Regulation of the cell type-specific expression of the gene encoding glial fibrillary acidic protein

Glial fibrillary acidic protein (GFAP) is a specific marker in the development of the central nervous system, where its presence distinguishes astrocytes from other glial cell types. Cell type-specific expression of the gene encoding GFAP is regulated principally at the level of transcription. The regulation of cell type-specific expression of this gene was examined by introducing various deletion mutants of the gene into GFAP expressing (U251 human astrocytoma) and non-expressing (HeLa) cell lines and measuring their transcriptional activity in a RNase protection assay. When the intact GFAP gene with 2 Kb of 5{dollar}spprime{dollar} flanking sequence and 2 Kb of 3{dollar}spprime{dollar} untranslated and 3{dollar}spprime{dollar} flanking sequences was transfected into U251 and HeLa cells, the GFAP specific mRNA was detected only in U251. Replacing sequences downstream from the GFAP transcriptional start site by a heterologous cDNA led to the expression of this chimeric gene in cells that are either permissive or nonpermissive for the expression of GFAP, implying the existence of an intragenic regulatory element. Analysis of the GFAP gene truncated at its 3{dollar}spprime{dollar} end showed the existence of negative regulatory elements located in the first and third introns.; Two positive regulatory elements were identified: the proximal element (located between nucleotide {dollar}-{dollar}94 and {dollar}-{dollar}78) and the distal element (located 1.5kb upstream from the GFAP transcriptional start site). The distal element can confer cell type specificity on a heterologous promoter. Gel retardation assays and DNaseI footprinting showed that an ubiquitous protein factor binds to sequences {dollar}-{dollar}96 to {dollar}-{dollar}80. Deletion of this sequence abolished the expression of GFAP in vivo. An AP-1 site was identified within the distal element which contributed to the cell type-specific expression of GFAP. Thus, the cell type-specific expression of the GFAP gene is regulated by the concerted action of multiple regulatory elements located both upstream and downstream from the transcriptional start site.