Suppression of alternative lengthening of telomeres by Sp100-mediated sequestration of the MRE11/RAD50/NBS1 complex

Abstract

Approximately 10% of cancers overall use alternative lengthening of telomeres (ALT) instead of telomerase to prevent telomere shortening, and ALT is especially common in astrocytomas and various types of sarcomas. The hallmarks of ALT in telomerase-negative cancer cells include a unique pattern of telomere length heterogeneity, rapid changes in individual telomere lengths, and the presence of ALT-associated promyelocytic leukemia bodies (APBs) containing telomeric DNA and proteins involved in telomere binding, DNA replication, and recombination. The ALT mechanism appears to involve recombination-mediated DNA replication, but the molecular details are largely unknown. In telomerase-null Saccharomyces cerevisiae, an analogous survivor mechanism is dependent on the RAD50 gene. We demonstrate here that overexpression of Sp100, a constituent of promyelocytic leukemia nuclear bodies, sequestered the MRE11, RAD50, and NBS1 recombination proteins away from APBs. This resulted in repression of the ALT mechanism, as evidenced by progressive telomere shortening at 121 bp per population doubling, a rate within the range found in telomerase-negative normal cells, suppression of rapid telomere length changes, and suppression of APB formation. Spontaneously generated C-terminally truncated Sp100 that did not sequester the MRE11, RAD50, and NBS1 proteins failed to inhibit ALT. These findings identify for the first time proteins that are required for the ALT mechanism.

FIG. 1.

Suppression of ALT-associated PML bodies (APBs) by overexpression of Sp100. APBs were visualized in growth-arrested cells with antibodies against PML and either TRF1 or TRF2. Sp100 was detected by fluorescence (YFP-Sp100) or immunostaining (Sp100). (A) Most untransfected control IIICF/c cells exhibited APBs, visualized here as prominent TRF1 foci within PML bodies. (B) APB formation was inhibited in transfected IIICF/c cells overexpressing YFP-Sp100 but not in cells with undetectable levels of transgene expression. Similarly, APB formation was suppressed by overexpression of Sp100 in both IIICF/c (C) and Saos-2 cells (D).

FIG. 2.

Expression levels and mutation profiles of YFP-Sp100 in transfected IIICF/c clones. Clones IIICF-4, -9,-10, -11, -16, and -17 were all analyzed at population doubling 38 to 41. Western blot analyses of YFP-Sp100 (A) and Sp100 (B) with anti-GFP and anti-Sp100 antibodies, respectively, are shown. (C) Schematic representation of YFP-Sp100 and genomic PCR analysis of the integrated YFP-Sp100 plasmid. IIICF-11 and -16 had undergone C-terminal truncations of YFP-Sp100, and no YFP-Sp100 sequence was retained in IIICF-4. NLS, nuclear localization signal; HSR, homogeneous staining region; HP1, heterochromatin protein 1 binding domain; Trans, transactivation domain.

FIG. 3.

Suppression of APB formation in IIICF/c clones overexpressing full-length YFP-Sp100. YFP fluorescence and double immunostaining of growth-arrested IIICF-4, -9, -10, -11, -16, and -17 cells (population doubling 30 to 33) with anti-TRF1 and anti-PML antibodies are shown. APB formation was suppressed in IIICF-10 and -17, which express high levels of YFP-Sp100, whereas they formed normally in cells that had no residual expression of the transgene (IIICF-4), expressed a C-terminally truncated transgene (IIICF-11 and -16), or expressed full-length YFP-Sp100 at a lower level (IIICF-9).

FIG. 4.

Progressive telomere shortening in clones overexpressing YFP-Sp100. Terminal restriction fragment length was analyzed by Southern blotting of genomic DNA from parental IIICF/c cells and IIICF-4, -9,-10, -11, -16, and -17 cells at the population doubling (PD) indicated. The asterisk indicates the population doubling levels at which small patches of revertant cells appeared within the cultures.

FIG. 5.

Inhibition of telomere length fluctuation in IIICF/c cells overexpressing full-length YFP-Sp100. (A) Telomere fluorescence in situ hybridization analysis, with a peptide nucleic acid telomere probe, on individual metaphase nuclei from clones IIICF-16 and -17 at population doubling 27. The insets show the marker chromosome (with an interstitial telomere signal) that was used for the quantitative analysis. (B) Ratios of p-arm to q-arm telomere fluorescence in situ hybridization signals on the marker chromosome from 20 metaphase spreads for each clone were measured as described in the text. Each bar represents the ratio for an individual chromosome, normalized by the median. The standard deviation of the ratios for IIICF-17 (0.55) was significantly lower than that for IIICF-16 (1.45; F test, P = 0.0001).

FIG. 6.

Sequestration of NBS1 by YFP-Sp100 in cells with suppression of ALT activity. (A) NBS1 (detected by immunostaining) colocalized with YFP-Sp100 (detected by YFP fluorescence) in IIICF-17 cells but to a much lesser extent with the truncated YFP-Sp100 in IIICF-16 cells. (B) NBS1 colocalized much more extensively with APBs (visualized as foci of TRF2 immunostaining) than with the truncated YFP-Sp100 aggregates in IIICF-16 cells (population doubling 40). In contrast, in IIICF-17 cells (population doubling 39), NBS1 was sequestered in aggregates of full-length YFP-Sp100 and APBs were suppressed. (C) Coimmunoprecipitation analyses showed decreased association of the MRE11/RAD50/NBS1 complex with C-terminally truncated YFP-Sp100. Anti-GFP antibody was used to immunoprecipitate (IP) proteins from the total protein extracts of growth-arrested IIICF-16 and -17 cells (population doubling 33). Proteins separated by gel electrophoresis were either stained with SYPRO Ruby (left panel) or probed with the indicated antibodies (right panel). The arrows (left panel) indicate the bands corresponding to RAD50, NBS1, and MRE11 (detected by immunoblotting in the right panel). The arrowheads indicate the YFP-Sp100 bands. (D) Coimmunoprecipitation of NBS1 and MRE11 was performed as in panel C except that the protein was extracted from cells that were growing asynchronously (population doubling 39). For panels C and D, equal numbers of cells were used from each clone, and 10% of the lysate was used for the input lanes (I). For panels A and B, cells were subjected to nuclear extraction before fixation. Fluorescent images of each channel were acquired and processed in an identical manner for pairwise comparisons in panels A and B.

FIG. 7.

APB formation requires NBS1 but not Sp100. (A) The NBS1 protein level was downregulated in IIICF/c cells 48 h after NBS1 siRNA (NBS1-si2) transfection. NBS1 and β-actin were detected by immunoblotting. (B) Sp100 siRNAs (Sp100-1 and Sp100-2) downregulated Sp100 expression in IIICF/c cells 48 h after transfection. The blot was probed with the indicated antibodies. (C) APB formation was suppressed in cells that lack NBS1, whereas cells lacking Sp100 formed APBs normally, as demonstrated by colocalization of TRF2 with NBS1 (D) and PML (E). The arrows indicate cells lacking NBS1 in panel C or Sp100 in panels D and E. For panels C to E, IIICF/c cells were growth arrested 48 h after siRNA transfection. The Sp100-1 and Sp100-2 siRNAs were used in panels D and E, respectively.