**2. Telomere-Associated Protein Complexes (TAPs)**

#### **2.1. Shelterin**

damage response (DDR) followed by cell cycle arrest [4]. Telomeres are bound by nucleosomes and a specialized complex known as shelterin, which is composed of six core protein subunits [5]. Shelterin determines the structure of telomeres. It is implicated in the formation of t-loops and also regulates the synthesis of telomeric DNA [6]. Additional proteins capable of inter‐ acting with shelterin proteins, such as DNA damage proteins, also play a role in maintaining

**TCAB1**

**NOP10**

**GAR1**

**NHP2**

3'

5'

**Figure 1.** Schematic representation of telomere end structure (A) and Telomere associated protein complexes (B).

**TERT**

**DYSKERIN**

3'

*TR*

5'

**CAJAL body (Telomerase maturation)**

**Telomerase Holoenzyme** 

*TR*

**TERT**

telomere length and chromosomal stability [7].

46 Telomere - A Complex End of a Chromosome

**STN1**

**TEN1**

**CST Complex (Terminates telomere elongation)**

**RTEL1**

**Helicase (t-loop resolution)**

**TRF2 TRF1**

**TIN2**

**(Telomere protection)**

5'

**CTC1**

**TPP1**

**POT1**

**(Telomere elongation) Shelterin**

t-loop D-loop 3 '

B

5'

3'

**RAP1**

A

Shelterin, as the name suggests, provides shelter to the ends of linear chromosomes by repressing DNA damage-signaling responses, masking telomeres from DNA repair machi‐ nery, and regulating the length of telomeres [7, 14]. Shelterin is a highly specialized complex comprising six central components namely, TTAGGG repeat-binding factors TRF1 and TRF2, TRF1-interacting protein (TIN2), protection of telomeres protein (POT1), Pot1-interacting protein (TPP1), and repressor/activator protein (RAP1) [15]. All shelterin components are ubiquitously expressed and associated with telomeres throughout the cell division cycle. The complete abrogation of all shelterin subunits (except RAP1) in mice results in embryonic lethality, thereby implicating their essential roles in development [16].

#### **TRF1**

TRF1 was the first shelterin complex subunit to be discovered that specifically associates with double-stranded telomeric DNA, mainly as a dimer through its TRF homology domain. Recently, it has been shown that TRF1 efficiently associates with telomeric DNA in nucleoso‐ mal context and is capable of remodeling telomeric nucleosomal arrays [17]. TRF1 interacts with TIN2 in the shelterin complex as shown in Figure 1B. It also functions as a negative regulator of telomere length in telomerase-positive cells. In addition, some reports have demonstrated that it is essential for survival independent of its telomere length regulatory activity [18]. TRF1 genome-wide binding analysis revealed that it exclusively localizes at telomeres under normal conditions as well as under extreme telomere shortening unlike other shelterin members which have extra telomeric roles [19]. TRF1 has also been shown to assist Aurora-B recruitment to centromeres, thus contributing to appropriate chromosome segrega‐ tion and maintenance of genomic integrity [20].

#### **TRF2**

TRF2 is highly similar to TRF1 in terms of protein sequence except that the N-terminal domain in TRF2 is acidic while that in TRF1 is rich in glycine and arginine residues (forming GAR domain). It possesses TRF homology domain, which mediates its dimerization. This Nterminal domain of TRF1 and TRF2 has been shown to regulate their ability to condense telomeric DNA [21]. TRF2 has been proposed to stabilize the t-loop by invasion of the upstream TTAGGG double-stranded region [22]. TRF2 has also been shown to bind at internal genomic regions, mainly at TTAGGG repeats referred to as interstitial telomeric sequences (ITSs) [23, 24]. Recent evidence suggests the role of TRF2 in the formation of novel chromosome end structures, which involve telomeres interacting with nontelomeric DNA, forming long-range chromosome loop that encompasses several megabases of chromatin and are known as interstitial telomeric loops (ITLs) [25]. Telomere-bound TRF2 is necessary to suppress the ataxia telangiectasia mutated (ATM)-dependent DNA damage response pathway [26] and the nonhomologous end joining (NHEJ) DNA repair pathway, thus playing a major role in protecting chromosome ends [9]. TRF2 also assists telomere replication by limiting resolvase activities leading to accurate repair of stalled forks [27]. It has been demonstrated that both TRF1 and TRF2 are modified post-translationally; however, the physiological relevance of these modifications is not yet completely understood [7].

#### **TIN2**

TIN2 (encoded by *TINF2*) associates with both TRF1 and TRF2, thus forming a bridge that connects the double-stranded telomeric DNA-binding proteins to those bound to singlestranded telomeric overhang region [28, 29]. A recent study by Frank et al demonstrates that TIN2 facilitates the recruitment of telomerase to telomeres [30]. In this study, the authors discovered a novel mutation in *TINF2* gene (which encodes TIN2 protein) and used novel functional assays to demonstrate a direct role for TIN2 in regulating telomere length through telomerase. This role is completely independent of its role in telomere protection.

#### **TPP1**

TPP1 interacts with TIN2 and POT1 through its carboxyl terminus and central domain, respectively [31-33]. Structurally, TPP1 possesses four domains, namely, OB (oligonucleotide/ oligosaccharide-binding fold), RD (POT1 recruitment domain), S/T (serine-rich region), and TID (TIN2-interacting domain). It has been demonstrated that TPP1 recruits POT1 to telomeres [34, 35]. In addition, TPP1 has been shown to contain telomerase-interacting domain, suggest‐ ing a role for TPP1 in the recruitment of telomerase to chromosome ends [36]. Interestingly, it has been shown that TPP1–POT1 interacts with N-terminal region of telomerase protein subunit TERT (telomerase reverse transcriptase), while TPP1 alone is also capable of interact‐ ing with C-terminal TERT residues through TPP1-OB domain [37]. TPP1-OB domain has been shown to contain a TEL patch (TPP1 glutamate (E)- and leucine (L)-rich patch), which mediates telomerase recruitment and processivity independent of its role in telomere capping [38]. In human stem cells, it has been further proven that TPP1 indeed mediates recruitment and activation of telomerase and telomere length homeostasis [39].

### **POT1**

mal context and is capable of remodeling telomeric nucleosomal arrays [17]. TRF1 interacts with TIN2 in the shelterin complex as shown in Figure 1B. It also functions as a negative regulator of telomere length in telomerase-positive cells. In addition, some reports have demonstrated that it is essential for survival independent of its telomere length regulatory activity [18]. TRF1 genome-wide binding analysis revealed that it exclusively localizes at telomeres under normal conditions as well as under extreme telomere shortening unlike other shelterin members which have extra telomeric roles [19]. TRF1 has also been shown to assist Aurora-B recruitment to centromeres, thus contributing to appropriate chromosome segrega‐

TRF2 is highly similar to TRF1 in terms of protein sequence except that the N-terminal domain in TRF2 is acidic while that in TRF1 is rich in glycine and arginine residues (forming GAR domain). It possesses TRF homology domain, which mediates its dimerization. This Nterminal domain of TRF1 and TRF2 has been shown to regulate their ability to condense telomeric DNA [21]. TRF2 has been proposed to stabilize the t-loop by invasion of the upstream TTAGGG double-stranded region [22]. TRF2 has also been shown to bind at internal genomic regions, mainly at TTAGGG repeats referred to as interstitial telomeric sequences (ITSs) [23, 24]. Recent evidence suggests the role of TRF2 in the formation of novel chromosome end structures, which involve telomeres interacting with nontelomeric DNA, forming long-range chromosome loop that encompasses several megabases of chromatin and are known as interstitial telomeric loops (ITLs) [25]. Telomere-bound TRF2 is necessary to suppress the ataxia telangiectasia mutated (ATM)-dependent DNA damage response pathway [26] and the nonhomologous end joining (NHEJ) DNA repair pathway, thus playing a major role in protecting chromosome ends [9]. TRF2 also assists telomere replication by limiting resolvase activities leading to accurate repair of stalled forks [27]. It has been demonstrated that both TRF1 and TRF2 are modified post-translationally; however, the physiological relevance of

TIN2 (encoded by *TINF2*) associates with both TRF1 and TRF2, thus forming a bridge that connects the double-stranded telomeric DNA-binding proteins to those bound to singlestranded telomeric overhang region [28, 29]. A recent study by Frank et al demonstrates that TIN2 facilitates the recruitment of telomerase to telomeres [30]. In this study, the authors discovered a novel mutation in *TINF2* gene (which encodes TIN2 protein) and used novel functional assays to demonstrate a direct role for TIN2 in regulating telomere length through

TPP1 interacts with TIN2 and POT1 through its carboxyl terminus and central domain, respectively [31-33]. Structurally, TPP1 possesses four domains, namely, OB (oligonucleotide/ oligosaccharide-binding fold), RD (POT1 recruitment domain), S/T (serine-rich region), and TID (TIN2-interacting domain). It has been demonstrated that TPP1 recruits POT1 to telomeres [34, 35]. In addition, TPP1 has been shown to contain telomerase-interacting domain, suggest‐

telomerase. This role is completely independent of its role in telomere protection.

tion and maintenance of genomic integrity [20].

48 Telomere - A Complex End of a Chromosome

these modifications is not yet completely understood [7].

**TRF2**

**TIN2**

**TPP1**

POT1 interacts with TRF1 complex via protein–protein interaction with TPP1, and this interaction is believed to affect its loading on the single-stranded TTAGGG telomeric repeat [40]. POT1 has been predicted to associate with telomeric sequences at t-loop as well as D-loop through its OB-fold domains [32]. POT1 serves a fundamental role in telomere length regula‐ tion, since it functions as a terminal transducer of telomere length control [40]. POT1–TPP1 complex has been shown to increase RAP (consecutive repeats that telomerase can add to telomere before dissociating) with minimal effect on telomerase activity [41]. Further, POT1– TPP1 has been shown to reduce the dissociation rate of telomerase from its telomeric substrate and assist in the translocation step [42].

#### **RAP1**

RAP1 associates with TRF2 and thus localizes to telomeres. As a component of the shelterin complex, it is dependent on TRF2 [43, 44]. RAP1 has been shown to be indispensable for telomere function in mouse and human systems [45, 46]. Further, RAP1 has been shown to possess extra telomeric roles that will be described in later part of this chapter.

#### **2.2. Telomerase complex**

Telomeres in human somatic cells shorten with each replication cycle due to end-replication problem consequently leading to genomic instability and cell death. Telomerase elongates telomeres and thus maintain their replicative potential. The minimal components of telomer‐ ase are a catalytic protein subunit termed as telomerase reverse transcriptase (TERT) and an RNA template known as *Terc* (*TR*) [47]. Telomerase catalyzes the addition of TTAGGG sequences to the ends of the chromosomes during each replication cycle, thus preventing telomere attrition and maintaining genomic integrity. Telomerase activity is detected pre‐ dominantly in stem cells and cancer cells [48]. In normal somatic cells, telomerase activity is almost undetectable, consequently leading to shortened telomeres, which limit their life span. Furthermore, while *TR* is highly expressed in all the cell types, the levels of TERT are highly regulated at transcriptional level [49]. In somatic cells, TERT is transcriptionally turned off while stem cells display high expression of TERT, resulting in high telomerase activity. Ninety percent of cancer cells reactivate TERT expression either by mutation in *TERT* promoter or by activating oncogenic transcription factors such as NF-κB, MYC, and β-catenin that are known to activate TERT transcription [50]. This reactivation of TERT confers cancer cells with unlimited replicative potential. Distinct from its telomeric function, TERT has also been shown to cross talk with various signaling pathways and impart several additional functions to cancer cells [51].

### **Terc (TR)**

Mature human *TR* is a small noncoding RNA consisting of 451 nucleotides and serves as a template for de novo telomeric repeat synthesis by telomerase. Structurally, it is subdivided into four domains namely the pseudoknot domain, the CR4-CR5 domain, the CR7, and H/ACA domain [52]. These domains perform various functions including RNA binding, dimerization, and recruitment of telomerase to telomeres. The pseudoknot domain and the CR4/5 domain along with TERT constitute the minimal requirement for reconstituted in vitro telomerase activity [53, 54]. The CR7 and H/ACA domains are required for stability and localization of telomerase [55]. H/ACA motif consists of two hairpins connected by a short single-stranded stretch, the H box, and a terminal ACA region [56] which is found in small nucleolar and small Cajal body (CB) RNAs (snoRNAs and scaRNAs). H/ACA small nucleolar ribonucleoprotein complex (H/ACA snoRNP) catalyzes pseudouridylation of small RNAs like ribosomal RNA, which may serve to stabilize their conformation [57]. The H/ACA domain of *TR* is essential for assembly into ribonucleoprotein (RNP) with four member H/ACA–RNP complex which include the core heterotrimer (Dyskerin, NHP2, and NOP10) and a fourth factor, GAR1 [56]. Dyskerin represents the catalytic subunit of H/ACA complex [57]. Major function for the association of *TR* with dyskerin is its stabilization and nuclear retention. However, no pseudouridylation of *TR* has been reported [58]. Structurally, dyskerin contains a TruB domain that functions in eukaryotic ribosomal RNA processing. The TruB domain consists of two motifs, TruB I and TruB II. In addition, it has two nuclear localization (NL) signals, N-terminal and C-terminal, and the PUA, pseudouridine synthase and archaeosine transglycosylase, domain involved in RNA modification [59]. Dyskerin and NOP10 form the stable core to which GAR1 and NHP2 subsequently bind [60]. Mutations in these proteins affect the stability of *TR* consequently affecting telomere synthesis and result in various human diseases discussed in a later section of the chapter.

Although *TR* is transcribed by RNA polymerase II, it is not polyadenylated; instead, its 3′ end is formed by exonucleolytic cleavage up to the boundary element formed by the H/ACA domain, where further cleavage is prevented by dyskerin associated with RNA [61]. The tetrameric complex of dyskerin, NOP10, NHP2, and the chaperone NAF1 associates with *TR* cotranscriptionally and is essential for its accumulation [62]. The significance of this complex formation is highlighted by a number of telomere length-associated disorders with mutations in these factors which results in reduced levels of *TR* and thus telomerase activity [63].

*TR* also harbors specialized sequence elements in the terminal loop of its 3′ hairpin namely BIO box, which stimulates *TR* stability by H/ACA RNP formation, and CAB box, which it shares with the scaRNAs. CAB box is required for trafficking *TR* to the CB, where it receives its 2,2,7-trimethyl guanosine (TMG) cap, and NAF1 is replaced by GAR1 [57, 64, 65].

#### **TERT**

Human TERT is a large protein consisting of 1133 amino acids and it is subdivided into four distinct domains: (a) N-terminal extension (TEN), (b) TERT RNA-binding domain (TRBD), (c) the reverse transcriptase domain (RT), and (d) the C-terminal extension (CTE) [66]. TEN domain is essential for telomerase activity and functions in proper localization and correct positioning of its catalytic site on telomeric DNA [67]. The TRBD domain functions in telo‐ merase RNP assembly as well as RNA binding [68]. The RT domain of TERT forms the catalytic center in telomerase complex and consists of seven universally conserved RT motifs [69–71]. RT domain can be divided into two putative subdomains namely the fingers and the palm domains where fingers domain interacts with nucleic acid substrate while the palm domain contains the catalytic site [72]. CTE possesses the thumb domain and is sequentially not conserved among species [66]. CTE serves a critical role in catalytic activity and processivity of telomerase [73].

to cross talk with various signaling pathways and impart several additional functions to cancer

Mature human *TR* is a small noncoding RNA consisting of 451 nucleotides and serves as a template for de novo telomeric repeat synthesis by telomerase. Structurally, it is subdivided into four domains namely the pseudoknot domain, the CR4-CR5 domain, the CR7, and H/ACA domain [52]. These domains perform various functions including RNA binding, dimerization, and recruitment of telomerase to telomeres. The pseudoknot domain and the CR4/5 domain along with TERT constitute the minimal requirement for reconstituted in vitro telomerase activity [53, 54]. The CR7 and H/ACA domains are required for stability and localization of telomerase [55]. H/ACA motif consists of two hairpins connected by a short single-stranded stretch, the H box, and a terminal ACA region [56] which is found in small nucleolar and small Cajal body (CB) RNAs (snoRNAs and scaRNAs). H/ACA small nucleolar ribonucleoprotein complex (H/ACA snoRNP) catalyzes pseudouridylation of small RNAs like ribosomal RNA, which may serve to stabilize their conformation [57]. The H/ACA domain of *TR* is essential for assembly into ribonucleoprotein (RNP) with four member H/ACA–RNP complex which include the core heterotrimer (Dyskerin, NHP2, and NOP10) and a fourth factor, GAR1 [56]. Dyskerin represents the catalytic subunit of H/ACA complex [57]. Major function for the association of *TR* with dyskerin is its stabilization and nuclear retention. However, no pseudouridylation of *TR* has been reported [58]. Structurally, dyskerin contains a TruB domain that functions in eukaryotic ribosomal RNA processing. The TruB domain consists of two motifs, TruB I and TruB II. In addition, it has two nuclear localization (NL) signals, N-terminal and C-terminal, and the PUA, pseudouridine synthase and archaeosine transglycosylase, domain involved in RNA modification [59]. Dyskerin and NOP10 form the stable core to which GAR1 and NHP2 subsequently bind [60]. Mutations in these proteins affect the stability of *TR* consequently affecting telomere synthesis and result in various human

Although *TR* is transcribed by RNA polymerase II, it is not polyadenylated; instead, its 3′ end is formed by exonucleolytic cleavage up to the boundary element formed by the H/ACA domain, where further cleavage is prevented by dyskerin associated with RNA [61]. The tetrameric complex of dyskerin, NOP10, NHP2, and the chaperone NAF1 associates with *TR* cotranscriptionally and is essential for its accumulation [62]. The significance of this complex formation is highlighted by a number of telomere length-associated disorders with mutations in these factors which results in reduced levels of *TR* and thus telomerase activity [63].

*TR* also harbors specialized sequence elements in the terminal loop of its 3′ hairpin namely BIO box, which stimulates *TR* stability by H/ACA RNP formation, and CAB box, which it shares with the scaRNAs. CAB box is required for trafficking *TR* to the CB, where it receives

Human TERT is a large protein consisting of 1133 amino acids and it is subdivided into four distinct domains: (a) N-terminal extension (TEN), (b) TERT RNA-binding domain (TRBD), (c)

its 2,2,7-trimethyl guanosine (TMG) cap, and NAF1 is replaced by GAR1 [57, 64, 65].

diseases discussed in a later section of the chapter.

cells [51]. **Terc (TR)**

50 Telomere - A Complex End of a Chromosome

**TERT**

TERT interacts with chaperones HSP90 and p23 as well as with AAA+ ATPases pontin and reptin [74, 75]. HSP90 and p23 interact with active telomerase; however, reptin and pontin are reported to interact with a pool of TERT, which is not assembled into active complex suggesting their role in telomerase assembly [75]. In addition, reptin and pontin are known to interact with dyskerin and are necessary for H/ACA RNP assembly, which is an essential step in TR stability [75, 76].

TERT transcription and telomerase activity is highest in the S phase of the cell cycle [77], and telomerase recruitment to telomeres has been shown to be restricted to the S phase [78, 79].

TERT interacts with two TR elements. The TRBD associates with CR4/5 region of TR and RT domain of TERT associates with pseudoknot region of TR [53, 80]. The human telomerase RNP purified from HEK293T cells overexpressing TERT and TR has been shown to be a dimeric structure, which is around 28 nm in length [81]. Although many studies have suggested the existence of multiple copies of dyskerin, NOP10, NHP2, and GAR1 with human telomerase RNP, the presence of two catalytically active TERT has been controversial since its biological significance is not clearly understood [82].

Telomerase activity in cells is limited by the levels of TERT protein (reported to be around 600 molecules/cell). All the other components of telomerase RNP are abundant [77, 83]. In normal somatic cells, TERT expression is repressed epigenetically or due to lack of activating tran‐ scription factors such as MYC, NF-κB, NFAT, RAS/RAF pathway, Ets factor steroids, and HIF [84]. Thus, transcriptional reactivation of TERT represents one of the major mechanisms responsible for activating telomerase and thus achieving replicative immortality in cancers. Recently, many cancers have been reported to harbor *TERT* promoter mutations resulting in high TERT expression and telomerase activity [85]. This is further discussed in later sections of the chapter.

#### **2.3. Accessory proteins/complexes/factors for proper telomere maintenance**

#### **Regulator of telomere elongation helicase 1 (RTEL1)**

RTEL1 is a DNA helicase, which contains N-terminal helicase domains and a C-terminal extension [86]. TRF2 recruits RTEL1 to telomeres during S phase of cell cycle to assist t-loop disassembly. RTEL1 is required for stability, protection, and elongation of telomeres [87]. RTEL1 has also been shown to interact with proliferating cell nuclear antigen (PCNA) and this interaction is important to prevent telomere fragility [87].

#### **CST complex**

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 approximately one binding and extension event per cell cycle.

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

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**

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 by directly binding to telomerase [94, 95].
