Activity, function, and gene regulation of the catalytic subunit of telomerase (hTERT)

Gene 269 (2001) 1±12

www.elsevier.com/locate/gene

Review

Activity, function, and gene regulation of the catalytic subunit of telomerase (hTERT) Joseph C. Poole, Lucy G. Andrews, Trygve O. Tollefsbol* Department of Biology, University of Alabama at Birmingham, Birmingham, AL 35294-1170, USA Received 29 December 2000; received in revised form 1 March 2001; accepted 14 March 2001 Received by A.J. van Wijnen

Abstract Recent interest in the regulation of telomerase, the enzyme that maintains chromosomal termini, has lead to the discovery and characterization of the catalytic subunit of telomerase, hTERT. Many studies have suggested that the transcription of hTERT represents the ratelimiting step in telomerase expression and key roles for hTERT have been implied in cellular aging, immortalization, and transformation. Before the characterization of the promoter of hTERT in 1999, regulatory mechanisms suggested for this gene were limited to speculation. The successful cloning and characterization of the hTERT 5 0 gene regulatory region has enabled its formal investigation and analysis of potential mechanisms controlling hTERT expression. Although these studies have provided important information about hTERT gene regulation, there has been some confusion regarding the nucleotide boundaries of this region, the location, number, and importance of various transcription factor binding motifs, and the results of promoter activity assays. We feel that this uncertainty, combined with the sheer volume of recent publications on hTERT regulation, calls for consolidation and review. In this analysis we examine recent advances in the regulation of the hTERT gene and attempt to resolve discrepancies resulting from the nearly simultaneous nature of publications in this fastmoving area. Additionally, we aim to summarize the extant knowledge of hTERT gene regulation and its role in important biological processes such as cancer and aging. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Telomerase; Human telomerase reverse transcriptase (hTERT); Gene regulation; Telomeres; Cancer

1. Introduction The role of telomerase, the ribonucleoprotein responsible for maintaining the ends of chromosomes, has been the subject of intense investigation in recent years due to its potential role in cellular aging, cancer, and immortalization (Counter et al., 1992; Kim et al., 1994; Harley et al., 1990). In adult somatic cells, the ends of chromosomes contain tandem telomeric repeats (e.g. 5 0 -TTAGGG-3 0 in humans) (Greider, 1996) that act to preserve chromosomal integrity by preventing degradation, end-to-end fusions, rearrangements, and chromosome attrition (Greider, 1991). In normal somatic cells, each cell division is associated with the loss of Abbreviations: hTERT, human telomerase reverse transcriptase; hTER, human telomerase RNA; MZF-2, myeloid-speci®c zinc ®nger protein 2; WT1, Wilms' tumor protein 1; ERE, estrogen response element; ER, estrogen receptor; TSA, trichostatin A * Corresponding author. Department of Biology, 175A Campbell Hall, 1300 University Boulevard, University of Alabama at Birmingham, Birmingham, AL 35294-1170. Tel.: 11-205-9344573; fax: 11-2059756097. E-mail address: [email protected] (T.O. Tollefsbol).

30±150 bp of telomeric DNA (Vaziri et al., 1993; Harley et al., 1990; Allsopp et al., 1992). This loss of non-coding DNA at the ends of chromosomes eventually results in the reduction to a certain critical length associated with growth arrest and cellular senescence (Chiu and Harley, 1997; Autexier and Greider, 1996) (Fig. 1). In the presence of telomerase, an RNA-dependent DNA polymerase (Greider and Blackburn, 1987; Morin, 1989), telomere lengths are extended or maintained and replicative senescence is avoided (Vaziri and Benchimol, 1998; Bodnar et al., 1998) (Fig. 1). While most normal human somatic cells exhibit no detectable telomerase activity, highly proliferative cells such as germline cells, trophoblasts, hematopoietic cells, endometrial cells, and up to 95% of cancer cells express telomerase to varying degrees (Kim et al., 1994; Counter et al., 1994a,b, 1995; Shay and Bacchetti, 1997; Wright et al., 1996; Yasumoto et al., 1996; Broccoli et al., 1995; HaÈrle-Bachor and Boukamp, 1996) (Fig. 1). Control of telomerase activity has been widely studied and mechanisms for post-translational regulation by phosphorylation have been suggested (Li et al., 1998; Kharbanda et al., 2000). However, the identi®cation of differentially

0378-1119/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(01)00440-1

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Fig. 1. Diagram showing the expression of hTERT in various cell types over the time course of differentiation, aging, stimulation, and immortalization. The expression of hTERT decreases to undetectable levels in most differentiated adult somatic cells but increases in highly proliferative somatic cells such as endometrial tissue and mitogen-stimulated lymphocytes. High levels of hTERT are present in most immortalized and cancer cells, while a lack of detectable hTERT expression in most normal somatic cells results in telomeric attrition, ultimately leading to cellular senescence. In the presence of a transforming event, such as the expression of an oncogenic virus, some cells may escape from senescence to a condition of further cell division referred to as pre-crisis. Still telomerase-negative, these cells may continue dividing until chromosomal termini are essentially devoid of telomeres. This state of crisis is characterized by massive cell death (indicated by the arrow connected to the bold end bar). Alternatively, the upregulation of hTERT expression/telomerase activity and the subsequent extension of telomeric ends at this stage enable escape from crisis and in cases of prolonged hTERT expression, cellular immortalization (Harley et al., 1992).

expressed subunits of telomerase have indicated a major control point at the level of transcription. Studies of the telomerase enzyme complex have revealed the presence of two major subunits contributing to enzymatic activity: an RNA component (hTER) that serves as the template for the polymerase activity of this enzyme and a catalytic subunit with reverse transcriptase activity (hTERT) (Nakayama et al., 1998; Harrington et al., 1997; Feng et al., 1995). Both hTER and hTERT are necessary for reconstitution of telomerase activity in vitro (Weinrich et al., 1997; Beattie et al., 1998). While hTER is widely expressed in embryonic and somatic tissue, hTERT is tightly regulated and is not detectable in most somatic cells (Meyerson et al., 1997; Nakamura et al., 1997). hTERT mRNA expression temporally parallels changes in telomerase activity during cellular differentiation (Bestilny et al., 1996; Savoysky et al., 1996; Xu et al., 1999) and neoplastic transformation (Takakura et al., 1998; Wu et al., 1999a) (Fig. 1). Further support for the essential role of hTERT comes from recent studies showing that ectopic expression of hTERT is suf®cient for restoring telomerase activity in a number of telomerase-negative cell lines, including foreskin ®broblasts, mammary epithelial cells, retinal pigment epithelial cells, and umbilical endothelial cells (Weinrich et al., 1997; Vaziri and Benchimol, 1998; Wen et al., 1998; Bodnar et al., 1998; Counter et al., 1998). Although the additional inactivation of the RB/p16 pathway is required for the hTERT-mediated immortalization of keratinocytes and mammary epithelial cells (Kiyono et al.,

1998), the relationship between hTERT expression and the capacity for cellular immortalization is well established. Taken together, these ®ndings point to the expression of hTERT as the rate-limiting step in telomerase activity and bring the study of hTERT gene expression to the forefront of telomerase regulation research. Elucidation of the mechanisms governing hTERT expression will likely have wide ranging effects on the study and treatment of cancer and other age-related diseases. Approximately 85±95% of tumorigenic tissues express hTERT (Shay and Bacchetti, 1997), making it a valuable tool in cancer diagnosis. Moreover, in some of the most common and lethal cancers, including thyroid (Umbricht et al., 1997), breast (Poremba et al., 1998), cervical (Kyo et al., 1998; Shroyer et al., 1998; Iwasaka et al., 1998), and prostate cancer (Zhang et al., 1998), hTERT expression is detectable in the early stages of malignancy. Additionally, the quanti®cation of in vivo hTERT levels holds promise for cancer prognosis as high telomerase activity has been correlated with a poor prognosis for a number of cancers, including neuroblastoma (Hiyama et al., 1995b, 1997), acute myelogenous leukemia (Xu et al., 1998), breast (Clark et al., 1997), and gastrointestinal cancers (Hiyama et al., 1995c; Okusa et al., 1998). Since hTERT appears to be linked to the early progression as well as the severity of cancer, telomerase/hTERT inhibitors are currently being investigated for their therapeutic potential. Recent work with inducible dominant-negative mutants of hTERT and antisense telo-

J.C. Poole et al. / Gene 269 (2001) 1±12

merase has led to a marked reduction of endogenous telomerase activity in tumor cell lines and eventual death of these cells in vitro (Kondo et al., 1998; Zhang et al., 1999; Hahn et al., 1999a). Additional studies involving hammerhead ribozymes have identi®ed a ribozyme (13ribozyme) that is capable of targeting the 5 0 -end of hTERT mRNA and strongly inhibiting telomerase activity (Yokoyama et al., 2000). Further understanding of the mechanisms involved in hTERT regulation are necessary for the development of anticancer therapies in vivo, as well as for the study of aging and the treatment of other age-related diseases. 2. Establishment of a consensus nucleotide numbering system for the hTERT regulatory region Though recent studies have examined the alternate splicing of the hTERT transcript (Kilian et al., 1997; Ulaner et al., 1998; 2000), the search for mechanisms governing the regulated activity of telomerase has focused on the level of transcription. The sequencing and characterization of the hTERT promoter region has enabled direct study of the molecular mechanisms involved in the regulation of hTERT gene expression (Cong et al., 1999; Horikawa et al., 1999; Takakura et al., 1999; Wick et al., 1999). However, the near simultaneous nature of these publications has yielded some disagreement concerning the sequence numbering system of the hTERT promoter region. The basis for this discrepancy lies in the determination of a consensus transcription start site (Fig. 2). The hTERT promoter region lacks a traditional TATA box or a CAAT box and, as is common with TATA-less promoters (Boisclair et al., 1993), possesses a relatively high density of CpG dinucleotides and Sp1 sites (Cong et al., 1999; Horikawa et al., 1999; Takakura et al., 1999; Wick et al., 1999). Using a CapSite Hunting technique the transcriptional start site for hTERT was assigned to a cytosine located 77 bp upstream of the ATG translational start site (Takakura et al., 1999), while RNase protection assays revealed a transcriptional start site at a guanine found 22 bp further downstream (Horikawa et al., 1999). Both studies identi®ed the transcriptional start sites as the 11 position, while Cong et al. (1999) and Wick et al. (1999) designated the adenine of the initiating ATG as the 11 position. Wick et al. (1999) narrowed the start site for transcription to the region 60±112 bp upstream of the ATG translational start site, though the use of RT-PCR analysis de®ned no exact position for transcription initiation. It is worth noting that this range includes the start site documented by Takakura et al. (1999) described above, but excludes the 255 position corresponding to the transcriptional start site established by Horikawa et al. (1999). The use of different methods for determining transcriptional start sites, combined with the use of different cell lines likely contributed to this discrepancy. Alternatively, the possibility of multiple transcrip-

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tional start sites in a generalized or in a cell-speci®c pattern has been well documented in many TATA-less promoters (Clark et al., 1998; Rudge and Johnson, 1999; Dong et al., 2000). In order to provide a basis for making comparisons between studies, we have chosen to denote the adenine of the initiating ATG as the 11 position. This translational start site for hTERT is accepted by all groups analyzing the hTERT gene control region (Cong et al., 1999; Horikawa et al., 1999; Takakura et al., 1999; Wick et al., 1999), and therefore represents a consensus regarding the numbering of this sequence. All co-ordinates for transcription factor binding motifs and positions of individual nucleotides in our study assume that 11 indicates the translational start site (Fig. 2). Until a consensus transcriptional start site for hTERT is found that applies to all cell types or the lack of a single site is con®rmed, the convention we have proposed is intended to standardize nucleotide numbering within the hTERT 5 0 regulatory region. 3. The hTERT promoter region is rich in transcription factor binding sites Recent studies of the hTERT 5 0 gene regulatory region have identi®ed both canonical and non-canonical motifs for the binding of numerous transcription factors (Tables 1 and 2). While the exact location and number of these motifs varies according to the particular study, the presence of sites for multiple activators and repressors suggests a complex system of regulation. Of the transcription factors known to upregulate hTERT, the oncoprotein c-Myc is the most widely studied, though roles for Sp1, estrogen, and progesterone have also been suggested (Takakura et al., 1999; Kyo et al., 1999; Misiti et al., 2000; Wu et al., 1999b; Kyo et al., 2000; Wang et al., 2000a). The ®rst repressor of the hTERT gene regulatory region identi®ed is the product of the Wilms' tumor 1 suppressor gene (WT1) that may repress hTERT expression in a cell-speci®c manner (Oh et al., 1999). Recent studies have suggested negative regulation of hTERT gene expression by additional transcription factors including MZF-2, p53, and Mad1. The antagonistic control of gene expression by c-Myc- and Mad1-containing complexes has been investigated (Ayer and Eisenman, 1993; Cultraro et al., 1997) and holds promise as a major regulatory mechanism in hTERT expression (Takakura et al., 1999; GuÈnes et al., 2000; Oh et al., 2000). In this review we will attempt to summarize the studies that have investigated the regulatory functions of binding motifs within the hTERT promoter including the potential role of methylation effects on the binding of transcription factors. 3.1. Activators of hTERT gene expression 3.1.1. c-Myc Prior to the characterization of the hTERT promoter

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J.C. Poole et al. / Gene 269 (2001) 1±12

Fig. 2. Nucleotide sequence and features of the hTERT gene regulatory region. Canonical and non-canonical binding sites for known activators and repressors of hTERT are shown in boxes. The core promoter as identi®ed by various groups is underlined as follows: solid line, Cong et al., 1999; dashed line, Horikawa et al., 1999; dotted line, Takakura et al., 1999. Start sites for transcription that have been reported are indicated by arrows: open arrow, Takakura et al., 1999; gray®lled arrow, Horikawa et al., 1999; black-®lled arrow range, Wick et al., 1999. Methylatable CpG sites are italicized. The ATG translational start site is in bold and is designated as nucleotide 11. The deleted region from 16 to 1348 is CpG rich and may participate in hTERT regulation; however, this region lacks binding sites for important transcription factors and is therefore excluded.

region, a correlation between c-Myc oncogene expression and telomerase activity had been established (for review see Cerni, 2000). Using a retrovirus that directs c-Myc expression, Wang et al. (1998) induced telomerase expression in human mammary epithelial cells (that are normally telomerase negative) to levels similar to those measured in breast carcinoma cell lines. Northern blots performed on these cells indicated a 50-fold increase in hTERT mRNA following transfection with the c-Myc vector (Wang et al., 1998). Following the characterization of the hTERT promoter region, identi®cation of binding sites for c-Myc facilitated investigations of potential mechanisms responsible for this regulation (Cong et al., 1999; Horikawa et al., 1999; Takakura et al., 1999; Wick et al., 1999). The c-Myc oncoprotein forms a complex with the Max protein that binds as a heterodimer to activate gene transcription (Blackwood and Eisen-

man, 1991). This c-Myc/Max dimer recognizes and binds the consensus sequence, 5 0 -CACGTG-3 0 , known as an `Ebox' (Wu et al., 1999b; Kyo et al., 2000). Additionally, a related sequence, 5 0 -CA(C/T)GCG-3 0 , also binds c-Myc/ Max heterodimers providing further potential for regulation through binding at these non-canonical sites (Wu et al., 1999b). Of 29 potential binding sites for c-Myc complexes that have been identi®ed in the region of the hTERT gene, 18 are canonical (Wu et al., 1999b). Of these, the main focus has been on two E-boxes (5 0 -CACGTG-3 0 ) located centrally within the hTERT minimal promoter (Horikawa et al., 1999; Takakura et al., 1999; Greenberg et al., 1999) (Fig. 2). Direct activation of hTERT by c-Myc at these E-boxes has been suggested by gene reporter analyses using the luciferase gene (Fig. 3) and mobility shift assays (Table 2) (Horikawa et al., 1999; Takakura et al., 1999; Greenberg

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Table 1 Characterization of hTERT 5 0 -regulatory region transcription factor binding sites by activity, methylation sensitivity, and position a Ttranscription factor

Activator or repressor of hTERT

Methylation sensitivity b,c

Position in hTERT 5 0 regulatory region relative to ATG with number of CpGs given in parentheses d

AP2

Activator

1

AP4 CCAC c-Ets-2 c-Myb c-Myc CREB/ATF/ AP1 ER ER/AP1 IK2 Mad1 MAZ MAZ/Sp1 MyoD MZF-2 NF1 NF-E2/Sp1 NFkB/T3Ra/ NMYC p53 PR Sp1

Activator Activator Activator Activator Activator Activator

? ? ? ? 1 1

2620(1) 1, 2559(2) 2, 2322(2) 2, 2228(0) 3, 2167(1) 4, 2151(1) 4, 2139(1) 4, 2131(0) 4, 2127(1) 4, 2113(0) 4, 2108(0) 4, 25(2) 2, 112(2) 2, 198(0) 2, 1129(2) 2, 1193(2) 2, 1315(1) 2 2853(0) 2, 2618(2) 2, 2531(0) 2, 2509(1) 2, 2315(1) 2, 2192(1) 2, 256(1) 2, 2118(1) 2, 1289(1) 2 2827(0) 1 2247(0) 3, 229(0) 3 2894(1) 2, 1242(1) 2 2242(1) 1±4, 234(1) 1±3 2728(1) 2

Activator Activator Activator Repressor Activator Activator Activator Repressor Activator Activator Activator

? ? ? ? ? ? ? ? ? ? 1

22754(0) 5 2794(0) 10 2815(2) 2, 2647(0) 2, 2377(2) 2, 2227(1) 2, 1121(2) 2, 2175(1) 2 2242(1) 6,7, 234(1) 6,7 2117(0) 3 2151(0) 3, 2108(0) 3 2483(1) 2, 1214(1) 2, 1319(1) 2 2764(0) 8, 2696(2) 8, 2620(0) 8, 2594(0) 8 2929(0) 2, 2841(0) 2, 2458(1) 2, 2232(0) 2, 296(0) 3, 217(1) 2, 168(3) 2, 1149(0) 2, 1343(1) 2 2206(0) 3 2669(4) 2

Repressor ? Activator

? ? 2

Sp1/ER WT1

Activator Repressor

? ?

21954(0) 9, 21317(0) 9 2477(0) 2 2953(2) 2, 2806(1) 1, 2358(3) 1,2, 2323(1) 1, 2262(1) 3, 2206(0) 3, 2188(1) 3,4, 2168(1) 1,3,4, 2151(0) 3, 2133(0) 4, 2113(0) 4, 2108(0) 3, 284(1) 1,3,4, 1254(2) 2 2950(1) 5,10 2358(3) 11

a Superscript numbers represent the following reference citations: 1 Wick et al., 1999; 2 Cong et al., 1999; 3 Horikawa et al., 1999; 4 Takakura et al., 1999; 5 Kyo et al., 1999; 6 Oh et al., 2000; 7 GuÈnes et al., 2000; 8 Fujimoto et al., 2000; 9 Kanaya et al., 2000; 10 Misiti et al., 2000. b Methylation sensitivities described by `?' are currently inconclusive or have not been reported. c Known methlation sensitivities as reviewed by Tate and Bird (1993) and Zingg and Jones (1997). d Positions in italics refer to sites known to speci®cally bind the indicated transcription factor.

et al., 1999; Wu et al., 1999b; Wang et al., 1998). Further evidence of direct c-Myc activation of hTERT was provided by studies revealing that c-Myc-induced upregulation of hTERT occurs in the absence of new protein synthesis (Greenberg et al., 1999). There have been con¯icting reports regarding the relative importance of these two E-boxes for full activity of the hTERT promoter. Abrogation of the proximal E-box (at position 234) resulted in a 10-fold decrease in promoter activity, similar to expression levels for promoterless constructs (Greenberg et al., 1999). This ®nding combined with the determination that the distal E-box is not conserved in the mouse TERT promoter suggests that the ability of cMyc to upregulate hTERT is mediated primarily through the proximal E-box (Greenberg et al., 1999). However, results from luciferase assays where the distal E-box (at position 2242) had been deleted or abrogated revealed a signi®cant reduction in activity in all cell types studied (Fig. 3) (Takakura et al., 1999; Horikawa et al., 1999; Kyo et al., 2000). These contrasting results may suggest a synergistic system when both of these E-boxes are occupied by c-Myc and thus a requirement for both the distal and proximal E-boxes for

maximal activity of the hTERT promoter. Regulation of hTERT at these two E-boxes will be discussed in greater detail later in conjunction with the negative regulation of hTERT by another E-box binding factor, Mad1. 3.1.2. Sp1 The ability of Sp1 to act in co-operation with c-Myc has been reported (Kyo et al., 2000). Co-transfection of a c-Myc or c-Myc/Max expression vector with an Sp1 expression vector induces a two to seven-fold increase in hTERT transcription, depending on cell type (Kyo et al., 2000). Further analysis in the absence of Sp1 revealed only marginal activation, suggesting a reliance on Sp1 for full activity of c-Myc (Kyo et al., 2000). Though widely considered to be a constitutively-expressed factor, Sp1 expression has been reported to vary up to 100-fold in different tissues (Saffer et al., 1991). These ®ndings suggest that the cell type-speci®c induction of hTERT expression by c-Myc/Max may relate to varying levels of endogenous Sp1 (Kyo et al., 2000). Examination of Myc and Sp1 levels in normal and transformed cells has provided further support for Myc-Sp1 co-operation. Cancer cells express high levels of c-Myc and Sp1 while these levels

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Table 2 Mobility shift assay results for known activators and repressors of hTERT Transcription factor a

Recognition sequence within hTERT 5 0 regulatory region

c-Myc c-Myc Sp1 Sp1 Sp1 Sp1 Sp1 ER/Sp1 ER

2242 234 2187 2165 2133 2113 284 2950 22754

WT1 p53 p53 MZF-2 MZF-2 MZF-2 MZF-2 Mad1 Mad1

2358 21954 21317 2764 2696 2620 2594 2242 234

Binding to hTERT 5 0 regulatory region

Binding inhibition by wildtype / mutated competitor

References

CACGTG* CACGTG* CTCCGCCTC* CCGCCC* CCCAGCCCC* CCCAGCCCC* CCGCCC* TGACC/GGGCGGG GGTCAGGCTGATC

1 1 1 1 1 1 1 15 / 26 1

1/2 1/2 1/2 1/2 1/2 1/2 1/2 1 5,? 6 / 2 5,? 6 1/2

1±3 1±3 4 4 4 4 4 5,6 5

GCGCGGGCG AGGCTGGTCT AGGCCTGTTC GGTGGGGA* CGCGGGGA* TCCCCAGC* TCCCCTTC* CACGTG* CACGTG*

1 ? ? 1 1 1 1 1 1

1/2 ?/? ?/? 1/2 1/2 1/2 1/2 1/2 1/2

7 8 8 8 8 9,10 9,10

a Reference and superscript numbers represent the following citations: 1Wu et al., 1999b; 2 Greenberg et al., 1999; 3 Wang et al., 1998; 4 Horikawa et al., 1999; 5 Kyo et al., 1999; 6 Misiti et al., 2000; 7 Oh et al., 1999; 8 Fujimoto et al., 2000; 9 GuÈnes et al., 2000; 10 Oh et al., 2000. Repressors are shown in italics. Asterisks denote binding sites with canonical sequences.

are relatively low in normal cells (Kyo et al., 2000). This variation in expression parallels ®ndings for both hTERT expression and telomerase activity (Kyo et al., 2000). Moreover, during SV-40-induced transformation of normal ®broblasts, levels of Sp1, Myc, and hTERT rise in a co-ordinate manner (Kyo et al., 2000). In contrast to the relatively constant expression of the constitutively-expressed Max protein, Sp1 expression is markedly upregulated during cellular transformation (Kyo et al., 2000). While the exact mechanism by which Sp1 contributes to hTERT transcription is unknown, its role as a co-operative factor with c-Myc is well supported (Horikawa et al., 1999; Kyo et al., 2000). Moreover, recent reports examining the role of estrogen as an activator of hTERT transcription have indicated that Sp1 may assist in the effects of estrogen in ovary epithelial cells (Kyo et al., 1999; Misiti et al., 2000). 3.1.3. Estrogen Though certainly the exception, some normal somatic cells do exhibit telomerase activity. These include highly proliferative cells such as trophoblasts, endometrial cells, and hematopoietic cells (Kyo et al., 1997a,b; Tanaka et al., 1998; Hiyama et al., 1995a) (Fig. 1). Study of telomerase expression in endometrial tissue has linked rising estrogen levels to increased telomerase expression during the proliferative phase of the uterine cycle (Kyo et al., 2000; Tanaka et al., 1998). Moreover, treatment of breast cancer cells and normal ovarian epithelium with estrogen in vitro has been shown to induce an elevation in hTERT mRNA levels and telomerase activity (Kyo et al., 1999; Misiti et al., 2000). A

prior report by Kyo et al. (1999) found two potential estrogen response elements (EREs) within the hTERT promoter region. One of the EREs, located 2754 bp upstream of the translational start codon, binds estrogen and its receptor (ER) in mobility shift assays (Table 2). Moreover, luciferase assays using this putative ERE sequence cloned into a gene reporter construct yield an ,10-fold increase in promoter activity following treatment with estrogen (Kyo et al., 1999). However, a similar assay performed with ER-negative cells failed to induce promoter activity, suggesting a regulatory role for estrogen that is limited to ER-positive cells (Kyo et al., 1999). The more downstream ERE, found at 2950 in the hTERT gene regulatory region, allows for potential coregulation by estrogen and Sp1. This site contains an Sp1 recognition sequence (5 0 -GGGCGGG-3 0 ) adjacent to an ER half site (5 0 -TGACC-3 0 ) ± a motif type known to function as an ERE (Klein-Hitpass et al., 1986; Dubik and Shiu, 1992; Krishnan et al., 1994; Rishi et al., 1995). While Kyo et al. (1999) found this potential ERE to be less important than the ERE further upstream, recent reports indicate direct activation of hTERT expression through the binding of ER at this site (Misiti et al., 2000). Moreover, mutation at this potential ERE drastically reduced hTERT promoter activity, indicating that activation of the hTERT gene by estrogen is direct, and not secondary to the upregulation of c-Myc by estrogen (Misiti et al., 2000). 3.2. Repressors of hTERT gene expression 3.2.1. WT1 With the characterization of the 5 0 regulatory region for

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hTERT (Cong et al., 1999; Horikawa et al., 1999; Takakura et al., 1999; Wick et al., 1999), came evidence from gene reporter assays suggesting the possibility of repressor sites between ,300 and 700 bp upstream of the translation start site (Horikawa et al., 1999; Takakura et al., 1999) (Fig. 2). Shortly thereafter, the identi®cation of the Wilms' Tumor 1 tumor suppressor gene product (WT1) as a potential transcriptional repressor of hTERT marked the ®rst report of a negatively-acting transcription factor for hTERT (Oh et al., 1999). The discovery of repressors of hTERT is of high interest since hTERT gene expression is down-regulated in normal somatic cells during early embryogenesis (Ulaner and Giudice, 1997). Previously, the anti-oncogenic protein, WT1, had been shown to be involved in growth regulation of kidney cells (Englert, 1998; Rauscher, 1997; Coppes et al., 1993; Hastie, 1992; Haber and Buckler, 1992). Interestingly, additional expression of WT1 appears to be limited to gonad and spleen. Such a narrow range of WT1-expression suggests the potential for WT-mediated repression of hTERT in a tissue-speci®c manner. Support for such speci®city comes from gene reporter assays in which mutating the WT1 binding site of the hTERT 5 0 regulatory region

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affected promoter activity only in cells known to express WT1 (Oh et al., 1999). 3.2.2. p53 The possibility for transcriptional control of hTERT by the anti-oncogenic protein, p53, was ®rst suggested by ®ndings indicating an inverse relationship between p53 levels and telomerase activity. To determine whether the activation of telomerase associated with transformation could be due to the loss of p53-mediated hTERT repression, Kanaya et al. (2000) analyzed the effects of p53 overexpression in SiHa cervical cancer cells. Signi®cant repression of hTERT transcription was observed, and two p53 binding sites, located at 21954 and 21317 bp upstream of the ATG, were implicated (Kanaya et al., 2000). Notably, the transcriptional repression effects of exogenous p53 expression preceded any cell growth inhibition traditionally associated with p53 activity (Kanaya et al., 2000). Thus, these ®ndings offer an additional pathway for cell proliferation control by p53, perhaps through the direct repression of hTERT gene expression. As previously described for the regulation of hTERT by c-Myc and estrogen, the transcription factor,

Fig. 3. Depiction of results of luciferase assays using expression constructs containing portions of the hTERT 5 0 gene regulatory region. (a) Distribution of binding sites for known activators and repressors of hTERT. X-axis co-ordinates are given as the number of bp upstream of the initiating ATG. Activators are shown in green; repressors are shown in red. For transcription factors with multiple adjacent sites (e.g. MZF-2 and Sp1), the number of sites is shown in parenthesis. (b) Lines represent assays of six different cancer cell lines known to express hTERT: yellow, ME180 (cervical cancer); green, HeLa (cervical cancer); orange, C33A (cervical cancer); red, SiHa (cervical cancer), from Takakura et al., 1999; violet, RCC23 (renal cell carcinoma); blue, SiHa (cervical cancer), from Horikawa et al., 1999. Each triangle represents an hTERT promoter construct that contains a portion of the hTERT 5 0 gene regulatory region having an upstream limit given by the x-axis. Each cell type assayed has been standardized by designating peak activity at 100%. (c) Diagram of mean of results from all six cell lines given in (b).

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Sp1, is thought to play a co-operative role in p53-based regulation as well (Kanaya et al., 2000). Mutation of Sp1 binding sites completely eliminated the repressive effects of p53 indicating absolute dependence on Sp1 (Kanaya et al., 2000). Interestingly, Sp1 seems to contribute to the activity of both activators and repressors of hTERT suggesting an increasingly complex model, perhaps involving proteinprotein interactions between Sp1 and other transcription factors (Kanaya et al., 2000; Ohlsson et al., 1998; Kyo et al., 2000). Further investigations will be necessary to determine the extent of p53-mediated repression of hTERT across a range of cell types and to reveal possible mechanisms of action requiring co-operation of Sp1. 3.2.3. MZF-2 Gene reporter assays that suggested the presence of repressor sites upstream of the hTERT core promoter (Fig. 3) lead to the discovery and characterization of WT1 as a repressor of hTERT gene expression (Oh et al., 2000). However, as a factor whose expression is limited to kidney, spleen, and gonad, WT1 cannot represent the only repressor of hTERT transcription. The search for a more universal repressor has lead to the discovery of four binding sites for myeloid-speci®c zinc ®nger protein 2 (MZF-2) within a 200 bp region just upstream of the hTERT core promoter region (Fujimoto et al., 2000). hTERT promoter constructs that have their 5 0 boundaries within this 200 bp region (from approximately 2800 to 2600) have characteristically lower expression (Fig. 3) (Takakura et al., 1999; Horikawa et al., 1999). The repression of hTERT transcription associated with cellular differentiation was shown to be enhanced by promoter constructs containing the two proximal MZF-2 sites, suggesting a mechanism of MZF-2-dependent repression of hTERT (Fujimoto et al., 2000). Conversely, MZF-2 levels have been found to remain relatively stable during cellular differentiation, thus casting doubt that MZF-2 acts alone in this repression (Fujimoto et al., 2000). This feature of MZF-2 expression, combined with the identi®cation of telomerase-positive cells that express MZF-2, suggests that MZF-2 may be a minor factor in the regulation of hTERT gene expression at least in most cell types (Fujimoto et al., 2000). 3.2.4. Mad1 The myc/max/mad network encompasses a group of transcription factors that dimerize and bind to both consensus and non-consensus E-box motifs (GuÈnes et al., 2000). Speci®cally, c-Myc or Mad1 dimerizes with the ubiquitously produced Max factor to form a heterodimer capable of either activation or repression, respectively (GuÈnes et al., 2000). Undifferentiated cells, as well as most neoplastic and transformed cell lines, are characterized by high levels of c-Myc, while Mad1 expression is generally minimal in these cells (GuÈnes et al., 2000). In cellular contexts characterized by minimal expression of hTERT, such as is found in most normal somatic cells, the situation is reversed and c-Myc

levels are low while Mad1 levels are high (GuÈnes et al., 2000; Oh et al., 2000). The direct antagonism between cMyc and Mad1 in the control of hTERT gene expression suggests a regulatory model whereby hTERT expression relates directly to the relative levels of c-Myc and Mad1 (Oh et al., 2000). In support of this model, it has recently been demonstrated that ectopic expression of c-Myc in normal somatic cell lines under Mad1-mediated repression can activate hTERT gene expression, presumably by outcompeting endogenous Mad1 levels in dimerizing with Max and binding to the hTERT 5 0 gene regulatory region (Oh et al., 2000). The possibility of reversible control of hTERT gene expression involving the interplay between c-Myc and Mad1 at one or both of the E-box sequences in the hTERT core promoter provides an exciting prospect for the mechanism of hTERT repression during early embryogenesis, as well as the derepression associated with neoplastic transformation. 4. Transcriptional control of hTERT by differential methylation Analysis of the hTERT 5 0 gene regulatory region revealed the presence of a CpG island and a high overall GC content (Cong et al., 1999; Horikawa et al., 1999; Takakura et al., 1999; Wick et al., 1999). This feature, combined with the presence of binding sites for methylation-sensitive transcription factors within the hTERT core promoter (Table 1), suggests a possible role for methylation in the regulation of hTERT gene expression. Methylationmediated control of gene expression is most often associated with gene silencing which may result from randomlydispersed or region-speci®c CpG methylation (Jones and Laird, 1999). In the regulation of genes linked to cancer and development, including the von Hippel-Lindau retinoblastoma gene, the tumor suppressor gene p16INK4a and the hMLH1 gene, promoter-speci®c CpG methylation acts to inhibit gene expression (Merlo et al., 1995; GonzalezZulueta et al., 1995; Deng et al., 1999; Laird and Jaenisch, 1996). Early studies investigating possible mechanisms for methylation-mediated control of hTERT gene expression have not revealed a generalized mode of methylationbased regulation. However, trends in methylation patterns of the hTERT 5 0 gene regulatory region among cells of similar type and activity suggest a possible role for methylation in the de-repression of hTERT during oncogenesis (Devereux et al., 1999; Dessain et al., 2000). Several cancer cell lines, known to express hTERT, possess hypermethylated hTERT promoter regions (Devereux et al., 1999; Dessain et al., 2000) suggesting a role for methylation in the blocking of negatively-acting transcription factors. Similarly, normal somatic cells that do not express the hTERT gene are commonly characterized as having unmethylated or hypomethylated hTERT promoter regions

J.C. Poole et al. / Gene 269 (2001) 1±12

(Devereux et al., 1999; Dessain et al., 2000). While no methylation-sensitive repressor with binding sites within the hTERT 5 0 regulatory region has yet been reported, the binding site for the repressor WT1 has three CpGs, methylation of which could contribute to de-repression of hTERT in a limited range of tissue-speci®c cancers (Oh et al., 1999). Additionally, the MZF-2 recognition sequence at 696 bp upstream from the hTERT translational start site contains two methylatable CpGs. Although the methylation sensitivity of MZF-2 has not yet been reported, the presence of CpGs within their binding sequences has potential importance for methylation-mediated derepression of hTERT transcription. Moreover, the potential for methylationmediated control of hTERT gene expression is not limited to direct inhibition of transcription factor binding, since indirect control mechanisms involving the overlapping of binding or ®ngerprint motifs or acetylation-dependent chromatin restructuring may also contribute to transcriptional control of hTERT (Siegfried and Cedar, 1997; Hendrich and Bird, 1998; Meehan et al., 1989; Boyes and Bird, 1992). While dramatic changes in hTERT promoter methylation between normal and transformed cells support the validity of methylation-mediated control, further work in this area will be necessary to elucidate speci®c mechanisms responsible for this regulation. 5. Conclusion Recent reports addressing hTERT regulation present multiple and wide-ranging mechanisms potentially involved in the transcriptional control of hTERT. From the identi®cation of activators and repressors that bind to the hTERT 5 0 regulatory region to the role of CpG methylation and histone acetylation, an abundance of regulatory models have been suggested. Further work will be necessary to determine how hTERT expression and telomerase activity are regulated both in vitro and in vivo. Currently, investigations into the role of hTERT in cancer and immortalization are providing new avenues for the treatment of cancer and the study of cellular aging and age-related diseases. Gene therapy involving an apoptotic agent combined with a segment of the hTERT promoter region holds promise for treatment of nearly all cancers (Koga et al., 2000; Gu et al., 2000). Early work on the immortalization of normal cells by the ectopic expression of hTERT has allowed for the establishment of non-transformed, immortalized cell lines. Although the recent creation of both transformed and non-transformed immortalized cells using hTERT has increased understanding of the factors that mediate neoplastic transformation (Hahn et al., 1999b; Ouellette et al., 2000), the immortalization of cells with hTERT in vivo is considered risky due to potential increased susceptibility to neoplastic transformation (Wang et al., 2000b). Additional study will likely be necessary before in vivo applications are practicable. Further

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study of the relationship between hTERT and c-Myc expression will also be necessary in order to resolve the question of the potential oncogenicity of hTERT-mediated immortalization (Wang et al., 2000b). This work also promises to elucidate the mechanisms for the reversible control of hTERT expression by the myc/mad/max network that could represent a generalized regulatory model. Additionally, mechanisms involving Mad1-mediated recruitment of histone deacetylases (Cong and Bacchetti, 2000), may lead to new insight regarding chromatin structure in the control of hTERT expression. From the recent studies that have analyzed hTERT regulation in normal, neoplastic, and immortalized cells, the case for tissue and cell-speci®c regulation of hTERT expression seems strong. Further work in the area of hTERT/telomerase control will likely produce several mechanisms of reversible control involving multiple transcription factors, methylation changes and chromatin restructuring necessary for the variation in telomerase activity found during the course of human development and aging. Acknowledgements We thank Joyce Haskell for critical reading of the manuscript and Jason Key for technical assistance in preparation of the ®gures. This work was supported in part by grants from the John A. Hartford Foundation, the Southeast Center for Excellence in Geriatric Medicine, the American Cancer Society, and the UAB Center for Aging, Department of Biology and Natural Sciences and Mathematics Graduate School. References Allsopp, R.C., Vaziri, H., Patterson, C., Goldstein, S., Younglai, E.V., Futcher, A.B., Greider, C.W., Harley, C.B., 1992. Telomere length predicts replicative capacity of human ®broblasts. Proc. Natl. Acad. Sci. 89, 10114±10118. Autexier, C., Greider, C.W., 1996. Telomerase and cancer: revisiting the telomere hypothesis. Trends Biochem. Sci. 21, 387±391. Ayer, D.E., Eisenman, R.N., 1993. A switch from Myc:Max to Mad:Max heterocomplexes accompanies monocyte/macrophage differentiation. Genes Dev. 7, 2110±2119. Beattie, T.L., Zhou, W., Robinson, M.O., Harrington, L., 1998. Reconstitution of human telomerase activity in vitro. Curr. Biol. 8, 177±180. Bestilny, L.J., Brown, C.B., Miura, Y., Robertson, L.D., Riabowol, K.T., 1996. Selective inhibition of telomerase activity during terminal differentiation of immortal cell lines. Cancer Res. 56, 3796±3802. Blackwood, E.M., Eisenman, R.N., 1991. Max: a helix±loop±helix zipper protein that forms a sequence-speci®c DNA-binding complex with Myc. Science 251, 1211±1217. Bodnar, A.G., Ouellette, M., Frolkis, M., Holt, S.E., Chiu, C.-P., Morin, G.B., Harley, C.B., Shay, J.W., Lichtsteiner, S., Wright, W.E., 1998. Extension of life-span by introduction of telomerase into normal human cells. Science 279, 349±352. Boisclair, Y.R., Brown, A.L., Casola, S., Rechler, M.M., 1993. Three clustered Sp1 sites are required for ef®cient transcription of the TATA-less promoter of the gene for insulin-like growth factor-binding protein-2 from the rat. J. Biol. Chem. 268, 24892±24901.

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