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Molecular Cancer Research 1:739-746 (2003)
© 2003 American Association for Cancer Research

Cell Cycle, Cell Death, and Senescence

Telomerase Reverse Transcriptase Promoter Regulation During Myogenic Differentiation of Human RD Rhabdomyosarcoma Cells1

Hongwen Ma1, Virginia Urquidi2, Jeremy Wong1, Jeanine Kleeman1 and Steve Goodison2

1 UCSD Cancer Center and 2 Department of Pathology, University of California, San Diego, La Jolla, CA

Requests for reprints: Steve Goodison, UCSD Cancer Center, 9500 Gilman Drive, La Jolla, CA 92093-0064. Phone: (858) 822-2083; Fax: (858) 822-1111. E-mail: sgoodison{at}ucsd.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
During terminal differentiation of human and murine cells, telomerase activity and parallel transcription of telomerase reverse transcriptase (hTERT) are inhibited. In this study, we used in vitro and in vivo analyses to determine the role of hTERT promoter elements and associated factors during differentiation-induced inhibition of telomerase expression in RD, a human rhabdomyosarcoma cell line. Assay of telomerase enzyme activity, hTERT mRNA, and reporter gene assays confirmed that the hTERT promoter was silenced during 12-O-tetradecanoylphorbol-13-acetate-induced myogenic differentiation of telomerase-positive RD cells. Promoter deletion and mutation analyses revealed that two E-boxes and an AP-2 site present in a 320-bp region of the promoter were essential for the transcriptional activity of the hTERT gene. Electrophoretic mobility shift assays identified several factors that interact with this region of DNA, including the muscle-specific transcription factors Myf5, Myf6, and myogenin and the ubiquitously expressed factors Sp1 and AP-2. Ectopic expression of the E-box binding factors c-Myc and Mad did influence promoter activity in these cells; indeed, the presence of endogenous c-Myc protein was altered after differentiation. Our findings suggest that the acute regulation of hTERT transcription is primarily controlled by E-box elements, which bind a series of factors during the phased phenotypic changes occurring during the differentiation of RD human muscle cells.

Key Words: Myc • AP-2 • E-box • hTERT promoter


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Progressive telomere erosion has been proposed to be a limiting factor in replicative capacity and to elicit a signal for the onset of cellular senescence (1). For human cells to proliferate beyond this senescent checkpoint, they need to restore telomere length, a process that can be achieved by the activity of the enzyme telomerase (2). Accordingly, while most adult human somatic cells lack detectable telomerase, germline cells, stem cells, and most tumor cells thus far tested exhibit telomerase expression (3, 4). The human telomerase holoenzyme is a RNA-dependent DNA polymerase comprising three major components: a RNA component (hTR) that provides the template for complementary telomere synthesis, a telomerase-associated protein (TP1) that coordinates the assembly of the complex, and the telomerase reverse transcriptase (hTERT) (5). Cells with undetectable telomerase activity most often also lack hTERT mRNA, although the presence of hTR and the telomerase-associated proteins remains (6). Furthermore, ectopic expression of hTERT is sufficient to immortalize some cell types (7, 8), indicating that hTERT expression is the limiting factor for telomerase activation and that induction of hTERT transcription is a key regulatory mechanism in the extension of cellular replicative life span.

Telomerase activity is detectable in early gestational age human embryonic cells, but enzyme activity and hTERT mRNA are concomitantly down-regulated in most cell types, as the fetus develops beyond 21 weeks (9, 10). Telomerase activity and parallel hTERT transcription are also inhibited during terminal differentiation of human and murine immortalized cells (11, 12). Recent studies have identified the E-box sequences present in the proximal promoter region as being directly involved in this regulation (13, 14), and a role for a Myc/Mad molecular switch for hTERT regulation in differentiation has been proposed (13). However, several other HLH/LZ proteins are known to bind E-boxes and to be capable of transactivation from the same element (15). The complex interactions between family members and between cis-acting elements are not yet fully understood and depend on the cellular context.

In this study, we used in vitro and in vivo analyses to determine the role of hTERT promoter elements and associated factors in the differentiation-induced inhibition of telomerase expression in RD, a human rhabdomyosarcoma cell line. Reporter gene assays revealed that two E-boxes present in a 320-bp region (320 bp upstream of the translational ATG site, designated +1) of the hTERT gene were involved in the regulation of promoter activity in telomerase-positive RD. We show that ectopic expression of c-Myc and Mad could influence promoter activity in these cells. However, the overall regulation appears to be more complex than just a c-Myc/Mad antagonism, with a number of other E-factors and unrelated transcription factors being involved in the control of hTERT transcription in these human muscle cells.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Telomerase Activity Is Regulated During RD Cell Differentiation
RD cells were induced to differentiate by incubation with 12-O-tetradecanoylphorbol-13-acetate (TPA). Marked changes in morphology were observed, with an increasing population of elongated and multinucleated cells appearing, as described previously (16, 17). Proliferation was progressively inhibited, as these myofiber-like structures became the majority population after 5–7 days of TPA treatment. Differentiation status was also monitored via the expression of muscle myosin heavy chain protein (Fig. 1A), an indicator of muscle cell differentiation (16). The presence of telomerase activity was analyzed before and after differentiation using the telomere repeat amplification protocol (TRAP) (18). RD cells, being derived from a human rhabdomyosarcoma, were, as expected, found to be telomerase positive in proliferative culture. Both telomerase activity and hTERT transcription were repressed when RD cells were differentiated (Fig. 1, B and C).



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  		FIGURE 1. A. Transcription factor expression during RD cell differentiation. RD cells were cultured in differentiation medium (±100 nM TPA) for 7 days as described in "Materials and Methods." Five micrograms of nuclear extracts were analyzed by immunoblotting for the detection of the muscle-specific (Myosin, MyoD, and Myogenin) and E-box binding (USF-1, USF-2, Myc, and Mad) transcription factors. B. Telomerase activity is inhibited during RD cell differentiation. Cellular extracts containing 0.6-µg protein were analyzed for telomerase activity using the TRAPeze detection kit. Telomerase activity produces a 6-bp ladder of amplification products (upper panel). No lysate (NL) and HeLa cell lysate (HeLa) reactions served as negative and positive controls, respectively. C. RNA was isolated from the same cells and subjected to nested reverse transcription-PCR designed to detect human hTERT mRNA (lower panel) as described in "Materials and Methods."

 
Transcription Factor Expression During Differentiation
Differentiation of RD cells was associated with the regulated expression of a number of transcription factors that are capable of interacting with elements present in the hTERT promoter. The muscle-specific protein MyoD was expressed before but not after differentiation (Fig. 1A). Conversely, myogenin, a factor expressed in growth arrested myoblasts, although down-regulated, remained expressed during RD differentiation. The presence and absence of these factors is as expected during progression to myogenic differentiation (19). The ubiquitously expressed factors USF-1 and USF-2 were present before and after differentiation of RD cells (Fig. 1A), as was the generally repressive factor Mad, whereas levels of the oncogene c-Myc appeared to be down-regulated during differentiation. None of the transcription factors monitored were up-regulated during RD differentiation.

hTERT Promoter Regulation During Differentiation
A PCR product containing 989 bp (nucleotides [nt] +1 to -989) was derived from a human genomic DNA template, sequenced, and cloned into a luciferase reporter gene vector. The PCR-derived sequence was essentially the same as those previously reported (20, 21). RD cells were transiently transfected with a series of unidirectionally 5'-deleted and specific motif-deleted hTERT promoter reporter gene constructs (Fig. 2A). The "full-length" promoter (p989) exhibited transcriptional activity in RD cells (Fig. 3), paralleling the observed telomerase activity measured by the TRAP assay (Fig. 1). In undifferentiated RD cells, deletion of the promoter from -989 to -820 (p820) had no significant effect on activity. However, promoter deletion to -620 (p620) resulted in increased reporter activity (Fig. 3), implying that there is a "silencing" element between positions -820 and -620. A "core" promoter construct deleted to 320 bp (p320) contained all the necessary elements (22–24) for transcriptional activity in RD cells. Two E-box elements (CACGTG) are contained within the 320-bp core promoter, one at positions -242 to -237 (E1) and one at positions -34 to -29 (E2) (Fig. 2A). Evaluation of the promoter activity of p620 constructs with either individual E-box (E1 or E2) or both E-boxes (E1E2) deleted revealed that either E-box alone could support promoter activity in RD cells (Fig. 3), but deletion of both E-boxes significantly reduced transcription. As expected, overall activity of all constructs was reduced to very low levels in differentiated RD cells, with promoter activity being reduced ~10-fold. However, the role of the E-boxes in promoter activity was markedly altered in the differentiated state. Neither single nor double E-box deletions had any effect in differentiated RD cells, suggesting that the loss of a positive mechanism for hTERT transcription had occurred at this stage (i.e., the necessary factor(s) for positive E-box-mediated regulation was no longer present) rather than a switch to the presence of an overriding negative factor.



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  		FIGURE 2. A. Schematic representation of the hTERT promoter sequences contained in luciferase reporter constructs. Unidirectional deletion of the promoter to 820, 620, and 320 bp upstream of the translational start site was achieved using PCR as described in "Materials and Methods." Single and double deletion of E-boxes (E1 and E2) are represented in the p620 constructs. B. Design of overlapping DNA fragments (1–5) used in EMSA, and the relative position of consensus motifs for transcription factor binding sites in the hTERT promoter. The transcriptional start site is indicated by an arrow, and the translational start site (ATG codon at position +1) is shown in bold. The E-boxes (E1 and E2) are shown, and the Sp1 and AP-2 sites, which were mutated for functional analyses, are indicated by asterisks.

 


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  		FIGURE 3. Deletion analysis of the hTERT promoter and comparison of activity before and after RD cell differentiation. RD cells were transfected with a series of luciferase reporter constructs containing different lengths of promoter (989, 820, 620, and 320 bp upstream of the translational start site; see Fig. 2) and p620 constructs containing single (E1 and E2) or double (E1E2) E-box deletions. All constructs were tested in RD cells before (A) and after (B) differentiation. Dual-luciferase assays were performed on cell lysates as described in "Materials and Methods." Firefly luciferase activity of the promoter-less pGL3-basic plasmid (control) was normalized to 1, and luciferase activity of test constructs is shown relative to control. Bars, SD.

 
Transcription Factor Binding Profiles During Differentiation of RD Cells
Electrophoretic mobility shift assays (EMSA) were performed using the hTERT core promoter (-320 to +1) and RD nuclear protein extracts obtained before and after muscle cell differentiation. The 320-bp region was divided into five fragments by synthesizing overlapping double-stranded oligonucleotides (Fig. 2B). The first lane (unlabeled) of each panel in Fig. 4 displays the complexes revealed after incubation of the DNA fragment with nuclear protein extracts prepared from RD cells before (Undiff.) and after differentiation (Diff.). Fragment 1 (containing the E-box E2) exhibited several band shifts (Fig. 4), one (of unknown identity) of which disappeared during differentiation (band X). The identity of the factors present in these complexes was revealed by supershift assay, whereby antibodies specific for transcription factors (indicated above each lane in Fig. 4) were preincubated with nuclear extracts before addition of the radiolabeled DNA fragment. A supershift of a band in the presence of the specific antibody identifies the factor present in that DNA-protein complex. Supershift assays revealed that the E-box binding factors Myf5, Myf6, USF-1, USF-2, and myogenin all formed complexes with this promoter region, and one of the USF complexes appeared reduced after differentiation. Assays using fragment 2 revealed a Sp1 complex, but no alteration in band shift profile was observed after differentiation. Fragment 3 EMSA also revealed a Sp1 complex, in this case appearing to be more prevalent before differentiation. An AP-2{gamma} antibody caused a weak band shift of fragment 3, which disappeared on differentiation. Fragment 4 revealed no notable complexes with RD cell extracts (data not shown). Fragment 5 (containing E-box E1) produced several bands of high mobility, but no bands were shifted by antibodies to muscle-specific E-box factors. This fragment was retarded by both USF-1 and USF-2 antibodies in a similar manner to fragment 1. A c-Myc complex with fragment 5 was also revealed, but no alteration of any of these complexes occurred relative to differentiation status.



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  		FIGURE 4. Identification of transcription factors present in RD cells that are capable of binding to specific regions of the hTERT core promoter. EMSAs were performed with double-stranded DNA fragments 1–5 (see Fig. 2B). Three micrograms of nuclear extracts were incubated with radiolabeled probe before electrophoresis as described in "Materials and Methods." Representative autoradiograms are shown. The first lane (unlabeled) of each panel displays the complexes revealed after incubation of the DNA fragment with nuclear protein extracts prepared from RD cells before (Undiff.) and after (Diff.) differentiation. The identity of the factors present in these complexes was revealed by supershift assay, whereby antibodies specific for transcription factors (indicated above each lane) were preincubated with nuclear extracts before addition of the radiolabeled DNA fragment. A supershift of a band in the presence of the specific antibody identifies the factor present in that DNA-protein complex.

 
Role of AP-2 Binding Sites in RD Cell hTERT Promoter Activity
Adjacent consensus AP-2 and Sp1 sites exist between the two E-boxes in the hTERT promoter, and both AP-2 and Sp1 antibodies were shown to retard hTERT DNA fragments in EMSA experiments (Fig. 4). Mutations in the Sp1 site at positions -136 to -128 (GGGCCCC) and at the only AP-2 consensus site (CCCNNNGGG) within the hTERT promoter at nt -129 to -121 (25) were created to test the functionality of these sites in hTERT regulation. A number of nucleotides in the AP-2 consensus site were altered and tested for their ability to bind AP-2 factors in EMSA assays using HeLa cell nuclear extracts (data not shown). Altering nucleotides CT at position -127 to TT resulted in the loss of AP-2 binding, so this mutation was used in subsequent analyses. These mutations were introduced into the p620 promoter constructs and into the constructs containing the single and dual E-box deletions. Reporter gene assay data obtained after transfection of these constructs into RD cells are shown in Fig. 5. The inability of Sp1 to bind at the mutated site appeared to have no effect on the promoter activity of the p620 construct before or after RD differentiation. However, the p620AP-2mt construct had significantly reduced (3-fold) promoter activity in undifferentiated RD cells (Fig. 5). The combination of the AP-2 site mutation with deletion of either E-box alone had no additive or subtractive effect, but most notably, the mutation of the AP-2 site negated the previously observed double E-box deletion repressive effect (Fig. 3). These experiments show that maximal transcription of the hTERT promoter in telomerase-positive RD cells requires a functional E-box and the consensus AP-2 site centered at position -125. In differentiated RD cells, the p620AP-2mt construct maintained the same low level of activity as the p620 construct, and no E-box deletions had any further inhibitory or enhancing effects.



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  		FIGURE 5. A functional AP-2 site is required for hTERT promoter activity in RD cells. RD cells were transfected with p620 luciferase reporter constructs (with and without E-box deletions; see Fig. 3) containing Sp1 (Sp1mt) or AP-2 (AP2mt) mutations. All constructs were tested in RD cells before (A) and after (B) differentiation. Dual-luciferase assays were performed on cell lysates as described in "Materials and Methods." The promoter-less pGL3-basic plasmid (control) was normalized to 1. Promoter activities are expressed as described in Fig. 3.

 
Transcription Factor Overexpression Can Modulate the hTERT Promoter
To further assess the role of specific elements and their associated factors in hTERT promoter activity, RD cells were cotransfected with constructs p620, p320, and p620Sp1mt and expression vectors coding for the transcription factors c-Myc, Mad, Max, USF-1, USF-2, AP-2{alpha}, and AP-2{gamma}. Ectopic expression of c-Myc resulted in a 3-fold induction of reporter gene transcription driven by the p620 construct (Fig. 6). This fold induction was observed in both predifferentiated and postdifferentiated RD cells. Overexpression of the transcription factor Mad had a 2.5-fold inhibitory effect in undifferentiated cells but had no significant effect in telomerase-negative differentiated RD cells. It is difficult to evaluate a potential negative effect in differentiated cells, because the promoter activity is already reduced to levels approaching those of the promoter-less control vector. Experiments evaluating the effect of cotransfection of the transcription factors with the p320 construct gave essentially the same result as that observed with the p620 construct. The Sp1 site centered at position -132 was not necessary for Myc/Mad effects, as evidenced by the activity of the p620Sp1mt promoter (Fig. 6). Reporter assays using the p620E1E2 construct were not altered by the overexpression of any tested exogenous transcription factors (data not shown), confirming that the observed Myc/Mad factor effects are specifically mediated via these E-boxes. The expression of the USF-1, AP-2 (Fig. 7), USF-2, and Max (data not shown) transcription factors had no effect on wild-type or Sp1-mutated p620 or p320 promoter activities.



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  		FIGURE 6. Exogenous transcription factor regulation of hTERT promoter activity in RD cells. RD cells were transfected with p620, p320, and p620Sp1mt (Sp1mt) luciferase reporter constructs alone (NoTF) or with expression vectors for the transcription factors c-Myc, Mad, USF-1, AP-2{alpha} (AP2a), and AP-2{gamma} (AP2g). All cotransfections were tested in RD cells before (A) and after (B) differentiation. Dual-luciferase assays were performed on cell lysates as described in "Materials and Methods." Promoter activities are expressed relative to that of the promoter-less pBasic construct as described in Fig. 3.

 


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  		FIGURE 7. Regulation of the hTERT promoter by endogenous Myc does not require a functional AP-2 site. RD cells were transfected with wild-type p620 constructs (p620) and p620 containing single (E1 and E2) or double (E1E2) E-box deletions or a mutated AP-2 consensus site (AP2mt). These constructs were cotransfected with a c-Myc expression vector (+) or a null expression vector (-), and dual-luciferase assays were performed on cell lysates as described in "Materials and Methods." Promoter activities are expressed relative to that of the promoter-less pBasic construct as described in Fig. 3.

 
Does hTERT Promoter Regulation by Myc Require Interaction With the AP-2 Site?
Having shown that Myc can positively override regulation of the hTERT promoter in RD cells, we tested the role of both E-boxes and the AP-2 site in this enhancement. Although the AP-2 site mutation decreased the activity of all promoters tested, the observed 2-fold induction of promoter activity by Myc was not abrogated by this functional mutation (Fig. 7). Independent deletion of the two E-boxes, in conjunction with the AP-2 mutation, revealed that the response to exogenous Myc was specifically mediated by E2 in undifferentiated RD cells. Although the AP-2 site clearly has a role in the regulation of the hTERT promoter, there does not appear to be an absolute requirement for interaction between the AP-2 site and the E-box binding factor Myc, at least when Myc expression is manipulated to be in excess.


   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
We have demonstrated that a loss of telomerase enzyme activity during differentiation of RD rhabdomyosarcoma cells is paralleled by the loss of detectable hTERT mRNA and the silencing of the hTERT promoter. Deletion of the proximal promoter region revealed that the changes in hTERT regulation during TPA-induced myogenesis were largely mediated by a core promoter extending 320 nt upstream of the ATG translational start site. EMSA analyses revealed that the RD cells express the E-box binding factors Myf5, Myf6, myogenin, USF-1/2, and Myc. Specific mutation of the E-box elements revealed that either of the two E-boxes could sustain hTERT core promoter activity in RD cells, but as expected, double mutation of these two E-boxes resulted in significant reduction of transcriptional activity, confirming that these elements are essential in the regulation of the hTERT core promoter. However, E-box regulation does not constitute the entire regulatory mechanism because removal of both elements does not fully restore or repress hTERT promoter activity to wild-type levels in any predifferentiation or postdifferentiation comparison. Upon differentiation, the E-box deletions appeared to be inconsequential, suggesting that acute promoter inhibition is not mediated via the binding of a negative factor to an E-box. Long-term hTERT promoter silencing may be mediated by the binding of the basic helix-loop-helix protein Mad, an E-box factor that is associated with negative regulation (22). Mad regulates transcription via recruitment of histone deacetylases to the binding site, altering chromatin structure and making the promoter inaccessible (23, 24). It is possible that in these studies RD cell differentiation was not yet advanced enough to employ this longer-term silencing mechanism.

While there are exceptions (26), most studies show that Myc can elicit expression of hTERT (13, 27); consequently, loss of Myc is a good candidate mechanism for the reduction of hTERT transcription during differentiation. However, while c-Myc and Mad overexpression could influence hTERT promoter activity in this study, there appears to be other factors and/or elements involved in hTERT transcriptional regulation in RD cells. Both the ectopic expression of specific E-factors and the deletion of the elements known to mediate E-factor effects resulted only in 2–3-fold changes. Therefore, there must be other factors and/or elements involved in hTERT transcriptional regulation in RD cells. It is conceivable that the expression of some of these "other factors" may also be perturbed in the differentiated state. The ability of E-factors to interact with the transcriptional RNA polymerase complex may be modulated by interactions with a number of other factors (28), including AP-2 (29, 30). The observation that the AP-2 site mutation caused a down-regulation of the promoter similar to that of the double E-box deletion suggested that the E-box regulatory factors either interact with or require the AP-2 site binding factors to exert their effects in RD cells. High-affinity binding sites for AP-2 sometimes overlap Myc response elements in bona fide Myc target genes. However, this is not the case in the hTERT promoter, with the only consensus AP-2 binding site (CCCNNNGGG centered at -125). Mutation of the AP-2 site did not restrict the ability of ectopic c-Myc expression to up-regulate the hTERT promoter via the E2-box. The ectopic expression of AP-2 factors had no additive effect on reporter gene activity either, so although both AP-2 and E-box sites both appear to be essential for hTERT promoter regulation in RD cells, it remains to be determined whether they act in combination or in an independent fashion.

Previous studies have shown that telomerase activity decreases during C2C12 mouse myoblast differentiation (31, 32), and a role for Sp1 in rodent TERT promoter regulation has been reported. Vinals et al. described MyoD activation and subsequent down-regulation of Sp1 during mouse C3H10T1/2 cell myogenesis (33), and Nozawa et al. have shown that the loss of both Sp1 and Sp3 binding correlates with the down-regulation of mTERT expression in C2C12 cells in myogenesis (14). However, in our study, it was the loss of MyoD expression that correlated with differentiation and the absence of telomerase activity and hTERT promoter activity in RD cells. Furthermore, Sp1 protein levels appeared unchanged during differentiation and hTERT inhibition. There may be fundamental differences between TERT regulation in humans and rodents, although there are similarities between promoter sequences.

It is evident from this study that multiple factors capable of binding E-box elements are present throughout differentiation (USF-1/2, Myf5/6, Myc/Mad, etc.). Therefore, a subtle interplay between these factors and with other cis-acting elements (e.g., AP-2 site) is inferred, with competitive binding being dependent on availability of DNA, factor abundance, and complex protein-protein interactions. A recent study reporting the dominant-negative effect of a truncated USF on the hTERT promoter during lymphocyte activation confirms that any E-box binding factor may have a regulatory effect in a specific cellular context (34). Indeed, as described in many other cell types (35), in this study, USF-1 and USF-2 were found to be the major hTERT E-box binding proteins, but their role in gene-specific regulation is unclear. The USF proteins share with Myc similar structure and affinity for E-box binding, yet the cellular functions of USF and Myc are very different. Overexpression of Myc, in collaboration with a second oncogene, is sufficient to trigger the transformation of primary fibroblasts, whereas overexpression of USF has instead been found to inhibit growth in a number of cancer cell lines (36, 37). The overexpression of USF proteins in RD cells did not affect promoter activity in RD cells. Perhaps USF acts in a more general way as an "occupying" factor, weakly competing out more overtly positively and negatively acting E-box binding factors while keeping the promoter chromatin accessible for acute regulation.

The redundancy of E-box binding may enable concurrent positive and negative regulation of multiple target genes during the cell cycle or phased phenotypic changes such as differentiation. During muscle cell differentiation, distinct regulatory factors are expressed serially to coordinate the expression of progressive structural components, but hTERT is required to be silenced relatively early in this process. During differentiation, there may be a "swapping out" of E-factors binding to the hTERT E-boxes, with the common effect of preventing the binding of a positive regulator. Long-term effects, such as Mad-mediated epigenetic changes, may be brought about only when it is pertinent to shift the balance to an overall shutdown status, i.e., on terminal differentiation. In a transformed tumor cell, perhaps the complexity of these competing interactions is reduced or compromised, because the cells were previously fully differentiated and quiescent. In this scenario, the inappropriate expression of just one positive factor, e.g., Myc, could override the silencing of the hTERT promoter, resulting in telomerase reactivation.

In summary, our results show that two E-boxes and an AP-2 element have critical roles in hTERT transcriptional regulation during human RD cell differentiation. During the phased changes that occur during differentiation, these E-boxes bind a series of transcription factors, which combine through complex interactions to silence the hTERT gene. A better understanding of the factors and mechanisms that can achieve and maintain silencing of the hTERT promoter would enable manipulation of hTERT expression, a goal relevant to the amelioration of tumor cell proliferation.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Cell Line Culture
RD cells were purchased from the American Type Culture Collection (Rockville, MD) and were propagated using DMEM medium supplemented with 10% fetal bovine serum. Cell cultures were incubated at 37°C in a humidified atmosphere of 5% CO2-95% air. All culture reagents were purchased from Life Technologies, Inc. (Gaithersburg, MD).

Induction of Muscle Cell Differentiation
RD cells (used at passages 5–12) were induced to differentiate by adding 100-nM TPA (Sigma Chemical Co., St. Louis, MO) to the medium for 7 days (16). Differentiation was assessed by microscopic observation and by monitoring the skeletal muscle myosin heavy chain expression profile (16) (antibody MHC [G-4]; Santa Cruz Biotechnologies, Santa Cruz, CA). Progressive fusion and myotubule formation occurred 2 days after TPA treatment. Analyses were performed after 5–7 days, when 60–80% of the cells had fused.

Recombinant Plasmid Constructs
The 5' hTERT gene flanking region (990 nt upstream from the translational initiation site, designated +1) was amplified from human genomic DNA by PCR, ligated into a pCR2.1-TOPO vector (Invitrogen, San Diego, CA), and sequence verified. Variable lengths (989, 820, 620, and 320 bp) of hTERT promoter DNA fragments were subcloned into the luciferase reporter pGL3-basic vector (Promega, Madison WI; Fig. 3). The Myc/Mad/Max (38) and USF (35) expression constructs expression constructs have been described previously.

Site-Specific Deletions
E-box E1 (CACGTG, position -242) was deleted using PCR primer pairs 5' nt -99/XhoI and E1R (reverse primer with deletion of E1), E1F (forward primer containing deletion of E1) and 3' nt -1/HindIII using p989 as template. Second-round amplification used flanking primers at 5' position -620/XhoI and 3' position -1/HindIII, and PCR products were subcloned into the pGL3-basic vector producing p620E1. The deletion of the proximal E-box E2 (CACGTG at -34) was performed similarly, creating p620E2. The same reactions using p620E1 as template created the double E-box deletion p620E1E2. Sp1 (-136 to -128) and AP-2 site (-129 to -121; CCCNNNGGG) mutations were created using the following oligonucleotides: Sp1mt forward primer (5'-CGGCCCAGTTCCCTCCGGGCCCTCCC) and AP-2mt forward primer (5'-CGGCCCAGCCCCTTCCGGGCCCTCCC). Altered nucleotides are in bold italic. The Sp1 and AP-2 mutations were introduced into the p620 and E-box-deleted constructs to create p620Sp1mt, p620AP-2mt, etc. All constructs were sequence verified.

Telomerase Activity Assays
Telomerase activity was evaluated using the PCR-based TRAP assay (18). Protein extractions and amplification assays were performed using the TRAPeze XL detection kit (Intergen Co., Norcross, GA). Assay products were visualized by acrylamide gel electrophoresis as described previously (39).

Reverse Transcription-PCR
Total cellular RNA isolation and cDNA synthesis were achieved as previously described (39). Nested PCR for hTERT mRNA used the following primers: forward 5'-CGGAAGAGTGTCTGGAGCAA and reverse 5'-TCAGTCCAGGATGGTCTTGAAGTC followed by forward 5'-CTCACCCACGCGAAAACCT and reverse 5'-CCACTGTCTTCCGCAAGTTCA. Amplification of ß-actin transcripts served as cDNA internal control.

Transient Transfections and Reporter Assays
Transient transfection were performed using TransFast reagent (Promega). Dual-luciferase assays (Promega) were performed according to manufacturer's protocol. Each reaction contained 700-ng hTERT promoter luciferase reporter, 200-ng cotransfected transcription factor (when applicable), and 100-ng Renilla luciferase control reporter vector, pRL-SV40. The promoter-less pGL3-basic vector (Promega) was used to make equal amounts of total DNA per reaction. All experiments were performed in triplicate.

Electrophoretic Mobility Shift Assay
Labeled, overlapping double-stranded DNA fragments (Fig. 3) were created by PCR using p989 as template, incorporating [33P]dCTP during the reaction. Nuclear extracts were prepared from cell lines using NE-PER extraction reagents (Pierce Chemical Co., Rockford, IL). Binding reactions were performed at 4°C for 10 min in a 20-µl reaction containing 3-µg nuclear protein, 1-µg poly(deoxyinosinic-deoxycytidilic acid), 4% Ficoll 4000, 25-mM HEPES (pH 7.9), 35-mM KCl, 1-mM EDTA, 1-mM DTT, and 4-mg/ml BSA. About 1000–2000 counts/min of 33P-labeled DNA fragments were added, and the binding reaction continued at 25°C for 30 min. Complexes were electrophoresed on 6% native polyacrylamide gels in 0.5x Tris-borate EDTA buffer, and dried gels were subjected to autoradiography. Supershift antibodies against c-Myc, Mad, Max, USF-1, USF-2, AP-2{alpha}, AP-2{gamma}, MyoD, Myf5, Myf6, myogenin, and Sp1 were purchased from Santa Cruz Biotechnologies. Antibodies were preincubated with nuclear extracts on ice for 1 h before DNA binding reactions.

Western Blot Analysis
Five micrograms of nuclear protein were electrophoresed in 10% SDS-PAGE gels and blotted to polyvinylidine difluoride membranes. Specific primary antibodies (described above) were detected with peroxidase-labeled secondary antibodies (Amersham, Piscataway, NJ) using SuperSignal West Dura Extended Duration Substrate (Pierce Chemical) per manufacturer's instructions.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Prof. Hiro Ariga (Hokkaido University) for providing the Myc/Mad/Max expression constructs and Prof. Graham Packham (Southampton General Hospital, United Kingdom) for providing the USF expression constructs.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 Sidney Kimmel Foundation (S.G., Sidney Kimmel Scholar Award), California Cancer Research Program grant 570V-10189 (V.U.), and intramural support funds from the UCSD Cancer Center. Back

Received March 24, 2003; revised May 11, 2003; accepted June 11, 2003.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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