Identification of candidate alternative lengthening of telomeres genes by methionine restriction and RNA interference

Abstract

Telomerase-negative cancer cells can maintain their telomeres by a recombination-mediated alternative lengthening of telomeres (ALT) process. We reported previously that sequestration of MRE11/RAD50/NBS1 complexes represses ALT-mediated telomere length maintenance, and suppresses formation of ALT-associated promyelocytic leukemia (PML) bodies (APBs). APBs are PML bodies containing telomeric DNA and telomere-binding proteins, and are observed only in a small fraction of cells within asynchronously dividing ALT-positive cell populations. Here, we report that methionine restriction caused a reversible arrest in G 0 /G 1 phase of the cell cycle and reversible induction of APB formation in most cells within an ALT-positive population. We combined methionine restriction with RNA interference to test whether the following proteins are required for APB formation: PML body-associated proteins, PML and Sp100; telomereassociated proteins, TRF1, TRF2, TIN2 and RAP1; and DNA repair proteins, MRE11, RAD50, NBS1 and 53BP1. APB formation was not decreased by depletion of Sp100 (as reported previously) or of 53BP1, although 53BP1 partially colocalizes with APBs. Depletion of the other proteins suppressed APB formation. Because of the close linkage between ALT-mediated telomere maintenance and ability to form APBs, the eight proteins identified by this screen as being required for APB formation are also likely to be required for the ALT mechanism.

High level of telomerase RNA gene expression is associated with chromatin modification, the ALT phenotype and poor prognosis in liposarcoma

“…It has been shown that APBs are not always associated with the ALT phenotype (Fasching et al, 2005) and although candidate genes have been suggested (Jiang et al, 2007), no molecular markers for the detection of ALT have yet been confirmed. Previous work in cell line models highlighted an association of trimethyl K20 of histone H4 with both telomerase gene promoters in ALT cell lines, suggesting that chromatin modifications may have potential as novel markers of the ALT phenotype (Atkinson et al, 2005).…”

“…A recent study showed the requirement of PML, TRF1, TRF2, TIN2, RAP1 and the MRN complex proteins MRE11, RAD50 and NBS1 for APB formation in ALT cells. The close association of APB formation with ALT highlights these genes as candidate markers or regulators of the ALT pathway (Jiang et al, 2007); however, the mechanisms of regulation remain to be fully determined. We have recently shown that chromatin remodelling at the telomerase gene promoters is associated with a lack of expression of hTR and hTERT in some ALT cell lines, highlighting one potential mechanism for regulating the activation of ALT or telomerase (Atkinson et al, 2005).…”

The MUS81 endonuclease is essential for telomerase negative cell proliferation

“…TRF2 and MUS81 interaction suggests that TRF2 may regulate MUS81 access to APBs, since TRF2 affects APBs formation in G 0 /G 1 cells. 15 To test this hypothesis, we examine the localization of MUS81 in APBs after depletion of TRF2. However, depletion of TRF2 does not change APB formation and localization of MUS81 to APBs at G 2 phase in ALT cells (data not shown), excluding that TRF2 regulates the sequences, sister chromatids can find sites of homology anywhere to align two telomere strands asymmetrically.…”

“…14 A recent study reports that the proportion of ALT cells with APBs is increased following methionine starvation. 15 These induced APBs contain TRF1, TRF2, TIN2, RAP1, the PML protein, the MRN complex, 53BP1, SP100 and telomeric DNA. We have also observed induction of APBs containing telomeric DNA upon methionine restriction.…”

“…These proteins co-localize with telomeric (TTAGGG)n repeats at nuclear substructures known as ALT-associated promyelocytic leukemia (PML) nuclear bodies (APBs), which are present only in ALT cells. 6,[13][14][15] Imaging of live cells has revealed that an individual telomere may move in and out of contact with an APB, 16 suggesting possible on-going telomere extension in ALT cells. Additionally, APBs are enriched during the G 2 phase of the cell cycle when HR is most active and ALT activity is likely occurring.…”