Biomedical subjects

Titia de Lange

Publications and source records attributed to Titia de Lange.

At least 19 recordsLinked to original sources

PubMed2006

MDC1 accelerates nonhomologous end-joining of dysfunctional telomeres.

Here we document the role of MDC1 (mediator of DNA damage checkpoint 1) in the detection and repair of human and mouse telomeres rendered dysfunctional through inhibition of TRF2. Consistent with its role in promoting DNA damage foci, MDC1 knockdown affected the formation of telomere dysfunction-induced foci (TIFs), diminishing the accumulation of phosphorylated ATM, 53BP1, Nbs1, and to a lesser extent, gamma-H2AX. In addition to this effect on TIFs, the rate of nonhomologous end-joining (NHEJ) of dysfunctional telomeres was significantly decreased when MDC1 itself or its recruitment to chromatin was inhibited. MDC1 appeared to promote a step in the NHEJ pathway after the removal of the 3' telomeric overhang. The acceleration of NHEJ was unlikely to be due to increased presence of 53BP1 and Mre11 in TIFs, since knockdown of neither factor affected telomere fusions. Furthermore, relevant cell cycle effectors (Chk2, p53, and p21) of the ATM kinase pathway were unaffected and there was no change in the rate of cell cycle progression. We propose that the binding of MDC1 to gamma-H2AX directly affects NHEJ in a manner that is independent of the ATM-dependent cell cycle arrest pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Hepatocytes with extensive telomere deprotection and fusion remain viable and regenerate liver mass through endoreduplication.

We report that mouse liver cells are highly resistant to extensive telomere dysfunction. In proliferating cells, telomere dysfunction results in chromosome end fusions, a DNA damage signal, and apoptosis or senescence. To determine the consequences of telomere dysfunction in noncycling cells, we used conditional deletion of the telomeric protein TRF2 in hepatocytes. TRF2 loss resulted in telomeric accumulation of gamma-H2AX and frequent telomere fusions, indicating telomere deprotection. However, there was no induction of p53 or apoptosis, and liver function appeared unaffected. Furthermore, the loss of TRF2 did not compromise liver regeneration after partial hepatectomy. Remarkably, liver regeneration occurred without cell division involving endoreduplication and cell growth, thereby circumventing the chromosome segregation problems associated with telomere fusions. We conclude that nondividing hepatocytes can maintain and regenerate liver function despite substantial loss of telomere integrity.

Animals↗

PubMed2006

Lasker Laurels for telomerase.

This year the Lasker Foundation pays tribute to telomerase, a medically important enzyme required for chromosome stability and long-term cell proliferation.

Animals↗

PubMed2006

Ku70 stimulates fusion of dysfunctional telomeres yet protects chromosome ends from homologous recombination.

Ku70-Ku80 heterodimers promote the non-homologous end-joining (NHEJ) of DNA breaks and, as shown here, the fusion of dysfunctional telomeres. Paradoxically, this heterodimer is also located at functional mammalian telomeres and interacts with components of shelterin, the protein complex that protects telomeres. To determine whether Ku contributes to telomere protection, we analysed Ku70(-/-) mouse cells. Telomeres of Ku70(-/-) cells had a normal DNA structure and did not activate a DNA damage signal. However, Ku70 repressed exchanges between sister telomeres - a form of homologous recombination implicated in the alternative lengthening of telomeres (ALT) pathway. Sister telomere exchanges occurred at approximately 15% of the chromosome ends when Ku70 and the telomeric protein TRF2 were absent. Combined deficiency of TRF2 and another NHEJ factor, DNA ligase IV, did not elicit this phenotype. Sister telomere exchanges were not elevated at telomeres with functional TRF2, indicating that TRF2 and Ku70 act in parallel to repress recombination. We conclude that mammalian chromosome ends are highly susceptible to homologous recombination, which can endanger cell viability if an unequal exchange generates a critically shortened telomere. Therefore, Ku- and TRF2-mediated repression of homologous recombination is an important aspect of telomere protection.

Animals↗

PubMed2006

Recent expansion of the telomeric complex in rodents: Two distinct POT1 proteins protect mouse telomeres.

Human telomeres are protected by shelterin, a complex that includes the POT1 single-stranded DNA binding protein. We found that mouse telomeres contain two POT1 paralogs, POT1a and POT1b, and we used conditional deletion to determine their function. Double-knockout cells showed that POT1a/b are required to prevent a DNA damage signal at chromosome ends, endoreduplication, and senescence. In contrast, POT1a/b were largely dispensable for repression of telomere fusions. Single knockouts and complementation experiments revealed that POT1a and POT1b have distinct functions. POT1a, but not POT1b, was required to repress a DNA damage signal at telomeres. Conversely, POT1b, but not POT1a, had the ability to regulate the amount of single-stranded DNA at the telomere terminus. We conclude that mouse telomeres require two distinct POT1 proteins whereas human telomeres have one. Such divergence is unprecedented in mammalian chromosome biology and has implications for modeling human telomere biology in mice.

Animals↗

PubMed2006

Apollo, an Artemis-related nuclease, interacts with TRF2 and protects human telomeres in S phase.

Human chromosome ends are protected by shelterin, an abundant six-subunit protein complex that binds specifically to the telomeric-repeat sequences, regulates telomere length, and ensures that chromosome ends do not elicit a DNA-damage response (reviewed in). Using mass spectrometry of proteins associated with the shelterin component Rap1, we identified an SMN1/PSO2 nuclease family member that is closely related to Artemis. We refer to this protein as Apollo and report that Apollo has the ability to localize to telomeres through an interaction with the shelterin component TRF2. Although its low abundance at telomeres indicates that Apollo is not a core component of shelterin, Apollo knockdown with RNAi resulted in senescence and the activation of a DNA-damage signal at telomeres as evidenced by telomere-dysfunction-induced foci (TIFs). The TIFs occurred primarily in S phase, suggesting that Apollo contributes to a processing step associated with the replication of chromosome ends. Furthermore, some of the metaphase chromosomes showed two telomeric signals at single-chromatid ends, suggesting an aberrant telomere structure. We propose that the Artemis-like nuclease Apollo is a shelterin accessory factor required for the protection of telomeres during or after their replication.

Cell Line↗

PubMed2005

p16INK4a as a second effector of the telomere damage pathway.

Telomere damage resulting from telomere shortening can potentially suppress tumorigenesis by permanently arresting or eliminating incipient cancer cells. Dysfunctional telomeres activate the canonical DNA damage response pathway, resulting in a p53-mediated G(1)/S arrest and senescence or apoptosis. Experimental induction of telomere damage through inhibition of the telomeric protein TRF2 recapitulates aspects of telomere attrition, including a p53-mediated cell cycle arrest. Using this system, we have shown that telomere damage can also elicit a G(1)/S arrest through the RB-regulator p16INK4a, especially in cells lacking p53 function. Here we discuss the significance of p16INK4a as a second effector of the telomere damage response.

Animals↗

PubMed2005

Shelterin: the protein complex that shapes and safeguards human telomeres.

Added by telomerase, arrays of TTAGGG repeats specify the ends of human chromosomes. A complex formed by six telomere-specific proteins associates with this sequence and protects chromosome ends. By analogy to other chromosomal protein complexes such as condensin and cohesin, I will refer to this complex as shelterin. Three shelterin subunits, TRF1, TRF2, and POT1 directly recognize TTAGGG repeats. They are interconnected by three additional shelterin proteins, TIN2, TPP1, and Rap1, forming a complex that allows cells to distinguish telomeres from sites of DNA damage. Without the protective activity of shelterin, telomeres are no longer hidden from the DNA damage surveillance and chromosome ends are inappropriately processed by DNA repair pathways. How does shelterin avert these events? The current data argue that shelterin is not a static structural component of the telomere. Instead, shelterin is emerging as a protein complex with DNA remodeling activity that acts together with several associated DNA repair factors to change the structure of the telomeric DNA, thereby protecting chromosome ends. Six shelterin subunits: TRF1, TRF2, TIN2, Rap1, TPP1, and POT1.

Antigens, Surface↗

PubMed2005

POT1 protects telomeres from a transient DNA damage response and determines how human chromosomes end.

The hallmarks of telomere dysfunction in mammals are reduced telomeric 3' overhangs, telomere fusions, and cell cycle arrest due to a DNA damage response. Here, we report on the phenotypes of RNAi-mediated inhibition of POT1, the single-stranded telomeric DNA-binding protein. A 10-fold reduction in POT1 protein in tumor cells induced neither telomere fusions nor cell cycle arrest. However, the 3' overhang DNA was reduced and all telomeres elicited a transient DNA damage response in G1, indicating that extensive telomere damage can occur without cell cycle arrest or telomere fusions. RNAi to POT1 also revealed its role in generating the correct sequence at chromosome ends. The recessed 5' end of the telomere, which normally ends on the sequence ATC-5', was changed to a random position within the AATCCC repeat. Thus, POT1 determines the structure of the 3' and 5' ends of human chromosomes, and its inhibition generates a novel combination of telomere dysfunction phenotypes in which chromosome ends behave transiently as sites of DNA damage, yet remain protected from nonhomologous end-joining.

Alternative Splicing↗

PubMed2005

DNA processing is not required for ATM-mediated telomere damage response after TRF2 deletion.

Telomere attrition and other forms of telomere damage can activate the ATM kinase pathway. What generates the DNA damage signal at mammalian chromosome ends or at other double-strand breaks is not known. Telomere dysfunction is often accompanied by disappearance of the 3' telomeric overhang, raising the possibility that DNA degradation could generate the structure that signals. Here we address these issues by studying telomere structure after conditional deletion of mouse TRF2, the protective factor at telomeres. Upon removal of TRF2 from TRF2(F/-) p53-/- mouse embryo fibroblasts, a telomere damage response is observed at most chromosome ends. As expected, the telomeres lose the 3' overhang and are processed by the non-homologous end-joining pathway. Non-homologous end joining of telomeres was abrogated in DNA ligase IV-deficient (Lig4-/-) cells. Unexpectedly, the telomeres of TRF2-/- Lig4-/- p53-/- cells persisted in a free state without undergoing detectable DNA degradation. Notably, the telomeres retained their 3' overhangs, but they were recognized as sites of DNA damage, accumulating the DNA damage response factors 53BP1 and gamma-H2AX, and activating the ATM kinase. Thus, activation of the ATM kinase pathway at chromosome ends does not require overhang degradation or other overt DNA processing.

Animals↗

PubMed2004

Significant role for p16INK4a in p53-independent telomere-directed senescence.

Telomere attrition in primary human fibroblasts induces replicative senescence accompanied by activation of the p53 and p16(INK4a)/RB tumor suppressor pathways. Although the contribution of p53 and its target, p21, to telomere-driven senescence have been well established, the role of p16(INK4a) is controversial. Attempts to dissect the significance of p16(INK4a) in response to telomere shortening have been hampered by the concomitant induction of p16(INK4a) by cell culture conditions. To circumvent this problem, we studied the role of p16(INK4a) in the cellular response to acute telomere damage induced by a dominant negative allele of TRF2, TRF2(Delta B Delta M). This approach avoids the confounding aspects of culture stress because parallel cultures with and without telomere damage can be compared. Telomere damage generated with TRF2(Delta B Delta M) resulted in induction of p16(INK4a) in the majority of cells as detected by immunohistochemistry. Inhibition of p16(INK4a) with shRNA or overexpression of BMI1 had a significant effect on the telomere damage response in p53-deficient cells. While p53 deficiency alone only partially abrogated the telomere damage-induced cell cycle arrest, combined inhibition of p16(INK4a) and p53 led to nearly complete bypass of telomere-directed senescence. We conclude that p16(INK4a) contributes to the p53-independent response to telomere damage.

Bromodeoxyuridine↗

PubMed2004

Homologous recombination generates T-loop-sized deletions at human telomeres.

The t-loop structure of mammalian telomeres is thought to repress nonhomologous end joining (NHEJ) at natural chromosome ends. Telomere NHEJ occurs upon loss of TRF2, a telomeric protein implicated in t-loop formation. Here we describe a mutant allele of TRF2, TRF2DeltaB, that suppressed NHEJ but induced catastrophic deletions of telomeric DNA. The deletion events were stochastic and occurred rapidly, generating dramatically shortened telomeres that were accompanied by a DNA damage response and induction of senescence. TRF2DeltaB-induced deletions depended on XRCC3, a protein implicated in Holliday junction resolution, and created t-loop-sized telomeric circles. These telomeric circles were also detected in unperturbed cells and suggested that t-loop deletion by homologous recombination (HR) might contribute to telomere attrition. Human ALT cells had abundant telomeric circles, pointing to frequent t-loop HR events that could promote rolling circle replication of telomeres in the absence of telomerase. These findings show that t-loop deletion by HR influences the integrity and dynamics of mammalian telomeres.

Animals↗

PubMed2004

Human Rif1, ortholog of a yeast telomeric protein, is regulated by ATM and 53BP1 and functions in the S-phase checkpoint.

We report on the function of the human ortholog of Saccharomyces cerevisiae Rif1 (Rap1-interacting factor 1). Yeast Rif1 associates with telomeres and regulates their length. In contrast, human Rif1 did not accumulate at functional telomeres, but localized to dysfunctional telomeres and to telomeric DNA clusters in ALT cells, a pattern of telomere association typical of DNA-damage-response factors. After induction of double-strand breaks (DSBs), Rif1 formed foci that colocalized with other DNA-damage-response factors. This response was strictly dependent on ATM (ataxia telangiectasia mutated) and 53BP1, but not affected by diminished function of ATR (ATM- and Rad3-related kinase), BRCA1, Chk2, Nbs1, and Mre11. Rif1 inhibition resulted in radiosensitivity and a defect in the intra-S-phase checkpoint. The S-phase checkpoint phenotype was independent of Nbs1 status, arguing that Rif1 and Nbs1 act in different pathways to inhibit DNA replication after DNA damage. These data reveal that human Rif1 contributes to the ATM-mediated protection against DNA damage and point to a remarkable difference in the primary function of this protein in yeast and mammals.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2004

The telomeric protein TRF2 binds the ATM kinase and can inhibit the ATM-dependent DNA damage response.

The telomeric protein TRF2 is required to prevent mammalian telomeres from activating DNA damage checkpoints. Here we show that overexpression of TRF2 affects the response of the ATM kinase to DNA damage. Overexpression of TRF2 abrogated the cell cycle arrest after ionizing radiation and diminished several other readouts of the DNA damage response, including phosphorylation of Nbs1, induction of p53, and upregulation of p53 targets. TRF2 inhibited autophosphorylation of ATM on S1981, an early step in the activation of this kinase. A region of ATM containing S1981 was found to directly interact with TRF2 in vitro, and ATM immunoprecipitates contained TRF2. We propose that TRF2 has the ability to inhibit ATM activation at telomeres. Because TRF2 is abundant at chromosome ends but not elsewhere in the nucleus, this mechanism of checkpoint control could specifically block a DNA damage response at telomeres without affecting the surveillance of chromosome internal damage.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2004

TIN2 binds TRF1 and TRF2 simultaneously and stabilizes the TRF2 complex on telomeres.

Human telomeres contain two related telomeric DNA-binding proteins, TRF1 and TRF2. The TRF1 complex contains the TRF1 interacting partner, TIN2, as well as PIP1 and POT1 and regulates telomere-length homeostasis. The TRF2 complex is primarily involved in telomere protection and contains the TRF2 interacting partner human (h)Rap1 as well as several factors involved in the DNA damage response. A prior report showed that conditional deletion of murine TRF1 reduced the presence of TRF2 on telomeres. Here we showed that TRF2 is also lost from human telomeres upon TRF1 depletion with small interfering RNA prompting a search for the connection between the TRF1 and TRF2 complexes. Using mass spectrometry and co-immunoprecipitation, we found that TRF1, TIN2, PIP1, and POT1 are associated with the TRF2-hRap1 complex. Gel filtration identified a TRF2 complex containing TIN2 and POT1 but not TRF1 indicating that TRF1 is not required for this interaction. Co-immunoprecipitation, Far-Western assays, and two-hybrid assays showed that TIN2, but not POT1 or PIP1, interacts directly with TRF2. Furthermore, TIN2 was found to bind TRF1 and TRF2 simultaneously, showing that TIN2 can link these telomeric proteins. This connection appeared to stabilize TRF2 on the telomeres as the treatment of cells with TIN2 small interfering RNA resulted in a decreased presence of TRF2 and hRap1 at chromosome ends. The TIN2-mediated cooperative binding of TRF1 and TRF2 to telomeres has important implications for the mechanism of telomere length regulation and protection.

Antigens, Surface↗

PubMed2004

POT1-interacting protein PIP1: a telomere length regulator that recruits POT1 to the TIN2/TRF1 complex.

Human telomere length is controlled by a negative feedback loop based on the binding of TRF1 to double-stranded telomeric DNA. The TRF1 complex recruits POT1, a single-stranded telomeric DNA-binding protein necessary for cis-inhibition of telomerase. By mass spectrometry, we have identified a new telomeric protein, which we have named POT1-interacting protein 1 (PIP1). PIP1 bound both POT1 and the TRF1-interacting factor TIN2 and could tether POT1 to the TRF1 complex. Reduction of PIP1 or POT1 levels with shRNAs led to telomere elongation, indicating that PIP1 contributes to telomere length control through recruitment of POT1.

Carrier Proteins↗

PubMed2004

TIN2 is a tankyrase 1 PARP modulator in the TRF1 telomere length control complex.

Telomere length in humans is partly controlled by a feedback mechanism in which telomere elongation by telomerase is limited by the accumulation of the TRF1 complex at chromosome ends. TRF1 itself can be inhibited by the poly(ADP-ribose) polymerase (PARP) activity of its interacting partner tankyrase 1, which abolishes its DNA binding activity in vitro and removes the TRF1 complex from telomeres in vivo. Here we report that the inhibition of TRF1 by tankyrase is in turn controlled by a second TRF1-interacting factor, TIN2 (ref. 6). Partial knockdown of TIN2 by small hairpin RNA in a telomerase-positive cell line resulted in telomere elongation, which is typical of reduced TRF1 function. Transient inhibition of TIN2 with small interfering RNA led to diminished telomeric TRF1 signals. This effect could be reversed with the PARP inhibitor 3-aminobenzamide and did not occur in cells overexpressing a PARP-dead mutant of tankyrase 1. TIN2 formed a ternary complex with TRF1 and tankyrase 1 and stabilized their interaction, an effect also observed with the PARP-dead mutant of tankyrase 1. In vitro, TIN2 protected TRF1 from poly(ADP-ribosyl)ation by tankyrase 1 without affecting tankyrase 1 automodification. These data identify TIN2 as a PARP modulator in the TRF1 complex and can explain how TIN2 contributes to the regulation of telomere length.

HeLa Cells↗

PubMed2004

Telomerase regulation at the telomere: a binary switch.

Telomerase is known to preferentially elongate the shortest telomeres in a cell. Using an elegant yeast assay, Texeira et al. (2004 [this issue of Cell]) address what aspect of telomerase action is regulated by telomere length: the frequency or the extent of telomere elongation. They show that short telomeres are elongated more frequently than long telomeres, arguing that telomeres switch between two states, one that allows telomere extension and one that does not.

Crosses, Genetic↗