Telomere Rapid Deletion Regulates Telomere Length in <em>Arabidopsis thaliana</em><sup><a href="#fn1" rid="fn1" class=" fn">▿</a></sup>

Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128

^{Corresponding author. Mailing address: Department of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, TX 77843-2128. Phone: (979) 862-2342. Fax: (979) 845-9274. E-mail:}ude.umat@neppihsd.

Received 2006 Nov 3; Revised 2006 Dec 2; Accepted 2006 Dec 11.

Abstract

Telomere length is maintained in species-specific equilibrium primarily through a competition between telomerase-mediated elongation and the loss of terminal DNA through the end-replication problem. Recombinational activities are also capable of both lengthening and shortening telomeres. Here we demonstrate that elongated telomeres in Arabidopsis Ku70 mutants reach a new length set point after three generations. Restoration of wild-type Ku70 in these mutants leads to discrete telomere-shortening events consistent with telomere rapid deletion (TRD). These findings imply that the longer telomere length set point is achieved through competition between overactive telomerase and TRD. Surprisingly, in the absence of telomerase, a subset of elongated telomeres was further lengthened, suggesting that in this background a mechanism of telomerase-independent lengthening of telomeres operates. Unexpectedly, we also found that plants possessing wild-type-length telomeres exhibit TRD when telomerase is inactivated. TRD is stochastic, and all chromosome ends appear to be equally susceptible. The frequency of TRD decreases as telomeres shorten; telomeres less than 2 kb in length are rarely subject to TRD. We conclude that TRD functions as a potent force to regulate telomere length in Arabidopsis.

Abstract

Telomeres are dynamic nucleoprotein complexes at the end of eukaryotic chromosomes that consist of long stretches of a simple G-rich repeat and sequence-specific DNA binding proteins. The primary function of telomeres is to protect chromosome termini from being recognized as a double-strand break. The extreme 3′ terminus of the chromosome is single stranded and can undergo a protein-assisted conformational change, folding back upon and invading the duplex region to form a structure termed the t-loop (18). The t-loop is thought to physically sequester the chromosome end, masking the telomere from DNA repair machinery (13).

Telomeric DNA is maintained through a variety of mechanisms that compensate for loss of terminal DNA sequences that occurs as a consequence of nucleolytic processing or the end-replication problem (19, 38). Slow loss of telomeric sequences during DNA replication can be offset by the action of telomerase, a ribonucleoprotein reverse transcriptase that extends the 3′ overhang through reiterative copying of its internal RNA template (reviewed in reference 11). Telomerase is subjected to both positive and negative regulation in cis on the chromosome terminus, mitigating its ability to extend any given telomere (1, 31, 37, 42, 49, 50, 52).

The protein counting model posits that the primary means of telomere length regulation is through an ability to “count” the number of telomeric binding proteins (37). If too many proteins are bound, the telomere will be recalcitrant to extension by telomerase, while if too few proteins are bound, the telomere will be in a more open conformation and will be accessible to telomerase activity. Accordingly, telomerase extension results in an increase in the number of binding sites for telomere proteins and hence an increase in protein occupancy. On the other hand, telomere loss due to the end-replication problem or nuclease attack results in a decrease in the number of sites and fewer proteins bound. This model is strongly supported by studies in yeast (50), mammals (1, 21), and Arabidopsis (48), where telomerase has been shown to act preferentially on the shortest telomeres in the population. Competition between the end-replication problem and telomerase results in a range of telomere lengths that fluctuate between species-specific boundaries. For example, telomeres in Saccharomyces cerevisiae are approximately 300 bp (34), those in Arabidopsis are from 2 to 8 kb (48), and those in mice are from 10 to 60 kb (55).

Positive and negative regulators of telomere length include the double-strand telomere binding proteins TRF1 (49), Rap1 (36), and Taz1 (12) and the single-strand telomere binding protein Pot1 (31). Additionally, telomere length is influenced by KU, a heterodimer of 70- and 80-kDa subunits that is an integral component of the nonhomologous end-joining (NHEJ) DNA double-strand break repair pathway (44). KU is a strong negative regulator of telomerase in Arabidopsis (7, 16, 47); its deletion results in rapid telomerase-dependent extension of telomere tracts (16, 46). Interestingly, in S. cerevisiae and humans, deletion of KU leads to telomere shortening (5, 40), indicating that KU's influence on telomere length regulation is evolving.

In the absence of telomerase, telomeres progressively shorten until they reach a critical length that elicits a DNA damage checkpoint response (14). If cells are forced to continue dividing, telomeres will become uncapped and fuse together. The resulting dicentric chromosomes may then break during the next mitosis only to fuse in the next cell cycle. The resulting breakage-fusion-bridge cycle leads to genomic instability (39). Strong selective pressure against genome instability results in the formation of different types of survivors in yeast (33), whose chromosome ends are maintained through alternate means (28, 51). Several different types of survival have been identified, including recombinational elongation and rolling circle amplification (22, 32). In humans, this form of telomere maintenance is termed alternative lengthening of telomeres (ALT), and is characterized by extremely heterogenous telomeres and the presence of ALT-associated promyelocytic leukemia (PML) bodies.

Cells with elongated telomeres do not face the same selective pressure as cells with extremely short telomeres. Indeed, Arabidopsis ku70 mutants maintain telomeres much longer than wild type, with no apparent affect on growth, development, or genome stability (47). However, studies in yeast indicate that elongated telomeres are quickly returned to wild-type length in a single-step event termed telomere rapid deletion (TRD) (29). These deletion events are intrachromosomal and result in loss of the most terminal sequences (6). A similar phenomenon has been described in humans and Kluyveromyces lactis. Human cells expressing a mutant form of the telomere double-strand binding protein TRF2 undergo catastrophic telomere deletions, concomitant with the formation of extrachromosomal telomere circles (ECTCs) the size of t-loops (53). Similarly, in K. lactis mutants with elongated telomeres due to a mutation in Stn1p, reintroduction of Stn1p results in rapid loss of the elongated telomeres and a return to wild-type length (23).

It has been proposed that branch migration of the displacement loop formed by the invading G-overhang within the t-loop structure results in a Holliday junction (HJ). This structure is then resolved, leading to the formation of a shortened telomere and an extrachromosomal telomeric DNA fragment (35), which in mammals is a circle (ECTC). In S. cerevisiae, TRD and the two major types of survivors are dependent upon Rad52, indicating both processes are recombinational in nature (29, 33). Telomere lengthening in stn1 K. lactis mutants is similarly dependent upon Rad52 (23). Sequestration of the MRX complex in human ALT cells results in slow loss of telomeric DNA and repression of the ALT mechanism of elongation (24). Additionally, the Rad51 paralog Xrcc3, which may be a mammalian Holliday junction resolvase (30), is required for the TRD events observed in TRF2 mutants (53).

Arabidopsis is a genetically tractable model that has been exploited for studies of telomere dynamics (39). One important feature of this organism is that 8 of the 10 chromosome arms are abutted by unique subtelomeric sequences, making it possible to study the fate of individual telomeres in different genetic backgrounds. Here we examine the fate of ultralong telomeres in Arabidopsis ku70 mutants. We demonstrate that elongated telomeres in this background can be rapidly shortened by TRD, either upon reintroduction of KU70 or through loss of telomerase. In addition, we provide evidence for an ALT-like mechanism in plants with elongated telomeres, which we term telomerase-independent lengthening of telomeres (TILT). Finally, we show that wild-type-length telomeres are subject to both TRD and TILT, arguing that recombinational mechanisms play a role in regulating telomere length in wild-type plants.

Acknowledgments

We thank Barbara Zellinger and Karel Riha for their invaluable help in providing unpublished materials and sharing unpublished data. We are also grateful to Elizabeth Summer for the use of her CHEF Mapper and Laurent Vespa, Michelle Heacock, and Yulia Surovtseva for critically reading the manuscript.

This work was supported by NIH GM65383 to D.E.S.

Acknowledgments

Footnotes

^{Published ahead of print on 22 December 2006.}

Footnotes

REFERENCES

References

1. Ancelin, K., M. Brunori, S. Bauwens, C.-E. Koering, C. Brun, M. Ricoul, J.-P. Pommier, L. Sabatier, and E. Gilson. 2002. Targeting assay to study the cis functions of human telomeric proteins: evidence for inhibition of telomerase by TRF1 and for activation of telomere degradation by TRF2. Mol. Cell. Biol.22:3474-3487.
2. Blanc, G., A. Barakat, R. Guyot, R. Cooke, and M. Delseny. 2000. Extensive duplication and reshuffling in the Arabidopsis genome. Plant Cell12:1093-1101.
3. Bleuyard, J. Y., M. E. Gallego, F. Savigny, and C. I. White. 2005. Differing requirements for the Arabidopsis Rad51 paralogs in meiosis and DNA repair. Plant J.41:533-545. [[PubMed]
4. Borevitz, J. O., D. Liang, D. Plouffe, H. S. Chang, T. Zhu, D. Weigel, C. C. Berry, E. Winzeler, and J. Chory. 2003. Large-scale identification of single-feature polymorphisms in complex genomes. Genome Res.13:513-523.
5. Boulton, S. J., and S. P. Jackson. 1998. Components of the Ku-dependent non-homologous end-joining pathway are involved in telomeric length maintenance and telomeric silencing. EMBO J.17:1819-1828.
6. Bucholc, M., Y. Park, and A. J. Lustig. 2001. Intrachromatid excision of telomeric DNA as a mechanism for telomere size control in Saccharomyces cerevisiae. Mol. Cell. Biol.21:6559-6573.
7. Bundock, P., H. van Attikum, and P. Hooykaas. 2002. Increased telomere length and hypersensitivity to DNA damaging agents in an Arabidopsis KU70 mutant. Nucleic Acids Res.30:3395-3400.
8. Cesare, A. J., and J. D. Griffith. 2004. Telomeric DNA in ALT cells is characterized by free telomeric circles and heterogeneous t-loops. Mol. Cell. Biol.24:9948-9957.
9. Cheung, I., M. Schertzer, A. Rose, and P. M. Lansdorp. 2006. High incidence of rapid telomere loss in telomerase-deficient Caenorhabditis elegans. Nucleic Acids Res.34:96-103.
10. Ciudad, T., E. Andaluz, O. Steinberg-Neifach, N. F. Lue, N. A. Gow, R. A. Calderone, and G. Larriba. 2004. Homologous recombination in Candida albicans: role of CaRad52p in DNA repair, integration of linear DNA fragments and telomere length. Mol. Microbiol.53:1177-1194. [[PubMed]
11. Cohen, S., and MMechali. 2002. Formation of extrachromosomal circles from telomeric DNA in Xenopus laevis. EMBO Rep.3:1168-1174. [Google Scholar]
12. Cooper, J. P., E. R. Nimmo, R. C. Allshire, and T. R. Cech. 1997. Regulation of telomere length and function by a Myb-domain protein in fission yeast. Nature385:744-747. [[PubMed]
13. de Lange, T. 2002. Protection of mammalian telomeres. Oncogene21:532-540. [[PubMed]
14. Ferreira, M. G., K. M. Miller, and J. P. Cooper. 2004. Indecent exposure: when telomeres become uncapped. Mol. Cell13:7-18. [[PubMed]
15. Fitzgerald, M. S., K. Riha, F. Gao, S. Ren, T. D. McKnight, and D. E. Shippen. 1999. Disruption of the telomerase catalytic subunit gene from Arabidopsis inactivates telomerase and leads to a slow loss of telomeric DNA. Proc. Natl. Acad. Sci. USA96:14813-14818.
16. Gallego, M. E., N. Jalut, and C. I. White. 2003. Telomerase dependence of telomere lengthening in Ku80 mutant Arabidopsis. Plant Cell.15:782-789.
17. Grant, J. D., D. Broccoli, M. Muquit, F. J. Manion, J. Tisdall, and M. F. Ochs. 2001. Telometric: a tool providing simplified, reproducible measurements of telomeric DNA from constant field agarose gels. BioTechniques31:1314-1316, 1318. [[PubMed]
18. Griffith, J. D., L. Comeau, S. Rosenfield, R. M. Stansel, A. Bianchi, H. Moss, and T. de Lange. 1999. Mammalian telomeres end in a large duplex loop. Cell97:503-514. [[PubMed]
19. Harley, C. B., A. B. Futcher, and C. W. Greider. 1990. Telomeres shorten during ageing of human fibroblasts. Nature345:458-460. [[PubMed]
20. Heacock, M., E. Spangler, K. Riha, J. Puizina, and D. E. Shippen. 2004. Molecular analysis of telomere fusions in Arabidopsis: multiple pathways for chromosome end-joining. EMBO J.23:2304-2313.
21. Hemann, M. T., M. A. Strong, L. Y. Hao, and C. W. Greider. 2001. The shortest telomere, not average telomere length, is critical for cell viability and chromosome stability. Cell107:67-77. [[PubMed]
22. Henson, J. D., A. A. Neumann, T. R. Yeager, and R. R. Reddel. 2002. Alternative lengthening of telomeres in mammalian cells. Oncogene21:598-610. [[PubMed]
23. Iyer, S., A. D. Chadha, and M. J. McEachern. 2005. A mutation in the STN1 gene triggers an alternative lengthening of telomere-like runaway recombinational telomere elongation and rapid deletion in yeast. Mol. Cell. Biol.25:8064-8073.
24. Jiang, W.-Q., Z.-H. Zhong, J. D. Henson, A. A. Neumann, A. C.-M. Chang, and R. R. Reddel. 2005. Suppression of alternative lengthening of telomeres by Sp100-mediated sequestration of the MRE11/RAD50/NBS1 complex. Mol. Cell. Biol.25:2708-2721.
25. Joseph, I., D. Jia, and A. J. Lustig. 2005. Ndj1p-dependent epigenetic resetting of telomere size in yeast meiosis. Curr. Biol.15:231-237. [[PubMed]
26. Kilian, A., C. Stiff, and A. Kleinhofs. 1995. Barley telomeres shorten during differentiation but grow in callus culture. Proc. Natl. Acad. Sci. USA92:9555-9559.
27. Larson, D. D., E. A. Spangler, and E. H. Blackburn. 1987. Dynamics of telomere length variation in Tetrahymena thermophila. Cell50:477-483. [[PubMed]
28. Le, S., J. K. Moore, J. E. Haber, and C. W. Greider. 1999. RAD50 and RAD51 define two pathways that collaborate to maintain telomeres in the absence of telomerase. Genetics152:143-152.
29. Li, B., and A. J. Lustig. 1996. A novel mechanism for telomere size control in Saccharomyces cerevisiae. Genes Dev.10:1310-1326. [[PubMed]
30. Liu, Y., J. Y. Masson, R. Shah, P. O'Regan, and S. C. West. 2004. RAD51C is required for Holliday junction processing in mammalian cells. Science303:243-246. [[PubMed]
31. Loayza, D., and Tde Lange. 2003. POT1 as a terminal transducer of TRF1 telomere length control. Nature423:1013-1018. [[PubMed][Google Scholar]
32. Lundblad, V. 2002. Telomere maintenance without telomerase. Oncogene21:522-531. [[PubMed]
33. Lundblad, V., and E. H. Blackburn. 1993. An alternative pathway for yeast telomere maintenance rescues est1− senescence. Cell73:347-360. [[PubMed]
34. Lundblad, V., and J. W. Szostak. 1989. A mutant with a defect in telomere elongation leads to senescence in yeast. Cell57:633-643. [[PubMed]
35. Lustig, AJ. 2003. Clues to catastrophic telomere loss in mammals from yeast telomere rapid deletion. Nat. Rev. Genet.4:916-923. [[PubMed][Google Scholar]
36. Lustig, A. J., S. Kurtz, and D. Shore. 1990. Involvement of the silencer and UAS binding protein RAP1 in regulation of telomere length. Science250:549-553. [[PubMed]
37. Marcand, S., E. Gilson, and D. Shore. 1997. A protein-counting mechanism for telomere length regulation in yeast. Science275:986-990. [[PubMed]
38. Maringele, L., and DLydall. 2002. EXO1-dependent single-stranded DNA at telomeres activates subsets of DNA damage and spindle checkpoint pathways in budding yeast yku70Delta mutants. Genes Dev.16:1919-1933. [Google Scholar]
39. McKnight, T. D., and D. E. Shippen. 2004. Plant telomere biology. Plant Cell16:794-803.
40. Myung, K., G. Ghosh, F. J. Fattah, G. Li, H. Kim, A. Dutia, E. Pak, S. Smith, and E. A. Hendrickson. 2004. Regulation of telomere length and suppression of genomic instability in human somatic cells by Ku86. Mol. Cell. Biol.24:5050-5059.
41. Natarajan, S., and M. J. McEachern. 2002. Recombinational telomere elongation promoted by DNA circles. Mol. Cell. Biol.22:4512-4521.
42. Pennock, E., K. Buckley, and V. Lundblad. 2001. Cdc13 delivers separate complexes to the telomere for end protection and replication. Cell104:387-396. [[PubMed]
43. Polotnianka, R. M., J. Li, and A. J. Lustig. 1998. The yeast Ku heterodimer is essential for protection of the telomere against nucleolytic and recombinational activities. Curr. Biol.8:831-834. [[PubMed]
44. Riha, K., M. L. Heacock, and D. E. Shippen. 2006. The role of the nonhomologous end-joining DNA double-strand break repair pathway in telomere biology. Annu. Rev. Genet.40:237-277. [[PubMed]
45. Riha, K., T. D. McKnight, L. R. Griffing, and D. E. Shippen. 2001. Living with genome instability: plant responses to telomere dysfunction. Science291:1797-1800. [[PubMed]
46. Riha, K., and D. E. Shippen. 2003. Ku is required for telomeric C-rich strand maintenance but not for end-to-end chromosome fusions in Arabidopsis. Proc. Natl. Acad. Sci. USA100:611-615.
47. Riha, K., J. M. Watson, J. Parkey, and D. E. Shippen. 2002. Telomere length deregulation and enhanced sensitivity to genotoxic stress in Arabidopsis mutants deficient in Ku70. EMBO J.21:2819-2826.
48. Shakirov, E. V., and D. E. Shippen. 2004. Length regulation and dynamics of individual telomere tracts in wild-type Arabidopsis. Plant Cell16:1959-1967.
49. Smogorzewska, A., B. van Steensel, A. Bianchi, S. Oelmann, M. R. Schaefer, G. Schnapp, and T. de Lange. 2000. Control of human telomere length by TRF1 and TRF2. Mol. Cell. Biol.20:1659-1668.
50. Teixeira, M. T., M. Arneric, P. Sperisen, and J. Lingner. 2004. Telomere length homeostasis is achieved via a switch between telomerase-extendible and -nonextendible states. Cell117:323-335. [[PubMed]
51. Teng, S. C., J. Chang, B. McCowan, and V. A. Zakian. 2000. Telomerase-independent lengthening of yeast telomeres occurs by an abrupt Rad50p-dependent, Rif-inhibited recombinational process. Mol. Cell6:947-952. [[PubMed]
52. van Steensel, B., and Tde Lange. 1997. Control of telomere length by the human telomeric protein TRF1. Nature385:740-743. [[PubMed][Google Scholar]
53. Wang, R. C., A. Smogorzewska, and T. de Lange. 2004. Homologous recombination generates T-loop-sized deletions at human telomeres. Cell119:355-368. [[PubMed]
54. Watson, J. M., P. Bulankova, K. Riha, D. E. Shippen, and B. Vyskot. 2005. Telomerase-independent cell survival in Arabidopsis thaliana. Plant J.43:662-674. [[PubMed]
55. Zijlmans, J. M., U. M. Martens, S. S. Poon, A. K. Raap, H. J. Tanke, R. K. Ward, and P. M. Lansdorp. 1997. Telomeres in the mouse have large inter-chromosomal variations in the number of T2AG3 repeats. Proc. Natl. Acad. Sci. USA94:7423-7428.