Gene 340 (2004) 1 – 10
www.elsevier.com/locate/gene
Review
Genetic and epigenetic modulation of telomerase activity in
development and disease
Liang Liu a, Serene Lai a, Lucy G. Andrews a, Trygve O. Tollefsbol a,b,c,*
a
Department of Biology, University of Alabama at Birmingham, Birmingham AL 35294-1170, USA
b
Center for Aging, University of Alabama at Birmingham, Birmingham, AL 35294, USA
c
Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35294, USA
Received 15 March 2004; received in revised form 18 May 2004; accepted 3 June 2004
Available online 25 July 2004
Received by A.J. van Wijnen
Abstract
Telomerase activity is one of the most important factors that have been linked to multiple developmental processes, including cell
proliferation, differentiation, aging and senescence. Dysregulation of telomerase has often been found in developmental abnormalities, such
as cancer, loss of function in the hematopoietic system, and low success rate of somatic cloning. A comprehensive network of transcription
factors has been shown to be involved in the genetic control of telomerase expression and activity. Epigenetic mechanisms have recently been
shown to provide an additional level of regulation, and may be responsible for the diverse expression status of telomerase that is manifested
in a tissue and cell-type-dependent manner. This article summarizes the recent developments in the field of telomerase research with a focus
on the coregulation of the telomerase gene by both genetic and epigenetic pathways. Developmental consequences of aberrant telomerase
activity will also be summarized and discussed.
D 2004 Elsevier B.V. All rights reserved.
Keywords: hTERT; DNA methylation; Chromatin remodeling; Transcription; Histone methylation; Histone acetylation
1. Telomerase and replication of telomeric DNA
Eukaryotic chromosomes are capped by telomeres at
each end which consist of tandem repeats of DNA-protein
complexes. In humans, the repetitive DNA sequence is 5VAbbreviations: ALT, alternative lengthening of telomeres; A-T, ataxia
telangiectasia; 5-aza-dc, 5-aza-2V-deoxycytidine; DHS, DNaseI hypersensitivity site; DKC, dyskeratosis congenita; DMSO, dimethyl sulfoxide;
DNMT, DNA methyltransferase; ERE, estrogen response element; ES,
embryonic stem; H3 – Lys9, histone3 – lysine9; HAT, histone acetyltransferase; HDAC, histone deacetylase; HMTase, histone methyltransferase;
hTERC, human telomerase RNA component; hTERT, human telomerase
reverse transcriptase; MEF, mouse embryonic fibroblast; MZF-2, myeloidspecific zinc finger protein 2; RNAi, RNA interference; SIP1, Smadinteracting protein-1; TERC, telomerase RNA component; TERT,
telomerase reverse transcriptase; TSA, trichostatin A; WT1, Wilm’s
Tumor 1.
* Corresponding author. Department of Biology, University of
Alabama at Birmingham, 175 Campbell Hall, 1300 University Boulevard,
Birmingham AL 35294-1170, USA. Tel.: +1-205-934-4573; fax: +1-205975-6097.
E-mail address: trygve@uab.edu (T.O. Tollefsbol).
0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.gene.2004.06.011
TTAGGG-3V (Moyziz et al., 1988), and the human telomeres usually extend 10 – 15 kb in length. During DNA
replication, the conventional DNA-polymerase is unable to
complete replication of the 5V-end of the new DNA strand,
which renders the newly synthesized strand slightly
shorter than the parental strand (reviewed by Klapper et
al., 2001). This phenomenon, often referred to as the
‘‘end-replication problem’’, leads to attrition of the chromosome ends (telomeres) during cell division. Telomerase
is a cellular ribonucleoprotein with reverse transcriptase
activity and is widely employed by eukaryotic systems to
counteract the ‘‘end-replication problem’’. This enzyme
stabilizes telomere length by adding hexameric repeats to
the telomeric ends of the linear chromosomes, thus compensating for the continued erosion of telomeres. Maintenance of telomeres is required for cells to escape from
replicative senescence and proliferate indefinitely. Telomere length is normally maintained by a balance between
processes that lengthen telomeres (mainly through telomerase activity) and processes that shorten telomeres (the
end-replication problem).
2
L. Liu et al. / Gene 340 (2004) 1–10
Currently identified components of telomerase consist
of a telomerase RNA (TERC) molecule with a welldefined secondary structure; the conserved catalytic subunit, telomerase reverse transcriptase (TERT); a number
of additional protein subunits, including Est1p and Est3p,
the two subunits of the Ku heterodimer, and then a large
variety of proteins contributing to the assembly and
maturation of the telomerase enzymatic complex. Among
the various components of the human telomerase, only
human telomerase RNA component (hTERC) and human
telomerase reverse transcriptase (hTERT) are essential for
the reconstitution of telomerase activity (Ishikawa, 1997;
Weinrich et al., 1997; Beattie et al., 1998). Studies have
shown that hTERC is widely expressed in most cell
types, and even in telomerase-negative cells, such as
differentiated somatic cells (Meyerson et al., 1997; Nakamura et al., 1997). hTERT, on the other hand, is tightly
regulated during differentiation and is not expressed or is
expressed at a very low level in most somatic cells. A
positive correlation has been found between the amount
of hTERT mRNA and telomerase activity, therefore suggesting that telomerase is primarily regulated at the level
of transcription of the hTERT gene (Yi et al., 1999; Li et
al., 2003; Cong et al., 1999; also reviewed by Ducrest et
al., 2002).
cases, it has been reported that certain telomerase-negative
immortalized cell lines have overextended telomeres. This
is due to a mechanism of alternative lengthening of
telomeres (ALT), which is not fully understood but
believed to be important for the lengthening of telomeres
independent of telomerase (Reddel et al., 1997; Henson et
al., 2002). Leukemic cells and most breast cancer cells
are telomerase positive, but they possess predominantly
short telomeres (Artande, 2003; Januszkievicz et al.,
2003). Telomerase activity may thus not always correlate
with telomeric length and telomerase activity in these
cancer cells may only maintain stable telomere length to
support the rapid proliferation of these cells. Furthermore,
telomere shortening in somatic cells may not necessarily
be due to complete repression of telomerase activity. A
low level of hTERT expression and telomerase activity
has recently been detected in cycling human fibroblasts
using the immunopurification method with enhanced
sensitivity (Masutomi et al., 2003). Disruption of this
low level of activity by ectopic expression of a catalytically inactive mutant of hTERT or by RNA interference
(RNAi) of hTERT leads to premature senescence (Masutomi et al., 2003). Some cells that lack telomerase
activity, on the other hand, still have a high level of
hTERT transcription. In these cases, regulation at the
level of alternative splicing may lead to the skipping of
exons that encode reverse transcriptase function, so any
2. Activity of telomerase in cellular proliferation,
differentiation and senescence
Telomerase activity is detected at different levels in
various cell types and correlates with the proliferative
potential of the cells. Human cells that retain readily
detectable telomerase activity include germ cells and other
self-renewing tissues, such as basal epidermal cells, lymphocytes and other hematopoetic cells (see reviews by
Forsyth et al., 2002 and Mason, 2003). By contrast,
telomerase activity is downregulated in most somatic cells
contributing to telomeric attrition. This attrition continues
as somatic cells divide until a critical minimum telomeric
length is reached at which time the cells undergo cellular
senescence (Nakayama et al., 1998; Wright and Shay,
1992; Fig. 1). Senescent cells usually have short telomeres and have lost their proliferative ability. In contrast,
70% of immortalized human somatic cell lines and 90 –
95% of human cancer cells express high levels of
telomerase and have stable telomere length as compared
to the cells from which they originate, which suggests a
strong correlation between telomere length maintenance
and tumorigenesis or immortalization (Bryan et al., 1997;
Shay and Gazdar, 1997; also reviewed by Saldanha et al.,
2003).
Because telomerase is responsible for replicating the
telomeres, it is plausible to hypothesize that the activity
of telomerase would positively correlate with the length
of telomeres. This may be true in most cases; but in some
Fig. 1. Depiction of changes in human telomere length during cellular
differentiation and division. As indicated, embryonic stem cells and
germline cells possess relatively stable telomere length regardless of the
number of cell divisions, which is consistent with the constitutive
expression of telomerase activity in these cells. During somatic cell
division, telomeres shorten as a result of early embryonic downregulation of
telomerase activity. Continuous telomere shortening will eventually trigger
cellular senescence that leads to cellular crisis and apoptosis. Aberrant
activation of telomerase activity will enable somatic cells to escape crisis or
senescence states and to achieve indefinite proliferation although the
telomeres are not restored to the full-length level of native immortal
germline cells.
L. Liu et al. / Gene 340 (2004) 1–10
translation product would not produce an active enzyme
(Ulaner et al., 1998).
3. Epigenetic mechanisms in regulation of gene activity
Epigenetics is a fast-growing field of study which focuses on the regulatory network of gene activities during
development that is beyond the level of the gene sequence
itself. Major epigenetic mechanisms include methylation
modification of DNA and the packaging of DNA by histone
proteins into chromatin structure. Methylation of regulatory
DNA sequences often alters the binding ability of transcription factors, which consequently changes gene activity.
During DNA packaging, modifications of the histone proteins by methylation or acetylation can remodel the conformation of the chromatin DNA affecting the accessibility of a
gene to transcription factors. Such epigenetic features may
be created de novo or be erased during cellular proliferation
depending on the presence of the factors involved in the
epigenetic modification pathways. The plasticity of these
epigenetic features confers a dynamic functional status to
the same gene sequence.
DNA methylation occurs in most eukaryotes, and methylation reactions are catalyzed by the DNA methyltransferases (DNMTs). Three functional DNMT groups have
been reported in both mouse and human, including
DNMT1, DNMT3a and DNMT3b. Knockout of each of
these DNMTs in mice is embryonic-lethal in association
with vast loss of genomic methylation (Okano et al., 1999).
Furthermore, genomic DNA tends to become hypomethylated with aging and the exact mechanism is not well
understood. Histone modification is another universal epigenetic mechanism employed by eukaryotes for gene regulation (reviewed by Jenuwein and Allis, 2001). Adding an
acetyl group to the lysine residue located in the N-terminus
of histones changes the charge status of the histone tails,
which decreases the attraction between DNA and histone
tails and thus confers an open conformation of the chromatin DNA for transcription factors to bind (Krajewski, 2002).
Methylation modification of the histone lysine residues may
exist in three different forms: mono-, di- and trimethylation.
Using antibodies specific for each of these methylated states
at Histone3 –Lysine9 (H3 –Lys9), it has been demonstrated
that mono- and dimethylation are associated with inactive
genes in silent euchromatin domains, whereas trimethylated
H3 – Lys9 is enriched at pericentric heterochromatin (Rice et
al., 2003). It is not yet understood what causes this distribution pattern of each form of the methylated histones in the
genome. One possibility may be that these different forms of
methylation modification may occur in situ by target-specific histone methyltransferase (HMTase) activities after the
incorporation of the nonmethylated histones into the chromatin. G9a HMTase, for example, is indicated to be responsible for all detectable dimethylation and a significant
amount of monomethylation within the silent euchromatin,
3
while Suv39h1 and Suv39h2 HMTases may direct trimethylation specifically at pericentric heterochromatin (Rice et
al., 2003).
Histone acetyltransferase (HAT) and histone deacetylase
(HDAC) are two antagonistic players involved in acetylation and deacetylation of the chromatin. It remains in
dispute whether there exists any active demethylation
mechanism for both DNA and histones. One DNA demethylase and several potential histone demethylases have
been reported, but none of them has gained firm experimental support (Bhattacharya et al., 1999; and reviewed by
Bannister et al., 2002). Alternative mechanisms underlying
the reversible biological methylation process have therefore
been proposed that include replacement of the methylated
molecules with nonmethylated molecules, or through clipping of the methyl group (Bannister et al., 2002; Vairapandi,
2004).
Interestingly, telomere length in mice is recently reported
to be directly regulated by histone methylation (Garcia-Cao
et al., 2004 and references therein). Telomeres are normally
enriched in trimethylated H3 – Lys9. In embryonic stem (ES)
cells and embryonic fibroblast cells (MEF) derived from
HMTase null mice, however, telomeres appear to have less
trimethylated H3 – Lys9 but more monomethylated H3 –
Lys9. In addition, HMTase-mutant mice seem to have
abnormally long telomeres relative to wild-type controls.
Surprisingly, there is no change in telomerase activity in
HMTase-mutant cells that harbors such an abnormal telomere elongation. A possible contributing factor may be due
to an increased recruitment of the telomerase to telomeres
(Garcia-Cao et al., 2004). Based on these interesting observations, it will also be worthwhile to determine whether
telomere length changes after knockout of the DNMTs in
mice or human cancer cells. Furthermore, it is observed
recently that telomerase activity in normal human fibroblasts is required for stabilizing DNMT1 activity (Young et
al., 2003), which contributes to the maintenance of a young
state of the cells. If this intriguing interaction between
telomerase and DNMT1 could be confirmed in a wide range
of cell types and tissues, the loss of genomic DNA methylation during aging in somatic cells can then be attributed
to the downregulation of telomerase activity that leads to
reduced DNMT activity during the aging process.
4. Regulation of telomerase activity by transcription
factors
Telomerase activity is significantly reduced in many
somatic cells due to an early embryonic downregulation
(Wright et al., 1996), which can be mainly attributed to the
transcriptional repression of the hTERT gene as previously
mentioned. Transcriptional control of hTERT has thus
emerged as the focus of regulation of telomerase activity.
Characterization of the 5V-hTERT gene regulatory region,
which is depicted in Fig. 2, reveals that it contains numerous
4
L. Liu et al. / Gene 340 (2004) 1–10
Fig. 2. Schematic depiction of the 5Vregulatory region of hTERT containing the recognition sites of selected transcription factors (not drawn to scale). The cMyc/Mad binding sites are often referred to as ‘‘E-boxes’’, and the interaction of c-Myc/Mad with the hTERT promoter has been clearly demonstrated. The 3V
end of this region also depicts the transcription start site of the hTERT gene.
binding sites for transcription factors. These factors are
divided into two categories: repressors and activators.
Repressors include the tumor suppressor protein p53,
Mad1, myeloid-specific zinc finger protein 2 (MZF-2),
Wilms’ Tumor 1 (WT1), TGF-h and Menin. Menin can
bind directly to the hTERT promoter, whereas TGF-h acts
through Smad-interacting protein-1 (SIP1; Lin and Elledge,
2003). The presence of MZF-2 significantly represses
hTERT transcription (Fujimoto et al., 2000), but it is
assumed to play a minor role in the regulation of hTERT.
Binding of WT1 protein to the hTERT gene regulatory
region causes a downregulation of hTERT transcription,
and these effects are specific for WT1-positive cells (Oh
et al., 1999). Overexpression of p53 in SiHa cervical
carcinoma cells, or activation of endogenous p53 at a
physiological level in MCF-7 breast carcinoma cells, can
trigger a rapid downregulation of hTERT mRNA expression
(Kanaya et al., 2000; Xu et al., 2000). Inhibition of p53
activity by means of siRNA-treatment in U2OS cells or in
p53-deleted HCT116 cells, however, failed to reactivate
hTERT expression (Lin and Elledge, 2003), which raises
the question whether p53 is a bona fide repressor of hTERT.
The use of different cell lines may partly account for the
discrepancy between these studies. The epigenetic states of
the hTERT promoter in different cell types may well dictate
the responsive status of hTERT to the level of p53 activity.
In addition to these tumor suppressor pathways identified
as negative regulators of hTERT transcription, activators of
hTERT transcription have also been identified, including cMyc, Sp1 and estrogen. c-Myc is an oncogene and its
product complexes with Max protein as a heterodimer to
activate gene transcription (Blackwood and Eisenman,
1991). Sp1 is another activator of hTERT although the exact
mechanism of its function is unclear. Two estrogen response
element (ERE) sites located in the hTERT promoter bind the
hormone estrogen and its receptor as they translocate into
the nucleus, causing an increased transcription of hTERT.
This activation is ER dependent and cells without ERs are
unresponsive to this pathway (Kyo et al., 1999).
Table 1 shows the major transcription factors known to
interact with the hTERT promoter. Among them, Mad1 and
c-Myc play antagonistic roles in the regulation of hTERT.
They both bind to the consensus sequence 5V-CACGTG-3V,
also called an ‘‘E-box’’ (Wu et al., 1999a,b; Kyo et al.,
2000; Fig. 2). In undifferentiated cells and most neoplastic
and transformed cells, the levels of c-Myc protein are
elevated, while Mad1 protein levels are depressed. Conversely, in differentiated somatic cells, Mad1 levels are
elevated and c-Myc levels are minimal (Günes et al.,
2000). Interestingly, these data correlate with the finding
that cells expressing high levels of c-Myc often express high
levels of hTERT, and high levels of Mad1 are observed in
cells with repressed hTERT. Recent studies have shown that
the preferential binding of the Max protein to Mad1 at the Eboxes of the hTERT promoter in untransformed cells is
characteristically switched to high levels of c-Myc and cMyc/Max heterodimer protein binding at the E-boxes after
Table 1
Key transcription factors that regulate the hTERT genea
Transcription
factor
Role
Number of
binding sites
Reference
p53
MZF-2
Repressor
Repressor
(Kanaya et al., 2000)
(Fujimoto et al., 2000)
TGF-h
Menin
RAK/BRIT1
BRCA1
WT1
Mad1
Repressor
Repressor
Repressor
Repressor
Repressor
Repressor
Tax
E2F-1
Two
(Won et al., 2002b)
c-Myc
Repressor
Repressor in
cancer cells
Activator in
normal cells
Activator
Two
Four
(canonical)
–
Two
–
–
One
Two
(canonical)
–
Two
Estrogen
Activator
Two
(canonical)
Two
USF1/2
Activator
Two
(canonical)
Sp1
Activator
Five
(canonical)
(Wu et al., 1999a,b;
Kyo et al., 2000)
(Kyo et al., 1999;
Misiti et al., 2000)
(Goueli and Janknecht,
2003;
Yago et al., 2002)
(Kyo et al., 2000)
E2F-1
(Yang et al., 2001)
(Lin and Elledge, 2003)
(Lin and Elledge, 2003)
(Li et al., 2002)
(Oh et al., 1999)
(Oh et al., 2000;
Günes et al., 2000)
(Gabet et al., 2003)
(Crowe et al., 2001)
a
This table presents some of the key transcription factors controlling
hTERT activity. Canonical sequences of the binding sites in the hTERT
regulatory region for some of these factors have been confirmed by
mobility shift assays as noted. ‘‘ – ’’ Indicates that these factors have no
direct binding sites identified so far in the hTERT regulatory region and
may act through interaction with other factors, such as c-Myc, Sp1, etc.
More exhaustive lists of transcription factors have been previously
reviewed (Poole et al., 2001; Ducrest et al., 2002).
5
L. Liu et al. / Gene 340 (2004) 1–10
induction of cellular transformation (Casillas et al., 2003a).
This switching pattern of preferential complexing and
binding at the E-boxes is also reported during the induction
of cellular differentiation (Xu et al., 2001).
5. DNA methylation and telomerase activity
Telomerase activity is known to be regulated mainly at
the level of transcription of the hTERT gene, but the exact
molecular mechanism underlying the tumor-specific expression of telomerase remains unclear. Located within the
hTERT promoter are clusters of CpG dinucleotides (Horikawa et al., 1999). These CpG sites are targets for DNA
methylation. Methylation at CpG sites within the promoter
and surrounding regulatory region generally leads to gene
silencing. The promoter regions of some tumor suppressor
genes (e.g., p16 and hMLH1) become methylated during
tumorigenesis and their repression is associated with tumorassociated phenotypes, such as genomic instability and
metastasis of tumor cells (reviewed by Esteller, 2002).
The presence of abundant CpG sites in the hTERT promoter
region has triggered an increasing interest in examining the
possible role of DNA methylation in regulation of hTERT
transcription in normal and cancer cells. Using a bisulfite
genomic sequencing method and a methylation-specific
PCR-based assay, several groups have debated the correlation of hTERT promoter methylation with hTERT activity
(Devereux et al., 1999; Dessain et al., 2000; Guilleret et al.,
2000; Bechter et al., 2002; Lopatina et al., 2003; Shin et al.,
2003). Hypomethylation of the hTERT promoter is seen in
undifferentiated and untransformed cells which are hTERTnegative, suggesting that these cells have a mechanism(s) to
tightly repress the hTERT transcription independent of
promoter methylation (Dessain et al., 2000; Lopatina et
al., 2003; Shin et al., 2003). Methylation of the hTERT
promoter is also observed in differentiated and senescent
cells that do not express hTERT (Lopatina et al., 2003; Shin
et al., 2003), whereas in some transformed and neoplastic
cells, hTERT is reactivated and transcribed regardless of its
densely methylated promoter (Guilleret et al., 2000). This
inconsistent correlation between hTERT expression and its
promoter methylation may be due to the involvement of a
large variety of transcription factors interacting with the
hTERT promoter, which determines the activity of hTERT
depending on the final balance among all the involved
factors. The establishment of a nucleation site methylation
at the hTERT promoter may also be influenced by a
particular transcription factor which recruits (or repels) the
DNMTs to the hTERT promoter in a cell-type-specific
manner (Casillas et al., 2003b).
Retinoic acid-induced differentiation of human teratocarcinoma (HT) cells and human leukemia HL60 cells is
accompanied by silencing of the hTERT gene and increased
methylation of the hTERT promoter (Lopatina et al., 2003;
Liu et al., 2004). Treatment of the differentiating HT cells
with 5-aza-2V-deoxycytidine (5-aza-dC), a common demethylating agent, can reactivate the hTERT gene, suggesting a
direct control of hTERT activity by DNA methylation in
these cells (Lopatina et al., 2003). Other studies have
reported that 5-aza-dC caused inhibition (rather than induction) of hTERT expression and telomerase activity in human
prostate cancer cells and immortal oral dysplasia cultures
(Kitagawa et al., 2000; McGregor et al., 2002; Table 2). The
methylation status of hTERT promoter in these cells was not
analyzed, and the inhibition of hTERT expression by 5-azadC was proposed to be due to an indirect effect from altered
expression of other factors affecting the hTERT transcription, such as inhibition of c-Myc by upregulation of p16
(Kitagawa et al., 2000). Due to the impact of genome-wide
demethylation by 5-aza-dC, direct correlation of demethylation of a specific gene promoter with gene reactivation
would be required to further support the results from such
studies.
Table 2
Chromatin and DNA methylation studies of hTERT regulation in normal cells and cancer cell lines using compounds TSA and 5-aza-dC
Cell lines/chemical compound used
Effect on hTERT/telomerase
activitya
Proposed mechanism of action
Reference
HA1-IM cells/TSA
Pre-crisis lung fibroblast IMR90/TSA
Renal cortical epithelial cells/TSA
Human dermal fibroblasts,
T lymphocytes/TSA
SUSM-1/TSA/5-aza-dC
Differentiated HT cells/5-aza-dC
NHOF, NHOK/5-aza-C
U2OS and GM847 Cells/5-aza-C
Activation
Activation
Activation
Activation
Promoter hyperacetylation
Inhibition of histone deacetylation
Disruption of Sp1/HDAC1 complex
Promoter hyperacetylation
Cong and Bacchetti, 2000
(Wang and Zhu, 2003)
(Takakura et al., 2001)
(Hou et al., 2002)
Activation
Activation
Activation
Increased hTERT expression,
but no changes in
telomerase activity
Repression
Repression
Repression
No effect
Promoter
Promoter
Promoter
Promoter
(Devereux et al., 1999)
(Lopatina et al., 2003)
(Shin et al., 2003)
(Dessain et al., 2000)
TSU-PR1 (prostate cancer)/5-aza-C
Immortal dysplasia/5-aza-dC
Lan-1, HeLa, Co115/5-aza_dC
Cervical cancer cells ME180/TSA
a
demethylation/hyperacetylation
demethylation
demethylation
demethylation
Activation of p16/repression of c-Myc
Activation of RAR-beta and p16
Promoter demethylation
—
(Kitagawa et al., 2000)
(McGregor et al., 2002)
(Guilleret and Benhattar, 2003)
(Takakura et al., 2001)
Activation or repression of hTERT expression generally leads to an elevated or diminished telomerase activity, respectively, unless otherwise specified.
6
L. Liu et al. / Gene 340 (2004) 1–10
The promoter of both the human and mouse telomerase
RNA (hTERC) genes contains a CpG island and may also be
under regulation by DNA methylation (Zhao et al., 1998).
Early studies have shown that the hTERC promoter is
methylated in three of five ALT cell lines, and is associated
with a total absence of hTERC expression in these three cell
lines (Hoare et al., 2001). This strong correlation between
hTERC promoter hypermethylation and lack of hTERC
expression appears to exist only in ALT cell lines. In
addition, these studies also suggest that methylation of the
hTERC promoter may be implicated only in telomerasenegative cell lines, but not in telomerase-negative normal
tissues nor in telomerase-positive tumor tissues (Hoare et al.,
2001). Consistent with these findings, results from a separate
study report that the hTERC promoter is not methylated in
any of 22 telomerase-negative soft tissue sarcomas regardless of the expression status of hTERC, but hypermethylated
in three out of eight telomerase-positive cell lines, indicating
that hTERC expression is not strictly regulated by promoter
methylation (Guilleret et al., 2002).
6. Chromatin remodeling of the hTERT gene during
differentiation and senescence
In addition to methylation modification of the hTERT
promoter, the chromatin environment is another important
epigenetic factor actively involved in hTERT regulation.
Direct evidence comes from transient transfection studies
using luciferase reporters controlled by hTERT promoter
sequences, which showed similar levels of luciferase activity regardless of the expression status of the endogenous
hTERT in the transfected cells (Wang and Zhu, 2003).
Analysis of the endogenous hTERT chromatin susceptibility
to DNaseI digestion consistently reveals a DNaseI hypersensitivity site (DHS) near the hTERT transcription initiation site only in telomerase-positive cells but not in
telomerase-negative cells, which further supports the idea
that differential chromatin conformation at the endogenous
hTERT promoter is directly involved in the control of
telomerase activity (Wang and Zhu, 2003).
Histone acetylation/deacetylation has been implicated as
a common mechanism underlying the hTERT trans-activation/repression in human normal and malignant cells (Cong
and Bacchetti, 2000; Table 2). As previously mentioned,
reversible acetylation of histones remodels the chromatin
structure, which represents an important means in hTERT
regulation. The histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), has been applied to activate the transcription of many genes by increasing the acetylation levels
of nucleosomal histones. Several studies have examined the
effect of TSA on hTERT gene transcription and demonstrated that TSA can indeed reactivate hTERT transcription in
normal somatic cells (Takakura et al., 2001; Cong and
Bacchetti, 2000; Hou et al., 2002; Lopatina et al., 2003;
Wang and Zhu, 2003). Treatment with TSA in normal
telomerase-negative cells leads to an activation of telomerase activity and upregulation of hTERT mRNA, and this
activation effect is not observed in cancer cells (Takakura et
al., 2001). TSA can also induce the formation of a DNaseI
hypersensitivity site at the hTERT promoter that is normally
present in telomerase-positive cells, which provides a mechanistic explanation for the function of TSA in activating
hTERT expression via remodeling its chromatin structure
(Wang and Zhu, 2003). In addition, through transient
transfection experiments, it has also been confirmed that
the hTERT promoter is a target of TSA action and the region
responsible for this TSA-mediated action is localized within
the 181 bp-proximal core promoter containing two E-boxes
and five GC boxes (Takakura et al., 2001; Fig. 2). Due to the
potential effects of TSA treatment on a wide range of genes,
it is not yet clear whether TSA directly affects the chromatin
remodeling of the transfected plasmid, or if this effect is
mediated through other cellular factors in response to TSA
treatment.
In addition to the mechanisms discussed above, a few
transcription factors have been linked to the regulation of
the chromatin structure at the hTERT gene locus. The E-box
binding activator c-Myc and repressor Mad1, for example,
have been shown to regulate hTERT transcription through
modulating the acetylation status of nucleosomal histones at
the hTERT promoter in proliferating versus differentiated
leukemia cells (Xu et al., 2001). Binding of the endogenous
c-Myc/Max complex to the hTERT promoter in proliferating
leukemia cells correlates with acetylation of the histones and
hTERT expression. Following differentiation induced by
dimethyl sulphoxide (DMSO), the endogenous Mad1/Max
complex replaces the c-Myc/Max complex at the hTERT
promoter and this replacement is associated with deacetylation of the histones and inactivation of hTERT. It has also
been demonstrated in a separate study that repression of the
hTERT promoter by the Mad protein requires HDAC
activity, whereas derepression by TSA is independent of
the E-boxes located in its core region (Cong and Bacchetti,
2000). In addition to its normal function in transcription
activation, Sp1 has also been implicated as a recruiting
factor of HDAC to the hTERT promoter to silence telomerase activity in normal fibroblasts and T lymphocytes (Hou et
al., 2002; Won et al., 2002a).
Reactivation of hTERT by 5-aza-dC treatment through
demethylation of the hTERT promoter can be enhanced by
combination with TSA (Devereux et al., 1999), suggesting a
synergistic role of DNA methylation and histone acetylation
in regulating hTERT transcription. Although histone methylation modification also causes chromatin restructuring,
this specific effect has not been explored in the regulation
hTERT promoter. The substantial changes in the state of
telomeric heterochromatin structure in HMTase-mutant
mouse cells were observed to be independent of any
significant alterations in telomerase activity, partly indicating that hTERT activity may not be a direct target of the
disrupted HMTases (Garcia-Cao et al., 2004). Additional
L. Liu et al. / Gene 340 (2004) 1–10
investigations of the interactions among transcription factors
and these epigenetic modulators are warranted for further
understanding of the chromatin structure-mediated regulation of the hTERT transcription.
7. Telomerase dysfunction in cancer and other human
diseases
Telomerase is closely associated with the proliferation
and senescence of normal and malignant cells. Currently,
most attention is focused on telomerase activity in tumorigenesis. Studies performed in telomerase RNA null
(mTERC / ) mice, which lack telomerase activity and
therefore harbor progressive telomere dysfunction, have
revealed after several generations that telomere dysfunction
leads to an increase in initiation of tumor lesions due to
enhanced genomic instability. Progression of these tumor
lesions, however, may be abrogated due to cellular crisis
triggered by the absence of telomerase activity (Wong et al.,
2000; Rudolph et al., 2001). It is further demonstrated that
telomerase reconstitution in cells derived from mTERC /
mice can restore genomic integrity and chemoresistance
(Lee et al., 2001). Based on these observations, it is
proposed that telomere dysfunction may first promote
chromosomal instability that drives early carcinogenesis,
and telomerase activation can late restore genomic stability
to a level permissive for tumor progression. These data
provide intriguing functions of telomerase and telomeres
during tumorigenesis and will have important implications
in cancer therapy.
It is clear that telomerase is required for continuous
tumor cell proliferation and malignant progression, but it
is not yet clear whether the telomerase present in 90% of
human cancers exists as a consequence of selection of
preexisting telomerase-positive cells during carcinogenesis
or through induction of hTERT expression in cells which
normally lack telomerase. The initial idea that telomerase is
present only in cancers and germ cells turns out to be an
incomplete view. Relatively low levels of telomerase activity have also been detected in the proliferative cells of
certain self-renewing tissues, including the bone marrow,
trachea and bronchi, skin (basal layer) and gut (lower crypt;
Forsyth et al., 2002; Masutomi et al., 2003). Such levels of
telomerase may be sufficient to slow down, but not to
prevent, telomere shortening during tissue renewal. Given
the prevalence of reactivation of telomerase manifested in
human cancers, it is well accepted that telomerase represents
an attractive target for new anticancer drugs. Results with a
variety of telomerase inhibitory strategies in human cancer
cells have confirmed that its functional inactivation results
in progressive telomere shortening, leading to growth arrest
and/or cell death through apoptosis (Hahn et al., 1999;
Rezler et al., 2002; Mittal et al., 2004). Because telomerase
activity is primarily regulated through hTERT expression,
understanding hTERT regulation in normal cells is crucial
7
for the understanding of carcinogenesis and may be important in future cancer therapies that target telomerase. Given
that active telomerase activity has been found to exist in the
proliferative parts of self-renewing tissues, the physiological consequences of inhibiting telomerase in these normal
tissues by antitelomerase drugs used to control the proliferation of cancer cells are unclear and await further
investigation.
In addition to cancer, aberrant telomere shortening or
telomerase activity has been implicated in other diseases,
including Down syndrome and atherosclerosis (reviewed by
Klapper et al., 2001), dyskeratosis congenita (DKC; Bessler
et al., 2004), haemopoietic proliferative failure and chronic
infections (reviewed by Wong and Collins, 2003), human
cerebral microvascular disease (Auerbach et al., 2003) and
Alzheimer’s disease (Zhu et al., 2000). Most of the affected
tissues in the above-listed diseases display reduced proliferative capacity, which may be due to abnormal loss of
telomerase activity and accelerated telomere shortening that
leads to loss of function of the cells. DKC, for example, is a
rare genetic disease that is associated with mutations in
hTERC, resulting in reduced telomerase activity in the
haemopoietic system and development of bone marrow
failure typically prior to age 50 years (reviewed by Greenwood and Lansdorp, 2003). Ataxia telangiectasia (A-T) is a
recessive hereditary disorder which is characterized by
progressive neurodegeneration, genomic instability, cancer
susceptibility and accelerated aging. The progression of A-T
appears to be hastened in mice doubly null for the A-T
mutated gene and mTERC in association with accelerated
telomere erosions and an overall proliferation defect in most
cell types, which indicates that telomerase activity may be
an important factor in determining the progression of this
disease (Wong et al., 2003). In most of the disease conditions mentioned above, restoration of hTERC/hTERT
expression or telomerase activity has been proposed as a
therapeutic approach to recover the normal proliferation or
growth of the affected cells. As tempting as the idea may
sound, it may be extremely challenging in practice to restore
the telomerase activity just enough to maintain sufficient
cell proliferation without risk of adverse effects on the cells.
8. Conclusions
The regulation of telomerase activity and consequential
maintenance of telomere length is a complex and dynamic
process that is tightly linked to regulation of cell proliferation. A variety of mechanisms exist to control the
transcription of the hTERT gene, leading to repression or
reactivation of telomerase activity in normal and cancer
cells in a context-dependent manner. In addition to the
regulatory network of transcription factors, epigenetic
mechanisms confer another level of regulation of functional states of the telomerase gene under different settings
(Fig. 3). Although telomerase reactivation is frequently
8
L. Liu et al. / Gene 340 (2004) 1–10
Fig. 3. A model illustrating the synergistic control of hTERT promoter
activity by transcription factors and epigenetic modulators. Epigenetic
modification may affect the accessibility of hTERT by a specific
transcription factor. Alternatively, excess amounts of a particular transcription factor in a specific cell type or aberrant recruitment of that transcription
factor to the hTERT promoter may interfere with the epigenetic stability of
the hTERT promoter that may affect telomerase activity. These interactions
between genetic and epigenetic factors and among different transcription
factors will form a permissive or inhibitive condition for hTERT
transcription depending on the specific cellular context.
observed in neoplastic cells, it does not mean that it is the
factor which causes cancer. The activities of key regulators
of telomerase may first be altered prior to tumor initiation,
which could subsequently trigger the reactivation of telomerase; however, reactivation of telomerase is apparently
the most prevalent means employed by cancer cells to
achieve indefinite growth and its activation generally
occurs early in tumorigenesis. Unraveling the complexities
of the functional control of telomerase should provide
further avenues for targeting telomerase activity as a
common therapy for the great majority of cancer patients.
Acknowledgements
This work was supported in part by grants from the
National Institute on Aging, the Ovarian SPORE Program,
the American Cancer Society, the Purdue-UAB Botanicals
Center, the Geriatric Education, Research and Clinical
Center, and the UAB Postdoc Career Development Award
to Liang Liu.
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