Identification of Ets-1 as an important transcriptional activator of CTP:phosphocholine cytidylyltransferase alpha in COS-7 cells and co-activation with transcriptional enhancer factor-4.
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 CTalpha 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 CTalpha transcription, we made CTalpha 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 CTalpha 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 CTalpha promoter constructs was greatly enhanced. Co-transfection experiments with Ets-1 and TEF-4 showed enhanced expression of reporter constructs as well as CTalpha mRNA. These results suggest that Ets-1 is an important transcriptional activator of the CTalpha gene and that Ets-1 activity is enhanced by TEF-4.
Sugimoto
Hiroyuki
H
Department of Biochemistry, Gunma University School of Medicine, Maebashi 371-8511, Japan.
Sugimoto
Sayaka
S
Tatei
Kazuaki
K
Obinata
Hideru
H
Bakovic
Marica
M
Izumi
Takashi
T
Vance
Dennis E
DE
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D019916:Choline-Phosphate Cytidylyltransferase / Q000235:genetics / Q000378:metabolism*; D004247:DNA; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D050783:Proto-Oncogene Proteins c-ets; D000091262:TEA Domain Transcription Factors; D014157:Transcription Factors / Q000378:metabolism* / Q000502:physiology*
D004268:DNA-Binding Proteins; C494042:Ets1 protein, mouse; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D000091262:TEA Domain Transcription Factors; C486310:Tead2 protein, mouse; D014157:Transcription Factors; D004247:DNA; D008156:Luciferases; D019916:Choline-Phosphate Cytidylyltransferase