Epigenetic Regulation of Human Telomerase ReverseTranscriptase Promoter Activity during CellularDifferentiation

Liang Liu,1 Sabita N. Saldanha,1 Mitchell S. Pate,1 Lucy G. Andrews,1 and Trygve O. Tollefsbol1–3*1Department of Biology, University of Alabama at Birmingham, Alabama2Center for Aging, University of Alabama at Birmingham, Alabama3Comprehensive Cancer Center, University of Alabama at Birmingham, Alabama

The human telomerase reverse transcriptase (TERT) gene is transcriptionally inactivated in most differentiated cells but isreactivated in the majority of cancer cells. To elucidate how TERT is inactivated during differentiation, we applied all-transretinoic acid (ATRA) to induce the differentiation of human teratocarcinoma (HT) cells and human acute myeloid leukemia(HL60) cells. We first showed that TERT promoter activity decreased rapidly, which preceded a gradual loss of endogenoustelomerase activity following ATRA induction. To elucidate the underlying mechanisms of the reduced TERT promoter activityduring differentiation, we performed epigenetic studies on the TERT promoter and found a progressive histone hypoacety-lation coupled with a gradual accumulation of methylated cytosines in the TERT promoter. We also observed that the TERTpromoter was less methylated in pluripotent HT cells than in multipotent HL60 cells throughout a 12-day differentiationprocess. This origin-dependent epigenetic change was also confirmed in histone acetylation studies, indicating that the TERTpromoter was more resistant to deacetylation in HT cells than in HL60 cells. Taken together, our results demonstratesynergistic involvement of DNA methylation and histone deacetylation in the down-regulation of TERT promoter activity thatmay be dependent on the origin of the cell types, and they add new insight into the way telomerase activity may be regulatedduring differentiation. © 2004 Wiley-Liss, Inc.

INTRODUCTION

Early development is characterized by rapidcell division and a fast growth rate of the organ-ism. One of the prominent players in this processis telomerase, the enzyme responsible for syn-thesizing the telomeric DNA at the ends of chro-mosomes that is essential to stabilizing them(Greider, 1990; Bachand, 2000). Telomeresshorten gradually in somatic cells with each cellcycle from late development through aging (Har-ley et al., 1990; Vaziri et al., 1993) because ofearly embryonic down-regulation of telomeraseactivity. Critically short, dysfunctional telomereseventually induce cells to senesce or die throughmechanisms not yet fully understood (Campisi etal., 2001). Human telomerase consists of an RNAcomponent and a catalytic subunit with reverse-transcriptase activity (TERT; Weinrich et al.,1997). Interestingly, expression of TERT mRNAhas been shown to parallel temporally cellularchanges in telomerase activity throughout devel-opment (Poole et al., 2001; Granger et al., 2002).Thus, the absence of telomerase activity in ter-minally differentiated somatic cells can be attrib-uted to down-regulation of TERT expressionduring early development.

In contrast to the inactivation of TERT and theresulting loss of telomerase activity during earlydevelopment, about 90% of tumor tissues and can-cer cells display high telomerase activity andreadily detectable TERT expression (Meyerson,2000; Granger et al., 2002). Inappropriate regula-tion of TERT is often associated with harmful de-velopmental consequences such as that observed inmalignant transformation. Alternative mechanismsof maintaining telomere length have been reportedin both mouse and human cells (Bryan et al., 1997;Jaco et al., 2003), but most tumors examined so farappear to employ telomerase activation as a meansof escaping senescence and retaining a high prolif-erative potential. These observations suggest that

Supported by: National Institute on Aging; Grant number: 1 R03AG20375 01; American Cancer Society; Grant number: IRG-60-001-41; John A. Hartford Foundation (Southeast Center for Excellencein Geriatric Medicine); Leukemia Research Foundation.

*Correspondence to: Dr. Trygve O. Tollefsbol, University ofAlabama at Birmingham, Department of Biology, 175 CampbellHall, 1300 University Boulevard, Birmingham, AL 35294-1170.E-mail: trygve@uab.edu

Received 4 November 2003; Accepted 5 March 2004DOI 10.1002/gcc.20058Published online 19 May 2004 in

Wiley InterScience (www.interscience.wiley.com).

GENES, CHROMOSOMES & CANCER 41:26–37 (2004)

© 2004 Wiley-Liss, Inc.

the control of TERT expression is a reversible pro-cess that has a significant impact during normaldevelopment and in disease conditions. Under-standing the control mechanisms of TERT expres-sion therefore has become a prominent topic ofresearch and under certain circumstances mayprove to be effective in the unraveling of the com-plicated tumorigenesis process.

A growing number of transcriptional factors arebeing studied as potential TERT regulators, includ-ing growth factors, protein kinases, and the onco-genic BMI1 protein, which appears to activateTERT transcription (Wang et al., 2000; Dimri et al.,2002; Maida et al., 2002). Among the various tran-scriptional factors, MYC (oncogenic protein) andMAD (putative tumor-repressor protein) are con-sidered major competitors that interact with theTERT promoter, which is roughly 2 kb in lengthand rich in transcription factor–binding sites(Takakura et al., 1999). The TERT minimal pro-moter, which is defined as the region of about 500bp upstream of the transcriptional starting site,contains two MYC/MAD recognition sites, oftenreferred to as E-boxes (Poole et al., 2001). It isgenerally believed that the binding of MYC to theE-boxes will enhance transcription initiation,whereas replacement of MYC by MAD is associ-ated with down-regulation of TERT (Amati et al.,1994; Greenberg et al., 1999; Gunes et al., 2000; Ohet al., 2000). WT1 is another transcriptional repres-sor with binding sites in the TERT promoter re-gion, but its function is thought to be tissue-spe-cific, and it may function mainly in specific tissuessuch as spleen, gonad, and kidney (Haber andBuckler, 1992; Oh et al., 1999).

In addition to the regulation of TERT by varioustrans-acting protein factors, epigenetic control ofTERT has recently come into focus. The TERTpromoter has a high GC content, particularlywithin the minimal promoter region, indicatingthat DNA methylation may be involved in control-ling TERT expression (Oh et al., 2000; Poole et al.,2001). Methylation of the promoter frequently canaffect the binding of transcriptional factors thatinteract with the TERT promoter (Tollefsbol andAndrews, 2001). Switching of the binding of tran-scriptional factors to the promoter, such as fromMYC to MAD, will often lead to a different func-tional state of the same gene (Xu et al., 2001). Inaddition to DNA methylation, histone modificationis another potent and widespread epigenetic regu-latory mechanism that can silence gene expressionin a reversible manner, through modifying thechromatin environment of the target gene and thus

affecting the accessibility of the regulatory se-quences to the transcriptional factors. Furthermore,it has been shown that DNA methylation can in-duce histone deacetylation through various methyl-CpG binding proteins or via direct interactionsbetween DNA methyltransferase and histonedeacetylase (HDAC) to recruit HDAC to deacety-late the methylated DNA (Fuks et al., 2000, 2001;Geiman and Robertson, 2002).

To determine further how DNA methylationand histone acetylation are involved in inactivatingTERT expression during early development, weassessed the changes in DNA methylation andacetylation of histone H3, and their effects onTERT promoter activity and telomerase activityduring differentiation of human stem cells. TheNCCIT human embryonic teratocarcinoma (HT)cell line is a pluripotent stem-cell line derived fromnonseminomatous germ-cell tumors (NSGCTs),with features intermediate between seminoma andembryonic carcinoma, that is capable of somaticand extraembryonic differentiation (Damjanov etal., 1993). HL60 is a myeloid multipotent cell linethat was derived from peripheral-blood leukocytesfrom a patient with acute myeloid leukemia. Thesecell lines were chosen for our studies because bothhave been shown to be telomerase positive and tobe able to differentiate in response to all-transretinoic acid (ATRA) induction. After differentia-tion, we monitored the kinetic changes in TERTpromoter activity and also in the DNA methylationand histone acetylation status of the TERT pro-moter, which allowed us to assess the epigeneticeffects on TERT expression during cellular differ-entiation.

MATERIALS AND METHODS

Cell Culture, Immunocytochemistry, andFlow-Activated Cell-Sorting Analyses

HT and HL60 cells (both from the AmericanType Culture Collection, Manassas, VA) weregrown in a 5% CO2 humidified incubator at 37°C inRPMI 1640 medium (Gibco, Carlsbad, CA) supple-mented with 1% L-glutamine, antibiotics (ampho-tericin B, penicillin, and streptomycin; Gibco), and10% heat-inactivated fetal bovine serum (Hyclone,Logan, UT). For ATRA treatment, 4 � 105 cells/ml were seeded in fresh medium. ATRA (Sigma,St. Louis, MO) was added to make a final optimalconcentration of 1 �M for HL60 cells, and of 2 �Mfor HT cells. The cells were passaged and refedwith fresh ATRA medium every 3 days to maintainlog-phase growth. Control cells were grown in par-

27EPIGENETIC CONTROL OF TERT PROMOTER ACTIVITY

allel in the same conditions as those for the ATRA-treated cells, except without the addition of ATRA.

For immunocytochemistry, HT cells were grownin two-well tissue culture chamber slides (NalgeNunc International, Rochester, NY) and treatedwith 2 �M of ATRA for 12 days. Cells were thenfixed in absolute acetone for 10 min, washed threetimes with phosphate-buffered saline (PBS; Gibco,Carlsbad, CA) and then blocked in 250 �l of block-ing buffer (1 ml of heat-inactivated goat serum and9 ml of PBS) for 20 min. Antifibronectin antibody(Santa Cruz Biotechnology, Inc., Santa Cruz, CA)was added to the cells at a 1:100 dilution in block-ing buffer, in which the cells were incubated for 1hr at 4°C, followed by having three 5-min washeswith cold PBS. Fluorescein isothiocyanate (FIT-C)–labeled secondary antibody (Santa Cruz Bio-technology, Inc., Santa Cruz, CA) was added at adilution of 1:100 in 1.5% PBS-goat serum and in-cubated for 45 min at 4°C, followed by three 5-minwashes with cold PBS. Two drops of gel mount(Biomeda, Foster City, CA) was added, and theslides were left to dry for 1 hr. Images were ana-lyzed on a Leitz fluorescence microscope.

For flow-activated cell-sorting (FACS) analysisof CD11b expression in differentiating HL60 cells,fluorochrome conjugates of monoclonal antibodyagainst human CD11b antigen, which is the matu-ration marker for neutrophils, were used for detect-ing CD11b expression in ATRA-treated and -un-treated HL60 cells according to the protocol fromthe manufacturer (Sigma, St. Louis, MO). Briefly,about 0.5 � 106 cells were harvested, washed twicewith cold PBS, and then incubated with 2 �g of theFITC-conjugated antibody for 30 min at room tem-perature. After incubation, the cells were washedtwice with diluent solution (PBS containing 1%bovine serum albumin and 0.1% NaN3), then fixedwith 2% paraformaldehyde, and analyzed on a Bec-ton Dickinson FACScalibur flow cytometer. Thefluorescence intensity of the viable cells was ana-lyzed with CellQuest software. Untreated HL60cells incubated only with diluent solution wereincluded as a control set for gating the thresholdvalue (1.0%).

Reporter Gene Assay of TERT Promoter Activity

The TERT promoter construct P-330 (a deletionfragment from the P-3996 construct, kindly pro-vided by Dr. Silvia Bacchetti) was cloned andtransformed into competent JM109 Escherichia colicells (Promega, Madison WI). Positive colonieswere selected and verified by sequencing, and theplasmid DNA was prepared by use of the QIAfilter

Plasmid Midi kit (Qiagen, Valencia, CA). Beforetransfection, HT cells were grown in 12-well cul-ture plates. One microgram of promoter-luciferaseconstruct was transiently transfected into the HTcells by use of FuGENE 6 (Roche, Indianapolis,IN). One microgram of pSV-beta-galactosidase (�-gal) control vector (Promega) was cotransfected tocontrol for transfection efficiency and to normalizethe values of the experimental vector. The nega-tive and positive controls were pGL2-basic vectorand pGL2-enhancer vector (Promega, MadisonWI), respectively. They were used for comparisonwith the luciferase values of the experimental sam-ples. Mock-transfected cells were used to controlfor the presence of endogenous �-gal activity in thecultured cells.

Forty-eight hours after transfection, cells werewashed with PBS and lysed in the reporter lysisbuffer (Promega, Madison WI). Luciferase activitywas assayed and measured at 560 nm with theLuciferase Reporter Assay System (Promega, Mad-ison WI) in a Monolight 1500 Luminometer (Bec-ton Dickinson, Microbiology Systems, Cock-eysville, MD). The �-gal assay was performed bymixing 100 �l of sample with 50 �l of 1� reporterlysis buffer (Promega). Standard assay buffer (150�l) was added to the mixture and incubated at37°C for 1 hr. The reaction was stopped by theaddition of 500 �l of 5 M sodium bicarbonate, anda spectrophotometric reading was taken at 420 nm.The �-gal values of the sample were extrapolatedfrom a standard �-gal curve. Luciferase activity wasobtained by normalization of the luciferase activityto the �-gal values. The normalized luciferase val-ues were represented as n-fold luciferase activityrelative to the pGL2-basic and the promoterlesspGL2-enhancer constructs.

Telomeric Repeat Amplification Protocol Assay

ATRA-treated and -untreated HT cells wereharvested on days 3, 6, 9, and 12 for telomeraseactivity analysis. The telomeric repeat amplifica-tion protocol (TRAP) assay was performed with theTRAPEZE XL kit (Intergen, Temecula, CA) asreported previously (Casillas et al., 2003). The fol-lowing controls were prepared: positive control(PC), minus-telomerase control, minus-Taq poly-merase, and standard internal control TSR8 (IC).All samples were incubated at 30°C for 30 minfollowed by polymerase chain reaction (PCR) am-plification (94°C for 30 sec, 59°C for 30 sec, and72°C for 1 min) for a total of 36 cycles. The PCRproducts were resolved on a 10% polyacrylamidegel, stained with 1� SYBR Green (Molecular

28 LIU ET AL.

Probes, Eugene, OR), and analyzed with KodakDigital Science software (Kodak, New Haven,CT).

Chromatin Immunoprecipitation (ChIP) Assay

ATRA-treated and -untreated cells weregrown for 12 days. Approximately 2 � 106 cellswere cross-linked, with a 1% final concentrationof formaldehyde (37%, Fisher Chemicals, Fair-lawn, NJ), and incubated for 10 min at 37°C,followed by addition of an equal molarity ofammonia and incubated for 5 min at room tem-perature. ChIP assays were performed with aChIP assay kit according to the manufacturer’sprotocol (Upstate Biotechnology, Inc., LakePlacid, NY). Briefly, cells were lysed and incu-bated in a protease inhibitor cocktail (1 �g/ml,Sigma, St. Louis, MO) on ice for 15 min. Celllysates were sonicated, then diluted in ChIP di-lution buffer (provided in the kit), and centri-fuged for 15 min at 4°C. An aliquot (one-tenth)of the supernatant was saved and used for DNAinput control, and the rest of the supernatant wasprecleared by incubation with a slurry of salmon-sperm DNA–protein A agarose for 30 min at 4°Cwith agitation. The agarose beads were pelleted,and the supernatant was collected. Ten micro-grams of antiacetylated H3 lysine 9 antibody(Upstate Biotechnology, Inc., Lake Placid, NY)was added to the supernatant and incubatedovernight at 4°C with agitation. A sample lackingprimary antibody but with rabbit IgG was used asa negative control. The antibody-histone com-plex was precipitated by incubation with salmon-sperm DNA–protein A agarose. After standardwashes with low-salt buffer, high-salt buffer,lithium chloride wash buffer, and TE buffer, DNAwas recovered from the immune complex by phenol-chloroform extraction and ethanol precipitation.ChIP-purified DNA was reconstituted in 15 �l ofDNase-free water.

PCR of the ChIP-purified DNA was performedwith use of primers specific for the TERT promotercontaining the two E-boxes. The primer sequencesused were: 5�-CAGGACCGCGCTTCCCACG-3�for the forward primer and 5�-GGCTTC-CCACGTGC GCAGC-3� for the reverse primer.The PCR reaction was performed under the fol-lowing conditions: 94°C for 5 min, followed by 30cycles at 94°C for 30 sec, 61.5°C for 45 sec, and72°C for 45 sec; and then incubated at 72°C for anadditional 5 min. The PCR reaction was preopti-mized to maximize the PCR amplification in thelinear range. The products were resolved on a 1.5%

agarose gel, stained with ethidium bromide, andanalyzed with Kodak Digital Science software.

DNA Methylation Analysis

To assess the changes in the methylation statusof the TERT promoter in differentiating HT andHL60 cells, sodium bisulfite methylation sequenc-ing was performed as described previously (Liu etal., 2004). Briefly, 3 �g of purified genomic DNA(Wizard Genomic DNA extraction kit, Promega)was treated with bisulfite. A total of 25 CpG sites inthe TERT promoter region were assessed for meth-ylation changes at days 3, 6, 9, and 12 for ATRA-treated and at day 12 for ATRA-untreated controlcells. The examined region contained the distalE-box and the WT1 recognition site. Nested PCRamplifications were performed with primers F1 (5�-GTTTTTAGGGTTTTTATATTATGG-3�), R1(5�-AACTAAAAAATAAAAAAA CAAAAC-3�), F2(5�-GGGTTATTTTATAGTTTAGGT-3�), andR2 (5�-AATCCCCAATCCCTCC-3�). The Sure-Start Taq PCR kit (Stratagene, Cedar Creek, TX) wasused for PCR amplification under the following con-ditions: 94°C for 15 min; followed by 35 cycles at94°C for 30 sec, 52°C for 45 sec, and 72°C for 45sec; and then incubation at 72°C for an additional 5min. One microliter of the product from the first PCRreaction (with the F1–R1 primer pair) was used as thetemplate for the nested PCR reaction (with theF2–R2 primer pair). PCR products were purified us-ing a gel extraction kit (Qiagen, Valencia, CA) andwere directly sequenced with primer F2 on an auto-mated DNA sequencer. We sequenced each samplethree times to determine the site-specific methyl-ation changes in the amplified regions.

RESULTS

ATRA-Induced Morphologic Changes DuringDifferentiation of HT and HL60 Cells

After ATRA treatment, both HT and HL60 cellsdisplayed gradual morphologic changes toward ter-minal differentiation within 12 days. Morphologi-cally, the differentiated HT cells were flattenedand extended (Fig. 1A). Differentiated HL60 cellsdisplayed morphologic features typical of neutro-phils, as indicated by their segmented nuclei inaddition to the formation of cell clumps from thesingle-cell suspension in culture (Fig. 1C). Identi-cal morphologies and growth properties of un-treated HL60 cells were observed throughout theexperiment, and, thus, only one control sample forday 12 is shown for all ATRA experiments. Duringthe differentiation process, a decrease in the pro-

29EPIGENETIC CONTROL OF TERT PROMOTER ACTIVITY

liferation rate of both HT and HL60 cells wasclearly visible by day 6, as indicated by cell count-ing (data not shown). Terminal differentiation ofHT cells was further confirmed by the abundantpericellular expression of fibronectin protein inATRA-treated cells by days 9 and 12 (Fig. 1B).The percentage of HL60 cells expressing CD11b,an adhesion molecule most likely responsible forthe cell clumps in the culture and a marker ofdifferentiation of HL60 cells, reached around 90%by 12 days after ATRA treatment (Fig. 1D).

A Significant Decrease in TERT Promoter ActivityPreceded Loss of Telomerase Activity inATRA-Treated HT Cells

To determine the effect of differentiation on theactivity of the TERT promoter, we performed re-porter gene assays in differentiating HT cellsthrough transient transfection, using a plasmid con-struct containing the luciferase gene under thecontrol of the TERT minimal promoter (Fig. 2A).The promoter activity was determined by compar-

Figure 1. Morphologic changes (A and C) andexpression analyses of differentiation markers (Band D) in HT and HL60 cells after addition ofATRA to the growth medium. (A) Untreated (leftpanel) and ATRA-treated (right panel; 2 �M finalATRA concentration) HT cells in culture. (B) Peri-cellular expression of fibronectin in ATRA-treatedHT cells (right panels; control in left panels) by days9 and 12 as determined by antifibronectin antibodystaining. (C) Untreated (left panel) and ATRA-treated (middle and right panels; 1 �M final ATRAconcentration) HL60 cells over 12 days of culture.(D) Representative FACS results showing expres-sion of CD11b in untreated (upper panels) andATRA-treated (lower panels) HL60 cells at days 6and 12. No RA, no FITC: untreated control cellsstained with diluent solution only; no RA, FITC:untreated control cells stained with FITC-conju-gated antibody. The values indicate the percentageof CD11b-positive differentiated cells. Magnifica-tion is 200� for A and C and 250� for B.

30 LIU ET AL.

ison of the luciferase values in ATRA-treated cells with those in untreated cells at 3, 6, 9, and 12 days.

Figure 1. (Continued.)

31EPIGENETIC CONTROL OF TERT PROMOTER ACTIVITY

As shown in Figure 2B, a reduction in TERT pro-moter activity in ATRA-treated HT cells wasclearly visible by day 3 when compared with activ-ity in untreated cells. Dramatic decreases in pro-moter activity continued during extended ATRAtreatment. By days 9 and 12, the promoter activitywas almost undetectable compared to that in theuntreated cells (Fig. 2B). To determine when dur-ing the differentiation process the telomerase ac-tivity would be lost, we performed TRAP assays onuntreated control and ATRA-treated HT cells atdays 3, 6, 9, and 12. As shown in Figure 3, thepresence of telomerase activity was demonstratedby the 6-bp-ladder banding pattern that started at61 bp. All control cells clearly displayed a ladder ofPCR products with 6-bp increments throughoutthe experimental period, which indicated that ahigh level of telomerase activity was maintained inundifferentiated HT cells. By contrast, the de-crease in telomerase activity in ATRA-treated cellswas a gradual process and was readily apparent byday 12 (indicated by a diminished ladder pattern).This diminished telomerase activity is consistentwith the loss of TERT expression in ATRA-treatedHT cells by day 9 as determined by quantitativeRT-PCR analysis (Lopatina et al., 2003). Similarchanges in telomerase activity and TERT expres-sion were also observed in ATRA-treated HL60cells during ATRA treatment (data not shown).

Differentiation Induces Accumulation of DNAMethylation at TERT Promoter

To determine whether the decreased TERT pro-moter activity could be attributed to DNA hyper-methylation, we exploited bisulfite genomic se-quencing to map the methylation status of 25 CpGsites within the TERT promoter, including the E-box1 site (Fig. 4A) in ATRA-treated and -untreatedcells. As illustrated in Figure 4B, the TERT promoterin undifferentiated HL60 cells appeared hypomethy-lated, which conformed to the general observationthat DNA hypomethylation is correlated with highlevels of TERT activity in these cells. In differentiat-ing HL60 cells, however, an increase in promotermethylation was visible, and this was a progressiveprocess in the presence of ATRA over the 12-dayperiod. DNA methylation analysis of the same regionin differentiating HT cells (Lopatina et al., 2003)demonstrated a similar gradual accumulation ofmethylation (Fig. 4B). Comparing the methylationchanges between HT and HL60 cells revealed thatHL60 cells tended to acquire higher levels of meth-ylation than did HT cells, which was also true inundifferentiated and early differentiating cells (Table

1, Fig. 4B). The difference may be because the plu-ripotent HT cells are of embryonic origin and are lessdifferentiated than the multipotent tissue stem-cellHL60 cells, which will be discussed in detail later.

Hypoacetylation of TERT Promoter Occurs duringDifferentiation

To assess whether histone deacetylation wasan alternative or additional pathway involved inTERT regulation in differentiating HT andHL60 cells, we exploited ChIP assays to analyzethe acetylation status of histone H3 lysine 9 inthe TERT promoter in both ATRA-treated and-untreated cells. As shown in Figure 5, the acet-ylation status in untreated control cells appearedto be high and thus consistent with the positivetelomerase activity in the undifferentiated cells.Stable levels of histone H3 acetylation in un-treated HT and HL60 cells were observedthroughout the experiment, and, thus, only onecontrol sample for day 12 was presented for allATRA-treated cells at different experimentaldays. Upon differentiation, H3 became progres-sively hypoacetylated, and this change was morepronounced in HL60 cells than in HT cells dur-ing early differentiation (Fig. 5A and B). HTcells appeared, nevertheless, to be more resistantto the induction effect of ATRA during the first6 days, which changed more rapidly by day 9. Byday 12, the TERT promoter appeared to be com-pletely hypoacetylated in both HT and HL60cells. These data indicated that histone deacety-lation was indeed involved in regulating TERTpromoter activity during differentiation of thesecells. The embryonic origin of HT cells againslowed the progress of this epigenetic change inHT cells in comparison to that in HL60 cells.

DISCUSSIONEpigenetic changes in the TERT promoter re-

gion appear to be important components of thedifferentiation process. We used both HT andHL60 cells to study the chromatin restructuringeffect on TERT promoter activity during differen-tiation. Our studies indicated that the TERT pro-moter became synergistically hypermethylated andhypoacetylated. Both modifications confer a closedchromatin conformation that is typical of inactivegenes. Consistent with these epigenetic changes,TERT promoter activity and endogenous telomer-ase activity were both decreased during the differ-entiation process.

The different origins of HT and HL60 cellsenable us to assess epigenetic plasticity of the

32 LIU ET AL.

TERT promoter in response to differentiation atdifferent developmental stages. An especiallynovel finding in this study is that we observed adelay of acetylation changes in HT cells comparedwith that of HL60 cells in response to ATRA in-duction (Fig. 5A and B). This cell-type specificityin response to ATRA treatment may be related to

the pluripotent status of the HT cells versus themultipotent status of the HL60 cells. The embry-onic origin of the HT cells may confer more abun-dant cellular factors, which will initially assist HTcells in counteracting the induction effect ofATRA. We propose that these factors graduallydecay and become more diluted during late orga-

Figure 2. Promoter activity analysis in differen-tiating HT cells. (A) Schematic drawing of the P-330TERT promoter–luciferase construct used in thetransfection assays; drawing contains the two E-boxes as depicted. (B) Graphic presentation of lu-ciferase reporter gene activity (directly under thecontrol of the TERT promoter) relative to the �-galinternal control in both undifferentiated and differ-entiating HT cells. The value for each sample wasderived from three independent experiments, andthe error bars represent standard errors of themean.

Figure 3. TRAP assay showing changes in telomerase activity during differentiation of HT cells. Thepresence of telomerase activity was demonstrated by 6-bp ladders starting from 61 bp. An 56-bp internalcontrol was used for each sample (PC: positive control; IC: internal control). ATRA-treated (�) and-untreated control (�) cells on the same experimental days (3, 6, 9, and 12) were run next to each otheron the gel.

33EPIGENETIC CONTROL OF TERT PROMOTER ACTIVITY

nogenesis and formation of the blood progenitorcells, which could render the HL60 cells relativelymore sensitive to ATRA induction.

Our initial effort was focused on epigenetic in-vestigations of both E-box1 and E-box2 siteswithin the TERT promoter, which have been des-ignated as pivotal recognition sites of MYC/MADproteins. We would like to point out that, althoughwe studied the histone H3 lysine acetylation statuson both E-boxes, we analyzed only E-box1 formethylation changes because of the high GC con-tent of the TERT promoter, which currently makesit difficult to accomplish methylation analysis ofE-box2 (Fig. 4A). Nevertheless, we observed thathypermethylation appeared to occur preferentiallyin the upstream segment of the promoter, at leastin differentiating HL60 cells, indicating that thissegment may harbor a methylation signal center(Fig. 4B). A close examination of the E-box1 site(Fig. 4B) indicated that it may not be the primarytarget of DNA methylation, which was contrary toour initial expectation. A plausible explanation forlower-than-expected levels of methylation may berelated to the frequent binding of transcriptionalfactors, such as MYC/MAX in undifferentiatedcells and MAD/MAX in differentiated cells, whichmay protect this site from becoming methylated.By 12 days of ATRA treatment, this E-box becamemethylated in HT cells but not in HL60 cells.Could this be because of MAD protein in differ-entiated HL60 cells being more abundant relativeto HT cells, thereby interfering with methylationof this site? It remains to be explored whether anyother protein factors may be involved in interactingwith the E-box1 site. The WT1 site, on the otherhand, appeared to be more easily methylated afterinduction of differentiation. The significance ofthis hypermethylation is not clearly understood,because earlier studies suggested that the regula-tory function of WT1 is restricted to certain tissue

types (Harber et al., 1992). It is possible, however,that hypermethylation of the WT1 site serves asthe nucleus for later methylation spreading to theneighboring CpG sites. This idea is supported bythe observation that the surrounding CpG sites ofthe WT1 site became progressively methylatedduring later differentiation (Fig. 4B). This interest-ing mechanism awaits further experimental explo-ration.

The functional significance of DNA methylationin the regulation of TERT activity is further com-plicated by conflicting observations on the correla-tion between promoter methylation and the ex-pression of TERT among different tissues(Bechter et al., 2002; Guilleret et al., 2002). Ourresults on methylation of the TERT promoter inundifferentiated cells conform to the general para-digm that a hypomethylated promoter correlateswith high transcriptional activity of TERT. To ourknowledge, this is the first demonstration that cellsof embryonic or stem-cell origins may possess adifferent TERT functional status associated with adifferent epigenetic status. In addition, a closecomparison of the methylation results between HTcells and HL60 cells revealed that the TERT pro-moter appeared to be more methylated in HL60cells than in HT cells at both the undifferentiatedand differentiating stages (Fig. 4). This differencemay again be related to HT cells being of embry-onic origin whereas HL60 cells are blood stemcells, which is consistent with observations thatgenomewide methylation tends to increase duringearly differentiation and organogenesis to restrainthe differentiation potential during late develop-ment (Reik et al., 2001).

From comparing the kinetics of changes in pro-moter activity with those of epigenetic modifica-tions (Figs. 2B, 4, and 5), it is clear that down-regulation of TERT promoter activity precedes theepigenetic changes during differentiation. Thisraises a question about what role such epigeneticeffects may play in silencing TERT. Both DNAmethylation and histone deacetylation have beenshown to be prime players in gene silencing duringnormal development and tumorigenesis, but thismay not be true when TERT is silenced duringdifferentiation, considering the relatively delayedchanges in DNA methylation and histone hy-poacetylation at the TERT promoter. It is quitepossible that some other trans-acting repressors,such as MAD, are involved first to repress TERTpromoter activity before the epigenetic factorscome to stabilize the repression effect (Xu et al.,2001; Lin and Elledge, 2003). A recent study has

TABLE 1. Percentage of DNA Methylation in the TERTPromoter Region in Differentiating HT and HL60 Cells*

Days of ATRA treatment HT Cells HL60 Cells

No ATRA 0 16%Day 3, ATRA 0 16%Day 6, ATRA 8% 36%Day 9, ATRA 22% 52%Day 12, ATRA 40% 64%

*Each percentage value is the percentage of methylated CpGs of all 25CpGs on each experimental day. The DNA methylation status remainedstable in untreated cells throughout the experiment regardless of theexperimental day.

34 LIU ET AL.

Figure 4. Methylation analysis of the TERT promoter in differenti-ating HT and HL60 cells. (A) Schematic illustration of TERT promoterregions subjected to methylation analysis (fragment A) and ChIP analysis(fragment B). The vertical bars on fragment A represent the approxi-mate location of CpG sites along the DNA. (B) Illustration of methyl-

ation data of fragment A in undifferentiated and differentiating HT andHL60 cells. The first CpG site corresponds to the base at �419upstream of ATG (�1) in the actual sequence. The undeterminedsequences (indicated by shaded boxes) are possibly a result of partialDNA methylation at these specific sites.

Figure 5. ChIP analysis of histone H3 acetylation in ATRA-treated and -untreated cells. (A) PCR amplification of ChIP-purified DNA from ATRA-treated and -untreated HT cells byuse of rabbit anti-acetylated histone H3 antibody. (B) PCRamplification of ChIP-purified DNA from ATRA-treated and-untreated HL60 cells by use of rabbit anti-acetylated histoneH3 antibody. For each experiment, one sample was incubatedonly with rabbit IgG to serve as negative control. One-tenth ofthe supernatant prior to immunoprecipitation with antibodywas used for DNA input control. The PCR product was 229bp in length.

35EPIGENETIC CONTROL OF TERT PROMOTER ACTIVITY

indeed provided supporting evidence for the main-tenance rather than initiating role in gene silencingby epigenetic factors (Mutskov and Felsenfeld,2004). Direct involvement of histone acetylationand DNA methylation in controlling TERT activityis supported by earlier studies that used trichosta-tin A, a histone deacetylase inhibitor, or 5-azacyti-dine, a demethylating agent (Hou et al., 2002;Lopatina et al., 2003; Wang and Zhu, 2003). Anal-ysis of endogenous TERT chromatin susceptibilityto DNaseI digestion consistently revealed a DNa-seI hypersensitivity site (DHS) near the TERTtranscription initiation site only in telomerase-pos-itive cells but not in telomerase-negative cells.TSA can induce reactivation of TERT that is ac-companied by the formation of a DHS at the TERTpromoter. These studies clearly suggest that chro-matin remodeling at the endogenous TERT pro-moter plays an important role in controlling telom-erase activity in addition to regulation bytranscription factors (Wang and Zhu, 2003).

In conclusion, our results add new insights intothe regulatory pathways of telomerase activity, therelative role of different epigenetic processes suchas DNA methylation and histone hypoacetylationon the control of the TERT, the kinetics of theseprocesses, the importance of the origin of cell typesin TERT gene control, and the involvement oftelomerase in cell differentiation. Considering theimmortalized status of the HT and HL60 cells,these studies also indicate that the chemotherapeu-tic effect of retinoic acid may function primarilythrough repressing telomerase activity in cancercells. Further elucidation of TERT transcriptionalregulation, including identification and character-ization of endogenous repressor proteins and thekinetics of their binding to the TERT promoterduring cellular differentiation, will shed new lighton the complex regulation of human telomerase innormal and cancerous cells and may lead to newavenues for future cancer treatment.

ACKNOWLEDGMENTS

L. Liu and S. N. Saldanha contributed equally tothis work. We thank Dr. Steve Watts for generousaccess to his research facility.

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