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J. Biol. Chem., Vol. 280, Issue 32, 28867-28876, August 12, 2005

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Sp1/Sp3-dependent Regulation of Human Telomerase Reverse Transcriptase Promoter Activity by the Bioactive Sphingolipid Ceramide^*

Leslie G. Wooten and Besim Ogretmen

From the Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425

In this study, the roles of Sp1/Sp3 transcription factors in the regulation of the activity of human telomerase reverse transcriptase (hTERT) promoter in response to ceramide were examined in the A549 human lung adenocarcinoma cells. The activity of the N-terminal truncated hTERT promoter, lacking the c-Myc recognition (E-box) region but containing multiple Sp1/Sp3 sites, was also significantly inhibited by C₆-ceramide, indicating a role for ceramide in the regulation of Sp1/Sp3 function. Partial inhibition of Sp1 expression using small interfering RNA resulted in a significant inhibition of the hTERT promoter. Treatment with C₆-ceramide inhibited the trans-activation function of overexpressed Sp1, whereas it induced the repressor effects of exogenous Sp3 on the hTERT promoter. The interaction between Sp1 and hTERT promoter DNA was significantly reduced in response to ceramide as assessed by chromatin immunoprecipitation analysis. In contrast, the promoter DNA-binding activity of Sp3 was slightly increased in response to C₆-ceramide, resulting in the increased ratio of Sp3/Sp1 on the hTERT promoter, which was concomitant with the reduced recruitment of RNA polymerase II to the promoter. Furthermore, mutations of various Sp1/Sp3 recognition sequences significantly attenuated the activity of the promoter in the presence or absence of ceramide, demonstrating the importance of multiple Sp1/Sp3 recognition sites for the promoter activity. Mechanistically, the data demonstrated that C₆-ceramide reduced the acetylation of Sp3 protein and partially blocked the activation of the hTERT promoter by the histone deacetylase inhibitor trichostatin A. The roles of endogenous long chain ceramide generated in response to gemcitabine in the inhibition of hTERT promoter activity and the regulation of Sp3 acetylation were also demonstrated.

Received for publication, November 30, 2004 , and in revised form, June 2, 2005.

^* This work was supported by National Institutes of Health (NIH) Grant CA-88932 and National Science Foundation/EPSCoR Grant EPS-0132573 (to B. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of the minority student research award from the Comprehensive Minority Biomedical Branch of NIH.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Ave., BSB-518E, Charleston, SC 29425. E-mail: ogretmen{at}musc.edu.

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