Regular article
Role of Ets/Id proteins for telomerase regulation in human cancer cells

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Abstract

Most human cancers express telomerase but its activity is highly variable and regulated by complex mechanisms. Recently, we have proposed that Ets proteins may be important for regulation of telomerase activity in leukemic cells. Here we provide further evidence for the role of Ets family members and related Id proteins in telomerase regulation and characterize the underlying molecular mechanisms. By using PCR-based and gel shift assays we demonstrated specific binding to a core hTERT promoter of Ets2, Fli1, Id2, c-Myc, Mad1, and Sp1 in lysates from subclones of U937 cells. Further analysis of binding of purified proteins and various mutants of the hTERT promoter suggested the existence of a trimolecular Ets–Id2–DNA complex, and Ets inhibitory activity mediated by c-Myc and the Ets binding site on the core hTERT promoter at −293 bp from the transcription initiation site as well as a positive Ets regulatory effect mediate through another Ets binding site at −36 bp. This analysis provided evidence for the existence of negative and positive Ets regulatory site and suggested a complex interplay between Ets/Id family members and c-Myc that may be an important determinant of the diversity of telomerase activity in leukemia and other cancers.

Introduction

Telomerase is a RNA-dependent ribonucleoprotein polymerase that elongates telomeric repeats at the chromosome ends and may have other functions that are currently under investigation Fu et al., 2002, Greider and Blackburn, 1989. Most human cancers express telomerase but its activity is highly variable and regulated by complex mechanisms Cong et al., 2002, Ducrest et al., 2002, Kyo and Inoue, 2002, Shay and Bacchetti, 1997. The transcriptional regulation of the expression of the human telomerase catalytic subunit with reverse transcriptase activity (hTERT) appears to be a major determinant of the telomerase activity regulation Ducrest et al., 2001, Kyo and Inoue, 2002. The hTERT promoter does not contain a TATA box, and the 200- to 400-bp region proximal to the transcription initiation site is responsible for most of its transcriptional activity Cong et al., 1999, Horikawa et al., 1999, Takakura et al., 1999. Multiple E-boxes, Sp1, Ets, and other binding sites for transcription factors are located in this core promoter region. c-Myc binds to these E-boxes through heterodimer formation with Max proteins and directly activates the hTERT transcription Greenberg et al., 1999, Wu et al., 1999. Binding of the c-Myc antagonists, Mad proteins, as Mad/Max complexes decreases the activity of the hTERT promoter Gunes et al., 2000, Kyo et al., 2000, Xu et al., 2001. Sp1 also binds to the core promoter and activates hTERT transcription (Kyo et al., 2000). Although overexpression of c-Myc is frequently observed in a wide variety of tumor types, and in some cases expression levels of c-Myc and Sp1 correlate with the levels of telomerase activity at different stages of transformation (Kyo et al., 2000), some tumors lack c-Myc overexpression despite the presence of telomerase activity. Sp1 protein is abundant in some types of normal cells that do not have high telomerase activity. Thus, the wide divergence of telomerase activity in cancer cells and the cancer-specific telomerase activation may require additional factors yet to be discovered.

Recently, we have found that Ets2 binds specifically to the hTERT promoter and proposed that these proteins and their partners, Id proteins, can play a role in the regulation of telomerase activity (Xiao et al., 2002). Independently, Maida et al. (2002) found that Ets proteins are important for telomerase regulation—they can enhance epidermal growth factor–mediated telomerase up-regulation in cancer cells. The Ets family of transcriptional factors, which is defined by a highly conserved 85-amino acid Ets DNA binding domain (EtsDBD), has more than 30 members and is involved in many important cellular functions, including differentiation, senescence, apoptosis, and tumor suppression Maroulakou and Bowe, 2000, Sharrocks, 2001, Suzuki et al., 1995. Ets family members have more than 200 targets identified, and the number is increasing. Id is a family of proteins that function as positive regulators of cell growth and inhibitors of cell differentiation Lasorella et al., 2001, Zebedee and Hara, 2001. They form heterodimers with other helix–loop–helix transcription factors, which drive cell differentiation and lineage commitment upon expression, thus interfering with their DNA binding ability. Id2 was shown to be the oncogenic effector of N-Myc in human neuroblastoma Lasorella et al., 2000, Lasorella et al., 2002. Myc expression resulted in increased levels of Id2 that can neutralize the function of Rb to block cell cycle progression. Id1 inhibited the senescence induced by high expression of p16INK4a, which was transcriptionally up- regulated by Ets1,2; the inhibitory effect of Id1 on Ets1,2-induced senescence was through direct Id1/Ets association (Ohtani et al., 2001).

The complex interplay of Ets/Id proteins and Myc proteins in the regulation of processes such as cell proliferation, differentiation, and senescence, which correlates to certain extent with telomerase activity, the presence of multiple Ets consensus binding sequences -GGAA- in the hTERT promoter, and recent data Maida et al., 2002, Xiao et al., 2002 indicating a role of Ets proteins in telomerase regulation prompted us to extensively characterize the role of Ets/Id, Myc, and Sp1 proteins in telomerase regulation. Here we provide evidence that Ets family members and their antagonistic Id proteins are directly involved in negative and positive telomerase transcriptional regulation, suggesting the existence of a complex regulatory network controlling telomerase activity in diverse cancer cells.

Section snippets

Cells and viruses

Frozen cell pellets and cell cultures from the NCI 60 cancer cell line panel were obtained from the Biological Test Branch, Developmental Therapeutics Program (DTP), NCI. The human promonocytic histiocytic lymphoma U937 line (Sundstrom and Nilsson, 1976) clones 10 (plus clone) and 17 (minus clone) were provided by H. Moriuchi (National Institute of Allergy and Infectious Diseases (NIAID), NIH, Bethesda, MD) and propagated in RPMI 1640 with 10% FCS (Moriuchi et al., 1997). The human myeloid

Specific interactions between Ets family members, Id2, and a core hTERT promoter

We have previously shown that Ets1 and Ets2 bind to a core hTERT promoter by using a sensitive PCR-based assay for detection of protein–DNA interaction that are below the sensitivity of the gel shift assay. By using the same assay we found that another Ets family member, Fli1, and Id2 also bind specifically to a core hTERT promoter in cell lysates from two subclones of U937 cells (Fig. 1a). The relative amount of bound promoter DNA was proportional to the amount of Fli1 or Id2 in the cell

Discussion

The regulation of telomerase activity is a multifactorial process that involves a number of transcriptional factors (Kyo and Inoue, 2002). This work adds another component to this complex mechanism—Ets proteins and their interplay with c-Myc and Id proteins. Not only do Ets proteins bind to the hTERT promoter but the activity of the different binding sites can inhibit or enhance in dependence on the position of the binding site and the presence of other transcription factors, specifically

Acknowledgements

We are grateful to Dr. Satoru Kyo for helpful comments. We thank Richard Camalier of Biological Test Branch, DTP, NCI for providing frozen pellets and cell cultures from the NCI 60 cell line panel, Dr. Sanjay Phogat for helping with the RT–PCR cloning of Id2, and Dr. David Waugh for constructing the ETS-DBD/ID2 coexpression vector. This work is supported by the NCI intramural program. The publication has been funded in part with Federal funds from the National Cancer Institute, National

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