Astrocytes play important roles in the development, maintenance, and response to injury of the CNS. To understand and manipulate astrocyte function, this project addresses transcriptional control of the GFAP gene, which encodes the astrocyte-specific intermediate filament protein glial fibrillary acidic protein (GFAP). Using transfection of astrocytoma cells with reporter gene constructs, multiple segments within the GFAP basal promoter and upstream region have been identified that interact to control expression of the gene. Site- directed mutagenesis is being used to pinpoint the critical specific sequences within these segments. This will be followed by isolating and studying the regulatory proteins acting at these sites. The activity of reporter constructs is also being examined in transgenic mice. A 2,000 base pair 5-flanking fragment of the GFAP gene has been found sufficient to drive expression of a beta-galactosidase reporter gene in astrocytes throughout the CNS. Deleting from this construct a GFAP segment found unimportant for expression in cultured cells also produces expression exclusively in astrocytes, but activity is largely restricted to the cortex. These results indicate that astrocytes are heterogeneous in gene expression and that different regulatory regions of the GFAP gene are utilized by different types of astrocytes. Projects are also under way to use the GFAP regulatory sequence to express other genes of interest in astrocytes to study brain development and function and to produce models for human diseases. Genes currently being expressed include those encoding the amyloid precursor protein associated with Alzheimer's disease, somatostatin, TGF-beta1, herpes simplex virus thymidylate kinase (HSV- TK), and a putative dominant negative GFAP.
