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J. Biol. Chem., Vol. 280, Issue 49, 40857-40866, December 9, 2005

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Sp1 Is a Co-activator with Ets-1, and Net Is an Important Repressor of the Transcription of CTP:Phosphocholine Cytidylyltransferase ^*

Hiroyuki Sugimoto, Koichi Okamura, Sayaka Sugimoto, Motoyasu Satou, Tomoyasu Hattori, Dennis E. Vance¶1, and Takashi Izumi2

From the Department of Molecular Biochemistry, Gunma University Graduate School of Medicine, Maebashi 371-8511, Japan, the Department of Biochemistry, Dokkyo University School of Medicine, Mibu 321-0293, Japan, 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) encoded by the Pcyt1a and Pcyt1b genes. Previously, we identified an Ets-1-binding site located at -49/-47 in the promoter of Pcyt1a as an important transcriptional element involved in basal CT transcription (Sugimoto, H., Sugimoto, S., Tatei, K., Obinata, H., Bakovic, M., Izumi, T., and Vance, D. E. (2003) J. Biol. Chem. 278, 19716-19722). In this study, we determined whether or not there were other important elements and binding proteins for basal CT transcription in the Pcyt1a promoter, and if other Ets family proteins bind to the Ets-1-binding site. The results indicate the formation of a ternary complex with Ets-1 binding at -49/-47 and Sp1 binding at -58/-54 of the Pcyt1a promoter that is important for activating CT transcription. When nuclear extracts of COS-7 cells expressing various Ets family repressors were incubated with DNA probes, binding of Net to the probes was observed. Net dose-dependently depressed the promoter-luciferase activity by 98%, even when co-expressed with Ets-1. RNA interference targeting Net caused an increase of endogenous CT mRNA. After synchronizing the cell cycle in NIH3T3 cells, CT mRNA increased at the S-M phase corresponding to an increase of Ets-1 mRNA and a decrease of Net mRNA. These results indicated that Net is an important endogenous repressor for CT transcription.

Received for publication, April 1, 2005 , and in revised form, August 26, 2005.

^* This work was supported in part by grants-in-aid for creative scientific research from the Ministry of Education, Science and Culture, Japan, 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.

¹ 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.: 780-492-8286; Fax:780-492-3383; E-mail: dennis.vance{at}ualberta.ca.

² 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.

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