Transcription Factor Pax3 Negatively Regulate The Expression Of Glial Fibrillary Acidic Protein Gene

Objective Glial fibrillary acidic protein (GFAP) is known as a characteristic marker during the important biological processes such as astrocyte maturation and astrocytic reaction. However, the invlolved transcription factors and the mechanisms underlying the transcriptional activation are not well depicted yet. In this study, we aimed to find new transcription factor involved in the regulation of GFAP transcription and its biological effect on the process of astrocyte differentiation from neural stem cell and astrocytic activation.Methods The promoter of GFAP was amplified from rat genomic DNA by PCR and cloned into pGL3- basic vector. The rat expression plasmid encoding Pax3 was constructed as well. The sequences of the 5′end flanking 2000 bp from this transcriptional start site of GFAP gene was used for putative transcriptional factor binding site analyses. The binding of Pax3 to the putative promoter region of the rat GFAP gene was determined by the electrophoretic mobility shift assay(EMSA). The promoter activity of GFAP gene was analyzed using dural luciferase reporter systems in RSC96 cell line. RT-PCR and Western blot assay were used to determine the effect of Pax3 overexpression on the GFAP expression in RSC96 cells. Immunocytochemistry and RT-PCR were further used to demonstrate whether Pax3 negatively regulated GFAP expression during the differentiation and activation of astrocytes. Chromatin immunoprecipitation(ChIP) analysis was applied to reveal whether transcription factor Pax3 could interact with the putative promoter region of the rat GFAP gene during the differentiation of astrocytes.Moreover, overexpression and suppression of Pax3 in NSCs and astrocytes were used to investigate the biological effect of the transcriptional mechanism .Results EMSA assay revealed that transcription factor Pax3 could bind the putative promoter region of the rat GFAP gene in vitro. The luciferase assay showed that the transcriptional activity of rat GFAP promoter was negatively regulated by transcription factors Pax3. RT-PCR and Western blot assay further demonstrated that overexpression of Pax3 negatively regulated GFAP expression in RSC96 cells. Immunocytochemistry and RT-PCR results demonstrated that Pax3 negatively regulated GFAP expression during the differentiation and activation of astrocytes. ChIP analysis confirmed that Pax3 occupies specific sites on the GFAP promoter during the serum- induced astrocyte differentiation from neural stem cells(NSCs). Moreover, overexpression of Pax3 inhibited astrocyte maturation and astrocytic activation, and suppression of Pax3 by shRNA promoted the above process.Conclusion The expression of the rat GFAP gene was negatively regulated by transcription factor Pax3, and this effect may play an important role during the process of astrocyte differentiation from NSCs and astrocytic activation.