N-Myc and SP regulate phosphatidylserine synthase-1 expression in brain and glial cells.
Phosphatidylserine (PS) is an essential constituent of biological membranes and plays critical roles in apoptosis and cell signaling. Because no information was available on transcriptional mechanisms that regulate PS biosynthesis in mammalian cells, we investigated the regulation of expression of the mouse PS synthase-1 (Pss1) gene. The Pss1 core promoter was characterized in vitro and in vivo through gel shift and chromatin immunoprecipitation assays. Transcription factor-binding sites, such as a GC-box cluster that binds Sp1/Sp3/Sp4 and N-Myc, and a degenerate E-box motif that interacts with Tal1 and E47, were identified. Pss1 transactivation was higher in brain of neonatal mice than in other tissues, consistent with brain being a major site of expression of Pss1 mRNA and PSS1 activity. Enzymatic assays revealed that PSS1 activity is enriched in primary cortical astrocytes compared with primary cortical neurons. Site-directed mutagenesis of binding sites within the Pss1 promoter demonstrated that Sp and N-Myc synergistically activate Pss1 expression in astrocytes. Chromatin immunoprecipitation indicated that Sp1, Sp3, and Sp4 interact with a common DNA binding site on the promoter. Reduction in levels of Sp1, Sp3, or N-Myc proteins by RNA interference decreased promoter activity. In addition, disruption of Sp/DNA binding with mithramycin significantly reduced Pss1 expression and PSS1 enzymatic activity, underscoring the essential contribution of Sp factors in regulating PSS1 activity. These studies provide the first analysis of mechanisms that regulate expression of a mammalian Pss gene in brain.
Department of Medicine, University of Alberta, Edmonton, Alberta T6G 2S2, Canada.
Guergana
G
Tasseva
Laura
L
Cole
Jean E
JE
Vance
D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000201:enzymology*; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D001921:Brain / Q000166:cytology*; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000166:cytology / Q000201:enzymology; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology / Q000201:enzymology*; D019884:Nitrogenous Group Transferases / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering; D051381:Rats; D017207:Rats, Sprague-Dawley; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D000073942:T-Cell Acute Lymphocytic Leukemia Protein 1; D058127:Transcription Factor 3 / Q000378:metabolism
D051792:Basic Helix-Loop-Helix Proteins; D019884:Nitrogenous Group Transferases; D011518:Proto-Oncogene Proteins; D016271:Proto-Oncogene Proteins c-myc; D034741:RNA, Small Interfering; D016329:Sp1 Transcription Factor; D000073942:T-Cell Acute Lymphocytic Leukemia Protein 1; D058127:Transcription Factor 3; C489366:Tal1 protein, mouse; C022370:phospholipid serine base exchange enzyme