**3. Cross talk of telomere-associated proteins with cellular signaling pathways**

#### **3.1. TAPs and DNA damage response**

When the genomic DNA undergoes any damage such as single-strand DNA breaks, doublestrand breaks (DSBs), nicks, or chromosome fusions, cells activate DNA repair pathway depending on the type of damage. During this process, cells seize to grow and initiate the repair, and once the repair is completed growth resumes; otherwise, they undergo apoptosis. Telomeres can also be sensed as breaks by cellular machinery. However, telomerase and shelterin complex cap the telomeres and thus maintain telomere integrity by inhibiting DNAdamage-response pathway at telomeres. Further, some of these DNA repair proteins play an essential role in telomere maintenance by directly associating with various TAPs. Recently, these TAPs have been shown to play a role in DDR at locations distinct from telomeres. TRF1, which is predominantly localized to telomeres, has been shown to interact with proteins implicated in DNA damage response such as ATM and Mre11/Rad50/Nbs1 [96]. Further, it has been shown that TRF1 is phosphorylated by Cdk1 and this form is incapable of binding to telomeres [97]. It has been shown that this phosphorylated TRF1 in a telomere-independent way facilitates end resection and homology repair (HR), activates G2/M checkpoint, and enables cell survival following double-strand break induction [98]. TRF2 is known to prevent activation of ATM-dependent DDR pathway at telomeres [26, 99, 100]. TRF2 has also been shown to localize at DNA double-strand breaks during early stages (within 2 s) of cellular response to DSBs and leaves those sites during repair processing [101]. Further reports suggested that TRF2 may also participate in HR of the extra telomeric damaged DNA [102].

It has been shown that telomerase localizes to mitochondria and protects cancer cells from nuclear DNA damage and apoptosis [103]. Further, TERT has also been shown to function in DNA damage response pathway and in regulating histone-dependent chromatin remodeling [104]. *TR* knockout mice display impaired DDR in response to damaging agents; however, the effects are evident clearly in late-generation TR null mice, which show significant telomere shortening and dysfunction [105, 106].

#### **3.2. TAPs and NF-κB pathway**

RTEL1 has also been shown to interact with proliferating cell nuclear antigen (PCNA) and this

The human CST complex consisting of CTC1, STN1, and TEN1 proteins plays a role in telomere protection and DNA metabolism [88]. Each telomerase RNP is believed to add 50–60 nucleo‐ tides to most telomeres following a single initiation event [89]. The CST complex has been proposed to set the upper limit of telomere elongation by binding to telomeric single-stranded DNA (ssDNA) and displacing telomerase, once telomeric overhang has reached certain length [90]. Study reported by Chen et al showed that CST competes with POT1–TPP1 for telomeric DNA [90]. It terminates telomerase activity through primer sequestration and physical association with the POT1–TPP1 subunits, which functions as a telomerase processivity factor [91]. CST–telomeric-DNA binding increases during late S/G2 phase following telomerase activity, concurrently with telomerase turn-off. Attenuation of CST enables excessive telomer‐ ase activity, fostering telomere elongation. It is suggested that through binding of the telo‐ merase-extended telomere, CST limits telomerase activity at individual telomeres to

TCAB1 (encoded by gene *WDR79*) contains a proline-rich region and WD40 motif and is localized in CBs. CBs have been shown to accumulate telomerase as well as associate with telomeres [78]. TCAB1 has been demonstrated to be a component of active telomerase and is necessary for the telomerase holoenzyme to accumulate in CB and thus regulate telomere elongation [92]. TCAB1 interacts with CAB box motif of *TR* and functions in telomerase assembly by driving telomerase to CB. Subsequent report by Stern et al showed that TCAB1 and CB are required for telomerase recruitment to telomeres independent of each other [93].

TERRA functions as a negative regulator of telomere length. It is transcribed from subtelomeric regions of telomeres that consist of UUAGGG repeats. TERRA may inhibit telomerase in cis

When the genomic DNA undergoes any damage such as single-strand DNA breaks, doublestrand breaks (DSBs), nicks, or chromosome fusions, cells activate DNA repair pathway depending on the type of damage. During this process, cells seize to grow and initiate the repair, and once the repair is completed growth resumes; otherwise, they undergo apoptosis. Telomeres can also be sensed as breaks by cellular machinery. However, telomerase and

**3. Cross talk of telomere-associated proteins with cellular signaling**

interaction is important to prevent telomere fragility [87].

approximately one binding and extension event per cell cycle.

**Telomerase Cajal body protein 1 (TCAB1)**

by directly binding to telomerase [94, 95].

**3.1. TAPs and DNA damage response**

**CST complex**

52 Telomere - A Complex End of a Chromosome

**TERRA**

**pathways**

NF-κB transcription factors are key mediators of various cellular, inflammatory, and devel‐ opment pathways [107]. NF-κB family consists of five transcription factors namely RelA (p65), RelB, c-Rel, and also includes p50 and p52 (processed from p105 and p100, respectively). Rel family proteins possess REL homology domain, which harbors DNA-binding domain, dimerization, and IκB-binding domain. NF-κB family members are held inactive in cytoplasm by IκB family of proteins. Upon stimulation, IkB proteins are phosphorylated by IκB kinase (IKK) complex and thus degraded or processed to produce active dimers, which can enter the nucleus and activate the transcription response [107]. TAFs, which have been shown to modulate NF-κB signaling, independent of their role at telomeres, include shelterin complex protein RAP1 and telomerase complex protein TERT [108, 109].

A fraction of cytosolic RAP1 associates with functional IKK complex. RAP1 increases the efficiency of IKK complex in phosphorylating p65 subunit at serine 536; however, it has no effect on the degradation of IκBa inhibitory protein, thus functioning as an adaptor in the IKK complex [110]. In line with this, it was recently shown that RAP1 regulates cytokine levels followed by fine healing of corneal injury by effective modulation of NF-κB signaling [111]. Since RAP1 is also localized in cytoplasm, there are possibilities that it might function as an adaptor in various other complexes under different stimuli.

TERT has also been shown to associate with p65 in the nuclear compartment, thus directly regulating its transcriptional response. Akiyama et al demonstrated that TERT associates with p65 to mediate its translocation to nucleus in multiple myeloma cells [112]. Recently, our group showed that TERT associates with p65 to directly affect its transcriptional output [113]. We showed that *TR-*knockout mice are more resistant to inflammatory agent lipopolysaccharide (LPS), which majorly functions by activating NF-κB pathway. Ectopic expression of TERT led to increased proliferation of cancer cells as well as xenograft model, which could be abrogated by inhibiting p65. Subsequently, another group showed that TERT regulates matrix metallo‐ proteinase (MMP) expression independently of telomerase activity via activation of NF-κBdependent transcription [114].

#### **3.3. TAPs and WNT/β-catenin pathway**

WNT pathway plays a key role in development processes like cell-fate determination, progenitor cell proliferation, and cell polarity [115]*.* In canonical WNT pathway, WNT ligand binds to its receptor leading to stabilization of β-catenin in the cytoplasm. Stabilized β-catenin then enters nucleus to activate transcription through its interaction with TCF/Lef family members. It further recruits chromatin remodelers like BRG1 to facilitate transcription.

TERT is the only TAP shown to modulate WNT pathway independent of telomeres and telomerase catalytic activity. The first evidence demonstrating a link between TERT and WNT pathway came from Choi et al who showed that knock in of catalytically inactive TERT in hair follicle stem cells led to their proliferation [116]*.* Changes in gene expression as analyzed by microarray, revealed differential expression of genes involved in development/morphogene‐ sis, signal transduction, and cytoskeleton/cell adhesion signaling pathways. Modulated gene expression pattern strongly correlated with transcriptional program of MYC and WNT, suggesting existence of a potential association of TERT with the WNT and MYC pathways. Subsequently, Park et al demonstrated the first evidence of the direct regulation of Wnt/βcatenin signaling by telomerase in mouse embryonic stem cells and *Xenopus laevis* embryos. The study reported that TERT functions as a cofactor in the β-catenin transcriptional complex through its interaction with Brg1, a chromatin-remodeling factor [117]. Ectopic expression of TERT or catalytically inactive TERT led to the activation of WNT-dependent reporters in vitro and in vivo, while chromatin immunoprecipitation assays uncovered TERT localization at WNT target gene promoters. It was shown that TERT null mice display partially penetrant homeotic transformation of vertebrae, due to the loss of 13th rib of one or both the vertebrae. Recently, another group reported that TERT forms a complex with Brg1, together with nucleostemin (NS), a nucleolar GTP-binding protein and/or its family member GNL3L and is essential for maintenance of the tumor-initiating cell phenotype in human cancer cells [118].

#### **3.4. TAPs and MYC**

Recently, we reported that TERT regulates MYC transcription [119]. It is well known that MYC directly regulates TERT transcription [120]. However, our study illuminated the existence of a feed-forward loop between TERT and MYC in MYC-driven cancers such as lymphomas. Using genetic and biochemical approaches, we showed that the absence of TERT delayed MYC-dependent lymphomagenesis and strikingly, this effect was not observed when the RNA component of telomerase, *TR* was removed. Using in vivo and in vitro approaches, we established that TERT stabilizes MYC and thus results in increased MYC-dependent tran‐ scriptional output. Furthermore, we showed that this effect of TERT on MYC stability was independent of its catalytic activity. Mechanistically, we showed that TERT associates with MYC, preventing its proteasomal degradation, thus stabilizing its protein levels [119].

#### **3.5. TAPs and mitochondria**

showed that TERT associates with p65 to directly affect its transcriptional output [113]. We showed that *TR-*knockout mice are more resistant to inflammatory agent lipopolysaccharide (LPS), which majorly functions by activating NF-κB pathway. Ectopic expression of TERT led to increased proliferation of cancer cells as well as xenograft model, which could be abrogated by inhibiting p65. Subsequently, another group showed that TERT regulates matrix metallo‐ proteinase (MMP) expression independently of telomerase activity via activation of NF-κB-

WNT pathway plays a key role in development processes like cell-fate determination, progenitor cell proliferation, and cell polarity [115]*.* In canonical WNT pathway, WNT ligand binds to its receptor leading to stabilization of β-catenin in the cytoplasm. Stabilized β-catenin then enters nucleus to activate transcription through its interaction with TCF/Lef family members. It further recruits chromatin remodelers like BRG1 to facilitate transcription.

TERT is the only TAP shown to modulate WNT pathway independent of telomeres and telomerase catalytic activity. The first evidence demonstrating a link between TERT and WNT pathway came from Choi et al who showed that knock in of catalytically inactive TERT in hair follicle stem cells led to their proliferation [116]*.* Changes in gene expression as analyzed by microarray, revealed differential expression of genes involved in development/morphogene‐ sis, signal transduction, and cytoskeleton/cell adhesion signaling pathways. Modulated gene expression pattern strongly correlated with transcriptional program of MYC and WNT, suggesting existence of a potential association of TERT with the WNT and MYC pathways. Subsequently, Park et al demonstrated the first evidence of the direct regulation of Wnt/βcatenin signaling by telomerase in mouse embryonic stem cells and *Xenopus laevis* embryos. The study reported that TERT functions as a cofactor in the β-catenin transcriptional complex through its interaction with Brg1, a chromatin-remodeling factor [117]. Ectopic expression of TERT or catalytically inactive TERT led to the activation of WNT-dependent reporters in vitro and in vivo, while chromatin immunoprecipitation assays uncovered TERT localization at WNT target gene promoters. It was shown that TERT null mice display partially penetrant homeotic transformation of vertebrae, due to the loss of 13th rib of one or both the vertebrae. Recently, another group reported that TERT forms a complex with Brg1, together with nucleostemin (NS), a nucleolar GTP-binding protein and/or its family member GNL3L and is essential for maintenance of the tumor-initiating cell phenotype in human cancer cells [118].

Recently, we reported that TERT regulates MYC transcription [119]. It is well known that MYC directly regulates TERT transcription [120]. However, our study illuminated the existence of a feed-forward loop between TERT and MYC in MYC-driven cancers such as lymphomas. Using genetic and biochemical approaches, we showed that the absence of TERT delayed MYC-dependent lymphomagenesis and strikingly, this effect was not observed when the RNA component of telomerase, *TR* was removed. Using in vivo and in vitro approaches, we established that TERT stabilizes MYC and thus results in increased MYC-dependent tran‐

dependent transcription [114].

54 Telomere - A Complex End of a Chromosome

**3.4. TAPs and MYC**

**3.3. TAPs and WNT/β-catenin pathway**

Among TAPs, TERT and TIN2 have been shown to be involved in regulating mitochondrial activity. It has been shown that TERT translocates to mitochondria under certain stress conditions [121–123]. Mitochondrial TERT binds to and protects mitochondrial DNA from hydrogen peroxide-induced oxidative damage [103, 124]. Overexpression and knockdown studies involving TERT in cancer cells have shown that the role of TERT in mitochondrial pathway of apoptosis is independent of its catalytic activity [122, 125]. Interestingly, it has also been shown that TERT functions as a reverse transcriptase in mitochondria using mitochon‐ drial tRNA as a template [126]. Furthermore, it has been shown that TERT can interact with RNA component of mitochondrial RNA-processing endoribonuclease (*RMRP*) to form a complex similar to RNA-dependent RNA polymerase (RdRP). This complex then affects gene silencing at the post-transcriptional level [126].

TIN2 has also been shown to localize to mitochondria, where it results in altered mitochondrial structure. The group showed that the reduction of TIN2 levels led to augmented mitochondrial oxidative phosphorylation and reduced aerobic glycolysis in cancer cells [127].

#### **3.6. TAPs and miscellaneous associations**

Apart from the above-described associations of TAPs with cellular machinery, there are various reports about many more interacting partners. TRF2 has been shown to function as a transcriptional activator by directly binding to promoter of the angiogenic tyrosine kinase platelet-derived growth factor receptor β (PDGFRβ). This study highlighted the angiogenic role of TRF2 uncoupled from its telomere-capping role [128]. Telomerase was shown to regulate rDNA transcription by directly associating with RNA polymerase I upon hyperpro‐ liferative stimuli such as during liver regeneration and Ras-induced hyperproliferation [129].

Figure 2 summarizes the role of telomere-interacting proteins in cross talk with cellular signaling pathways.
