A role for Sp1 in transcriptional regulation of phosphatidylethanolamine N-methyltransferase in liver and 3T3-L1 adipocytes.
Phosphatidylcholine is made in all nucleated mammalian cells via the CDP-choline pathway. Another major pathway for phosphatidylcholine biosynthesis in liver is catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). We have now identified 3T3-L1 adipocytes as a cell culture model that expresses PEMT endogenously. We have found that PEMT mRNA and protein levels increased dramatically in 3T3-L1 cells upon differentiation to adipocytes. 5'-Deletion analysis of the PEMT promoter-luciferase constructs stably expressed in 3T3-L1 adipocytes identified a regulatory region between -471 and -371 bp (relative to the transcriptional start site). Competitive and supershift assays demonstrated binding sites for transcription factors Sp1, Sp3 (-408 to -413), and YY1 (-417 to -420). During differentiation of 3T3-L1 cells to adipocytes, the amount of Sp1 protein decreased by approximately 50% just prior to activation of PEMT. Transduction of 3T3-L1 adipocytes with retrovirus containing Sp1 cDNA demonstrated that Sp1 inhibited PEMT transcriptional activity. Similarly, short hairpin RNA directed against Sp1 in 3T3-L1 adipocytes enhanced PEMT transcriptional activation. Chromatin immunoprecipitation assays confirmed that Sp1 binds to the PEMT promoter, and this interaction decreases upon differentiation to adipocytes. These experiments directly link increased PEMT expression in adipocytes to decreased transcriptional expression of Sp1. In addition, our data established that Sp1 binding was required for tamoxifen-mediated inhibition of Pemt promoter activity.
Cole
Laura K
LK
Department of Biochemistry, University of Alberta, Edmonton, Alberta TG6 2S2, Canada.
Vance
Dennis E
DE
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002454:Cell Differentiation; D017931:DNA Primers / Q000235:genetics; D022781:Hepatocytes / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D050918:Phosphatidylethanolamine N-Methyltransferase / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051705:Sp3 Transcription Factor / Q000378:metabolism; D013629:Tamoxifen / Q000494:pharmacology; D014158:Transcription, Genetic; D050986:YY1 Transcription Factor / Q000378:metabolism
D017931:DNA Primers; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016329:Sp1 Transcription Factor; C497122:Sp3 protein, mouse; D050986:YY1 Transcription Factor; C494699:Yy1 protein, mouse; D013629:Tamoxifen; D051705:Sp3 Transcription Factor; D050918:Phosphatidylethanolamine N-Methyltransferase