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Opposing regulatory roles of E2F in human telomerase reverse transcriptase (hTERT) gene expression in human tumor and normal somatic cells¹

JAEJOON WON^*,, JEONGBIN YIM^* and TAE KOOK KIM^,2

^* National Creative Research Initiative Center for Genetic Reprogramming, Institute for Molecular Biology and Genetics, Seoul National University, Seoul 151–742, Korea;
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon 305–701, Korea; and
Institute of Chemistry and Cell Biology, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA

²Correspondence: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon 305–701, Korea. E-mail: tkkim{at}mail.kaist.ac.kr

SPECIFIC AIMS

Human cells with high proliferative potential, such as tumor cells or stem cells, exhibit telomerase activity whereas most normal human somatic cells do not. Telomerase activity is tightly regulated by the expression of its catalytic subunit, human telomerase reverse transcriptase (hTERT). Through an expression cloning approach, we identified E2F-1 as a repressor of the hTERT gene in human tumor cells. Detailed analyses of E2Fs indicated that: E2F-1, E2F-2, and E2F-3 (but not E2F-4 and E2F-5) repressed hTERT promoter through Sp sites in human tumor cells, whereas all five E2Fs activated hTERT promoter through E2F sites in normal human somatic cells. These contrasting effects of E2F transcription factors on the hTERT promoter may contribute to the paradoxical biological activities of E2F, which can both promote and inhibit cellular proliferation and tumorigenesis.

PRINCIPAL FINDINGS

1. E2F-1 is identified as a repressor of the hTERT promoter in human tumor cells through an expression cloning approach
For efficient identification of the transcriptional repressor of the hTERT gene, we developed an expression cloning approach. We prepared a cDNA expression library from the mRNA isolated from normal human kidney tissue, where hTERT gene expression is repressed. The library was divided into pools of 100 cDNAs each and cotransfected into 293T cells along with the reporter plasmid containing hTERT promoter. Fractionation of positive pools, among 1200 pools (120,000 total cDNAs) screened, led to two pure cDNA clones that can repress the hTERT promoter activity in 293T cells. From sequence analysis, we found that this cDNA encoded the transcription factor E2F-1. Ectopic expression of E2F-1 also repressed the hTERT promoter activity in other human tumor cells including 293, HeLa, and U2OS cells (Fig. 1 A).

View larger version (19K): [in this window] [in a new window]   Figure 1. E2F-1, E2F-2, and E2F-3, but not E2F-4 and E2F-5, repress the hTERT promoter in human tumor cells whereas all five E2Fs activate the hTERT promoter in normal human somatic cells. A) Analyses of hTERT promoter with E2Fs in tumor cells. The indicated cells were cotransfected with 2.5 µg of p-1003 and 1 µg of each of the E2F expression plasmids. B) Analyses of hTERT promoter with E2Fs in normal cells. Cells were cotransfected with 4 µg of p-1003 and 2.5 µg of each of the E2F expression plasmids. A, B) Luciferase activity was normalized by protein concentration. Results shown are the average of 3 experiments; bars indicate SD.

2. E2F-1 represses the hTERT promoter through Sp1 in human tumor cells
To understand the mechanism underlying E2F-1-mediated repression of the hTERT promoter in human tumor cells (Fig. 1A ), we exploited two E2F-1 mutants that are defective in specific aspects of transcriptional regulation: E2F-1 Y411C is specifically defective in its interaction with pRB; E2F-1 1–88 is intact in its pRB-dependent function but is specifically defective in transcriptional regulation through the DNA binding site for Sp. These mutational analyses indicated that E2F-1 1–88, but not E2F-1 Y411C, did not repress the hTERT promoter in human tumor cells. Consistent with these results, E2F-1 failed to repress hTERT promoter activity with mutations of the Sp sites within the hTERT promoter region. Among Sp family members, Sp1 was found to be strongly associated with the hTERT promoter in human tumor cells from EMSA and formaldehyde cross-linked chromatin immunoprecipitation (X-ChIP) analysis. Sp1-mediated activation of the hTERT promoter was sharply inhibited by the coexpression of E2F-1. Together, these results suggested that E2F-1 may repress the hTERT promoter in human tumor cells by suppressing Sp1-mediated transcriptional activation.

3. E2F-1 activates the hTERT promoter through a noncanonical E2F site in normal human somatic cells
In sharp contrast to the results in human tumor cells, E2F-1 dramatically activated the hTERT promoter in normal human somatic cells such as IMR90, WI38, and HFF cells (Fig. 1B ). To understand the mechanism underlying E2F-1-mediated activation of the hTERT promoter in normal human somatic cells, we used the specific E2F-1 mutants used in our previous analyses. An E2F-1 mutant (E2F-1 1–88) defective in the transcriptional regulation through the Sp site can support the trans-activation of the hTERT promoter in normal human somatic cells. Thus, E2F-1 may activate the hTERT promoter in normal human somatic cells through some site(s) other than Sp sites. Results from E2F-1 mutant analysis prompted us to search for the region within the hTERT promoter important for the E2F-1-mediated activation of the hTERT promoter in normal human somatic cells. Serial deletion analysis of the hTERT promoter showed that the region between -51 and -88 is important in the transcriptional activation of the hTERT promoter by E2F-1. When we mutated a noncanonical E2F binding site within that region, E2F-1 consistently failed to activate the hTERT promoter in normal human somatic cells. Together, these results suggested that the E2F site is crucial for activation of the hTERT promoter by E2F-1.

4. E2F-1, E2F-2, and E2F-3, but not E2F-4 and E2F-5, repress the hTERT promoter in human tumor cells, whereas all five E2Fs activate the hTERT promoter in normal human somatic cells
E2F-1 belongs to a family of transcription factors composed of six members that can be divided into three subfamilies—E2F-1/2/3, E2F-4/5, and E2F-6—based on structural and functional features. Since most of the cellular genes under transcriptional control by E2F-1 are susceptible to regulation by other E2F family members, we examined the regulatory functions of E2F-2, E2F-3, E2F-4, and E2F-5 in hTERT promoter activity in human normal cells and tumor cells. All five E2F members activated the hTERT promoter in normal human somatic cells to varying degrees (Fig. 1B ). On the contrary, E2F-1, E2F-2, and E2F-3 repressed hTERT promoter activity in human tumor cells (Fig. 1A ). However, E2F-4 and E2F-5 did not repress hTERT promoter activity in these tumor cells, but instead activated the hTERT promoter activity to varying degrees (Fig. 1A ). These results suggest that E2F-1, E2F-2, and E2F-3 in the E2F family specifically exhibit contrasting functions in the hTERT promoter regulation in human normal and tumor cells.

CONCLUSIONS

E2F has been described as a multifaceted transcription factor. E2F can mediate transcriptional activation and repression of target genes and can both promote and inhibit cell proliferation and tumorigenesis. However, the molecular mechanism underlying these paradoxical biological activities has not been explained. Our studies demonstrated contradictory effects of E2F-1/2/3 on the hTERT promoter (Fig. 2 ). Ectopic expression (and up-regulation) of E2F-1/2/3 repressed hTERT promoter activity in human tumor cells but activated the hTERT promoter activity in normal human somatic cells. Expression of the hTERT gene and telomerase activity is important for the progression and maintenance of human tumors, and abrogation of the hTERT function in human tumor cells can induce growth arrest or apoptosis. Thus, it is plausible that the E2F-mediated repression and activation of the hTERT promoter could be related to the tumor-suppressing and oncogenic properties of E2F, respectively (Fig. 2) . Taken together, these results raise an interesting possibility that the paradoxical effects of E2F might be attributed at least in part to its regulation of hTERT promoter activity. These demonstrated bifunctional activities of E2F are also consistent with the view that regulation of E2F could function as one of the fine-tuning mechanisms that tightly control hTERT transcription within a certain range in a variety of proliferative conditions. In highly proliferating tissues, telomerase activity must be carefully regulated within a certain range to avoid tissue atrophy or tumorigenesis.

View larger version (13K): [in this window] [in a new window]   Figure 2. Possible roles of activation and repression of hTERT promoter by E2F in oncogenic and anti-oncogenic pathways. Ectopic expression (and up-regulation) of E2F-1/2/3 represses the hTERT promoter in tumor cells, whereas it activates the hTERT promoter in normal human somatic cells. Since expression of the hTERT gene and telomerase activity is important for the progression and maintenance of human tumors, these observations on hTERT could be directly related to the anti-oncogenic and oncogenic properties of E2F, respectively.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0311fje; to cite this article, use FASEB J. (October 4, 2002) 10.1096/fj.02-0311fje

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