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 hu man malignant glioma cell lines. Most of the malignant glioma lines that ex press B-FABP also express GFAP, an intermediate filament protein found in m ature astrocytes. We are studying the regulation of the B-FABP gene to dete rmine 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, includin g two nuclear factor I (NFL)-binding sites at -35 to -58 bp (footprint 1, f p1) and -237 to -260 bp (fp3), respectively. Competition experiments, super shift experiments with anti-NFI antibody, and methylation interference expe riments 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 prox imal NFI site is essential for B-FABP promoter activity in transiently tran sfected 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 ob tained with B-FABP(+) extracts. All bands are converted to a faster migrati ng form with potato acid phosphatase treatment, indicating that NFI is diff erentially phosphorylated in B-FABP(+) and B-FABP(-) lines. Our results sug gest that B-FABP expression in malignant glioma lines is determined by the extent of NFI phosphorylation which, in turn, is controlled by a phosphatas e activity specific to B-FABP(+) lines.