Tải bản đầy đủ - 0 (trang)
2 Roles of the Shelterin Complex at the Telomeres: T-Loop Maintenance, Telomere Length Regulation and Suppression of DDR

2 Roles of the Shelterin Complex at the Telomeres: T-Loop Maintenance, Telomere Length Regulation and Suppression of DDR

Tải bản đầy đủ - 0trang

Telomeres and Chromosome Stability



131



length (Lu et al. 2013; Cheung et al. 2012). Structural and mutational studies have

demonstrated the importance of the TRFH domain in protein homodimerization,

high-affinity DNA binding and recruitment to telomeres (Fairall et al. 2001).

Extensive replicative telomere erosion causes telomere deprotection and the

activation of the tumor suppressor p53. Phosphorylation of p53 by the ATM (ataxia

telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3 related) kinases

results in cellular senescence or apoptosis (Karlseder et al. 1999). An elegant study

by de Lange’s group directly addressed the role of the telosome in telomere

end-protection, by completely removing all the components of the shelterin complex from mouse telomeres (Sfeir and de Lange 2012). They reported the recruitment of general DDR factors by the shelterin complex and the activation of six

distinct repair pathways: ATM, ATR, classical-NHEJ (non-homologous end joining), alternative-NHEJ, HR (homologous recombination) and resection (Sfeir and

de Lange 2012).

The function of TRF2 and POT1 in protection and preventing telomere end-toend fusion is well established (Lu et al. 2013). Removing TRF2 or the use of a

dominant negative TRF2 leads to spontaneous telomere deprotection (uncapping)

and cellular senescence. The association of TRF2 with Apollo (SMN1/Pso2-type

nuclease) promotes telomere end-processing and the generation of the 30 overhang

by the resection of the 50 strand, thus supporting the efficient formation and

stabilization of the T-loop (Longhese et al. 2012). The use of TRF2-depleted

mouse embryonic fibroblasts (MEFs) provided evidence for the function of TRF2

in the repression of the ATM pathway. In TRF2À/À MEFs, accumulation of the

MRN (Mre11, Rad50 and Nbs1) complex at the leading telomeric strand and

phosphorylation of H2AX in the subtelomeric and telomeric chromatin are readily

observable (de Lange 2010). High levels of telomere-dysfunction-induced foci

(TIFs) characterized by enrichment of DDR factors such as 53BP1 at the telomeres

are also induced (de Lange 2010). A TRF2-ATM-p53 positive-feedback loop

implicating the Siah1 E3 ubiquitin ligase was proposed to function in the amplification of the telomere uncapping-induced p53-mediated DDR (Horikawa

et al. 2011). Their results suggest that the reduced level of TRF2 induces ATM

pathway activation to phosphorylate p53. Activated p53 subsequently activates

Siah1 to target TRF2 for degradation, leading to a further decrease in TRF2 levels

at the telomeres. TRF2 is also important to prevent unwanted telomeric repair and

end-to-end fusion by the NHEJ pathway and the generation of lethal dicentric

chromosomes at mitosis. TRF2 hinders the association of the NHEJ proteins

Ku70/Ku80 with the telomere by promoting the formation and stabilization of the

T-loop structure (de Lange 2010). Thus, TRF2 plays a central role in telomere

end-protection.

Protection of the single-stranded region of the telomeres is mediated by POT1, a

single-stranded DNA binding protein with two oligosaccharide/oligonucleotide

binding (OB) folds. The localization of POT1 relies on the interaction of its

C-terminus with TPP1 to form a heterodimer linked to the rest of the shelterin

complex via the TPP1-TIN2 interaction. While the telomeres are in an extendible

state (open T-loop), POT1 coats the 30 G-rich overhang. However, when the



132



T.W. Chu and C. Autexier



telomeres are in a closed configuration, POT1 binds to the short single-stranded

DNA segment of the D-loop (de Lange 2010; Cristofari and Lingner 2006)

(Fig. 1a). POT1 regulates telomeric 50 -end resection by inhibiting nucleases such

as Apollo, and therefore, is a determinant of the 50 chromosomal end sequences

(Hockemeyer et al. 2005; Latrick and Cech 2010). POT1 negatively controls

telomere length by limiting the access of telomerase to the telomeres and inhibits

telomerase activity in vitro (Lei et al. 2004). Interestingly, when in a complex with

TPP1, POT1 acts synergistically to enhance telomerase activity and telomere

elongation, playing dual opposing roles in telomerase and telomere regulation

(Wang and Lei 2011). Studies of mouse POT1 demonstrated its importance in

repressing NHEJ and HR at telomeres (He et al. 2006). POT1 inhibits the ATR

pathway and its interaction with TIN2 and TPP1 allows it to effectively compete

with the ATR activation protein, RPA (replication protein A) for binding to

telomeres (de Lange 2010). RPA is a single-stranded DNA binding heterotrimer

which participates in several pathways of DNA metabolism, including DNA replication and damage repair (Zou et al. 2006).

TIN2 is the core subunit of the shelterin complex and cooperates with TRF2 to

inhibit the activation of the ATM pathway, facilitates TPP1-mediated recruitment

of telomerase and contributes to telomere length homeostasis (Lu et al. 2013).

Mammalian Rap1 does not bind to telomeric DNA and its association with the

shelterin complex relies on its interaction partner TRF2. Furthermore, using mass

spectrometry, Songyang’s group identified several DDR factors that interact with

the Rap1-TRF2 complex, such as Rad50, Mre11, PARP1 and Ku86/70 (O’Connor

et al. 2004). Another study identified the DNA repair protein BTBD12 (human

orthologue of yeast SLX4, a nuclease implicated in resolution of recombination

intermediates) as an interactor of Rap1-TRF2 and suggests a role for SLX4 in the

repression of HR (Svendsen et al. 2009).

In addition to its function in recruiting POT1 to the telomeres, TPP1 also directly

interacts with the telomerase essential N-terminal (TEN)-domain of hTERT, via

seven residues termed the TEL patch which are located within its OB fold, to recruit

telomerase to the telomeres (Zhong et al. 2012; Nandakumar et al. 2012; Schmidt

et al. 2014). Importantly, the TPP1-POT1 heterodimer acts to stimulate telomerase

enzyme processivity (discussed below), an important determinant of telomere

length maintenance (Cifuentes-Rojas and Shippen 2012). Furthermore, TPP1 also

interacts with the single-stranded DNA binding protein Stn1, a component of the

CST complex (discussed below) involved in 50 -end resection (Wan et al. 2009).

These, and new data, highlight the role of TPP1 in regulating chromosomal

end-protection, telomerase recruitment and activation, and telomere length homeostasis set point (Sexton et al. 2014).

Although individual telosome components regulate the telomere negatively or

positively, the telosome complex is a negative regulator of telomere length. Proper

control of telomere homeostasis sets the telomere length within a range of nucleotides. This is accomplished using a negative feed-back system dependent on the

number of telomere-bound shelterin proteins, principally the negative regulator

POT1 (Diotti and Loayza 2011). The number of shelterin complexes bound to the



Telomeres and Chromosome Stability



133



telomeres is directly proportional to telomere length and thus, dictates telomere

length regulation and homeostasis (Diotti and Loayza 2011). In the protein counting

model (Marcand et al. 1997), as telomeres lengthen, the number of binding sites

increases, and more shelterin proteins can bind, including POT1 that associates

with the 30 single-stranded telomeric DNA (Lei et al. 2004). Recruitment of POT1

enhances the inhibitory effect at the telomere by acting in cis to block the access of

telomerase to the telomeric 30 overhang (Diotti and Loayza 2011). In contrast, as

telomeres shorten, the number of bound inhibitory proteins decreases and consequently, drives the telomeres to switch into an open state to allow telomere

elongation by telomerase. TPP1 also controls stem cell telomere length homeostasis

via a feedback regulation of telomerase by telomere length (Sexton et al. 2014).



3.3



The CST Complex



The CST complex, composed of Cdc13, Stn1 and Ten1, was first identified in yeast

as a heterotrimer with structural similarity to the RPA protein complex (Chen and

Lingner 2013). The CST complex binds to the 30 telomeric overhang and contributes to its protection. Studies identified yeast homologs of Stn1 and Ten1 in humans

in addition to a third peptide, CTC1, which possesses no conservation with Cdc13

but still trimerizes with the other two components to localize to the telomeric 30 end.

Human CTC1 and STN1 enhance the telomere binding affinity and the activity of

DNA polymerase α-primase to facilitate telomere replication and stimulate

C-strand fill-in synthesis (Chen and Lingner 2013). Also, the human CST complex

competes with the heterodimer POT1-TPP1 for binding to the telomeres. The

interaction of CST with telomeres peaks in late S/G2 phase, which corresponds to

the timing of post-telomere replication by telomerase and therefore limits telomere

over-elongation by terminating telomerase’s action at telomeres (Fig. 1a).



4 Telomere Length Maintenance

Telomere homeostasis is a dynamic process and is the end result of two opposing

forces: telomere maintenance or lengthening and telomere erosion. The majority of

human stem, germ and cancer cells relies on the enzyme telomerase to counteract

telomere attrition (Calado and Dumitriu 2013) but in some human cancer and

immortalized cell lines, a homologous-recombination-based mechanism is used,

termed the alternative lengthening of telomeres (ALT) (O’Sullivan and Almouzni

2014).



134



4.1



T.W. Chu and C. Autexier



Enzyme-Based Telomere Maintenance: Telomerase



In the mid-1980s, Blackburn and Greider’s work using Tetrahymena themophila

extracts identified an enzyme, telomerase, with terminal transferase activity that

adds tandem TTGGGG repeats to synthetic telomeric substrates (Greider and

Blackburn 1985). Telomerase is a specialized reverse transcriptase that counteracts

telomere shortening during cell replication. Following birth, expression of telomerase is down-regulated and is virtually undetectable in normal differentiated human

somatic cells, with the exception of the highly proliferative stem cell compartments

or the germ cells. Consequently progressive telomere shortening occurs throughout

the lifetime of the organism. Ectopic expression of telomerase allows normal

human cells to bypass senescence and extends their replicative lifespan (Bodnar

et al. 1998), suggesting that telomerase expression is a limiting factor for cellular

proliferation. Additionally, telomerase preferentially extends short telomeres in

human cells (Britt-Compton et al. 2009) and a recent study demonstrated that the

onset of replicative senescence in Saccharomyces cerevisiae is controlled by the

length of the shortest telomere (Xu et al. 2013). Importantly, 85 % of human cancers

express telomerase to overcome telomere shortening (Shay et al. 2012) through the

addition of ~60 nucleotides at each round of extension (Zhao et al. 2011). During

the last three decades, significant resources were invested to understand the structure and function of the enzyme in anticipation of developing specific and effective

telomerase-based anti-cancer therapies.



4.2



Telomerase Structure



Human telomerase is minimally composed of the catalytic subunit hTERT (human

telomerase reverse transcriptase) and its integral RNA component hTR or hTERC

(human telomerase RNA; human telomerase RNA component), which serves as a

template for the de novo synthesis of telomeres (Fig. 1a). In vivo, the presence of

additional accessory proteins is essential for the proper biogenesis, cellular trafficking and catalytic activity of the telomerase holoenzyme. Telomerase associates

with the dyskerin complex, pontin, reptin and TCAB1. DKC1 (dyskerin), GAR1,

NHP2 and NOP10 are members of the dyskerin complex and are implicated in

holoenzyme assembly and the maintenance of telomerase RNA stability (Mitchell

et al. 1999). In vivo assembly of the active enzyme also requires the ATPases pontin

and reptin (Venteicher et al. 2008). TCAB1 (WDR79), a WD repeat containing

protein which binds to dyskerin and the telomerase RNA, is essential for the

accumulation of hTR in the Cajal bodies (CB), for the trafficking of the holoenzyme

to the telomeres (Venteicher and Artandi 2009) and for licensing of catalytically

active telomerase ribonucleoprotein (Vogan and Collins 2015).

The crystal structures of the Tribolium castaneum TERT provided tremendous

insights into the structural and functional organization of the catalytic subunit,



Telomeres and Chromosome Stability



135



despite the absence of the N-terminal TEN domain in the beetle TERT (Mitchell

et al. 2010). Based on this structure which highly resembles that of HIV-reverse

transcriptase (RT), TERT forms a ring that can accommodate up to 8 bases of the

DNA-RNA hybrid, whereby the 30 end of the telomeric substrate is positioned

within the active site to allow catalysis (Mitchell et al. 2010). Single particle

electron microscopy enabled the 3D visualization of the active full length human

telomerase as two molecules of hTERT associated with two molecules of hTR that

can function as a dimer to bind to two telomeric DNA substrates (Sauerwald

et al. 2013).

Unlike other conventional nucleic acid polymerases, telomerase possesses the

unique ability to reiteratively add long stretches of telomeric DNA to the ends of the

chromosomes using the short template (one and a half repeats in length for hTR)

within its integral RNA component. The addition of multiple telomeric repeats is

accomplished through repeated rounds of nucleotide addition, enzyme dissociation,

translocation and realignment with the newly synthesized telomeres. This complex

property, termed “repeat-addition processivity” (RAP), is tightly regulated and is an

important determinant of telomere length homeostasis (D’Souza et al. 2013).



4.3



Telomerase Regulation



Telomerase is under very strict molecular and cellular control to ensure proper

telomere length regulation, with the transcriptional regulation of the TERT component being the limiting component of enzyme activity (Cifuentes-Rojas and

Shippen 2012). Furthermore, cellular compartmentalization, post-transcriptional

and translational modifications, as well as interactions with associated proteins

also contribute to the multi-layer control of telomerase function.

The most essential requirement for human telomere maintenance in telomerasepositive cells is the recruitment and localization of both hTERT and hTR to their

site of action, the telomeres. Cellular trafficking of telomerase is cell cycle regulated (Tomlinson et al. 2006). TERT and TR are synthesized separately and

accumulate in distinct subnuclear compartments in G1 (Tomlinson et al. 2006).

hTR, through its interaction with TCAB1, accumulates in the CB for further

processing and maturation whereas hTERT is enriched in nucleoplasmic foci

termed TERT foci (Tomlinson et al. 2006). In early and mid-S phase, TERT foci

are found adjacent to hTR-containing CB and colocalize with telomeres

(Tomlinson et al. 2006). The mechanism by which hTERT associates with hTR

and is shuttled to telomeres remains to be elucidated. A recent work by Collins’s

group demonstrated that hTERT is stably associated with hTR throughout the cell

cycle but the association between hTR and TCAB1 is disrupted in M-phase cells,

thus ensuring localization of telomerase to the telomeres in a cell cycle-dependent

manner (Vogan and Collins 2015).

Numerous proteins are engaged in the recruitment of telomerase to the telomeres, including the shelterin protein TPP1, PinX1, and finally, the most recently



136



T.W. Chu and C. Autexier



identified double-stranded telomeric DNA binding protein HOT1, a positive regulator of telomere length (Kappei et al. 2013). Telomere repeat-containing non-coding RNAs (TERRA) (discussed below) inhibit telomerase action in cis by binding

to telomeres and interacting with telomerase through complementary base-pairing

with the RNA template. Thus, TERRA negatively impacts telomerase function and

telomere length (Redon et al. 2010). Another regulator of telomerase, the hPif1

DNA/RNA helicase, interacts with hTERT and preferentially binds to telomeric

DNA, negatively influencing telomere length (Cifuentes-Rojas and Shippen 2012).

A new study in yeast demonstrated that Pif1 can efficiently remove telomerase from

the telomeres in a length dependent manner, thereby regulating telomerase activity

and telomere length (Li et al. 2014).



4.4



Homologous Recombination Based: Alternative

Lengthening of Telomeres (ALT)



Although the majority of human cancers expresses telomerase as a mean to confer

unlimited replicative potential, at least 10 % of human cancers rely on a homologous recombination-based TMM, termed alternative lengthening of telomeres, or

ALT (O’Sullivan and Almouzni 2014). The exact mechanisms controlling

ALT-mediated telomere maintenance are unknown and therefore, targeting of

ALT cancer cells is not yet possible.



4.5



Characteristics of ALT Cells



ALT cells are distinguished by their highly heterogeneous telomere length resulting

from HR-based template copying from a sister chromatid or another chromosome.

ALT cells display noticeably higher incidences of telomeric-sister-chromatid

exchange events compared to telomerase-positive cells (O’Sullivan and Almouzni

2014). ALT cells also exhibit elevated levels of extrachromosomal telomeric repeat

DNA including c-circles which are composed of a full-length telomeric C-rich

strand hybridized to a partial G-rich strand (Henson et al. 2009). C-circles represent

the most specific and quantifiable marker of ALT reported to date, are 750-fold

more abundant in ALT-positive cells compared to ALT-negative cells and are

detected in blood and tumor tissue from ALT cancer patients (Henson

et al. 2009). The mechanism by which c-circles are generated is currently unknown.

ALT cells are also characterized by the presence of a special subtype of

promyelocytic leukemia (PML) nuclear body, termed ALT-associated PML body

(APB) (O’Sullivan and Almouzni 2014). PML bodies are usually involved in the

regulation of normal cellular functions such as DNA replication, transcription, and

DNA repair as well as tumor suppression. APBs contain the PML protein, telomeric



Telomeres and Chromosome Stability



137



DNA, telosome components, and factors implicated in DNA repair, replication and

recombination (O’Sullivan and Almouzni 2014). A recent study revealed a role for

PML bodies in telomeric chromatin compaction and telomeric repeat clustering,

leading to formation of APBs and depletion of TRF2 from the telomeres (Osterwald

et al. 2015). This subsequently promotes ATM autophosphorylation in the APBs

and activation of the DDR, whereby telomeres are replicated via the ALT pathway

(Osterwald et al. 2015).

In normal cells, three variants of the telomeric sequence (TGAGGG, TCAGGG,

TTGGGG) are detected at human telomeres and restricted to the telomeric proximal

end (Conomos et al. 2012). In ALT cells however, variant sequences are detectable

throughout the telomeres and are most likely the consequence of HR-based telomere replication (Conomos et al. 2012). The presence of non-cognate telomeric

sequences was proposed to decrease the sequence-specific binding of the telosome

and other telomere-associated proteins, and sequence-specifically recruit different

proteins such as nuclear receptors, potentially altering the architecture of ALT

telomeres to promote recombination (Conomos et al. 2012). For example, in ALT

cells, the ratio of TRF2/total amount of telomeric DNA is reduced compared to

ALT-negative cells (Conomos et al. 2013). Since TRF2 typically represses HR,

telomeres in ALT cells are therefore prone to HR (Conomos et al. 2013). A decrease

in the binding of the telosome in ALT cells could also explain the high levels of

genomic instability at ALT telomeres (TIFs) but nevertheless, ALT cells are able to

progress through the cell cycle, suggesting that the telomere uncapping is only a

transient state and that dysfunctional telomeres are a requirement for ALT

(Conomos et al. 2013).



4.6



Factors Regulating ALT



Numerous proteins associated with APBs, telomeres and HR are potentially implicated in the ALT pathway but only a few proteins or protein complexes are required

for ALT-mediated telomere length maintenance (Bhattacharyya et al. 2010; Cesare

and Reddel 2010). These include the MRN and SMC5/6 ‘structural maintenance of

chromosome’ recombination complexes, consistent with ALT being

HR-dependent, the MUS81 and FEN1 endonucleases, and the FANCD2 and

FANCA Fanconi anemia proteins.

No ALT inhibitors have been developed to date primarily because the molecular

mechanisms governing the ALT pathway are incompletely characterized. A better

understanding of the ALT mechanism will greatly contribute to the development of

therapeutic approaches to specifically target ALT. One major challenge in the

development of ALT specific inhibitors is that proteins with potential roles in

telomeric recombination and the ALT pathway also regulate DNA transactions,

including DDR proteins, recombination proteins, helicases and nucleases and are

present in normal cells (Cesare and Reddel 2010). Nonetheless, depletion of

MUS81, a DNA structure-specific recombination endonuclease induces decreased



Tài liệu bạn tìm kiếm đã sẵn sàng tải về

2 Roles of the Shelterin Complex at the Telomeres: T-Loop Maintenance, Telomere Length Regulation and Suppression of DDR

Tải bản đầy đủ ngay(0 tr)

×