Laboratory for Cell Biology and Genetics

Telomeres

Telomeres are nucleoprotein complexes that the natural ends of chromosomes that protect chromosome ends and ensure their complete replication. The loss of telomere protection is the root cause of the premature aging symptoms associated with Dyskeratosis congenita and other telomeropathies. Furthermore, telomere dysfunction plays an important role in the early stages of cancer and the activation of a telomere maintenance system (telomerase or ALT) is a hallmark of human cancer. We study how the telomeric shelterin complex prevents the activation of DNA damage signaling pathways and blocks various forms of double-strand break (DSB) repair.

Mammalian telomeres, composed of shelterin bound to the telomeric TTAGGG repeat array, can be maintained by the action of telomerase, which counteracts the loss of telomeric sequences during DNA replication. In many normal human cells, however, telomerase is not (sufficiently) expressed, resulting in telomere attrition with cell divisions. Eventually, the shortened telomeres become too short to fulfill their protective function, resulting in a block to further proliferation and a finite replicative life-span. At this point, the natural ends of chromosomes are recognized as sites DNA double-strand breaks (DSB), activating the two main DNA damage response (DDR) transducers, the ATM and the ATR kinases. The ATM and ATR signaling cascades then induce cell cycle arrest and senescence or apoptosis, depending on the cellular context. Dysfunctional telomeres are also threatened by DSB repair, including two forms of non-homologous end joining (classical and alternative NHEJ) and homology-directed recombination (HDR) and 5’ end resection.

2014 Figure 1

 

Shelterin
Relevant Publications

We have identified a telomere-specific protein complex, called shelterin, that is crucial for the protection of telomeres from the DNA damage response and regulates telomere maintenance by telomerase. Shelterin is composed of six proteins: TRF1, TRF2, Rap1, TIN2, TPP1, and POT1. TRF1 and TRF2 bind to the duplex telomeric repeat array and anchor shelterin on telomeres. POT1 binds to single-stranded TTAGGG repeats and is recruited to telomeres through its interaction with TPP1. TPP1 in turns binds to TIN2, which interacts with both TRF1 and TRF2. Due to its specificity for the sequence and structure of the telomeric DNA, shelterin accumulates at the ends of human and mouse chromosomes but does not bind elsewhere in the genome. Thus, shelterin constitutes a unique marker of telomeres that allows cells to distinguish natural chromosome ends from sites of DNA damage. Our main approach to understanding the function of shelterin is to generate mouse cells from which individual shelterin proteins can be removed using inducible systems, e.g. Cre-mediated deletion.



Independent repression of ATM and ATR by the TRF2 and POT1
Relevant Publications

DNA lesions can activate two transducing kinases, ATM and ATR, which enforce cell cycle arrest through phosphorylation of effector kinases and other targets, such as p53. When TRF2 is inhibited in human cells or deleted mouse cells, telomeres are recognized as sites of DNA damage and activate the canonical ATM kinase pathway. DNA damage response factors such as 53BP1 and MDC1 accumulate near telomeres and the histone H2AX becomes phosphorylated at chromosome ends. The ATM kinase becomes autophosphorylated on S1981 and its signaling activity results in a p53-dependent cell cycle arrest. These responses are entirely dependent on ATM. When TRF2 is deleted from ATM deficient cells, no DNA damage response is observed, indicating that the ATR kinase is not activated by telomeres lacking TRF2. Conversely, ATR but not ATM is activated when POT1 is removed from telomeres. Thus, telomeres use two distinct shelterin components to repress the two main DNA damage signaling pathways. These findings suggest how telomeres induce cell cycle arrest when they become critically shortened. Since the amount of shelterin at telomeres is dependent on telomere length, shortened telomeres are expected to carry diminished amounts of TRF2 and POT1, which will lead to insufficient repression of ATM and ATR.

The mechanism by which POT1 (POT1a in the mouse) represses ATR signaling is of obvious interest. We have proposed that POT1 primarily acts by preventing the accumulation of RPA on the single-stranded telomeric DNA. RPA is the ssDNA sensor in the ATR pathway and blocking its binding to telomeres is expected to be sufficient to prevent the activation of ATR. The POT1 proteins are proposed to be only effective at repressing RPA accumulation when they are tethered to the rest of shelterin. The repression of ATM signaling on the other hand is proposed to involve the formation of t-loops by TRF2 (see below).

2014 Figure 2

 

Inhibition of c-NHEJ by TRF2T1
Relevant Publications

Telomeres are threatened by the two major DSB repair pathways, NHEJ and HDR. Inappropriate NHEJ at telomeres can lead to unstable dicentric chromosomes and uncontrolled HDR can alter telomere length. NHEJ-mediated fusion of telomeres is rampant when TRF2 is deleted from mouse cells, resulting in long trains of joined chromosomes. The telomere fusions that occur in the absence of TRF2 are formed through the action of DNA ligase IV and the Ku70/80 heterodimer, indicating that they are due to classical NHEJ (c-NHEJ). An alternative (alt-) NHEJ reaction that is mediated by PARP1 and DNA ligase III can also take place at telomeres but only when the whole shelterin complex is removed and the cells lack the Ku70/80 heterodimer.

2014 Figure 3

 

Repression of ATM signaling and NHEJ by TRF2: t-loops
Relevant Publications

In collaboration with Jack Griffith (University of North Carolina, Chapel Hill) we found that telomeres can occur in a lariat conformation, referred to as the t-loop. T-loops are formed through the strand invasion of the 3’ telomeric overhang into the duplex part of the telomere. Since the discovery of t-loops in mammals, they have been found in many other eukaryotes, including protozoa, plants, and some fungi.

Given that the telomere terminus is sequestered in the t-loop configuration we proposed that this structure would protect telomeres. Specifically, the t-loop structure would render telomeres impervious to c-NHEJ, which requires the loading of the Ku70/80 complex on free DNA ends, and would prevent the activation of the ATM kinase, which involves binding of the MRN complex to DNA ends. TRF2, the only shelterin protein required for the repression of c-NHEJ and ATM signaling, has the ability to make t-loops in vitro. We tested the TRF2/t-loop model by using super-resolution STORM imaging to detect t-loops in relaxed chromatin from cells with and without TRF2. The results demonstrated that TRF2, but not the other components of shelterin, is required for the establishment and/or maintenance of t-loops.

2014 Figure 4

 

Inhibition of HDR and 5’ end resection
Relevant Publications

Telomeres are also threated by various types of HDR and by 5’ end resection. One of the HDR reactions observed at telomeres is related to the t-loop configuration. The base of the t-loop resembles an early step in HDR where a 3’ overhang is strand-invades double-stranded DNA. If a Holliday junction is formed, resolution of the structure by HJ resolvases could cleave off the loop of the t-loop. Indeed, this type of t-loop cleavage reaction (referred to as t-loop HR) is observed upon overexpression of a mutant form of TRF2 that lacks the N-terminal basic domain. A second HDR reaction can lead to inappropriate sequence exchanges between telomeres (referred to as Telomere-Sister Chromatid Exchanges or T-SCEs). The repression of T-SCEs requires both Rap1 and (one of) the POT1 proteins. Telomere exchanges are only readily detected when either Rap1 or both POT1 proteins are deleted from mouse cells that also lack the Ku70/80 heterodimer. In the presence of Ku70/80, telomere-telomere recombination is stringently repressed.

Finally, telomeres are threatened by 5’ end resection by the same nucleases that attack DSBs. Excessive end-resection occurs at dysfunctional telomeres when 53BP1 and/or Rif1 are inhibited. 53BP1 and Rif1 are general DDR factors that repress resection at DSBs and these factors also repress resection at dysfunctional telomeres. Using telomeres as a test system, we are studying how 53BP1 and Rif1 act.

 

Generation of the 3’ overhang
Relevant Publications

The protection of telomeres is in part dependent on the presence of a 3’ overhang at the telomere terminus. This overhang has to be regenerated every time telomeres are duplicated. Overhang generation is a complex process that involves multiple steps and the telomeres formed by leading- and lagging-strand DNA synthesis are processed differently as expected since their terminal structures are different immediately after DNA synthesis. The process of 3’ overhang formation is carefully controlled by shelterin. TRF2 recruits the Apollo nuclease, which is critical for an initial processing step at the leading-end telomeres. POT1b on the other hand, limits the length of the 3’ overhang by inhibiting Apollo. The Exo1 exonuclease also acts on telomere ends and excessive resection by Exo1 is counteracted also counteracted by POT1b. The latter regulation involves the interaction between POT1b and the CST complex which can promote a fill-in reaction at telomere ends. Mice lacking POT1b show excessive telomere shortening, especially when telomerase is limiting. Ultimately, this telomere shortening evokes phenotypes reminiscent of Dyskeratosis congenita.

 

TRF1 promotes telomere replication and prevents formation of fragile telomeres
Relevant Publications

Deletion of TRF1 from mouse embryo fibroblasts revealed that TRF1 functions to promote efficient replication of telomeric DNA. In absence of TRF1, replication fork stalling occurs in telomeric DNA tracts and the ATR kinase pathway is activated at telomeres. In metaphase, telomeres appear as broken or decondensed, resembling the common fragile sites (CFS) observed after treatment with aphidicolin. Indeed, aphidicolin treatment also induces the fragile telomere phenotype, indicating that telomeres are similar to the CFS. Experiments with the BLM and RTEL1 helicases indicated that TRF1 uses these factors to remove replication blocks from the telomeric DNA. We therefore proposed that TRF1 acts by removing G4 structures that can be formed in the TTAGGG repeats and might impeded replication fork progression.

2014 Figure 5

 

Using telomeres to study the DNA damage response
Relevant Publications

Telomeres offer distinct advantages for the study of the DDR. They represent molecularly marked sites in the genome that can be converted to sites of DNA damage by manipulation of shelterin. As a result, their structure and behavior can be studied before and after the induction of DNA damage signaling using imaging, DNA analysis, ChIP, etc. An additional advantage of telomeres is that they can be manipulated to activate either the ATM kinase (deletion of TRF2) or the ATR kinase (deletion of POT1a) or both (deletion of the whole shelterin complex). Using these telomeric tools, we have gained insight into several aspects of the DNA damage response such as: the lack of signaling requirement for the HDR at telomeres, indicating that many steps in HDR can take place independent of a DNA damage signal; the role of the MRN complex in the activation of the ATM kinase; the role of MDC1, 53BP1, and Rif1 in NHEJ and the control of end-resection; cell cycle aspects of NHEJ; the increase in mobility of dysfunctional telomeres and the role of 53BP1 in this process; and the induction of endoreduplication in the setting of irreparable DNA damage.