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J. Biol. Chem., Vol. 278, Issue 22, 19716-19722, May 30, 2003

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Identification of Ets-1 as an Important Transcriptional Activator of CTP:Phosphocholine Cytidylyltransferase in COS-7 Cells and Co-activation with Transcriptional Enhancer Factor-4^*

Hiroyuki Sugimoto  , Sayaka Sugimoto , Kazuaki Tatei  , Hideru Obinata  , Marica Bakovic ¶, Takashi Izumi   || ** and Dennis E. Vance ||   ¶¶

From the Department of Biochemistry, Gunma University School of Medicine, Maebashi 371-8511, Japan, the Japan Science and Technology Corporation (CREST), Tokyo 113-0033, Japan, the ^¶Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario N1G 2W1 Canada, and the Department of Biochemistry and Canadian Institutes of Health Research Group on Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, Alberta T6G 2S2, Canada

Phosphatidylcholine biosynthesis via the CDP-choline pathway is primarily regulated by CTP:phosphocholine cytidylyltransferase (CT). Transcriptional enhancer factor-4 (TEF-4) enhances the transcription of CT in COS-7 cells by interactions with the basal transcription machinery (Sugimoto, H., Bakovic, M., Yamashita, S., and Vance, D.E. (2001) J. Biol. Chem. 276,12338–12344). To identify the most important transcription factor involved in basal CT transcription, we made CT promoter-deletion and -mutated constructs linked to a luciferase reporter and transfected them into COS-7 cells. The results indicate that an important site regulating basal CT transcription is -53/-47 (GACTTCC), which is a putative consensus-binding site of Ets transcription factors (GGAA) in the opposite orientation. Gel shift analyses indicated the existence of a binding protein for -53/-47 (GACTTCC) in nuclear extracts of COS-7 cells. When anti-Ets-1 antibody was incubated with the probe in gel shift analyses, the intensity of the binding protein was decreased. The binding of endogenous Ets-1 to the promoter probe was increased when TEF-4 was expressed; however, the amount of Ets-1 detected by immunoblotting was unchanged. When cells were transfected with Ets-1 cDNA, the luciferase activity of CT promoter constructs was greatly enhanced. Co-transfection experiments with Ets-1 and TEF-4 showed enhanced expression of reporter constructs as well as CT mRNA. These results suggest that Ets-1 is an important transcriptional activator of the CT gene and that Ets-1 activity is enhanced by TEF-4.

Received for publication, February 14, 2003 , and in revised form, March 13, 2003.

^* This work was supported in part by grants-in-aid for Creative Scientific Research from the Ministry of Education, Science and Culture, Japan, the Naito Foundation, and from the Canadian Institutes of Health Research. 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.

^|| Both authors contributed equally to this research.

Medical Scientist of the Alberta Heritage Foundation for Medical Research and Canada Research Chair in Molecular and Cell Biology of Lipids.

^** To whom correspondence may be addressed. Tel: 81-27-220-7940; Fax: 81-27-220-7948; E-mail: takizumi{at}med.gunma-u.ac.jp. ¶¶ To whom correspondence may be addressed. Tel: 780-492-8286; Fax: 780-492-3383; E-mail: dennis.vance{at}ualberta.ca.

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