Sp1 is a co-activator with Ets-1, and Net is an important repressor of the transcription of CTP:phosphocholine cytidylyltransferase alpha.
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 CTalpha 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 CTalpha 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 CTalpha 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 CTalpha mRNA. After synchronizing the cell cycle in NIH3T3 cells, CTalpha 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 CTalpha transcription.
Sugimoto
Hiroyuki
H
Department of Molecular Biochemistry, Gunma University Graduate School of Medicine, Maebashi 371-8511, Japan.
Okamura
Koichi
K
Sugimoto
Sayaka
S
Satou
Motoyasu
M
Hattori
Tomoyasu
T
Vance
Dennis E
DE
Izumi
Takashi
T
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002453:Cell Cycle; D002467:Cell Nucleus / Q000737:chemistry; D002522:Chlorocebus aethiops; D019916:Choline-Phosphate Cytidylyltransferase / Q000235:genetics*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D017353:Gene Deletion; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic / Q000235:genetics; D050784:Proto-Oncogene Protein c-ets-1 / Q000235:genetics / Q000502:physiology*; D050783:Proto-Oncogene Proteins c-ets / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection
C086924:Elk3 protein, mouse; C494042:Ets1 protein, mouse; D050784:Proto-Oncogene Protein c-ets-1; D050783:Proto-Oncogene Proteins c-ets; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D016329:Sp1 Transcription Factor; D004247:DNA; D008156:Luciferases; D019916:Choline-Phosphate Cytidylyltransferase