Regulation of brain fatty acid-binding protein expression by differential phosphorylation of nuclear factor I in malignant glioma cell lines.
Brain fatty acid-binding protein (B-FABP) is expressed in the radial glial cells of the developing central nervous system as well as in a subset of human malignant glioma cell lines. Most of the malignant glioma lines that express B-FABP also express GFAP, an intermediate filament protein found in mature astrocytes. We are studying the regulation of the B-FABP gene to determine the basis for its differential expression in malignant glioma lines. By DNase I footprinting, we have identified five DNA-binding sites located within 400 base pairs (bp) of the B-FABP transcription start site, including two nuclear factor I (NFI)-binding sites at -35 to -58 bp (footprint 1, fp1) and -237 to -260 bp (fp3), respectively. Competition experiments, supershift experiments with anti-NFI antibody, and methylation interference experiments all indicate that the factor binding to fp1 and fp3 is NFI. By site-directed mutagenesis of both NFI-binding sites, we show that the most proximal NFI site is essential for B-FABP promoter activity in transiently transfected malignant glioma cells. Different band shift patterns are observed with nuclear extracts from B-FABP(+) and B-FABP(-) malignant glioma lines, with the latter generating complexes that migrate more slowly than those obtained with B-FABP(+) extracts. All bands are converted to a faster migrating form with potato acid phosphatase treatment, indicating that NFI is differentially phosphorylated in B-FABP(+) and B-FABP(-) lines. Our results suggest that B-FABP expression in malignant glioma lines is determined by the extent of NFI phosphorylation which, in turn, is controlled by a phosphatase activity specific to B-FABP(+) lines.
Bisgrove
D A
DA
Department of Oncology, Cross Cancer Institute and University of Alberta, 11560 University Avenue, Edmonton, Alberta T6G 1Z2, Canada.
Monckton
E A
EA
Packer
M
M
Godbout
R
R
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001923:Brain Chemistry; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I / Q000378:metabolism; D000074026:Fatty Acid Binding Protein 3; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D015972:Gene Expression Regulation, Neoplastic; D005910:Glioma / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051839:NFI Transcription Factors; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D014157:Transcription Factors; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins; D051840:Y-Box-Binding Protein 1
D022762:CCAAT-Enhancer-Binding Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C493408:FABP3 protein, human; C107608:FABP7 protein, human; D000074026:Fatty Acid Binding Protein 3; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D051839:NFI Transcription Factors; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D020033:Protein Isoforms; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D003850:Deoxyribonuclease I; D010749:Phosphoprotein Phosphatases