolution of recombination intermediates between telomeres located on different chromosomes. (A) Chromosome-oriented (CO-) FISH procedure. (B) The level of telomere sister chromatid exchange (T-SCE) remains unperturbed in VA13 cells upon expression of ICP0* as determined by CO-FISH using a probe against the G-rich strand (red) (See also Fig. S5). n = 40–42 metaphases. Error bars, SEM. (C and D) CO-FISH analysis revealing telomere recombination intermediates or telomere bridges (arrowheads) whose incidence increases upon transfection of VA13 cells with ICP0*. (Scale bars, 5 μm.) (E) Percentage of metaphase spreads with telomere bridges in VA13, VA13-C3-cl6 and HT1080 cells. Error bars represent the confidence interval. (F) Representative images for different categories of telomere bridges based on the pattern of parental G-rich (red) and C-rich (green) telomeric DNA strands. (G) Two sister chromatids forming two independent telomere bridges (arrowheads) with two different chromosomes. (H) Increased incidence of telomere bridges is not accompanied by an increase in nucleoplasmic bridges. Error bars represent the confidence interval. (I) We propose two, non-exclusive, models of how ICP0* interferes with the recombination of telomeres in enlarged (e-)APBs: ICP0* may prematurely promote the physical separation of telomeres or perturb the catalytic surface of APBs perhaps by inducing the relocation of recombination factors from APBs into the nucleoplasm.