The yeast homolog of human PinX1 is involved in rRNA and small nucleolar RNA maturation,not in telomere elongation inhibition

In human cells, PinX1 protein has recently been shown to regulate telomere length by repressing the telomerase. In this work, we show that the putative yeast homolog of PinX1, encoded by the YGR280c open reading frame (ORF), is a new component of the ribosomal RNA processing machinery. The protein has a KK(E/D) C-terminal domain typical of nucleolar proteins and bears a putative RNA interacting domain widespread in eukaryotes called the G-patch. The protein was hence renamed Gno1p (G-patch nucleolar protein). GNO1 deletion results in a large growth defect due to the inhibition of the pre-ribosomal RNA processing first cleavage steps at sites A(0), A(1), and A(2). Furthermore, Gno1p is involved in the final 3'-end trimming of U18 and U24 small nucleolar RNAs. A mutational analysis showed that the G-patch of Gno1p is essential for both functions, whereas the KK(E/D) repeats are only required for U18 small nucleolar RNA maturation. We found that PinX1 complemented the gno1-Delta mutation, suggesting that it has a dual function in telomere length regulation and ribosomal RNA maturation in agreement with its telomeric and nucleolar localization in human cells. Conversely, we found that Gno1p does not exhibit the in vivo telomerase inhibitor activity of PinX1.     

Increased Stability of Nucleolar PinX1 in the Presence of TERT

PinX1, a nucleolar protein of 328 amino acids, inhibits telomerase activity, which leads to the shortening of telomeres. The C-terminal region of PinX1 is responsible for its nucleolar localization and binding with TERT, a catalytic component of telomerase. A fraction of TERT localizes to the nucleolus, but the role of TERT in the nucleolus is largely unknown. Here, we report a functional connection between PinX1 and TERT regarding PinX1 stability. The C-terminal of PinX1^205–328, a nucleolar fragment, was much more stable than the N-terminal of PinX1^1–204, a nuclear fragment. Interestingly, PinX1 was less stable in TERT-depleted cells and more stable in TERT-myc expressing cells. Stability assays for PinX1 truncation forms showed that both PinX1^1–328 and PinX1^205–328, nucleolar forms, were more rapidly degraded in TERT-depleted cells, while they were more stably maintained in TERT-overexpressing cells, compared to each of the controls. However, PinX1^1–204 was degraded regardless of the TERT status. These results reveal that the stability of PinX1 is maintained in nucleolus in the presence of TERT and suggest a role of TERT in the regulation of PinX1 steady-state levels.     

The F-box protein FBX4 targets PIN2/TRF1 for ubiquitin-mediated degradation and regulates telomere maintenance

Pin2/TRF1 was identified previously as both a protein (TRF1) that binds to telomeric DNA repeats and as a protein (Pin2) that associates with the kinase NIMA and suppresses its mitosis inducing activity. Pin2/TRF1 negatively regulates telomere length and also plays a critical role in cell cycle checkpoint control. Pin2/TRF1 is down-regulated in many human cancers and may be degraded by the ubiquitin-proteasome pathway, but components of the pathway involved in Pin2/TRF1 turnover have not been elucidated. By using the two-hybrid system, we recently identified Pin2/TRF1-interacting proteins, PinX1-4, and we demonstrated that PinX1 is a conserved telomerase inhibitor and a putative tumor suppressor. Here we report the characterization of PinX3. PinX3 was later found to be identical to Fbx4, a member of the F-box family of proteins, which function as substrate-specific adaptors of Cul1-based ubiquitin ligases. Fbx4 interacts with both Pin2 and TRF1 isoforms and promotes their ubiquitination in vitro and in vivo. Moreover, overexpression of Fbx4 reduces endogenous Pin2/TRF1 protein levels and causes progressive telomere elongation in human cells. In contrast, inhibition of Fbx4 by RNA interference stabilizes Pin2/TRF1 and promotes telomere shortening, thereby impairing cell growth. These results demonstrate that Fbx4 is a central regulator of Pin2/TRF1 protein abundance and that alterations in the stability of Pin2/TRF1 can have a dramatic impact on telomere length. Thus, Fbx4 may play a critical role in telomere maintenance.     

C-terminal amino acids 290-328 of LPTS/PinX1 confer telomerase inhibition

LPTS/PinX1, a telomerase inhibitor composed of 328 amino acids, binds to the telomere associated protein Pin2/TRF1 and to the telomerase catalytic subunit hTERT. However, the mechanism by which LPTS/PinX1 regulates telomerase activity remains unclear. Here we show, for the first time, that LPTS/PinX1 uses different domains to interact with Pin2/TRF1 and hTERT. The LPTS/PinX1_254-289 fragment specifically binds to Pin2/TRF1, and LPTS/PinX1_290-328 can associate with hTERT. Compared with the full-length LPTS/PinX1 protein, LPTS/PinX1_290-328 shows stronger in vitro telomerase inhibitory activity. Moreover, the LPTS/PinX1 protein was recruited to telomeres for binding to Pin2/TRF1. Overexpression of LPTS/PinX1_290-328, which contains a nucleolus localization signal, in cells resulted in telomere shortening and progressive cell death. Conversely, telomere elongation was induced by expression of the dominant-negative LPTS/PinX1_1-289. Our results suggest that the C-terminal fragment of LPTS/PinX1 (LPTS/PinX1_290-328) contains a telomerase inhibitory domain that is required for the inhibition of telomere elongation and the induction of cell crisis. Our studies also provide evidence that LPTS/PinX1 interaction with Pin2/TRF1 may play a role in the stabilization of telomeres.     

Telomerase inhibitor PinX1 provides a link between TRF1 and telomerase to prevent telomere elongation

Telomere maintenance is essential for protecting chromosome ends. Aberrations in telomere length have been implicated in cancer and aging. Telomere elongation by human telomerase is inhibited in cis by the telomeric protein TRF1 and its associated proteins. However, the link between TRF1 and inhibition of telomerase elongation of telomeres remains elusive because TRF1 has no direct effect on telomerase activity. We have previously identified one Pin2/TRF1-interacting protein, PinX1, that has the unique property of directly binding and inhibiting telomerase catalytic activity (Zhou, X. Z., and Lu, K. P. (2001) Cell 107, 347-359). However, nothing is known about the role of the PinX1-TRF1 interaction in the regulation of telomere maintenance. By identifying functional domains and key amino acid residues in PinX1 and TRF1 responsible for the PinX1-TRF1 interaction, we show that the TRF homology domain of TRF1 interacts with a minimal 20-amino acid sequence of PinX1 via hydrophilic and hydrophobic interactions. Significantly, either disrupting this interaction by mutating the critical Leu-291 residue in PinX1 or knocking down endogenous TRF1 by RNAi abolishes the ability of PinX1 to localize to telomeres and to inhibit telomere elongation in cells even though neither has any effect on telomerase activity per se. Thus, the telomerase inhibitor PinX1 is recruited to telomeres by TRF1 and provides a critical link between TRF1 and telomerase inhibition to prevent telomere elongation and help maintain telomere homeostasis.     

Telomere shortening in human fibroblasts is not dependent on the size of the telomeric-3'-overhang

Telomeres shorten in human somatic cells with each round of DNA replication, and this shortening is thought to ultimately trigger replicative senescence. Telomere shortening is caused partly by the inability of semiconservative DNA replication to copy a linear strand of DNA to its very end. Post-replicative processing of telomeric ends, producing single-stranded G-rich 3' overhangs, has also been suggested to contribute to telomere shortening. This suggestion implies that a positive correlation should exist between the length of 3' overhangs and the rate of telomere shortening. We confirmed shortening of overhangs as human lung (MRC5) and foreskin (BJ) fibroblasts approach senescence by measuring overhang length using in-gel hybridization. However, a large study of fibroblast strains from 21 donors maintained under conditions which lead to two orders of magnitude of variation in telomere shortening rate failed to show any correlation between telomere overhang length and shortening rate, suggesting that overhang length is neither a cause nor a correlate of telomere shortening.     

Human PinX1 Mediates TRF1 Accumulation in Nucleolus and Enhances TRF1 Binding to Telomeres

Human PinX1 (hPinX1) is known to interact with telomere repeat binding factor 1 (TRF1) and telomerase. Here, we report that hPinX1 regulates the nucleolar accumulation and telomeric association of TRF1. In HeLa, HA-hPinX1 was co-localized with fibrillarin, a nucleolar protein, in 51% of the transfected cells and was present in the nucleoplasm of the remaining 48%. Mutant analysis showed that the C-terminal region was important for nucleolar localization, while the N-terminus exhibited an inhibitory effect on nucleolar localization. Unlike HA- and Myc-hPinX1, GFP-hPinX1 resided predominantly in the nucleolus. Nuclear hPinX1 bound to telomeres and other repeat sequences as well but, despite its interaction with TRF1, nucleolar hPinX1 did not bind to telomeres. Nucleolar hPinX1 forced endogenous TRF1 accumulation in the nucleolus. Furthermore, TRF1 binding to telomeres was upregulated in cells over-expressing hPinX1. In an ALT cell line, WI-38 VA-13, TRF1 did not co-localize with hPinX1 in the nucleoli. In summary, hPinX1 likely interacts with TRF1 in both the nucleolus and the nucleoplasm, and excess hPinX1 results in increased telomere binding of TRF1. The PinX1 function of mediating TRF1 nucleolar accumulation is absent from ALT cells, suggesting that it might be telomerase-dependent.     

Human MCRS2, a cell-cycle-dependent protein, associates with LPTS/PinX1 and reduces the telomere length

Human LPTS/PinX1 is a telomerase-inhibitory protein, which binds to the telomere protein Pin2/TRF1 and the catalytic subunit hTERT of telomerase. To explore the proteins that might be involved in the telomerase pathway, we performed a yeast two-hybrid screening with LPTS/PinX1 as the bait. A novel gene, MCRS2, encoding for an isoform of MCRS1/p78 and MSP58 was isolated. The expression of MCRS2 protein is cell-cycle dependent, accumulating in the very early S phase. MCRS2 interacts with LPTS/PinX1 in vitro, in vivo and colocalizes with LPTS/PinX1 in cells. MCRS2 and its amino terminus inhibit telomerase activity in vitro and long-term overexpression of MCRS2 in SMMC-7721 cells results in a gradual and progressive shortening of telomeres. Our findings suggest that MCRS2 might be a linker between telomere maintenance and cell-cycle regulation.     

Telomere shortening is proportional to the size of the G-rich telomeric 3'-overhang

Most normal diploid human cells do not express telomerase activity and are unable to maintain telomere length with ongoing cell divisions. We show that the length of the single-stranded G-rich telomeric 3'-overhang is proportional to the rate of shortening in four human cell types that exhibit different rates of telomere shortening in culture. These results provide direct evidence that the size of the G-rich overhang is not fixed but subject to regulation. The potential ability to manipulate this rate has profound implications both for slowing the rate of replicative aging in normal cells and for accelerating the rate of telomere loss in cancer cells in combination with anti-telomerase therapies.     

Accumulation of single-strand breaks is the major cause of telomere shortening in human fibroblasts

Telomere shortening triggers replicative senescence in human fibroblasts. The inability of DNA polymerases to replicate a linear DNA molecule completely (the end replication problem) is one cause of telomere shortening. Other possible causes are the formation of single-stranded overhangs at the end of telomeres and the preferential vulnerability of telomeres to oxidative stress. To elucidate the relative importance of these possibilities, amount and distribution of telomeric single-strand breaks, length of the G-rich overhang, and telomere shortening rate in human MRC-5 fibroblasts were measured. Treatment of nonproliferating cells with hydrogen peroxide increases the sensitivity to S1 nuclease in telomeres preferentially and accelerates their shortening by a corresponding amount as soon as the cells proliferate. A reduction of the activity of intracellular peroxides using the spin trap alpha-phenyl-t-butyl-nitrone reduces the telomere shortening rate and increases the replicative life span. The length of the telomeric single-stranded overhang is independent of DNA damaging stresses, but single-strand breaks accumulate randomly all along the telomere after alkylation. The telomere shortening rate and the rate of replicative aging can be either accelerated or decelerated by a modification of the amount of oxidative stress. Quantitatively, stress-mediated telomere damage contributes most to telomere shortening under standard conditions.     

Telomerase maintains telomere structure in normal human cells

In normal human cells, telomeres shorten with successive rounds of cell division, and immortalization correlates with stabilization of telomere length. These observations suggest that human cancer cells achieve immortalization in large part through the illegitimate activation of telomerase expression. Here, we demonstrate that the rate-limiting telomerase catalytic subunit hTERT is expressed in cycling primary presenescent human fibroblasts, previously believed to lack hTERT expression and telomerase activity. Disruption of telomerase activity in normal human cells slows cell proliferation, restricts cell lifespan, and alters the maintenance of the 3' single-stranded telomeric overhang without changing the rate of overall telomere shortening. Together, these observations support the view that telomerase and telomere structure are dynamically regulated in normal human cells and that telomere length alone is unlikely to trigger entry into replicative senescence.     

Mammalian Ku86 protein prevents telomeric fusions independently of the length of TTAGGG repeats and the G-strand overhang

Ku86 together with Ku70, DNA-PKcs, XRCC4 and DNA ligase IV forms a complex involved in repairing DNA double-strand breaks (DSB) in mammals. Yeast Ku has an essential role at the telomere; in particular, Ku deficiency leads to telomere shortening, loss of telomere clustering, loss of telomeric silencing and deregulation of the telomeric G-overhang. In mammals, Ku proteins associate to telomeric repeats; however, the possible role of Ku in regulating telomere length has not yet been addressed. We have measured telomere length in different cell types from wild-type and Ku86-deficient mice. In contrast to yeast, Ku86 deficiency does not result in telomere shortening or deregulation of the G-strand overhang. Interestingly, Ku86^–/– cells show telomeric fusions with long telomeres (>81 kb) at the fusion point. These results indicate that mammalian Ku86 plays a fundamental role at the telomere by preventing telomeric fusions independently of the length of TTAGGG repeats and the integrity of the G-strand overhang.     

Telomeric 3' overhangs derive from resection by Exo1 and Apollo and fill-in by POT1b-associated CST

A 3' overhang is critical for the protection and maintenance of mammalian telomeres, but its synthesis must be regulated to avoid excessive resection of the 5' end, which could cause telomere shortening. How this balance is achieved in mammals has not been resolved. Here, we determine the mechanism for 3' overhang synthesis in mouse cells by evaluating changes in telomeric overhangs throughout the cell cycle and at leading- and lagging-end telomeres. Apollo, a nuclease bound to the shelterin subunit TRF2, initiates formation of the 3' overhang at leading-, but not lagging-end telomeres. Hyperresection by Apollo is blocked at both ends by the shelterin protein POT1b. Exo1 extensively resects both telomere ends, generating transient long 3' overhangs in S/G2. CST/AAF, a DNA polα.primase accessory factor, binds POT1b and shortens the extended overhangs produced by Exo1, likely through fill-in synthesis. 3' overhang formation is thus a multistep, shelterin-controlled process, ensuring functional telomeric overhangs at chromosome ends.     

PinX1, a Telomere Repeat-binding Factor 1 (TRF1)-interacting Protein,Maintains Telomere Integrity by Modulating TRF1 Homeostasis,the Process in Which Human Telomerase Reverse Transcriptase (hTERT) Plays Dual Roles

TRF1, a telomere-binding protein, is important for telomere protection and homeostasis. PinX1 interacts with TRF1, but the physiological consequences of their interaction in telomere protection are not yet understood. Here we investigated PinX1 function on TRF1 stability in HeLa cells. PinX1 overexpression stabilized TRF1, but PinX1 depletion by siRNA led to TRF1 degradation, TRF1 ubiquitination, and less TRF1 telomere association. The depletion also induced DNA damage responses at telomeres and chromosome instability. These telomere dysfunctional phenotypes were in fact due to TRF1 deficiency. We also report that hTERT, a catalytic component of telomerase, plays dual roles in the TRF1 steady state pathway. PinX1-mediated TRF1 stability was not observed in hTERT-negative immortal cells, but was pronounced when hTERT was ectopically expressed in the cells, suggesting that hTERT may be needed in the PinX1-mediated TRF1 stability pathway. Interestingly, the knockdown of both PinX1 and hTERT in HeLa cells stabilized TRF1, suppressed DNA damage response activation, and restored chromosome stability. In summary, our findings suggested that PinX1 may maintain telomere integrity by regulating TRF1 stability and that hTERT may act as both a positive and a negative regulator of TRF1 homeostasis in a PinX1-dependent manner.     

Features that determine telomere homolog oligonucleotide-induced therapeutic DNA damage-like responses in cancer cells

Cancer is the second leading cause of death in the USA, with metastatic disease proving a particular management challenge. Treatment modalities for patients with metastatic disease are limited, and survival beyond 5 years is uncommon. We have reported that an 11-base DNA oligonucleotide 100% homologous to the telomere 3' overhang can induce apoptosis, senescence and/or differentiation of several types of malignant cells in vitro and in vivo, while having minimal effect on normal cells. We now report that 22 oligonucleotides, 9-20 bases in length, with or without a 5' phosphate group and with varying homology (40-100%) to the 3' overhang, inhibit growth and induce apoptosis of human cell lines derived from breast cancers, pancreatic and ovarian carcinomas, and malignant melanoma, lines that lack p53 and/or p16 and harbor a variety of other abnormalities in key regulatory signaling pathways. Cytosine (C) content adversely affected oligonucleotide efficacy, decreasing their effect on cellular apoptosis by > or =80%. These data confirm and expand our earlier work suggesting that such telomere homolog oligonucleotides (T-oligos) target an innate anti-cancer defense system in human cells and may provide an effective treatment for cancers of multiple different cellular origins and genetic profile.     

A critical role for Pin2/TRF1 in ATM-dependent regulation. Inhibition of Pin2/TRF1 function complements telomere shortening, radiosensitivity, and the G(2)/M checkpoint defect of ataxia-telangiectasia cells.

Cells derived from patients with the human genetic disorder ataxia-telangiectasia (A-T) display many abnormalities, including telomere shortening, premature senescence, and defects in the activation of S phase and G(2)/M checkpoints in response to double-strand DNA breaks induced by ionizing radiation. We have previously demonstrated that one of the ATM substrates is Pin2/TRF1, a telomeric protein that binds the potent telomerase inhibitor PinX1, negatively regulates telomere elongation, and specifically affects mitotic progression. Following DNA damage, ATM phosphorylates Pin2/TRF1 and suppresses its ability to induce abortive mitosis and apoptosis (Kishi, S., Zhou, X. Z., Nakamura, N., Ziv, Y., Khoo, C., Hill, D. E., Shiloh, Y., and Lu, K. P. (2001) J. Biol. Chem. 276, 29282-29291). However, the functional importance of Pin2/TRF1 in mediating ATM-dependent regulation remains to be established. To address this question, we directly inhibited the function of endogenous Pin2/TRF1 in A-T cells by stable expression of two different dominant-negative Pin2/TRF1 mutants and then examined their effects on telomere length and DNA damage response. Both the Pin2/TRF1 mutants increased telomere length in A-T cells, as shown in other cells. Surprisingly, both the Pin2/TRF1 mutants reduced radiosensitivity and complemented the G(2)/M checkpoint defect without inhibiting Cdc2 activity in A-T cells. In contrast, neither of the Pin2/TRF1 mutants corrected the S phase checkpoint defect in the same cells. These results indicate that inhibition of Pin2/TRF1 in A-T cells is able to bypass the requirement for ATM in specifically restoring telomere shortening, the G(2)/M checkpoint defect, and radiosensitivity and demonstrate a critical role for Pin2/TRF1 in the ATM-dependent regulation of telomeres and DNA damage response.