PERSpECTIvES
TIMELINE
Telomeres and telomerase: three
decades of progress
Jerry W. Shay
and Woodring E. Wright
Abstract | Many recent advances have emerged in the telomere and telomerase
fields. This Timeline article highlights the key advances that have expanded our
views on the mechanistic underpinnings of telomeres and telomerase and their
roles in ageing and disease. Three decades ago, the classic view was that telomeres
protected the natural ends of linear chromosomes and that telomerase was a
specific telomere-​t erminal transferase necessary for the replication of
chromosome ends in single-​celled organisms. While this concept is still correct,
many diverse fields associated with telomeres and telomerase have substantially
matured. These areas include the discovery of most of the key molecular
components of telomerase, implications for limits to cellular replication,
identification and characterization of human genetic disorders that result in
premature telomere shortening, the concept that inhibiting telomerase might be
a successful therapeutic strategy and roles for telomeres in regulating gene
expression. We discuss progress in these areas and conclude with challenges and
unanswered questions in the field.
Mammalian telomeres are composed
of tandem repeats of TTAGGGn DNA
sequences associated with a six-​member
protein shelterin complex that facilitates
the formation of a lariat-​like structure
(the t-​loop) to shield the exposed
chromosome ends of telomeric DNA from
DNA damage machinery1,2. Progressive
telomere shortening occurs in all dividing
normal cells owing to incomplete lagging-​
strand DNA synthesis, oxidative damage,
exonucleolytic processing events and
other factors (Fig. 1a), and this shortening
eventually results in cellular growth arrest
that is believed to be an initial proliferative
barrier to tumour formation in humans and
other large, long-​lived animals3–5. It is well
established that the length of the shortest
telomere is a key biomarker of the onset of
senescence6,7. However, the most common
techniques to measure telomere length
mostly provide information only about
average telomere length8,9. Thus, at present,
published studies indicating important
correlations with various age-​associated
pathologies based on very small differences
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in average telomere length should be viewed
with some degree of scepticism unless more
recent methods that measure the shortest
telomeres have been used10,11.
Although it is believed that one, or a few,
very short telomeres leads to replicative
senescence, initiating a dysfunctional
uncapped telomere response, many studies
using harsh cell culture conditions are likely
to be considerably nonphysiological, and
senescence from this type of culture has been
termed premature or culture-​shock-induced
senescence. However, it is known that
viral oncoproteins can bypass senescence,
leading to extension of cellular lifespan
despite continuing telomere shortening12,13.
In combination with other changes (such as
activation of oncogenes and loss of function
of tumour-​suppressor genes), genetic
instability can occur, and most cells do not
survive. However, a rare cell can activate
a telomere maintenance pathway either
by using a DNA recombination pathway
(alternative lengthening of telomeres (ALT)) or
by activating or upregulating the telomerase
reverse transcriptase (TERT) gene, which
encodes the catalytic component of the
telomerase enzyme (Fig. 1b). The ALT
pathway occurs in only ~10–15% of
cancers14, whereas telomerase activation
occurs in 85–90% of all human cancers15,16.
The TERT protein forms a complex with
another essential factor, the telomerase
RNA component (TERC) and accessory
proteins such as dyskerin (DKC1), TCAB1,
NHP2, NOP10 and GAR1 (ref.4). During
early human development, telomerase
is active but becomes transcriptionally
silenced between 12 weeks and 18 weeks
of gestation17,18. TERT alternative splicing
may also be involved in silencing telomerase
activity during development, thus limiting
the maximal length of human telomeres18,19,
but what regulates alternative splicing of
TERT is largely unknown. In addition to
alternative splicing, other mechanisms
such as epigenetic changes20 involving
telomere 3D looping21–25 may occur.
Finally, TERT promoter mutations have
emerged as another mechanism to activate
TERT transcription4.
This Timeline article provides our
personal perspective on the history of the
major advances in both the telomere and
telomerase fields. It is timely to review this
topic because there is mounting evidence of
a variety of genetic disorders associated with
mutations in either telomere maintenance
proteins or telomerase components that
impact an increasing number of medical
conditions (Table 1). Major events reviewed
include the discovery of telomeres and
telomerase and their roles in ageing and
cancer, the identification26 and cloning of
the core components of telomerase27,28 and
telomerase-​associated proteins (Fig. 1c).
In addition, we discuss the observation that
the introduction of the cDNA for TERT
into telomerase-​silent human cells is often
but not always sufficient to produce cell
immortalization29 and its implications for
stem cells and cancer. This article also covers
genetic telomere spectrum disorders30,31
and approaches to targeting telomerase for
cancer therapy32–36. In addition to providing
a historical timeline (Fig. 2), we cover more
recent discoveries such as the regulation of
gene expression by 3D telomere looping over
long distances37–40. We review the evidence
that gene expression can change with
progressive telomere shortening, as it
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Perspectives
a
Normal cells undergo progressive
telomere shortening with cell division
Earlier passage normal
Cell divisions
5′
3′
5′
3′
5′
3′
5′
3′
Late passage normal
b
ALT
Telomerase
TCAB1
NOP10
TERC
Similar to break-induced replication
NHP2
DKC1
or
3′
5′
Rolling circle amplification
Template
TERT
85–90% of tumours
10–15% of tumours
c
T-loop
POT1
ACD
TIN2
TRF1 TRF2
RAP1
5′
(TTAGGG)n
3′
(AATCCC)n
Fig. 1 | Human telomeres are repetitive DNA sequences at the ends of linear chromosomes.
a | Telomeres progressively shorten in normal cells with each division in the absence of a telomere
maintenance mechanism. When some chromosome ends become very short, an uncapped telomere
can elicit a DNA damage signal, resulting in growth arrest. b | Cells that become immortal as part of
cancer development generally activate telomerase (a ribonucleoprotein complex) (left panel).
However, in ~10–15% of tumours, a DNA homologous recombination mechanism, termed alternative
lengthening of telomeres (ALT) can be engaged (right panel). ALT cells use a telomeric DNA template
that is copied to a telomere of a non-​homologous chromosome, or it may involve extrachromosomal
telomere DNA in circular (illustrated) or even linear forms. This telomeric DNA could add TTAGGG
sequences to another region of the same telomere via loop formation or to the telomere of a sister
chromatid. c | The ends of telomeres are protected not only through invasion of the terminal
single-​stranded DNA overhang into duplex TTAGGG repeats to form a t-​loop so that there is no free
end but also through binding of a complex of proteins (termed the shelterin complex) that protects
the ends of telomeres to prevent the linear ends of chromosomes from being recognized as DNA
damage needing repair. ACD, adrenocortical dysplasia protein homologue; DKC1, dyskerin; POT1,
protection of telomeres 1; RAP1, repressor/activator protein 1; TERC, telomerase RNA component;
TERT, telomerase reverse transcriptase; TIN2, TRF1-interacting nuclear factor 2; TRF, telomeric
repeat-​binding factor.
300 | MAY 2019 | volume 20
provides a new understanding of why
human telomeres are fairly similar in length
at birth and how progressive telomere
shortening can change cell physiology and
affect diseases associated with ageing before
becoming terminally short and without
inducing a DNA damage signal.
Telomere discoveries
The early years in understanding
chromosome ends. Thomas Hunt Morgan,
an early cytogeneticist, was the first to
suggest a link between genetic traits
and the exchange of genetic material on
chromosomes. In 1911, he hypothesized that
genes were arranged on chromosomes like
“beads on a string” (ref.41) and in a particular
order with a beginning and an end. One of
Morgan’s students, Hermann Muller, would
later recognize, based in part on some of the
findings of Barbara McClintock, that the
‘free ends’ of linear chromosomes behaved
differently than X-​ray-induced broken ends
and called the ends of linear chromosomes
‘telomeres’, as did Haldane and Darlington,
from the Greek words for ‘end’ (telos) and
‘part’ (meros)42. McClintock, a pioneer
in the field of cytogenetics, was studying
the knobs of heterochromatin that she
could visualize at the ends of individual
chromosomes from maize (corn) cells. She
called these distinct knobs the ‘natural ends’
of the chromosomes43 and, by rupturing a
ring chromosome during mitosis, concluded
that these natural ends or telomeres behaved
much differently than the broken ends that
Muller had induced by X-​ray irradiation44.
Although McClintock was best known
for her work on transposable elements
(jumping genes) and the basic concept
of epigenetics, she was also the first to
recognize that induced chromosome ends
were distinctly different from natural ends
but that they could become altered such that
they became a stable free end much like a
natural telomeric end45. This observation
implied that there was some permanent
molecular change that occurred at the healed
ends and is consistent with our current
ideas regarding the actions of telomerase
during chromosome healing. Indeed, later
in her career, McClintock speculated based
on earlier observations that a specific corn
strain that could not heal the broken ends
supported the concept that there must be an
enzyme in the germ line that could normally
heal the broken ends of corn strains46.
These early cytogenetic studies paved the
way to the identification of telomeres as
heterochromatin with a unique structure and
function, but the molecular characterization
of telomeres took several decades to establish.
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Perspectives
Table 1 | Telomere spectrum disorders
Disease
Defective gene
Process or complex
Pathological features
Other genes reported
mutated
Primary telomere diseases
Coats plus
syndrome
CTC1 (ref.129)
CST complex
Bilateral retinopathy, intracranial
calcifications, leukodystrophy, osteopenia,
anaemia and gastrointestinal bleeding
STN1 and TEN1
Alazami disease
LARP7 (ref.152)
• TERT splicing
• Reduced TERC RNA
Developmental delays, axial hypotonia,
short distal phalanges and nails and
borderline anaemia
Homologue of
Tetrahymena thermophila
encoding p65
DKC, HHS
and Revesz
syndrome
TINF2 (refs132,133,147)
Shelterin, inhibits TRF1
PARylation
Nail dystrophy, oral leukoplakia, bone
marrow failure, immunodeficiency,
cancer and skin hyperpigmentation and
hypopigmentation
TERF1 (encoding TRF1),
TERF2 (encoding TRF2),
RAP1, TPP1, POT1, TNKS1
and TNKS1BP1
DKC, HHS and
IPF
RTEL1 (refs176–181)
T-​loop dissociation, an
essential iron–sulfur cluster-​
containing helicase
Bone marrow failure, immunodeficiency
and developmental defects and interstitial
lung disease
MMS19, MIP18, CIAO1
and IOP1
DKC
TCAB1 (ref.182)
Controls telomerase
localization and assembly at
Cajal bodies
COIL and HOT1
Nail dystrophy, oral leukoplakia,
bone marrow failure, cancer and skin
hyperpigmentation and hypopigmentation
DKC and IPF
PARN183,184
Reduced RNA of TERC, DKC1,
RTEL1 and TERF1
Interstitial lung disease, nail dystrophy,
oral leukoplakia, bone marrow failure,
cancer and skin hyperpigmentation and
hypopigmentation
TERF1
HHS
APOLLO (also known
as DCLRE1B)151
Overhang processing
Bone marrow failure, growth defects and
microcephaly
TERF2 and FANCD2
IPF, DKC
and aplastic
anaemia
TERT125,142,143, TERC128,
DKC1 (ref.126), NHP2
(ref.185) and NOP10
Telomerase
Interstitial lung disease, nail dystrophy,
oral leukoplakia, bone marrow failure,
cancer and skin hyperpigmentation and
hypopigmentation
GAR1
(ref.186)
Secondary telomere diseases
Ataxia
telangiectasia
ATM187–190
Signals uncapped telomeres
and recruits telomerase
Premature ageing of skin and hair,
immunodeficiency, interstitial lung disease
and cancer
NA
Bloom
syndrome
BLM191,192
Telomere replication, prevents
telomere fragility and ALT
Growth deficiency, immunodeficiency,
chronic lung disease and cancer
NA
Hutchinson–
LMNA118–123,161
Gilford progeria
Proper organization and
association of telomeres with
nuclear lamins
Growth deficiency, premature ageing
of skin and hair, osteoporosis and nail
atrophy119–123
NA
RECL4
disorders (RecQ
genes)
RECQL4 (refs193,194)
Telomere replication, prevents
telomere fragility
Growth deficiency, alopecia, premature
ageing of skin and hair, osteoporosis, nail
abnormalities, cataracts and cancer
NA
Werner
syndrome
WRN192,195–199
Telomere replication, prevents
chromatid telomere loss and
ALT
Growth deficiency, premature
ageing of skin and hair, osteoporosis,
immunodeficiency, nail atrophy and
cancer
NA
There are two types of telomere genetic diseases: those that have a defective gene in the maintenance of telomerase (primary telomere diseases) and those that
indirectly affect telomeres (secondary telomere diseases). The defective gene (or genes), the telomere-​related processes and symptoms shared with primary
telomeropathies are listed. See other reviews113–117. NOP10 and GAR1 are accessory proteins that interact with the telomerase RNA component (TERC). Although
structural analyses of TERC169,200, telomerase reverse transcriptase (TERT)170,173 and the telomerase complex174,175 may provide important insights into these telomere
diseases, the clinical manifestations of these telomere spectrum disorders can be highly variable even when the underlying mutation is identical30. ALT, alternative
lengthening of telomeres; DKC, dyskeratosis congenita; HHS, Hoyeraal–Hreidarsson syndrome; IPF, idiopathic pulmonary fibrosis; LMNA , lamins A and C, major
components of the nuclear lamina; NA , not available; PAR , poly(ADP-​ribose); PARN, poly(A)-specific ribonuclease; RTEL1, regulator of telomere elongation helicase 1;
TRF, telomeric repeat-​binding factor. Adapted with permission from ref.113, Elsevier.
The early years in cell culture and the
Hayflick limit. In 1881, the German
biologist August Weissman speculated
that death takes place because worn-​out
tissues could not renew forever47. However,
in 1921, the French Nobel prize-​winning
surgeon Alexis Carrel suggested that all cells
explanted in culture were immortal and
that the lack of continuous cell replication
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found in other laboratories was due to
ignorance on how best to cultivate the
cells48. This concept was generally accepted
until Leonard Hayflick and Paul Moorhead
overturned this dogma that all cells grown
in culture were immortal49,50. In hindsight,
it appears that Carrel’s team may have
infected the cell cultures with sarcoma
virus, thus immortalizing the cells, or by
using unfiltered chick embryo extracts that
may have inadvertently continued to reseed
the culture with living cells. Irrespective
of the explanation for Carrel’s continuous
cell culture observations, Hayflick sparked
investigations of the phenomenon known
as cellular senescence that is often referred
to as the Hayflick limit. Although the basic
Hayflick limit concept has mostly stood
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Perspectives
Telomeres
Telomerase
Muller – definition of telomere42
1938
McClintock: telomeres are natural ends44,45,46
1941
Hayflick limit: replicative senescence49
1961
Watson: telomere end replication problem53
1972
Blackburn: first telomere sequence55
1978
Two-stage model of
telomere loss12
Human telomeres sequenced59
1985
Terminal telomere transferase activity identified26
1988
Telomere regulation in yeast67
1989
Tetrahymena thermophila
telomerase RNA cloning89
Telomerase identified in HeLa cells91
Short telomeres
in tumours84
1990
RAP1 telomere-binding protein in yeast68,69
1994
TRAP assay; telomerase present in almost all human cancers15
1995
Cancers without telomerase regress100
1997
In vitro reconstitution of human telomerase99
qFISH of telomeres110
1998
TERT direct-immortalization of normal human cells29,98
TIN2 shelterin
protein identified78
1999
Direct inhibitors of telomerase35
2000
TERC secondary structure169
TPE in human cells159
2001
TERT splicing identified18
qPCR of telomeres104
2002
STELA assay for measuring some short telomeres107–108
2003
ACD identified79
2004
Telomere shortening in normal human cells83
Telomere shortening
in human tissues85
ALT14
First shelterin protein identified, TRF1 (REF.72,73)
TRF2, another shelterin
protein, identified74–77
Telomeres end in a looping
structure, t-loops81
Single-stranded telomere binding
protein, POT1, identified66
Report of first genetic telomere
human syndrome, DKC128
2008
Structure of TERT in beetles170
Universal STELA assay for measuring all short telomeres11
2010
2013
TERT promoter mutations171,172
2014
Telomerase structure in ciliates173
2015
Telomerase-mediated telomere uncapping36
2016
Telomere length regulation of the TERT gene22
2017
2018
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Absence of cancer in telomeraseexpressing normal cells101
Another genetically inherited telomere syndrome, IPF142,143
2009
TeSLA10
TERT cloned27,96
2007
TERRA168
Regulation of gene expression by telomere
looping, named TPE–OLD21,22,24
TERC cloned28
Cryo-EM structure of telomerase in humans174,175
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Perspectives
◀ Fig. 2 | Telomere and telomerase timeline. An abbreviated timeline of key advances in the telomere
(left panel) and the telomerase (right panel) fields. More details on advances in these areas can be
obtained at the following site: Telomerase Database. This site also includes annotation of the various
telomerase-​associated diseases, telomere sequences of various species and information on telomerase structure. ACD, adrenocortical dysplasia protein homologue; ALT, alternative lengthening of telomeres; Cryo-​EM, cryoelectron microscopy ; DKC, dyskeratosis congenita; IPF, idiopathic primary
fibrosis; POT1, protection of telomeres 1; qFISH, quantitative fluorescence in situ hybridization; qPCR,
quantitative PCR; RAP1, repressor/activator protein 1; STELA, single-​telomere length analysis; TERC,
telomerase RNA component; TERRA, telomere repeat-​containing RNA; TERT, telomerase reverse transcriptase; TeSLA, telomere shortest-​length assay; TIN2, TRF1-interacting nuclear factor 2; TPE-​OLD,
telomere positioning effect over long distances; TRAP, telomere repeat amplification protocol;
TRF, telomeric repeat-​binding factor.
the test of time, others remained sceptical.
For example, Harry Rubin argued that the
concept of a genetically predetermined
number of human fibroblast replications
was based on an artefact resulting from
the damage accumulated by the explanted
cells during their replication in the radically
foreign environment of cell culture51.
Actually, both Hayflick and Rubin were
partially correct, as we now know that the
harsh cell culture environment that most
scientists use often produces a premature
senescence that does not always reflect the
actual molecular-​counting mechanism52.
The onset of molecular biology and identity
of telomere sequences. With the elucidation
of the structure of DNA, the concept of the
double-​stranded helix of complementary
bases enabled scientists to begin to
understand how genetic material could be
copied and transmitted across generations
of organisms and across cell division
lineages. The study of the biochemistry of
DNA replication showed that the enzymatic
mechanism of DNA replication encountered
a unique problem when it came to the ends
of linear chromosomes. This mechanism of
DNA replication required a polynucleotide
primer with a free 3′-hydroxyl group to
initiate synthesis of the daughter strand.
Theoretically, this mechanism precluded
the complete replication of linear DNA
at the ends synthesized by lagging-​strand
mechanisms, and this theory led to the
proposal of the ‘end replication problem’
by James Watson in 1972 (ref.53). The
incomplete replication of linear DNA was
hypothesized to lead to a loss of genetic
information at the chromosome ends54.
Solving the end replication problem became
a central question in the field that eventually
connected telomere length dynamics to the
regulation of cellular senescence, ageing and
cancer biology.
It became necessary to better understand
the molecular details of telomeres to
address the challenges presented by the end
replication problem. The first telomeres
to be characterized were from the ciliated
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protozoa Tetrahymena thermophila55.
Variability at telomeres in yeast was then
described56, and more details about
T. thermophila chromosome ends emerged
showing that the telomere consisted
of 20–70 tandem repeats of a GC-​rich
hexanucleotide sequence. In the following
years, the sequences and structures of
telomeres from a variety of eukaryotic
organisms were identified, all with similar
but not identical characteristics to those
found in T. thermophila57,58. The sequence
of human tandem 5′-TTAGGG-3′ repeats at
telomeres was established in 198859, and this
same sequence was found to be conserved
among more than 90 eukaryotic species60
including all mammals61.
The conserved structure of telomere
sequences suggested a conserved function
for telomeres across species. The fact that
telomere DNA seemed to be protected from
nuclease degradation indicated that a
unique set of proteins might be involved in
packaging or associating with telomeric DNA.
A two-subunit telomere-​binding protein,
originally described by David Prescott’s
team62, was reported that recognized and
tightly bound the 3′ G-​rich single-​strand
overhang of telomeres in the ciliate Oxytricha
nova63. This protein, telomere end-​binding
protein (TEBP), has since been found to have
similarities to that in budding yeast (called
Cdc13 (refs64,65)) and in fission yeast, as well
as in humans (protection of telomeres 1
(POT1))66. The first protein found to bind
to duplex telomeric DNA was repressor/
activator protein 1 (Rap1) in the budding
yeast Saccharomyces cerevisiae67. Rap1 was
originally identified as a transcriptional
regulator, and the in vivo association with
telomeres provided evidence that proteins
involved in other cellular functions could also
play an integral role in telomere structure
and function68. Interestingly, Rap1 binding
at telomeres was found to be a negative
regulator of telomere length68,69. The number
of Rap1 molecules bound to the telomere
duplex DNA appeared to act as a counting
mechanism that regulated telomere length70,71,
at least in yeast.
A series of mammalian telomeric DNA-​
binding and associated proteins, later termed
the shelterin proteins2, were identified
following these discoveries about telomere-​
binding proteins in model organisms. POT1
(ref.66) (a telomere single-​stranded binding
protein) and telomeric repeat-​binding factor 1
(TRF1)72,73 and TRF2 (refs74–77) (both of
which bind to duplex telomeric sequences)
are now known to work in concert with
three additional proteins, TRF1-interacting
nuclear factor 2 (TIN2; also known as
TINF2)78, adrenocortical dysplasia protein
homologue (ACD; also known as TPP1,
PIP1 and PTOP)79 and RAP1, to form the
six-​protein shelterin complex that is essential
for telomeres to function and protects
the ends of mammalian chromosomes2,3
(Fig. 1c). Both TRF1 and TRF2 bind to
the canonical TTAGGG double-​stranded
telomeric repeats, and both interact with
TIN2 (ref.78). TRF2 also binds to double-​
stranded telomeric repeats and interacts
with RAP1. POT1 binds to single-​stranded
TTAGGG repeats and interacts with both
TRF1 and TRF2 through a binding partner,
ACD79, which also associates with TIN2
(ref.80). Finally, the single-​stranded telomere
overhang loops back and invades the
double-​stranded telomeric repeats to form a
t-​loop, so there are no exposed free ends that
may trigger DNA damage responses. This
further helps to preserve genomic integrity
and provide end protection81.
Telomeres in ageing and cancer. Even
though Hayflick49,50 and Olovnikov54
suggested that there was a counting
mechanism for replicative senescence, the
molecular mechanism was not determined
until much later. In 1984, Shampay et al.57
hypothesized that there must be an enzyme
at work on yeast telomeric DNA, similar
to the speculation by McClintock in corn
strains that could not repair their ends46.
This led in 1989, using the power of yeast
genetics, for Lundblad and Szostak82 to
demonstrate an ‘ever shortening telomere’
(EST) phenotype in budding yeast that led
to their senescence. In the same year, the
behaviour of cells expressing an inducible
SV40 large T antigen was interpreted
to suggest that human cells had two
independent steps that had to be bypassed
to become immortal12. In the following year,
three seminal papers were published almost
simultaneously that showed that telomeres
shortened during the ageing of human
fibroblasts in cell culture, that telomeres
shortened in normal tissues in vivo during
ageing, that some reproductive tissues had
longer telomeres than somatic tissues
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Perspectives
had and finally that telomeres were very
short in primary tumours83–86. These
observations and others86,87 reinforced a
hypothetical molecular mechanism for
the Hayflick limit, but all were based on
correlations, and the proof of a cause-​andeffect relationship had to wait until a few
years later.
Telomerase discoveries
Discovery of telomerase. As short telomeres
were proposed to limit the maximum
number of divisions in normal cells,
there had to be a solution to the telomere
end replication problem in immortal
organisms, in the germline cells of higher
organisms and in immortal cancer cells.
The solution came, once again, from
studies in T. thermophila, this time by Carol
Greider, a graduate student in Elizabeth
Blackburn’s laboratory. Greider and
Blackburn discovered enzymatic activity in
extracts of T. thermophila that synthesized
and elongated telomeres and termed this
activity terminal transferase1,26,88. This
would later become known as telomerase.
The observation that RNase inactivated
terminal transferase activity prompted the
characterization of an RNA species that
co-​purified with telomerase activity88. This
was the first evidence that telomerase existed
as a ribonucleoprotein complex. The
T. thermophila RNA template sequence was
identified and confirmed in 1989 (ref.89).
Telomerase activity was subsequently found
in a variety of species, which all generated
their species’ characteristic telomere repeat
sequence in an RNA-​dependent manner,
consistent with the results from
T. thermophila88–93.
Telomerase in humans. The discovery
of telomerase activity in human cancer
cell extracts provided additional evidence
that the reverse transcriptase activity was
widespread and suggested a mechanism
by which cancer cells could grow
indefinitely4,13,29. Although the presence of
the telomerase reverse transcriptase protein
and RNA template subunits was established
in the mid-​to-late 1980s, it would be another
decade before their genes were cloned.
The human TERC (also known as TR or
hTR for human telomerase RNA) gene was
cloned in 1995 (ref.28) and is ubiquitously
expressed in all normal human cells. The
human TERT (also known as hTERT) gene
encoding the catalytic subunit of telomerase
was cloned in 1997 (refs27,94–98). TERT was
not detected in most normal human cells
but was expressed in almost all cancer
cells and was rapidly appreciated to be
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an almost universal marker of advanced
human cancers15,16. As it was difficult to
obtain sufficiently large tumour samples
for the classic primer extension telomerase
activity assay of Greider and Blackburn,
a PCR-​based assay, the telomere repeat
amplification protocol (TRAP)15, was
eagerly adopted by pathologists to test
tumour types of interest from archival
stocks. As telomerase activity was absent in
most normal tissue, and almost all human
tumours (85–90%) not only constitutively
expressed telomerase but also had short
telomeres, the inhibition of telomerase
became, and remains, an attractive target
for cancer therapeutics32–36. However, so far,
there have not been any anti-​telomerase
therapies approved for any indication. This
was certainly not from lack of trying but
most likely a result of the long lag period
from inhibiting telomerase until telomeres
were short enough to cause cells to enter
crisis and undergo apoptosis33. In addition,
as normal cells such as haematopoietic
proliferative cells transiently express
telomerase activity, rate-​limiting toxic effects
have reduced the utility of direct telomerase
inhibitors33. In addition, in some rarer
cancer types, an ALT-​based maintenance
mechanism can be engaged that involves
DNA recombination14, so there is concern
that effective telomerase inhibitors might
engage this ALT-​based survival pathway.
Telomerase can directly immortalize
human cells. Of all the components that
have been found to interact with telomeres,
none inspired such fascination as that of the
holoenzyme complex known as telomerase.
Several lines of evidence suggested that
there had to be a mechanism for generating
de novo telomere sequences, the first of
which was McClintock’s observation that
broken chromosomes could be healed and
that the once-​broken ends then behaved
as natural chromosome ends45. However,
the idea that telomere shortening actually
caused human cell senescence and that
telomerase was the mechanism that
bypassed senescence required the cloning
of human TERT. First, TERT was cloned in
Euplotes on the basis of one of the Lundblad
EST genes82, then in yeast and finally in
humans27. One year after the cloning of
human TERT, the introduction of only
the TERT gene encoding the telomerase
catalytic protein component was shown to
be sufficient to produce telomerase activity29.
Even though human TERC was known
to be present in telomerase-​silent cells, it
was surprising that the introduction of
human TERT was sufficient to reconstitute
telomerase activity in cells. Certain normal
human cells stably expressing transfected
TERT were not only able to maintain or
grow the length of their telomeres but were
functionally immortal. The normal longevity
determination mechanism of telomere
shortening in human cells (the Hayflick
limit) was circumvented for the first time by
a known mechanism, and this established a
causal role of telomerase in human cell
immortalization, at least in certain cells29.
There were several immediate follow-up
reports showing that telomerase could be
reconstituted in vitro by combining the
RNA and protein components98,99 and that
some cancer cells that lacked telomerase
underwent spontaneous remission100
(suggesting that, in the absence of a telomere
maintenance mechanism, cancer cells could
not continue to divide indefinitely). This
also reinforced the idea that inhibiting
telomerase in most cancers might be a
successful therapeutic strategy32–36. The
introduction of telomerase into normal
cells also raised the concern that this might
transform the normal cells into cancer
cells. However, this concern was quickly
shown to be false101. Although telomerase
activity is permissive for cancer, it is not a
dominantly acting oncogene that can induce
transformation by itself101 unless greatly
and nonphysiologically overexpressed.
Finally, telomerase was detected in some
proliferating normal stem-​like cells such
as human T cells102, thus demonstrating
that some highly proliferative normal
cells could express regulated telomerase
activity, thus providing a mechanism for
highly reproductive tissues such as the skin,
intestines and bone marrow to divide much
longer than indicated by the Hayflick limit.
Methodological advances
New methods are often critical to progress
a given field, and this is also true for
quantitative methods of analysing telomeres
and telomerase. In some instances, new
methods may be faster but suffer limitations.
For example, terminal restriction fragment
analysis87,103 is the gold standard for
measuring telomere length, but it is also a
low-​throughput assay8,9. The quantitative
PCR (qPCR) assay was developed to
provide greater throughput and is widely
used104. Although it has been useful for large
population-​based studies, qPCR for telomere
length provides only relative lengths and
does not provide information on most of
the shortest telomeres8,105,106. It is now well
established that it is the shortest telomeres
that trigger senescent cell growth arrest6, and
thus new techniques were developed such as
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Perspectives
the single-​telomere length analysis (STELA)
assay, which can measure the telomere
length on individual chromosomes107,108.
Although there have been many advances
using this method, one limitation is that
not all chromosome ends have the unique
sequences that are required for the design of
primers for this assay, and this restricts the
number of chromosome ends that
can be followed8. The universal STELA
(U-​STELA) method was introduced in 2010
in an attempt to resolve this problem11 and
was improved upon in 2017 by the telomere
shortest-​length assay (TeSLA)8,10. These
methods can detect telomeres from every
chromosome end, making it possible to
monitor changes in all the shortest telomeres
in cells. Although these methods have
lower throughput than qPCR does, they
can potentially provide information about
how telomere shortening actually leads
to senescence or disease pathology. Other
telomere methods, such as flow fluorescence
in situ hybridization (FISH)109,110, can
provide somewhat higher throughput,
mostly for lymphocytes, but this method
depends on probe hybridization kinetics,
and there are likely to be telomere repeats
on the shortest telomeres that are below the
threshold for detection8,9.
For telomerase activity measurements,
the original primer extension assay
developed by Greider26 is robust but
requires large numbers of cells and a partial
purification step. In 1994, the TRAP assay
was developed15 and was widely adopted
because it could semiquantitatively measure
telomerase enzyme activity with a much
smaller number of input cells. More
recently, using the power of droplet digital
PCR, the telomerase assay has become
quantitative (ddTRAP), enabling the
determination of the number of telomerase
molecules per cell and even permitting
single-​cell analyses111,112.
Telomere disorders and gene expression
Primary and secondary telomere spectrum
diseases. There have been many reports of
monogenic inherited diseases that display
signatures of human premature ageing, and
cells from these patients often exhibit much
shorter telomeres than those from age-​
matched controls30,31,113. These premature-​
ageing syndromes have been termed
telomere maintenance spectrum disorders,
or telomeropathies. The well-​characterized
primary diseases (primary telomeropathies)
include inherited and sporadic aplastic
anaemia, dyskeratosis congenita and
familial idiopathic pulmonary fibrosis
(Table 1), and these diseases often show an
NAture Reviews | GeNeTics
early age of onset and genetic anticipation
in future generations113–117. The primary
telomeropathies are caused by mutations
resulting in defects in the telomere
maintenance machinery. By contrast, what
has been termed secondary telomeropathies
(such as ataxia telangiectasia, Bloom
syndrome, Werner syndrome, RECL4
disorders and Hutchinson–Gilford progeria)
have some overlapping symptoms with
primary telomeropathies (Table 1) but are
generally caused by mutations in DNA
repair proteins or structural proteins that
contribute to telomere preservation or
that compromise viability so that there
is increased cell turnover (reviewed
elsewhere113). For example, these studies on
secondary telomeropathies offer a clue into
and potential molecular mechanisms of how
a laminopathy such as Hutchinson–Gilford
progeria syndrome could affect telomere
length and be considered a premature
telomere-​associated ageing syndrome118–123.
As the shelterin protein TRF2 interacts with
lamins A and C but not progerin
(a truncated version of the lamin A protein),
the mutation could disrupt the normal
homeostasis and stability of telomeres118,119.
Mutations in a variety of genes directly
associated with telomeres and telomerase
function113–117,123–151 are considered primary
telomeropathies, but others are likely to
be identified in the future because the
network of genes associated with telomere
maintenance is very large30. For example,
a new disorder, Alazami disease, was
recently identified152. LARP7, which is
mutated in Alazami disease, may be the
human orthologue of the T. thermophila
p65 protein, which is required for
telomerase activity in the latter organism.
This discovery led to the identification of
a potential new primary telomeropathy in
two distinct families in separate areas of the
world that had Alazami syndrome (loss of
function of LARP7). A hallmark of these
telomere spectrum disorders is exceptionally
shortened telomeres compared with those
of age-​matched controls. A mechanistic
understanding of how very short telomeres
lead to tissue-​specific pathology remains
speculative30; hence, more work is needed on
these largely unexplored disorders.
The 3D genomic DNA landscape includes
telomeres. How chromosomes move and
overlap to form topologically associating
domains is still poorly understood but
involves CTCF and cohesins (that form
insulated neighbourhoods)153–156. In addition
to 3D interactions throughout the genome,
the ends of linear chromosomes can form
3D looping interactions21–25,37–39.
Furthermore, the telomere position
effect (TPE) is another mechanism
generally associated with transcriptional
repression of genes close to telomeres, as
previously demonstrated in both yeast and
humans157–159. The progressive erosion of
telomeres that occurs with cell division
in normal cells provides a ‘clocking’
mechanism that limits the maximal numbers
of cell divisions, and it is generally accepted
that age-​dependent telomere shortening
may generate DNA damage signals from a
too-​short telomere. Whereas the concept of
senescence from short telomeres has driven
the cellular-​ageing research field for decades,
much less is known about telomere length
modulating the expression of genes adjacent
Glossary
Alternative lengthening of telomeres
Senescence
(ALT). A telomerase-​independent mechanism of
maintaining telomere length that involves DNA
recombination events.
The process of cellular ageing generally thought
to be irreversible. Senescence can be initiated
by short telomeres and by genotoxic stressors
(in an occurrence often termed premature
senescence).
Chromosome
The thread-​like structure in the nucleus that carries
genetic information. A normal human cell has 23 pairs of
chromosomes (46 total chromosomes). Twenty-​two pairs
are called somatic or body chromosomes. The remaining
two chromosomes are called sex chromosomes and
determine whether a person is a male or a female.
Genetic anticipation
A genetic disorder that is passed on to the next
generation with an earlier age of disease and an increase
in severity of disease. In the telomere field, this can be
due to germline transmission of shorter telomeres in
succeeding generations.
Hayflick limit
The inability of cells to divide (replicate) indefinitely in
culture.
Shelterin
A six-​member protein complex associated with
telomeric DNA that protects the telomeres
from being recognized as damaged DNA needing
repair.
Telomerase
The ribonucleoprotein enzyme complex that adds
telomeric sequences to telomeres and has been
associated with cellular immortality.
Telomeres
The long natural end sequences of a chromosome
composed of repetitive DNA sequences (such as
hexameric, TTAGGGn repeats in mammals).
volume 20 | MAY 2019 | 305
Perspectives
to or over long distances from the telomeres
without initiating a DNA damage
signal. For example, it has been suggested
that telomere loops can interact with
interstitial telomere sequences by lamin
A/C and a shelterin protein, TRF2 (refs37,39).
There are thousands of interstitial telomere
sequences in the human genome, and some
contain enough TTAGGG repeats to interact
with shelterin proteins160–162, leading to stable
interactions with long telomeres that are
lost when telomeres become short. While
this is a fairly new area of research, a better
understanding of the topological chromatin
interactions involving telomeres should
provide new insights into how progressive
telomere shortening may be important in
gene regulation over long periods of time.
This shortening has the potential to be
mechanistically involved in normal human
ageing and disease progression.
TERT and telomere looping: an example
of antagonistic pleiotropy?. Telomerase
is detected in the early stages of human
development (for example, in the blastocyst
stage) but becomes silent in a tissue-​specific
manner during fetal development in all
somatic tissues and remains silenced in most
tissues unless cancer occurs. This pattern is
different in short-​lived small animals, such
as rodents, where telomerase continues to
be expressed in several tissues throughout
life. Thus, the location of the TERT gene
close to the telomere in large long-​lived
mammals22 may have evolved to ensure
sufficient cell divisions early in development
and then to silence TERT when telomeres
are sufficiently long. One mechanism to
explain this process would involve telomere
looping that changes the chromatin patterns
near the TERT and CLPTM1L loci at an
early age and silences TERT expression;
however, with ageing and progressive
telomere shortening, TERT could become
permissive for reactivation, increasing the
risk of cancer. The concept of antagonistic
pleiotropy163–166 was proposed to help explain
evolutionary theories of ageing applied to
long-​lived organisms. For example, silencing
the transcription of the human TERT
gene during fetal development minimizes
telomerase activity during childhood and the
child-​raising years of adulthood and could
be a potent mechanism to prevent the early
onset of telomerase-​expressing cancer cells.
However, with continued cell divisions and
progressive telomere shortening, the TERT
gene becomes permissive for transcriptional
activation and may be detrimental to the
organism’s fitness post-​reproduction late in
life. Short-​telomere-associated pathologies
306 | MAY 2019 | volume 20
(ageing and cancer) are often associated with
the potential to transcriptionally activate
telomerase. Thus, the spatiotemporal
expression of telomerase is tightly regulated
in human cells and may have evolved
to prevent the early onset of age-​related
diseases, whereas the detrimental effects of
this regulatory setup would generally occur
in post-​reproductive years and hence would
not have strong and lasting evolutionary
implications. For additional information on
mechanisms regulating telomerase activity
with progressive telomere shortening, see
the recent review167.
Conclusions and future directions
While there continues to be much
excitement in the telomere and telomerase
field, many challenges and unanswered
questions remain. For example, what are
the best approaches to correct telomere
genetic disorders? One suggestion is to
use telomerase to elongate the shortest
telomeres, perhaps ex vivo or in young
individuals who harbour mutations
that anticipate disease onset. In these
young individuals, the risk of turning on
telomerase in a premalignant cell would
be relatively small compared with the
likelihood of developing a telomere disorder.
Other questions that remain outstanding
include identifying factors that regulate the
maximal telomere length in humans during
development and how telomerase activity is
regulated in proliferating stem-​like cells and
in cancer. Telomeres appear to lose ~50 bp
per cell doubling in vitro, but why do they
lose much less in vivo? Is the end replication
problem really rate limiting for what most
scientists call replicative senescence, or is
it a result of stressful culture conditions
that accelerate telomere shortening or that
induce premature growth arrest? Finally,
are there new approaches to targeting
telomerase or telomeres in diseases such as
cancer? Time will tell.
Jerry W. Shay
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
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20.
21.
22.
23.
24.
* and Woodring E. Wright
Department of Cell Biology, UT Southwestern Medical
Center, Dallas, TX, USA.
25.
*e-​mail: Jerry.Shay@UTSouthwestern.edu
26.
https://doi.org/10.1038/s41576-019-0099-1
Published online 13 February 2019
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Author contributions
Acknowledgements
Springer Nature remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.
This work was supported by the National Institutes of Health
(NIH) (AG01228), the Harold Simmons National Cancer
Institute Designated Comprehensive Cancer Center support
grant (CA142543) and the Southland Financial Corporation
Distinguished Chair in Geriatric Research. This work was performed in laboratories constructed with support from the NIH
(C06 RR30414). Owing to limited space, the authors
­apologize for not including all the advances in this field.
J.W.S. researched content for the article. Both authors contributed to discussing the content, writing, reviewing and
editing the manuscript before submission.
Competing interests
The authors declare no competing interests.
Publisher’s note
Related links
Jerry W. shay and Woodring Wright’s homepage: http://
www4.utsouthwestern.edu/cellbio/shay-​wright/index.html
telomerase Database: http://telomerase.asu.edu/
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