International Journal of

Molecular Sciences

Review
Telomere Checkpoint in Development and Aging
Alla Kalmykova

Koltzov Institute of Developmental Biology, Russian Academy of Sciences, 119334 Moscow, Russia;
allakalm@idbras.ru

Abstract: The maintenance of genome integrity through generations is largely determined by the
stability of telomeres. Increasing evidence suggests that telomere dysfunction may trigger changes
in cell fate, independently of telomere length. Telomeric multiple tandem repeats are potentially
highly recombinogenic. Heterochromatin formation, transcriptional repression, the suppression of
homologous recombination and chromosome end protection are all required for telomere stability.
Genetic and epigenetic defects affecting telomere homeostasis may cause length-independent internal
telomeric DNA damage. Growing evidence, including that based on Drosophila research, points to
a telomere checkpoint mechanism that coordinates cell fate with telomere state. According to this
scenario, telomeres, irrespective of their length, serve as a primary sensor of genome instability that is
capable of triggering cell death or developmental arrest. Telomeric factors released from shortened or
dysfunctional telomeres are thought to mediate these processes. Here, we discuss a novel signaling
role for telomeric RNAs in cell fate and early development. Telomere checkpoint ensures genome
stability in multicellular organisms but aggravates the aging process, promoting the accumulation of
damaged and senescent cells.

Keywords: telomere; telomeric RNA; chromatin; germline; telomere dysfunction; lamins; aging;
Drosophila

1. Introduction
Linear chromosome ends are protected by nucleoprotein structures called telomeres,
Citation: Kalmykova, A. Telomere which maintain genome integrity through generations. Telomere research as a specific area
Checkpoint in Development and of the life sciences originates from the theoretical works of Alexey Olovnikov written in
Aging. Int. J. Mol. Sci. 2023, 24, 15979. 1971–1973, in which he described the phenomenon of the under-replication of chromosome
https://doi.org/10.3390/ ends, which he called marginotomy [1,2]. This phenomenon is associated with the inability
ijms242115979 of DNA polymerase to replicate the terminal DNA fragment, resulting in chromosome
shortening with each cell cycle. According to experimental estimates, chromosome ends are
Academic Editors: Hans Knecht and
Francesco Sessa
shortened by 50 base pairs per human cell division and by 2–5 kilobases per generation [3].
Alexey Olovnikov predicted the existence of a specialized enzyme that compensates for the
Received: 1 September 2023 loss of terminal DNA and elongates the chromosome end in the endlessly dividing germline,
Revised: 19 October 2023 stem, and tumor cells. The gradual shortening of terminal DNA during replication explains
Accepted: 2 November 2023 the Hayflick limit, which postulates a limited number of somatic cell divisions [4]. The
Published: 5 November 2023
telomere theory of aging proposed by Alexey Olovnikov explained how telomere shorten-
ing could be responsible for cell cycle arrest leading to replicative senescence of somatic
cells. The limitation of cell doubling potential was considered by Alexey Olovnikov as
Copyright: © 2023 by the author.
a tumor suppression mechanism [2]. Moreover, in 1973, he formulated the idea that the
Licensee MDPI, Basel, Switzerland. “artificial lengthening of the telogenes could be a means of delaying ageing in proliferating
This article is an open access article cell clones” [2].
distributed under the terms and These visionary predictions were confirmed decades later by the discovery of telom-
conditions of the Creative Commons erase, an enzyme that lengthens telomeres [5], and by studies demonstrating the effect of
Attribution (CC BY) license (https:// telomerase activation on the number of cell divisions [6] and the entire organism lifespan [7].
creativecommons.org/licenses/by/ Studies of the organization and functioning of the exceedingly complicated telomere com-
4.0/). plex have revealed new aspects of telomere biogenesis and their link with aging. This area

Int. J. Mol. Sci. 2023, 24, 15979. https://doi.org/10.3390/ijms242115979 https://www.mdpi.com/journal/ijms

 aging. This area of research continues to be at the cu ing edge of the life sciences 5
after Olovnikov’s discovery. In this review, we will discuss the ways in which telo
communicate with cell systems, since the consequences of this dialogue are extrem
portant for cell fate and development. The telomere checkpoint concept discusse
Int. J. Mol. Sci. 2023, 24, 15979 2 of 16
implies a pivotal role for telomere signaling in conditions threatening genome inte

2. A Telomere Is a Dynamic DNA-RNA-Protein Complex

of research continues to be at the cutting edge of the life sciences 50 years after Olovnikov’s
To understand
discovery. how
In this review, we telomeres
will discuss function
the ways ininwhich
different cells,communicate
telomeres let us briefly recall ho
with
are
cellorganized. Recent
systems, since reviews provide
the consequences detailed
of this dialogue are descriptions of thefor
extremely important structure
cell fate of telo
and development.
[8–10]. The telomereThe telomere
complexcheckpoint
includesconcept discussed
telomeric DNA,here implies a pivotal
a telomeric role protei
protective
for telomere signaling in conditions threatening genome integrity.
plex, and telomeric RNA, which forms an additional scaffold for binding telomer
teins. There are
2. A Telomere Is a several
Dynamictypes of telomericComplex
DNA-RNA-Protein repeats and mechanisms for their mul
tion. ToIn understand
most species,howthe mechanism
telomeres function ofintelomere
different lengthening is the
cells, let us briefly reverse
recall how transc
they are organized. Recent reviews provide detailed descriptions
reaction. The a achment of retroelements to the ends of linear DNA is thought of the structure of t
telomeres [8–10]. The telomere complex includes telomeric DNA, a telomeric protective
evolved as the primary way of protecting linear chromosomes [11]. Telomerase is th
protein complex, and telomeric RNA, which forms an additional scaffold for binding
totelomeric
be derived from
proteins. Therea retroelement
are several typesreverse transcriptase
of telomeric and later evolved
repeats and mechanisms for their to bec
highly specialized
multiplication. In mostenzyme formechanism
species, the maintaining telomeres
of telomere [12]. isUsing
lengthening a template
the reverse tran- RN
scription reaction.
lomerase The attachment
RNA component, of retroelements
TERC), telomerase to the ends of
reverse linear DNA is(TERT)
transcriptase thought length
to have evolved as the primary way of protecting linear chromosomes
ends of chromosomes by adding many short repeats, e.g., TTAGGG in mammals. [11]. Telomerase
is thought to be derived from a retroelement reverse transcriptase and later evolved to
Diptera insects, which are among the most prosperous and numerous species, ha
become a highly specialized enzyme for maintaining telomeres [12]. Using a template RNA
telomerase during
(telomerase RNA evolution,
component, returning
TERC), telomeraseto the most
reverse ancient way
transcriptase of maintaining
(TERT) lengthens telo
retrotransposon a achment
the ends of chromosomes to the
by adding many chromosome ends
short repeats, e.g., [13–15].
TTAGGG in Interestingly,
mammals. Manyin telom
Diptera insects,
encoding which
species, a are among the
achment most prosperous andtonumerous
of retrotransposons telomeres species,
is alsohave lost
observed, re
telomerase during evolution, returning to the most ancient way of maintaining telomeres:
in the formation of mixed-type telomeric repeats [16,17]. In mammals, the LIN
retrotransposon attachment to the chromosome ends [13–15]. Interestingly, in telomerase-
trotransposon
encoding species,isattachment
also ableoftoretrotransposons
a ach to unprotected
to telomerestelomeres [18,19].
is also observed, Finally,
resulting in satell
complex repeats
the formation were found
of mixed-type in mosquito
telomeric telomeres,
repeats [16,17]. In mammals, which are most
the LINE1 likely main
retrotrans-
poson is also able to attach to unprotected telomeres [18,19]. Finally,
through recombination [20,21] (Figure 1). The study of this mechanism of te satellite-like complex
repeats were found
maintenance in mosquito
in natural telomeres,
populations which
can are most
provide a newlikely maintained
impetus to thethrough
search for w
recombination [20,21] (Figure 1). The study of this mechanism of telomere maintenance in
combating the most
natural populations canaggressive
provide a new forms
impetusof cancer, whose
to the search telomeres
for ways are maintained
of combating the
recombination or theofalternative
most aggressive forms cancer, whose lengthening
telomeres areof telomeres
maintained (ALT)
using [22,23]. or
recombination
the alternative lengthening of telomeres (ALT) [22,23].

Figure
Figure 1. Different types
1. Different typesof of
telomeric repeats.
telomeric TAS, TAS,
repeats. telomere-associated sequences.
telomere-associated Arrowheads
sequences. Arrowhea
designate
ignate telomerase-generated repeats,
telomerase-generated repeats, different
differentcolored arrows
colored show telomeric
arrows retrotransposons,
show telomeric retrotransposo
different
ferent coloredsquares
colored squares represent
represent satellite-like repeats.
satellite-like repeats.
Int. J. Mol. Sci. 2023, 24, 15979 3 of 16

A special protein complex prevents the ends of chromosomes from being recognized
as DNA double-strand breaks by the DNA damage response (DDR) and prevents recom-
bination and chromosome fusion. The telomere protection complex in humans is termed
shelterin and consists of six components: TRF1 (Telomeric repeat-binding factor 1), TRF2,
RAP1, TIN2, POT1, and TPP1 [8,24]. A 30 overhang generated at the chromosome ends
as a result of incomplete end replication invades into a double-strand region forming
a t-loop (telomeric loop). DNA binding proteins of shelterin interact with double- and
single-strand telomeric DNA, protecting it from degradation and fusions. A functional
analog of shelterin, the terminin protein complex, performs similar functions in Drosophila
telomeres that are maintained by the retrotranspositions of telomeric retrotransposons [25].
In addition, functional telomeres are organized in heterochromatic domains with typical
epigenetic marks [26]. It is believed that both telomere compaction and shelterin protein
binding are required for the inhibition of ATM (ataxia telangiectasia mutated) and ATR
(ataxia telangiectasia and Rad3-related) checkpoint kinases and the prevention of activation
of DNA repair pathways at telomeres [24]. Shelterin proteins repress both nonhomologous
end joining and homologous recombination at the telomeres to prevent telomere fusions
and instability. Surprisingly, DNA repair factors interact with shelterin proteins to establish
telomere end protection and telomere transcriptional silencing [27]. DNA repair factors
are also required for the assembly of the telomere capping protein complex in Drosophila:
ATM and ATR kinases and the Mre11-Rad50-Nbs1 (MRN) complex facilitate the loading
of telomere protection and silencing proteins at Drosophila telomeres [28–30]. Most likely,
DNA repair proteins transiently associate with telomere ends or specific telomeric factors
to distinguish telomeres from dsDNA breaks and facilitate the binding of the telomere
protection complex. The epigenetic control of telomeric chromatin is a complex process,
which has not been fully understood to date and attracts particular attention due to its
impairment in aging.
Despite the heterochromatic features of telomeres, they are transcriptionally active.
Telomeric Repeat-containing RNA (TERRA) is a G-rich long non-coding RNA driven by
the subtelomeric promoters [31]. TERRA biogenesis regulated by numerous nuclear factors
resulted in the sequestration of the poly(A) minus TERRA fraction at the telomeres. Being
able to interact with shelterin components, TERRA serves as a scaffold at the telomeres.
TERRA is the most dynamic component of telomeres, the levels of which vary during the
cell cycle and with changes in the state of telomeric chromatin. The functions of TERRA are
being actively studied and it is already clear that telomeric RNAs have a huge regulatory
potential. TERRA is involved in the formation of R-loops (RNA-DNA hybrids) and G-
quadruplexes at the telomeres, both of which have regulatory significance [32]. It is believed
that high levels of TERRA in cancer cells of the ALT type ensure telomere lengthening
based on recombination [33].
A tight connection between the telomere and a network of cellular processes is sug-
gested by the identification of numerous protein and RNP (ribonucleoprotein) partners
of telomerase, components of shelterin complex and TERRA [34–37]. In-depth studies of
the organization of telomeres show that this structure is both highly dynamic and multi-
component, and many levels of regulation ensure the stable state of the telomere [8]. First
of all, it must be kept in mind that the telomere consists of multiple tandem repeats, the
same on all chromosomes, which carries an intrinsic recombinogenic danger, fraught with
genetic instability.
Telomere attrition is a significant contributor to aging [38,39]. The complexity and
dynamism of telomere organization revealed in recent studies suggest that telomere length
and telomerase activity are not the only parameters that may be associated with telomere
dysfunction during normal development and organismal aging. In fact, the relationship
between telomere length and chronological age in humans and 98 species of non-human
vertebrates, as determined by large sample sizes, is weak and varies based on tissue types
and telomere measurement methodology [40,41]. In addition to critically short telomeres,
Int. J. Mol. Sci. 2023, 24, 15979 4 of 16

long but defective telomeres contribute to cellular senescence and age-related diseases,
which are particularly noticeable in post-mitotic tissues [42–44].

3. The Signaling Role of Telomeric RNA

The disruption of the integrity of the telomere complex can lead to genetic instability,
aneuploidy, chromosomal abnormalities, and, ultimately, neoplastic transformation. In
a multicellular organism, cells with telomere dysfunction are eliminated, which serves
as an important anti-oncogenic mechanism in the development. Therefore, there must
be mechanisms for the transmission of signals from dysfunctional telomeres to cellular
processes. Such a mechanism is easy to imagine, given the numerous structural and
functional relationships between telomere components and biochemical and cell signaling
pathways. According to the present evidence, there are two main signaling pathways from
the telomeres. The first is the release of telomeric proteins or RNA from the telomeres
under certain conditions and their effect on cellular targets. The second is the accumulation
of damage at the telomeres above the threshold level, which activates DNA repair systems
and then the mechanisms of cell death. In this review, we will consider both mechanisms
as part of a single telomere checkpoint pathway.
The mechanisms of telomere signaling concerned with the regulation of non-telomeric
targets by telomeric proteins are described in comprehensive reviews [45–47]. The most il-
lustrative is the budding yeast model. During the shortening of yeast telomeres, the average
length of which is ~350 bp, telomeric proteins are released and interact with gene regulatory
regions in the nucleus. For example, Rap1 relocalization from the telomeres correlates with
the repression of core histone genes, which is typical for senescent cells [48]. Mammalian
shelterin proteins and telomerase have also been implicated in the transcriptional regu-
lation of non-telomeric genes [47]. Extratelomeric nuclear Rap1 regulates the expression
of energy metabolism [49] and immunity-related genes [50]. Cytoplasmic Rap1 induces
proinflammatory signaling, reducing lifespan in mice [51]. Thus, the signaling role of the
telomeric Rap1 protein is evolutionarily conserved. It remains an open question whether
the mechanism of release and relocalization of Rap1 in mammals is also determined by
telomere shortening, as it is in yeast. If this is the case, then such a “titration” mechanism
can explain the changes in the proliferative status of cells during replicative aging.
Telomerase reverse transcriptase, TERT, also regulates non-telomeric targets. For
example, TERT occupies promoters and regulates the expression of genes involved in the
Myc- and Wnt-signaling pathways via an interaction with the BRG1 chromatin remodeling
protein [52]. By controlling gene expression, TERT stimulates the proliferation of epidermal
progenitor cells, and that function does not require reverse transcriptase activity [53]. Im-
portantly, telomerase affects the expression of neurotrophins, which has a neuroprotective
effect and prevents neuronal degradation [54]. Non-canonical functions of telomerase and
shelterin proteins deserve close attention as they could provide a link between telomere
and cellular processes.
Mounting evidence suggests that TERRA is also involved in the telomere signaling
pathway (Figure 2). Indeed, TERRA expression is tightly linked to the state of telom-
eric chromatin [55], changes during the cell cycle [56] and is upregulated in ALT tumor
cells [57]. TERRA interacts with a large number of cellular proteins related to different
biochemical pathways [34,55,58,59]. An important finding is that TERRA can act in trans
as a transcriptional regulator of non-telomeric targets. This regulation is modulated by the
antagonistic interactions between TERRA and chromatin remodeler ATRX [60]. TERRA
molecules were found in extracellular exosomes during telomere uncapping and telom-
ere shortening [61]. TERRA-containing exosomes were strong inducers of inflammatory
response in mammalian cells. The proposed function of this inflammation is to recruit
macrophages to eliminate cells with telomeric repeat overexpression, which is the hallmark
of telomere disorder [62].
Int. J. Mol.
Int. J. Mol. Sci.
Sci. 2023, 24, x15979
2023, 24, FOR PEER REVIEW 6 5of
of 15
16

Figure 2. Signaling role of telomeric transcripts induced by overexpression from dysfunctional te-
Figure 2.The
lomeres. Signaling
schemerole
showsof telomeric transcripts
the reported examplesinduced by overexpression
of telomeric RNA-mediatedfrom dysfunctional
regulatory effects.
telomeres. The scheme shows the reported examples of telomeric RNA-mediated
In mammalian cells, TERRA can affect gene expression [60] (A) and induce innate immune regulatory ef-
activa-
fects.[63]
tion In (B)
mammalian cells, TERRAresponse
or cause inflammatory can affect[61]
gene
(C).expression [60] (A)
In the Drosophila and induce
germline, innatetelomeric
abundant immune
activation [63] (B) particles
ribonucleoprotein or cause inflammatory response
interact with Polo kinase,[61] (C). In the
affecting Drosophila
centrosome germline,during
biogenesis abundant
oo-
genesis
telomeric and early development
ribonucleoprotein [64] (D).interact with Polo kinase, affecting centrosome biogenesis
particles
during oogenesis and early development [64] (D).
4. Telomeres in Aging: Shortening or Dysfunction?
NassourOlovnikov
Alexey and co-authors recently
proposed reported
in 1973 a novel function
that telomere of TERRA
shortening in the
is counter to activation
the num-
of the autophagy program aimed at the elimination of cells with unstable telomeres
ber of cell divisions [2]. Human fibroblasts’ telomeres do, in fact, shorten with aging, lead- [63].
This mechanism is mediated by Z-DNA binding protein 1 (ZBP1], a nucleic acid
ing to replicative senescence [3], whereas telomerase expression in normal cells results in sensor
that induces innate immune activation and cell death. Abundant TERRA molecules, which
telomere lengthening and cell immortalization [6,70]. Despite the fact that telomere theory
accumulate as a result of telomere dysfunction, bind to ZBP1 leading to the formation
had been supported at the cellular level, Alexey Olovnikov recognized that it was insuffi-
of ZBP1 filaments on mitochondria, which triggers autophagy and cell death in human
cient to explain aging at the organismal level, viewing telomere shortening as a “witness”
fibroblasts and epithelial cells. These data emphasize the role of TERRA as the messenger
rather than the underlying cause of aging [71].
of dysfunctional telomeres that triggers cell death.
What causes cell division arrest when telomeres shorten? It is well established that
An analysis of telomeric RNA biogenesis in the Drosophila melanogaster model revealed
unprotected or critically shortened telomeres induce a persistent DNA damage response
the relationship between the telomeric transcripts and key protein regulators of the cell
leading to p53-mediated cellular senescence or apoptosis [72,73]. This happens because
cycle [64]. The depletion of the negative regulators of telomeric repeat expression (small
DNA repair mechanisms are inhibited at the telomeres to prevent telomere fusions and
RNA pathway component, deadenylase Ccr4 and RNA-binding protein Ars2) led to the
inter-chromosome recombination. Shortened telomeres induce persistent DNA damage
overexpression of the main telomeric retrotransposon HeT-A in the female germline and the
foci, marked by the phosphorylated histone γH2AX. These foci are termed telomere-
Int. J. Mol. Sci. 2023, 24, 15979 6 of 16

accumulation of HeT-A RNPs in the oocyte and early embryos. Aggregates of HeT-A RNPs
were found near the microtubule-organizing center, MTOC, in the oocyte, and around
centrosomes in the syncytial embryos. HeT-A overexpression in the germline, caused by
the depletion of transcription factors, chromatin components, RNA binding proteins, and
small RNA pathway genes, led to phenocopies associated with severe mitotic defects and
early embryonic lethality [64,65]. A mass spectrometry analysis of HeT-A RNP partners
in Drosophila syncytial embryos identified key proteins of the cell cycle and centrosome
components. The retention of Polo kinase in HeT-A RNP aggregates triggered centrosome
dysfunction and mitotic catastrophe, leading to early developmental arrest. We found
that the overexpression of telomeric repeats in Drosophila somatic cells was cytotoxic and
also led to the aggregation of HeT-A RNPs around centrosomes in mitotic cells. Thus,
telomeric RNAs produced by dysfunctional telomeres above the threshold level affect cell
cycle machinery and prevent abnormal development. Noteworthily, HeT-A RNP-mediated
signaling was not associated with telomere shortening or the loss of telomere-capping
proteins. In some cases, HeT-A overexpression was caused by chromatin changes and a
loss of transcriptional silencing in telomeres. However, HeT-A telomeric transcripts also
accumulated and affected the cell cycle machinery after the depletion of the Ccr4-Not
deadenylase complex that mediates the co-transcriptional degradation of telomeric RNAs
but does not change the telomeric chromatin state and protection [66]. Given that increasing
evidence supports a signaling role for telomeric RNA, the proper coordination of telomeric
repeat expression and biogenesis of telomeric RNA becomes more important.
The depletion of functionally different factors increased the transcription of telomeric
repeats in the Drosophila germline and showed an embryonic lethality phenotype likely
consistent with telomere dysfunction. These data strongly suggest that an activation
of transcription in altered but protected telomeres is one of the universal responses of
telomeres to various genetic stresses, and telomeric transcripts are able to trigger cell death
or developmental arrest to eliminate potentially dangerous cells. This pathway, activated
as the response to telomere damage, can be defined as a telomere checkpoint.
It will be interesting to see if TERRA can perform signaling functions in the mitotic
spindle of mammalian cells. TERRA extra-nuclear metabolism is not well understood. It
was observed that TERRA transcripts form distinct foci in the cytoplasm of tumor cells [67];
however, it is unknown which proteins associate with the TERRA foci. It was observed that
shelterin protein TRF1 localized to the mitotic spindle and interacted with Aurora kinase in
human cancer cells [68,69]; however, the functional outcome of such a localization remains
unknown. It is tempting to speculate that TRF1, which has been shown to interact with
TERRA [55], may be bound to TERRA at this location. How dysfunctional but protected
telomeres can induce cell cycle arrest in normal mammalian cells remains an open question.
Lessons from the fruit fly HeT-A RNP signaling mechanism could be helpful in elucidating
this pathway.

4. Telomeres in Aging: Shortening or Dysfunction?

Alexey Olovnikov proposed in 1973 that telomere shortening is counter to the number
of cell divisions [2]. Human fibroblasts’ telomeres do, in fact, shorten with aging, leading to
replicative senescence [3], whereas telomerase expression in normal cells results in telomere
lengthening and cell immortalization [6,70]. Despite the fact that telomere theory had been
supported at the cellular level, Alexey Olovnikov recognized that it was insufficient to
explain aging at the organismal level, viewing telomere shortening as a “witness” rather
than the underlying cause of aging [71].
What causes cell division arrest when telomeres shorten? It is well established that
unprotected or critically shortened telomeres induce a persistent DNA damage response
leading to p53-mediated cellular senescence or apoptosis [72,73]. This happens because
DNA repair mechanisms are inhibited at the telomeres to prevent telomere fusions and
inter-chromosome recombination. Shortened telomeres induce persistent DNA damage foci,
marked by the phosphorylated histone γH2AX. These foci are termed telomere-associated
Int. J. Mol. Sci. 2023, 24, 15979 7 of 16

foci (TAF), and their accumulation is considered to be a consequence of telomere shortening

in aging-related pathologies [42,74]. However, cellular senescence in normal organismal
aging occurs when telomere length is not critically short. For example, mouse telomere
length is around 100 kilobases, and senescence in mouse cells occurs at a telomere length
of several tens of kilobases. There is no correlation between telomere length/telomerase
activity and the lifespan of rodent species, among which there are extremely long-lived
species [75]. Mutations in the genes involved in telomere maintenance (TERC, TERT,
POT1, TIN2, TPP) lead to severe syndromes: telomere biology disorders (TBDs) with
signs of early senescence [42,76]. However, global telomere shortening is not necessarily
observed even in these syndromes. There are TBDs exhibiting excessive telomere elongation
or normal telomere length [76]. For example, POT1 mutations associated with longer
telomeres caused an increased risk for tumors and blood cancer [77]. Moreover, replicative
senescence occurs unequally in cell types with different mitotic capacities since the degree
of telomere shortening differs significantly among them. Despite the well-established fact
that telomeres do shorten during cell division and aging, these findings suggest that there
is no strong causal relationship between telomere length and normal organismal aging,
and, indeed, telomere shortening can be considered a “witness” of aging, but not its driver.
Instead, increasing evidence suggests that the accumulation of damage in telomeres,
regardless of their length, serves as a signal to trigger cell death. The accumulation
of unrepairable damage in telomeres occurs in the tissues of aging primates and mice;
noteworthily, the DDR signal is triggered at a normal average telomere length [78–80].
Indeed, up to 40% of all DDR foci in the nuclei of aging cells correspond to telomeres,
which account for only 0.02% of the genome [79]. Telomere-associated DDR foci have been
documented during aging in post-mitotic cells such as neurons, adipocytes, osteocytes, and
cardiomyocytes, irrespective of telomere length [81]. One of the striking examples of the
negative consequences of length-independent telomere dysfunction is cellular senescence
in human melanocytes [82]. Telomere quantitative FISH (Q-FISH) from young and older
skin demonstrated that telomeres in melanocytes do not undergo significant shortening
with aging, which is also confirmed by previously reported data about the low replicative
capacity of differentiated melanocytes [83]. However, during skin aging, an increase in
the number of signals of a protein marker of DNA damage, γH2AX, was recorded in the
telomeres of melanocytes. Senescent melanocytes were able to induce telomere dysfunction
and senescence in neighboring skin cells leading to epidermal atrophy.
Age-related disorders include genomic instability, telomere dysfunction, proteosta-
sis loss, mitochondrial dysfunction, cellular senescence and other metabolic and cellular
alterations [39,84]. Many aging-related hallmarks are associated with the generation of
DDR in telomeres and, accordingly, the accumulation of TAF. For example, telomere dys-
function has been linked to such signs of aging as mitochondrial dysfunction, chronic
inflammation, and loss of proteostasis, suggesting a telomere-centric mechanism of ag-
ing [85]. DDR should be able to be activated at both short and long telomeres to promote
cellular senescence and aging. It was previously reported that the overexpression of human
TERT leading to telomere elongation did not protect fibroblasts from stress-induced senes-
cence [86]. Treatment with general geroprotectors such as rapamycin, 17β-estradiol, and
senolytic drugs reduced the percentage of TAF-positive aging cells [87–89]. These studies
highlight how essential the telomere state is to aging and show that telomeric TAFs can be
considered a key hallmark of aging.
The reasons for length-independent telomere damage may be various influences that
change the organization of telomeres, the structure of telomeric chromatin, and the levels
of transcription. It was reported that telomeres of normal cells are extremely sensitive to
oxidative DNA damage. Oxidative stress in mouse neutrophils led to telomere dysfunction,
the accumulation of telomeric DDR signals, and the spread of the senescent phenotype to
surrounding cells, without affecting telomere length [90]. Oxidative lesions at the telomeres
of normal human fibroblasts and epithelial cells induced replication stress and telomeric
DDR activation in the absence of telomere shortening or deprotection [91].
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 8 of 15
Int. J. Mol. Sci. 2023, 24, 15979 8 of 16

Experimental evidence suggests that telomeric repeats are inherently prone to DNA
breakExperimental
accumulationevidence
(Figure suggests that
3). Internal telomeric
telomeric repeats
breaks are inherently
could be a result prone to DNA
of replication-
break accumulation
associated defects. In(Figure
addition,3).the
Internal telomeric
transcription breaks could
of telomeric be aisresult
repeats of replication-
strongly associated
associated
with DNAdefects.
damage.InThe
addition, the transcription
formation of telomeric
of TERRA RNA-DNA repeats(R-loops)
hybrids is stronglyinassociated
telomeres
with DNA
can lead to damage. The formation
the generation of TERRA
of DNA breaks RNA-DNA
through hybrids (R-loops)
several mechanisms in telomeres
[33,92,93]. R-loops
can lead to the generation of DNA breaks through several mechanisms [33,92,93].
cause replication fork stalling and, as a result, DNA breaks and recombination [32,94]. R-loops
can cause replication
Telomeric fork stallingincrease
R-loops considerably and, as homologous
a result, DNA breaks and recombination
recombination [32,94].
events, as discussed
Telomeric R-loops considerably
in recent extensive increase
reviews [33,95–97]. homologousrecombination
Homologous recombinationatevents, as discussed
the telomeres is in-
in recent
hibited byextensive reviews
the chromatin [33,95–97].
remodeling Homologous
factor ATRX, TERRA recombination
RNA-binding at the telomeres
proteins NONO is
inhibited
and SFPQ, byand
the chromatin remodeling
other factors factor R-loop
that suppress ATRX, TERRA RNA-binding
formation, proteins
recombination, andNONO
DNA
and SFPQ,
damage and other factors
in mammalian that [59,97,98].
telomeres suppress R-loop formation, recombination, and DNA
damage in mammalian telomeres [59,97,98].

Figure
Figure 3. Length-independentinternal
3. Length-independent internal telomeric
telomeric DNADNA lesions
lesions leadlead to DNA
to DNA damagedamage response.
response. Crit-
Critical telomere
ical telomere shortening
shortening and and deprotection
deprotection bothboth induce
induce cellular
cellular DNADNA damage
damage response
response (toleft).
(to the the
The scheme
left). to thetoright
The scheme showsshows
the right possible mechanisms
possible for thefor
mechanisms generation of internal
the generation telomeric
of internal DNA
telomeric
instability
DNA that induces
instability DDR in
that induces telomeres
DDR irrespective
in telomeres of their
irrespective of length. Broken
their length. arrows
Broken indicate
arrows DNA
indicate
damage.
DNA damage.

another, serve
Internal telomeric DNA breaks, generated in one way or another, serve as substrates
homologous recombination. ssDNA
for homologous ssDNA generated
generated as a result of dsDNA break resection
resection
can invade
can invade the
thetelomeric
telomericregions
regionsononanother
anotherchromosome,
chromosome, leading
leading to ectopic
to ectopic recombina-
recombination
tion genomic
and and genomic instability.
instability. The experimental
The experimental induction
induction of internal
of internal DSBsDSBs in mouse
in mouse telo-
telomeres
meres activated homologous recombination and the ALT pathway [99].
activated homologous recombination and the ALT pathway [99]. Recombination-based Recombination-
telomere elongation is observed in yeast [100,101], ALT cancer cells [57], and in mice in the
Int. J. Mol. Sci. 2023, 24, 15979 9 of 16

absence of Pot1 or Rap1 [102,103]. Noteworthily, telomere lengthening during the early
embryonic cleavages and in normal somatic tissues in mice is telomerase-independent
and requires a recombination-based mechanism [104,105]. Despite the functional role of
telomeric DNA breaks in recombination-based telomere maintenance, their levels and
appropriate processing appear to be strictly balanced during normal development to
prevent the activation of DDR or ALT cell proliferation. The accumulation of telomeric
DDR signals in protected, not critically shortened telomeres suggests that this balance is
severely impaired in senescent cells and in aging tissues.

5. Nuclear Topology and Epigenetics of Telomeres

The compromised functioning of the nuclear periphery is one of the main factors in
the destabilization of telomeres during senescence, laminopathies, and accelerated aging
syndromes [106–108].
The nuclear envelope plays a pivotal role in the 3D genome architecture. The disrup-
tion of nuclear lamina has a global effect on the compaction of chromatin domains and their
nuclear positioning [109]. Telomere positioning at the nuclear periphery is a key feature of
genome organization in yeast, Drosophila, and a subset of mammalian cell types [110–113].
The nuclear periphery provides a safe environment for the stable maintenance of
heterochromatin. The inhibition of homologous recombination at the nuclear lamina is a
means of suppressing recombination between repetitive sequences [114–116]. The main
feature of DNA break repair in heterochromatin is that homologous recombination is
temporarily blocked and then restarted after relocation to the nuclear periphery where
the DSB is isolated from homologous ectopic sequences [117]. Heterochromatin protein 1
(HP1) and histone H3 lysine 9 (H3K9) methyltransferases, typical components of silenced
chromatin, are required to prevent Rad51 recruitment to the heterochromatic DNA breaks,
and their loss results in the progress of ectopic recombination in Drosophila [118]. While
the overall mechanism of DNA repair in heterochromatin is unclear, the crucial role of
nuclear lamina in this process is a conserved feature. The translocation of heterochromatic
DSBs to the nuclear lamina assures their safe reparation in Drosophila [118]. In human
cells, the activation of the DDR at DSBs and homologous recombination are also inhibited
at the nuclear lamina [119]. The integrity of the nuclear periphery compartment and its
components ensures the safe repair of repeat-rich regions. The localization of telomeres
to the nuclear periphery also represents a way of reducing recombination between highly
repetitive telomeric sequences. The localization of yeast telomeres to the nuclear lamina
protects telomeric repeats from recombination [120], and the disruption of perinuclear
telomere anchoring led to a hyper recombinant telomeric state and a senescent-like pheno-
type [121]. The global effect of aging on nuclear periphery integrity and heterochromatin
structure is clearly linked to a relaxation of DNA repair control. High levels of telomeric
DNA damage observed in laminopathies emphasize the central role of telomeres in the
global epigenetic changes associated with accelerated aging syndromes [106,107].
Lamins are the main structural proteins associated with the nuclear lamina. Lamins
are involved in the DNA repair mechanisms [122] and interact with shelterin proteins [123].
A mutation in the lamin A gene, which produces a shortened form of the protein, causes a
premature aging syndrome, Hutchinson–Gilford progeria [124]. Structural defects of lamins
also occur during cellular aging, and laminopathy is one of the hallmarks of aging [125,126].
Consistently, factors that associate with lamins and stabilize heterochromatin of repetitive
elements delay cellular senescence [127–129]. Structural damage of the nuclear lamina
leads to systemic effects, the most important of which is a state of globally compromised
chromatin epigenetics that includes changes in heterochromatin structure, the misregulation
of gene activity, and telomere dysfunction [130–132].
Among the various signs of aging, the epigenetic changes are the most dramatic.
Aging is associated with the DNA hypomethylation of transposable elements and telomeric
regions, as well as the hypermethylation of promoters of coding genes [84,133]. Changes in
the DNA methylation pattern in aging have been called an epigenetic clock, owing to their
Int. J. Mol. Sci. 2023, 24, 15979 10 of 16

correlation with chronological age. The epigenetic clock of Steve Horvath includes 353
CpG sites in the human genome that methylation changes with age [134,135]. Importantly,
the DNA methylation pattern, but not telomere length, is associated with chronological
age in humans [136]. It is likely that telomere damage, rather than telomere shortening,
is a key sign of aging in a normal organism. In Hutchinson–Gilford syndrome, systemic
changes in the epigenetic status of the entire genome are also observed, but telomere
dysfunction and TAF accumulation are the main contributors to the manifestation of the
disease signs. Telomeropathy is equated with laminopathy in terms of the symptoms
of aging [107,137,138]. This point of view is supported by studies of a mouse model of
Hutchinson–Gilford syndrome where it was shown that the selective inhibition of the DDR
on the telomeres improves tissue homeostasis, reduces inflammation, and prolongs the
lifespan of the model animals [139].
The effects of lamin B mutation and physiological aging on the telomeres of D.
melanogaster germ cells are strikingly similar [140]. The appearance of DNA damage
foci, enriched in the γH2Av histone variant, and the accumulation of recombination marker
Rad51, which are particularly abundant in the telomeres of lamin B mutants and aged
flies, can be triggers of the observed germ cell death [140]. RAD51 is a known marker of
homologous recombination and break-induced replication [141]. In human cells, RAD51
associates with TERRA and promotes telomeric R-loop formation [92]. The accumulation
of recombinase Rad51 at Drosophila telomeres in lamin B mutants, as well as in aging germ
cells, suggests the activation of homologous recombination between telomeric repeats,
which elevates the risk of chromosomal rearrangements [140]. Telomeric chromatin decom-
paction observed in lamin B mutants can cause defective DNA repair and the accumulation
of γH2Av foci. Since the expression of the Drosophila telomeric retroelement HeT-A is
moderately increased in lamin B mutants and in aging, it cannot be excluded that Rad51
associates with HeT-A RNAs, thus facilitating homologous recombination and telomere
instability [140]. However, the mechanism of the inhibition of homologous recombination
in telomeres with the participation of lamin B is still unknown.
Taken together, these data point to a key role for telomere instability in both laminopa-
thy and aging, despite the dramatic changes in genome organization observed during
these processes.

6. Conclusions
Many lines of evidence suggest that not only telomere shortening, but also length-
independent telomere dysfunction, can be considered as “the heel of Achilles of the DNA
double helix” [2]. The telomere-centric mechanism of aging implies that telomeres collect
information about disorders in the cell and, at a critical level of dysfunction, trigger a re-
sponse leading to cell death. Such a mechanism, similar in principle to checkpoint systems,
explains the key role of telomeres in development and aging. Indeed, only telomeres can
provide a universal and rapid response to the enormous range of external and internal
stimuli that affect many targets in the cell and pose a threat to genetic stability. The causes of
telomere dysfunction can be genetic disorders, external influences, stress of various kinds,
physiological aging, laminopathy, and many others. As a result, disruptions in the structure
of telomeric chromatin, the appearance of telomeric DNA breaks, replicative stress, and
ectopic recombination occur; these serve as signals triggering the telomeric checkpoint,
which activates cell cycle arrest and cell death to protect genome stability (Figure 3). This is
a fundamental anti-oncogenic mechanism operating in multicellular organisms. However,
the upregulation of the DNA damage response system can also strongly stimulate the senes-
cent phenotype, leading to persistent inflammation, a typical characteristic of aging [142].
The molecular nature of length-independent telomere dysfunction and signaling is far from
understood. Basically, there are two such mechanisms: internal irreparable damage in
telomeres and signals coming from altered telomeres, e.g., telomeric proteins and RNA. The
study of telomere signaling is very important because normal aging occurs at an average
Int. J. Mol. Sci. 2023, 24, 15979 11 of 16

telomere length; the length of telomeres has been evolutionarily established with an excess
and is not physically exhausted in the course of normal development.
Telomere dysfunction and epigenetic changes, primarily those in telomeres, can serve
as important biomarkers of processes associated with age-related changes in normal tissues.
For example, detecting the level of telomeric DNA methylation associated with the activa-
tion of the DDR signal at telomeres can serve to detect signs of premature aging. Telomeric
RNAs emanating from non-shortened but functionally compromised telomeres are also an
important signature of telomere dysfunction, and TERRA levels can also be considered as a
potential diagnostic factor of telomere-associated pathologies. Such multilevel regulation
of telomeric homeostasis emphasizes the fundamental importance of this structure in main-
taining the integrity of the genome and provides us with potential tools for diagnosing and
treating cancer and premature aging.

Funding: This research was funded by the Ministry of Science and Higher Education of the Russian
Federation, No. 075-15-2020-773/9.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Acknowledgments: I would like to acknowledge Leonid Malaev for help with the illustrations.
Conflicts of Interest: The authors declare no conflict of interest.

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