Genetic and epigenetic modulation of telomerase activity in development and disease

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: [email protected] (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).

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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.

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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,

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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

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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 (Gu¨nes 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; Gu¨nes 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).

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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

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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.

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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

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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

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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

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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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