This proposal describes experiments that will characterize elements which regulate the astrocyte-specific expression of the glial fibrillary acidic protein (GFAP) gene. This gene encodes the major component of astrocyte intermediate filaments and is spatially and temporally regulated in the developing central nervous system. Astrocytes constitute nearly 40% of the CNS cell population and are responsible for structural, nutritional, developmental and biochemical support of the neurons. They also play an important role in CNS response to disease and injury and give rise to a significant portion of CNS tumors in adults. Understanding the mechanisms which regulate astrocyte growth and differentiation is important for future studies of CNS development and glial cell transformation. Transcriptional regulation of the GFAP gene can serve as a system for the identification of molecular signals which modulate glial cell differentiation: the gene is expressed upon differentiation of immature glial cells to mature astrocyte suggesting that factors regulating its transcription play a major role in astroglial differentiation. A 5'-flanking region of the gene was cloned land is able to direct glial-specific reporter gene expression in transfected cells. Transfection of deletion constructs revealed that a cell-specific regulatory region resides upstream of the gene promoter. Primer extension analysis verified transcriptional regulation of constructs in transfected cells. An upstream cis-acting element has been identified. Gel shift assays have identified glial-specific trans-acting factor which bind to the cis-acting element. DNA sequence analysis, coupled with DNase assays, will , locate and identify more precisely the sequence motifs which bind to cell-specific trans-acting factors. cDNA libraries was prepared from mRNA of transformed glial cells, and a similar library will be made from primary astrocytes. Sequence motifs which bind to cell-specific nuclear factors will be used for screening the cDNA expression libraries and isolating cDNA clones which encode DNA-binding proteins. These clones will be used for studies of their role in the process of astrocyte differentiation and oncogenic transformation.
